Die Wirkung einiger Cytostatica auf die DNA Synthese in einem oncogenen RNA Virus

Sowohl durch matrizeninaktivierende Cytostatica als auch durch Cytostatica, die am Enzymprotein angreifen, konnen in vitro die RNA-abhangige DNA-Polymerase und die DNA-abhangige DNA-Polymerase aus RML-Viren gehemmt werden. Von den getesteten Cytostatica, die die Matrize inaktivieren, wirken Daunoblastin (=Daunomycin) und Adriamycin selektiv hemmend auf die RNA-abhangige DNA-Polymerase, wahrend Olivomycin, Chromomycin und Actinomycin D bereits in niedrigen Konzentrationen die DNA-abhangige DNA-Polymerase aus RML-Viren selektiv inhibieren. Durch 2 Cytostatica konnen die enzymatischen Reaktionen der DNA-Polymerasen aus RML-Viren, durch Angriff am Enzymprotein beeinflust werden: Demethyl-Rifamp…

Purification and characterization of two exopolyphosphatases from the marine sponge Tethya lyncurium

Abstract Two exopolyphosphatases (exopolyphosphatase I and II; EC 3.6.1.11) which release orthophosphate from inorganic polyphosphates have been detected and purified for the first time from a marine sponge, Tethya lyncurium . Exopolyphosphatase I has a molecular mass of 45 kDa, a pH optimum of 5.0 and does not require divalent cations for activity, while exopolyphosphatase II has a molecular mass of 70 kDa, a pH optimum of 7.5 and displays optimal activity in the presence of Mg 2+ ions. Final purification of the enzymes could be achieved by affinity chromatography on polyphosphate-modified zirconia. The mode of action of both enzymes was found to be processive. Orthophosphate is the sole p…

Bleomycin, a selective inhibitor of DNA-dependent DNA polymerase from oncogenic RNA viruses.

Abstract Bleomycin, an antibiotic, inhibits the DNA-dependent DNA polymerase from Rauscher murine leukemia virus. Higher concentrations of BLM ∗ are required to inhibit it's RNA-dependent DNA polymerase. These inhibition effects of the non-competitive type are not altered by preincubation of the DNA with BLM. Under comparable conditions neither the DNA-dependent DNA polymerase activity from E. coli and mouse liver nor the DNA-dependent RNA polymerase activity from mouse lymphoma cells are affected by BLM.

Phylogenetic relationship of ubiquitin repeats in the polyubiquitin gene from the marine sponge Geodia cydonium

Ubiquitin is a 76-residue protein which is highly conserved among eukaryotes. Sponge (Porifera) ubiquitin, isolated from Geodia cydonium, is encoded by a gene (termed GCUBI) with six repeats, GCUBI-1 to GCUBI-6. All repeat units encode the same protein (with one exception: GCUBI-4 encodes ubiquitin with a change of Leu to Val at position 71). On the nt level the sequences of the six repeats differ considerably. All changes (except in GCUBI-4) are silent substitutions, which do not affect the protein structure. However, there is one major difference between the repeats: Codons from both codon families (TCN and AGPy) are simultaneously used for the serine at position 65. Using this characteri…

Evolutionary relationships of the metazoan βγ–crystallins, including that from the marine spongeGeodia cydonium

beta gamma-crystallins are one major component of vertebrate lenses. Here the isolation and characterization of a cDNA, coding for the first beta gamma-crystallin molecule from an invertebrate species, the marine sponge Geodia cydonium, is described. The size of the transcript as determined by Northern blotting was 0.7 kb in length. The deduced amino acid sequence consists of 163 aa residues and comprises four repeated motifs which compose the two domains of the beta gamma-crystallin. Motif 3 contains the characteristic beta gamma-crystallin 'Greek key' motif signature, while in each of the three other repeats, one aa residue is replaced by an aa with the same physico-chemical property. The…

Optical properties of in-vitro biomineralised silica

Silicon is the second most common element on the Earth's crust and its oxide (SiO(2)) the most abundant mineral. Silica and silicates are widely used in medicine and industry as well as in micro- and nano-optics and electronics. However, the fabrication of glass fibres and components requires high temperature and non-physiological conditions, in contrast to biosilica structures in animals and plants. Here, we show for the first time the use of recombinant silicatein-α, the most abundant subunit of sponge proteins catalyzing biosilicification reactions, to direct the formation of optical waveguides in-vitro through soft microlithography. The artificial biosilica fibres mimic the natural spon…

Anti-HIV-1 activity of inorganic polyphosphates.

Human blood plasma, serum, peripheral blood mononuclear cells, and erythrocytes contain significant amounts of inorganic polyphosphates (ranging from 53 to 116 microM, in terms of phosphate residues). Here we demonstrate that at higher concentrations linear polyphosphates display cytoprotective and antiviral activity. Sodium tetrapolyphosphate and the longer polymers, with average chain lengths of 15, 34, and 91 phosphate residues, significantly inhibited human immunodeficiency virus type 1 (HIV-1) infection of cells in vitro at concentrations > or = 33.3 microg/ml (> or = 283-324 microM phosphate residues), whereas sodium tripolyphosphate was ineffective. In the tested concentration range,…

Effect of hypoosmotic stress by low salinity acclimation of Mediterranean mussels Mytilus galloprovincialis on biological parameters used for pollution assessment

In the present study, we investigated the progressive acclimation of the mussel Mytilus galloprovincialis to different reduced seawater (SW) salinities and its effect on several biochemical markers and biotests. Mussels were purchased from a local mariculture facility during summer (SW temperature 27 degrees C, salinity 37.5 psu) and winter (13 degrees C, 37 psu) seasons, and transferred to the laboratory for acclimation to reduced SW salinities (37, 28, 18.5 and 11 psu). At the beginning and at the end of acclimation processes tests of mussel survival in air were provided. After 14 days of acclimation the DNA integrity, p38-MAPK activation, metallothionein induction, oxygen consumption rat…

Polyclonal antibodies to mannan from yeast also recognize the carbohydrate structure of gp120 of the AIDS virus: an approach to raise neutralizing antibodies to HIV-1 infection in vitro.

This study initiates a new method of developing an antigen which might be useful in the prevention of HIV-1 infection. Using a mannan preparation from Saccharomyces cerevisiae neutralizing antiserum was raised in rabbits which prevents HIV-1 infection in vitro up to a titre of 1:128. The corresponding antibody preparation neutralized the in vitro infectivity down to a concentration of 5 micrograms/ml. Analytical studies suggest that the antibodies are directed against the mannose residues of the HIV-1 glycoprotein (gp) 120 and its precursor gp 160.

Bioactive metabolites from the endophytic fungus Ampelomyces sp. isolated from the medicinal plant Urospermum picroides

Extracts of cultures grown in liquid or on solid rice media of the fungal endophyte Ampelomyces sp. isolated from the medicinal plant Urospermum picroides exhibited considerable cytotoxic activity when tested in vitro against L5178Y cells. Chromatographic separation yielded 14 natural products that were unequivocally identified based on their 1H and 13C NMR as well as mass spectra and comparison with previously published data. Six compounds (2, 4, 5, 7, 9 and 11) were natural products. Both fungal extracts differed considerably in their secondary metabolites. The extract obtained from liquid cultures afforded a pyrone (2) and sulfated anthraquinones (7 and 9) along with the known compounds …

Cytotoxic 14-Membered Macrolides from a Mangrove-Derived Endophytic Fungus, Pestalotiopsis microspora.

Seven new 14-membered macrolides, pestalotioprolides C (2), D-H (4-8), and 7-O-methylnigrosporolide (3), together with four known analogues, pestalotioprolide B (1), seiricuprolide (9), nigrosporolide (10), and 4,7-dihydroxy-13-tetradeca-2,5,8-trienolide (11), were isolated from the mangrove-derived endophytic fungus Pestalotiopsis microspora. Their structures were elucidated by analysis of NMR and MS data and by comparison with literature data. Single-crystal X-ray diffraction analysis was used to confirm the absolute configurations of 1, 2, and 10, while Mosher's method and the TDDFT-ECD approach were applied to determine the absolute configurations of 5 and 6. Compounds 3-6 showed signif…

A urochordate putative homolog of human EB1, the protein which binds APC1

Abstract The human EB1 protein has been cloned by virtue of its interaction with the C-terminus of the APC (adenomatous polyposis coli) protein, whose C-terminal truncated forms have been shown to accompany sporadic and familial forms of colorectal cancer. We have cloned a putative EB1 homolog from Botryllus schlosseri (Urochordata, Ascidiacea). The deduced protein is 287 amino acids long, and is identical with 48% of the residues in human EB1 and 24–25% in two yeast hypothetical proteins. We propose that such a high degree of conservation among EB1 homologs is indicative of an essential regulatory mechanism in eukaryotic cells.

Cultivation of primmorphs from the marine sponge Suberites domuncula: morphogenetic potential of silicon and iron.

Abstract Marine demosponges (phylum Porifera) are rich sources for potent bioactive compounds. With the establishment of the primmorph system from sponges, especially from Suberites domuncula , the technology to cultivate sponge cells in vitro improved considerably. This progress was possible after the elucidation that sponges are provided with characteristic metazoan cell adhesion receptors and extracellular matrix molecules which allow their cells a positioning in a complex organization pattern. This review summarizes recent data on the cultivation of sponges in aquaria and—with main emphasis—of primmorphs in vitro. It is outlined that silicon and Fe(+++) contribute substantially to the f…

Bioactive pyrrole alkaloids isolated from the Red Sea : marine sponge Stylissa carteri

Abstract Fifteen pyrrole alkaloids were isolated from the Red Sea marine sponge Stylissa carteri and investigated for their biological activities. Four of them were dibrominated [(+) dibromophakelline, Z-3-bromohymenialdisine, (±) ageliferin and 3,4-dibromo-1H-pyrrole-2-carbamide], nine compounds were monobrominated [(−) clathramide C, agelongine, (+) manzacidin A, (−) 3-bromomanzacidin D, Z-spongiacidin D, Z-hymenialdisine, 2-debromostevensine, 2-bromoaldisine and 4-bromo-1H-pyrrole-2-carbamide)] and finally, two compounds were non-brominated derivatives viz., E-debromohymenialdisine and aldisine. The structure elucidations of isolated compounds were based on 1D & 2D NMR spectroscopic …

Heat shock protein Hsp70 expression and DNA damage in Baikalian sponges exposed to model pollutants and wastewater from Baikalsk Pulp and Paper Plant

Abstract Lake Baikal, a unique habitat for a great number of endemic species, is the largest freshwater reservoir in the world which is still largely unaffected by anthropogenic pollution, except for some shore regions with industrial activity. The expressions of a biomarker of exposure (heat shock protein Hsp70) and a biomarker of effect (DNA single-strand breaks) were measured for the first time in endemic Baikalian sponge species (Baikalospongia intermedia, Lubomirskia fusifera, and Lubomirskia abietina). Tissue cubes of B. intermedia and dissociated cells of L. fusifera and L. abietina reacted to temperature stress (10–16 °C above ambient temperature) with a time-dependent increase in e…

Modular Small Diameter Vascular Grafts with Bioactive Functionalities.

We report the fabrication of a novel type of artificial small diameter blood vessels, termed biomimetic tissue-engineered blood vessels (bTEBV), with a modular composition. They are composed of a hydrogel scaffold consisting of two negatively charged natural polymers, alginate and a modified chitosan, N,O-carboxymethyl chitosan (N,O-CMC). Into this biologically inert scaffold two biofunctionally active biopolymers are embedded, inorganic polyphosphate (polyP) and silica, as well as gelatin which exposes the cell recognition signal, Arg-Gly-Asp (RGD). These materials can be hardened by exposure to Ca(2+) through formation of Ca(2+) bridges between the polyanions, alginate, N,O-CMC, and polyP…

EVOLUTION OF THE INNATE AND ADAPTIVE IMMUNE SYSTEMS

Porifera (sponge) form the lowest metazoan phylum and share a common ancestor with other metazoan phyla. In the present study, it is reported that sponges possess molecules that are similar in structure to those molecules involved in the immune system in mammals. Experiments with the marine sponges Geodia cydonium and Suberites domuncula have been performed on tissue (auto- and allografting) as well as on a cellular level. The studies revealed that sponges are provided with elements of the mammalian innate immune system, such as molecules containing scavenger receptor cysteine-rich domains. Furthermore, macrophage-derived cytokine-like molecules have been identified that are up-regulated du…

Spatial and temporal dynamics of bacterioplankton community composition in a subtropical dammed karst river of southwestern China.

Abstract River damming influences the hydro‐physicochemical variations in karst water; however, such disruption in bacterioplankton communities has seldom been studied. Here, three sampling sites (city‐river section, reservoir area, and outflow area) of the Ca2+–Mg2+–HCO 3 −–SO 4 2− water type in the dammed Liu River were selected to investigate the bacterioplankton community composition as identified by high‐throughput 16S rRNA gene sequencing. In the dammed Liu River, thermal regimes have been altered, which has resulted in considerable spatial‐temporal differences in total dissolved solids (TDSs), oxidation‐reduction potential (Eh), dissolved oxygen (DO), and pH and in a different microe…

Common genetic denominators for Ca++-based skeleton in Metazoa: role of osteoclast-stimulating factor and of carbonic anhydrase in a calcareous sponge.

Calcium-based matrices serve predominantly as inorganic, hard skeletal systems in Metazoa from calcareous sponges [phylum Porifera; class Calcarea] to proto- and deuterostomian multicellular animals. The calcareous sponges form their skeletal elements, the spicules, from amorphous calcium carbonate (ACC). Treatment of spicules from Sycon raphanus with sodium hypochlorite (NaOCl) results in the disintegration of the ACC in those skeletal elements. Until now a distinct protein/enzyme involved in ACC metabolism could not been identified in those animals. We applied the technique of phage display combinatorial libraries to identify oligopeptides that bind to NaOCl-treated spicules: those oligop…

Two-Armed Activation of Bone Mineral Deposition by the Flavones Baicalin and Baicalein, Encapsulated in Polyphosphate Microparticles

In this study, we investigated the effect of the two flavonoids, baicalin (baicalein 7-O-[Formula: see text]- d-glucuronic acid) and its aglycone, baicalein (5,6,7-trihydroxyflavone), after encapsulation into amorphous calcium polyphosphate (Ca-polyP) microparticles on mineralization of primary human osteoblasts (phOSB). Both flavonoids, which come from root extracts of Scutellaria baicalensis Georgi, are used in Traditional Chinese Medicine, and are nontoxic in cells up to a concentration of 3[Formula: see text][Formula: see text]g/ml. The morphogenetically active, energy-rich Ca-polyP particles with a stoichiometric P:Ca ratio of 1:2 are degraded by cellular alkaline phosphatase (ALP) to…

New Target Sites for Treatment of Osteoporosis

In the last few years, much progress has been achieved in the discovery of new drug target sites for treatment of osteoporotic disorders, one of the main challenging diseases with a large burden for the public health systems. Among these new agents promoting bone formation, shifting the impaired equilibrium between bone anabolism and bone catabolism in the direction of bone synthesis are inorganic polymers, in particular inorganic polyphosphates that show strong stimulatory effects on the expression of bone anabolic marker proteins and hydroxyapatite formation. The bone-forming activity of these polymers can even be enhanced by combination with certain small molecules like quercetin, or if …

From Single Molecules to Nanoscopically Structured Functional Materials: Au Nanocrystal Growth on TiO2 Nanowires Controlled by Surface-Bound Silicatein

Novel photoreception system in sponges?

Abstract Sponges (phylum Porifera) of the classes Hexactinellida and Demospongiae possess a skeleton composed of siliceous spicules, which are synthesized enzymatically. The longest spicules are found among the Hexactinellida, with the stalk spicules (length: 30 cm; diameter: 300 μm) of Hyalonema sieboldi as prominent examples. These spicules are constructed around a central axial filament, which is formed by approximately 40 siliceous layers. The stratified spicules function as optical glass fibers with unique properties. If free-spaced coupled with a white light source (WLS), the entire fiber is illuminated. Special features of the light transmission: (i) only wavelengths between 615 and …

Biocalcite and Carbonic Acid Activators

Based on evolution of biomineralizing systems and energetic considerations, there is now compelling evidence that enzymes play a driving role in the formation of the inorganic skeletons from the simplest animals, the sponges, up to humans. Focusing on skeletons based on calcium minerals, the principle enzymes involved are the carbonic anhydrase (formation of the calcium carbonate-based skeletons of many invertebrates like the calcareous sponges, as well as deposition of the calcium carbonate bioseeds during human bone formation) and the alkaline phosphatase (providing the phosphate for bone calcium phosphate-hydroxyapatite formation). These two enzymes, both being involved in human bone for…

The 2′-5′-oligoadenylate synthetase in the lowest metazoa: isolation, cloning, expression and functional activity in the sponge Lubomirskia baicalensis

Aquatic animals, especially filter feeders such as sponges [phylum Porifera], are exposed to a higher viral load than terrestrial species. Until now, the antiviral defense system in the evolutionary oldest multicellular organisms, sponges, is not understood. One powerful protection of vertebrates against virus infection is mediated by the interferon (IFN)-inducible 2'-5'-oligoadenylate synthetase [(2-5)A synthetase] system. In the present study we cloned from the freshwater sponge Lubomirskia baicalensis a cDNA encoding a 314 aa long ORF with a calculated size of 35748Da, a putative (2-5)A synthetase, and raised antibodies against the recombinant protein. The native enzyme was identified in…

2′,5′-oligoadenylate synthetase from a lower invertebrate, the marine sponge Geodia cydonium, does not need dsRNA for its enzymatic activity

AbstractRecently, the presence of 2′,5′-linked oligoadenylates and a high 2′,5′-oligoadenylate synthetase activity were discovered in a lower invertebrate, the marine sponge Geodia cydonium. It has been demonstrated that mammalian 2–5A synthetase isozymes require a dsRNA cofactor for their enzymatic activity. Our results show that, unlike mammalian 2–5A synthetases, the 2–5A synthetase from the sponge acts in a dsRNA-independent manner in vitro. A prolonged incubation of the G. cydonium extract with a high concentration of a micrococcal nuclease had no effect on the activity of the 2–5A synthetase. At the same time, the micrococcal nuclease was effective within 30 min in degrading dsRNA nee…

Formation of silicones mediated by the sponge enzyme silicatein-α

The sponge-restricted enzyme silicatein-α catalyzes in vivo silica formation from monomeric silicon compounds from sea water (i.e. silicic acid) and plays the pivotal role during synthesis of the siliceous sponge spicules. Recombinant silicatein-α, which was cloned from the demosponge Suberites domuncula (phylum Porifera), is shown to catalyze in vitro condensation of alkoxy silanes during a phase transfer reaction at neutral pH and ambient temperature to yield silicones like the straight-chained polydimethylsiloxane (PDMS). The reported condensation reaction is considered to be the first description of an enzymatically enhanced organometallic condensation reaction.

Bleomycin, an Antibiotic That Removes Thymine from Double-Stranded DNA

Publisher Summary This chapter reviews that bleomycins are members of a new class of DNA-modifying agents, the quasi-enzymes. In in vitro systems, bleomycin first removes thymines from native DNA by hydrolysis of the N-glycosidic bonds without modifying the deoxyribose moiety. In a second step, single-strand scissions occur at the sites of the nonglycosidic deoxyribose moieties, resulting in the formation of 3'-OH and 5'-P termini. It is suggested that bleomycin is bound to DNA by interaction of the positively charged terminal amine moiety with the negatively charged phosphate group in DNA; intercalation seems to be involved in binding. Bleomycin is inactivated by copper and zinc ions, prob…

Molecular cloning of a tyrosine kinase gene from the marine spongeGeodia cydonium: a new member belonging to the receptor tyrosine kinase class II family

We have isolated and characterized a cDNA from the marine sponge Geodia cydonium coding for a new member of the tyrosine protein kinase (TK) family. The cDNA encodes a protein of M(r) = 68,710, termed GCTK, which is homologous to class II receptor tyrosine kinases (RTKs). GCTK contains conserved amino acids (aa) characteristic of all protein kinases, and the sequences DLATRN and PIRWMATE which are highly specific for TKs. Furthermore, the sequence N-L-Y-x(3)-Y-Y-R is highly homologous to the sequence D-[LIV]-Y-x(3)-Y-Y-R found only in class II RTKs. The sponge TK, when compared with mammalian class II RTKs, shows maximum 31% homology in the TK domain indicating that this the oldest member o…

Chemosensitizers of the multixenobiotic resistance in amorphous aggregates (marine snow): etiology of mass killing on the benthos in the Northern Adriatic?

Periodically appearing amorphous aggregates, 'marine snow', are formed in the sea and if settled as mars on the sea bottom cause death of benthic metazoans. Especially those animals are killed which are sessile filter feeders, e.g, sponges, mussels, or Anthozoa. The etiology of the toxic principle(s) is not yet well understood. Gel-like marine snow aggregates occurred in the Northern Adriatic during summer 1997. Samples of these aggregates were collected during the period July to September and the outer as well as the inner zones were analyzed for (i) cell toxicity, and (ii) chemosensitizing activity of the multixenobiotic resistance (MXR)mechanism. Organic extracts were prepared an…

Towards an understanding of the molecular basis of immune responses in sponges: The marine demospongeGeodia cydonium as a model

The phylogenetic position of the phylum Porifera (sponges) is near the base of the kingdom Metazoa. During the last few years, not only rRNA sequences but, more importantly, cDNA/genes that code for proteins have been isolated and characterized from sponges, in particular from the marine demosponge Geodia cydonium. The analysis of the deduced amino acid sequences of these proteins allowed a molecular biological approach to the question of the monophyly of the Metazoa. Molecules of the extracellular matrix/basal lamina, with the integrin receptor, fibronectin, and galectin as prominent examples, and of cell-surface receptors (tyrosine kinase receptor), elements of sensory systems (crystallin…

IDENTIFICATION OF LECTINS IN THE KINETIDS OFTETRAHYMENA PYRIFORMIS

Previously we described lectin-like molecules in the ciliate Tetrahymena pyriformis; by application of synthetic neoglycoconjugates it is now shown that T. pyriformis contains considerable amounts of both a beta-D-glucose- and a lactose-specific lectin. No evidence for the presence of alpha-D-mannose-, alpha-D-galactose- or of alpha-L-fucose-specific lectins could be obtained. The two lectins, identified in T. pyriformis, are associated with the kinetids. During cell division the lectins disappear or become masked in the fission furrow. Therefore, we assume that these lectins are involved in the organization of the distribution pattern of the kinetids during cell division perhaps due to lec…

Sarcophytolide: a new neuroprotective compound from the soft coral Sarcophyton glaucum

Abstract Bioactivity-guided fractionation of an alcohol extract of the soft coral Sarcophyton glaucum collected from the intertidal areas and the fringing coral reefs near Hurghada, Red Sea, Egypt resulted in the isolation of a new lactone cembrane diterpene, sarcophytolide. The structure of this compound was deduced from its spectroscopic data and by comparison of the spectral data with those of known closely related cembrane-type compounds. In antimicrobial assays, the isolated compound exhibited a good activity towards Staphylococcus aureus, Pseudomonas aeruginosa , and Saccharomyces cerevisiae. Sarcophytolide was found to display a strong cytoprotective effect against glutamate-induced …

Engineering a morphogenetically active hydrogel for bioprinting of bioartificial tissue derived from human osteoblast-like SaOS-2 cells.

Abstract Sodium alginate hydrogel, stabilized with gelatin, is a suitable, biologically inert matrix that can be used for encapsulating and 3D bioprinting of bone-related SaOS-2 cells. However, the cells, embedded in this matrix, remain in a non-proliferating state. Here we show that addition of an overlay onto the bioprinted alginate/gelatine/SaOS-2 cell scaffold, consisting of agarose and the calcium salt of polyphosphate [polyP·Ca 2+ -complex], resulted in a marked increase in cell proliferation . In the presence of 100 μ m polyP·Ca2+ -complex, the cells proliferate with a generation time of approximately 47–55 h. In addition, the hardness of the alginate/gelatin hydrogel substantially i…

A Microplate Assay for DNA Damage Determination (Fast Micromethod)in Cell Suspensions and Solid Tissues

Abstract A rapid and convenient procedure for DNA damage determination in cell suspensions and solid tissues on single microplates was developed. The procedure is based on the ability of commercially available fluorochromes to interact preferentially with dsDNA in the presence of ssDNA, RNA, and proteins at high pH (>12.0), thus allowing direct measurements of DNA denaturation without sample handling or stepwise DNA separations. The method includes a simple and rapid 40-min sample lysis in the presence of EDTA, SDS, and high urea concentration at pH 10, followed by time-dependent DNA denaturation at pH 12.4 after NaOH addition. The time course and the extent of DNA denaturation is followed …

Bioactive and biodegradable silica biomaterial for bone regeneration.

Biosilica, a biocompatible, natural inorganic polymer that is formed by an enzymatic, silicatein-mediated reaction in siliceous sponges to build up their inorganic skeleton, has been shown to be morphogenetically active and to induce mineralization of human osteoblast-like cells (SaOS-2) in vitro. In the present study, we prepared beads (microspheres) by encapsulation of β-tricalcium phosphate [β-TCP], either alone (control) or supplemented with silica or silicatein, into the biodegradable copolymer poly(d,l-lactide-co-glycolide) [PLGA]. Under the conditions used, ≈5% β-TCP, ≈9% silica, and 0.32μg/mg of silicatein were entrapped into the PLGA microspheres (diameter≈800μm). Determination of …

Evidence for involvement of a nuclear envelope-associated RNA helicase activity in nucleocytoplasmic RNA transport

It seems well established that translocation of at least some mRNAs through the nuclear pore is (1) an energy-dependent process, and (2) dependent on the presence of the poly(A) segment attached to most mRNA species. We describe that RNA helicase (RNA duplex unwindase) activity is present in a nuclear envelope (NE) preparation, which also appears to be involved in nucleocytoplasmic RNA transport. This activity unwinds RNA: RNA hybrids. The helicase has a pH optimum of 7.5 and a temperature optimum of 30 degrees C. Applying the sealed NE vesicle system, it was shown that duplex RNA species are readily released from the vesicles in an unidirectional manner, in contrast to single-stranded RNA,…

Cytoprotective effect of NMDA receptor antagonists on prion protein (PrionSc)-induced toxicity in rat cortical cell cultures

Rat cortical cells were incubated with the Scrapie prion protein, PrionSc. At concentrations of 3 ng/ml of PrionSc and higher, the viability of the cells decreased significantly after a 12-h incubation period. Simultaneously, the degree of DNA fragmentation increased. In control experiments with antibodies against PrionSc, PrionSc lost its deleterious effect on neurons. PrionSc did not affect the viability of astrocytes. Drugs known to block NMDA receptor channels, such as memantine (1-amino-3,5-dimethyl-adamantane) (Mem), its analogue 1-N-methylamino-3,5-dimethyl-adamantane as well as (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) prevented the effect of …

Molecular evolution of the metazoan protein kinase C multigene family

Protein kinases C (PKCs) comprise closely related Ser/Thr kinases, ubiquitously present in animal tissues ; they respond to second messengers, e.g., Ca2+ and/or diacylglycerol, to express their activities. Two PKCs have been sequenced from Geodia cydonium, a member of the lowest multicellular animals, the sponges (Porifera). One sponge G. cydonium PKC, GCPKC1, belongs to the ''novel'' (Ca2+-independent) PKC (nPKC) subfamily while the second one, GCPKC2, has the hall-marks of the ''conventional'' (Ca2+-dependent) PKC (cPKC) subfamily. The alignment of the Ser/Thr catalytic kinase domains, of the predicted aa sequences for these cDNAs with respective segments from previously reported sequence…

Immunoglobulin-like domain is present in the extracellular part of the receptor tyrosine kinase from the marine sponge Geodia cydonium.

We have isolated and characterized two cDNAs from the marine sponge Geodia cydonium coding for a new member of a receptor tyrosine kinase of class II. The deduced amino acid sequence shows two characteristic domains: (i) the tyrosine kinase domain; and (ii) and immunoglobulin-like domain. The latter part shows high homology to the vertebrate C2 type immunoglobulin domain. This result demonstrates that immunoglobulin domains are not recent achievements of higher animals but exist also in those animals which have diverged from other organisms about 800 million years ago.

RNA dependent DNA polymerase in cells of xeroderma pigmentosum

Abstract Cells from X.P. ∗ skin contain an RNA dependent DNA polymerase, while in cells from normal skin this enzyme is lacking. This finding stimulates the thought that carcinogenesis in X.P. cells is due to an infection with an oncogenic RNA virus.

Shuttling of the autoantigen La between nucleus and cell surface after uv irradiation of human keratinocytes.

During the past years we have established that the nuclear autoantigen La shuttles between the nucleus and the cytoplasm in tumor cells after inhibition of transcription or virus infection. We reinvestigated this shuttling using primary human keratinocytes from both healthy donors and patients with xeroderma pigmentosum. Ultraviolet irradiation resulted in both an inhibition of transcription and a translocation of La protein from the nucleus to the cytoplasm. After a prolonged inhibition of transcription La protein relocated into the nucleus and assembled with nuclear storage regions. The uv-induced shuttling included a translocation to the cell surface, where La protein colocalized with ep…

Dithiodiketopiperazine derivatives from endophytic fungi Trichoderma harzianum and Epicoccum nigrum

A new epidithiodiketopiperazine (ETP), pretrichodermamide G (1), along with three known (epi)dithiodiketopiparazines (2-4) were isolated from cultures of Trichoderma harzianum and Epicoccum nigrum, endophytic fungi associated with medicinal plants Zingiber officinale and Salix sp., respectively. The structure of the new compound (1) was established on the basis of spectroscopic data, including 1D/2D NMR and HRESIMS. The isolated compounds were investigated for their antifungal, antibacterial and cytotoxic potential against a panel of microorganisms and cell lines. Pretrichodermamide A (2) displayed antimicrobial activity towards the plant pathogenic fungus Ustilago maydis and the human path…

Sponge aggregation factor and sponge hemagglutinin: possible relationships between two different molecules.

Abstract A lectin from the marine sponge GEODIA CYDONIUM was isolated and characterized. GEODIA lectin (GL) agglutinates human red blood cells irrespective of the ABO blood group and precipitates with a variety of D -galactose containing glycosubstances, i.e. certain snail galactans, bovine erythrocyte glycoprotein and PNEUMOCOCCUS type XIV polysaccharide. The only simple sugars inhibiting the GL-mediated hemagglutination were lactose and n -acetyl- D -galactosamine. GL was purified by affinity chromatography on Sepharose 4B almost to homogeneity as tested by polyacrylamide disc gel electrophoresis. Positive staining of the lectin band with Coomassie brilliant blue and PAS suggest that GL i…

Identification of a silicatein(-related) protease in the giant spicules of the deep-sea hexactinellid Monorhaphis chuni.

SUMMARYSilicateins, members of the cathepsin L family, are enzymes that have been shown to be involved in the biosynthesis/condensation of biosilica in spicules from Demospongiae (phylum Porifera), e.g. Tethya aurantium and Suberites domuncula. The class Hexactinellida also forms spicules from this inorganic material. This class of sponges includes species that form the largest biogenic silica structures on earth. The giant basal spicules from the hexactinellids Monorhaphis chuni and Monorhaphis intermedia can reach lengths of up to 3 m and diameters of 10 mm. The giant spicules as well as the tauactines consist of a biosilica shell that surrounds the axial canal, which harbours the axial f…

Silicatein conjugation inside nanoconfined geometries through immobilized NTA–Ni(ii) chelates

The chemical modification and bioconjugation processes inside confined geometries by His-tagged silicatein promote sensitive changes in the polarity and surface charge density that mainly contribute to the ionic current rectification properties of the single conical nanopores.

Biomimetic Alginate/Gelatin Cross-Linked Hydrogels Supplemented with Polyphosphate for Wound Healing Applications

In the present study, the fabrication of a biomimetic wound dressing that mimics the extracellular matrix, consisting of a hydrogel matrix composed of non-oxidized and periodate-oxidized marine alginate, was prepared to which gelatin was bound via Schiff base formation. Into this alginate/oxidized-alginate-gelatin hydrogel, polyP was stably but reversibly integrated by ionic cross-linking with Zn2+ ions. Thereby, a soft hybrid material is obtained, consisting of a more rigid alginate scaffold and porous structures formed by the oxidized-alginate-gelatin hydrogel with ionically cross-linked polyP. Two forms of the Zn-polyP-containing matrices were obtained based on the property of polyP to f…

Protection of HeLa‐T4 + cells against human immunodeficiency virus (HIV) infection after stable transfection with HIV LTR‐2‘,5‘‐oligoadenylate synthetase hybrid gene 1

An expression vector (pU3R-III/2-5AS) of human 2',5'-oligoadenylate (2-5A) synthetase was constructed in which a cDNA encoding an active form of the enzyme was located 3' to a 3'-long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1). The LTR-directed expression of this hybrid DNA could be activated in trans by the HIV tat gene product. This vector was used for transfection of HeLa-T4+ cells, which are permissive to HIV infection, as well as of normal HeLa cells. HIV replication after infection of the CD4-receptor-bearing HeLa-T4+ cells with HIV-1 was found to be strongly reduced when drug-selected cells cotransfected with pU3R-III/2-5AS and a hygromycin B resistance gene…

New amide and dioxopiperazine derivatives from leaves of Breynia nivosa

The first chemical investigation of leaves of Breynia nivosa from Nigeria resulted in the isolation of two new amide derivatives breynivosamides A and B (1 and 2) and two new dioxopiperazine derivatives breynivosines A and B (4 and 5) together with seven known compounds (3, 6-11). The structures of the new compounds were elucidated by 1D, 2D NMR and HRESIMS data as well as by comparison with the literature. All isolated compounds were tested for the cytotoxic and antimicrobial activities. Only cristatin A (6) showed cytotoxicity against the L5178Y mouse lymphoma cell line with an IC50 value of 13.9μM while breynivosamide A (1) exhibited moderate antimicrobial activity against Mycobacterium …

Inhibition of intrinsic protein tyrosine kinase activity of EGF-receptor kinase complex from human breast cancer cells by the marine sponge metabolite (+)-aeroplysinin-1

1. (+)-Aeroplysinin-1, a naturally occurring tyrosine metabolite from the marine sponge Verongia aerophoba, was found to inhibit the phosphorylation of lipocortin-like proteins by a highly purified preparation of the epidermal growth factor (EGF) receptor-tyrosine protein kinase complex from MCF-7 breast carcinoma cells. 2. (+)-Aeroplysinin-1 blocked the EGF-dependent proliferation of both MCF-7 and ZR-75-1 human breast cancer cells and inhibited the ligand-induced endocytosis of the EGF receptor in vitro. 3. Treatment with aeroplysinin-1 in the concentration range at 0.25-0.5 microM resulted in a time- and dose-dependent total tumor cell death in vitro. 4. At a 10-fold higher concentration…

Sponge proteins are more similar to those of Homo sapiens than to Caenorhabditis elegans

We compared 42 phylogenetically conserved proteins from four marine sponges [Porifera] with almost the complete set of Caenorhabditis elegans proteins and all known proteins from humans. The majority of the sponge proteins are significantly more similar to human than to C. elegans orthologues/homologues. This finding reflects the accelerated evolutionary rate in the C. elegans lineage, since sponges split off first from the common ancestor of all multicellular animals. Furthermore, three sponge/human proteins were not found in C. elegans: (2–5)A synthetase, DNA repair helicase and lens βγ -crystallin. Sponges are the source of the most ancient proteins already present in the common ancestor…

Detection of Human Immunodeficiency Virus-1 Nucleic Acid on Inactivated Filter Paper Disks by Polymerase Chain Reaction and Microtiter Plate Assay

Human immunodeficiency virus type 1 (HIV-1) in cultured cells, peripheral blood samples and sera were adsorbed on filter paper disks and inactivated by heat or ethanol. Two procedures, the polymerase chain reaction (PCR) and microtiter plate assay (HMPA) were used to detect the nucleic acid. The sensitivity after different heat treatments with nested PCR for HIV-1 DNA (or nested reverse transcription-PCR for HIV-1 RNA) was identical regardless of whether the samples were examined immediately or one month later. Inactivation by ethanol treatment resulted in a slight loss of sensitivity. The HMPA proved to be as reliable and specific as the conventional PCR technique. We conclude that the hea…

Osteogenic potential of a biosilica-coated P(UDMA-co-MPS) copolymer

A P(UDMA-co-MPS) copolymer was surface-functionalized through the polycondensation activity of the enzyme silicatein. The resulting biosilica coating significantly enhanced mineralization of osteoblastic cells, thereby revealing its osteogenic potential. Consequently, the functionalized copolymer may be explored as an alternative to conventionally used acrylics in applications where stable bone-material interfaces are required.

Molecular and functional analysis of the (6-4) photolyase from the hexactinellid Aphrocallistes vastus.

The hexactinellid sponges (phylum Porifera) represent the phylogenetically oldest metazoans that evolved 570-750 million years ago. At this period exposure to ultraviolet (UV) light exceeded that of today and it may be assumed that this old taxon has developed a specific protection system against UV-caused DNA damage. A cDNA was isolated from the hexactinellid Aphrocallistes vastus which comprises high sequence similarity to genes encoding the protostomian and deuterostomian (6-4) photolyases. Subsequently functional studies were performed. It could be shown that the sponge gene, after transfection into mutated Escherichia coli, causes resistance of the bacteria against UV light. Recombinan…

A novel member of an ancient superfamily: sponge (Geodia cydonium, Porifera) putative protein that features scavenger receptor cysteine-rich repeats

Proteins featuring scavenger receptor cysteine-rich (SRCR) domains are prominent receptors known from vertebrates and from one phylum of invertebrates, the echinoderms. In the present study we report the first putative SRCR protein from the marine sponge Geodia cydonium (Porifera), a member of the lowest phylum of contemporary Metazoans. Two forms of SRCR molecules were characterized, which apparently represent alternative splicing of the same transcript. The long putative SRCR protein, of 1536 aa, features twelve SRCR repeats, a C-terminal transmembrane domain and a cytoplasmic tail. The sequence of the short form is identical with the long form except that it lacks a coding region near th…

Prolidase in the Marine Sponge Suberites domuncula: Enzyme Activity, Molecular Cloning, and Phylogenetic Relationship

: The enzyme prolidase hydrolyzes the peptide bond that involves the imino nitrogen of proline or hydroxyproline; hence, it catalyzes the final step in collagen degradation. From mammals it is known that this enzyme plays a major role in the recycling of proline for collagen synthesis and can be considered to be essential for the control of cell growth. The dominant organic exoskeleton in sponges, especially in Demospongiae, is collagen and the collagen-related spongin. Here we demonstrate that crude extracts of the demosponge Suberites domuncula contain prolidase or prolidase-like activity. The complementary DNA encoding the putative prolidase was cloned from a library of the same animal. …

Bioorganic/inorganic hybrid composition of sponge spicules: matrix of the giant spicules and of the comitalia of the deep sea hexactinellid Monorhaphis.

The giant basal spicules of the siliceous sponges Monorhaphis chuni and Monorhaphis intermedia (Hexactinellida) represent the largest biosilica structures on earth (up to 3 m long). Here we describe the construction (lamellar organization) of these spicules and of the comitalia and highlight their organic matrix in order to understand their mechanical properties. The spicules display three distinct regions built of biosilica: (i) the outer lamellar zone (radius: >300 mu m), (ii) the bulky axial cylinder (radius: <75 mu m), and (iii) the central axial canal (diameter: <2 mu m) with its organic axial filament. The spicules are loosely covered with a collagen net which is regularly perforated …

Calcium Polyphosphate Nanoparticles Act as an Effective Inorganic Phosphate Source during Osteogenic Differentiation of Human Mesenchymal Stem Cells

The ability of bone-marrow-derived mesenchymal stem/stromal cells (BM-MSCs) to differentiate into osteoblasts makes them the ideal candidate for cell-based therapies targeting bone-diseases. Polyphosphate (polyP) is increasingly being studied as a potential inorganic source of phosphate for extracellular matrix mineralisation. The aim of this study is to investigate whether polyP can effectively be used as a phosphate source during the in vitro osteogenic differentiation of human BM-MSCs. Human BM-MSCs are cultivated under osteogenic conditions for 28 days with phosphate provided in the form of organic &beta

A Novel Biomimetic Approach to Repair Enamel Cracks/Carious Damages and to Reseal Dentinal Tubules by Amorphous Polyphosphate.

Based on natural principles, we developed a novel toothpaste, containing morphogenetically active amorphous calcium polyphosphate (polyP) microparticles which are enriched with retinyl acetate (“a-polyP/RA-MP”). The spherical microparticles (average size, 550 ± 120 nm), prepared by co-precipitating soluble Na-polyP with calcium chloride and supplemented with retinyl acetate, were incorporated into a base toothpaste at a final concentration of 1% or 10%. The “a-polyP/RA-MP” ingredient significantly enhanced the stimulatory effect of the toothpaste on the growth of human mesenchymal stem cells (MSC). This increase was paralleled by an upregulation of the MSC marker genes for osteoblast differ…

Chemical Mimicry: Hierarchical 1D TiO2@ZrO2 Core−Shell Structures Reminiscent of Sponge Spicules by the Synergistic Effect of Silicatein-α and Silintaphin-1

In nature, mineralization of hard tissues occurs due to the synergistic effect of components present in the organic matrix of these tissues, with templating and catalytic effects. In Suberites domuncula, a well-studied example of the class of demosponges, silica formation is mediated and templated by an axial proteinaceous filament with silicatein-α, one of the main components. But so far, the effect of other organic constituents from the proteinaceous filament on the catalytic effect of silicatein-α has not been studied in detail. Here we describe the synthesis of core-shell TiO(2)@SiO(2) and TiO(2)@ZrO(2) nanofibers via grafting of silicatein-α onto a TiO(2) nanowire backbone followed by …

Polyphosphate as a donor of high-energy phosphate for the synthesis of ADP and ATP.

Here, we studied the potential role of inorganic polyphosphate (polyP) as an energy source for ADP and ATP formation in the extracellular space. In SaOS-2 cells, we show that matrix vesicles are released into the extracellular space after incubation with polyP. These vesicles contain both alkaline phosphatase (ALP) and adenylate kinase (AK) activities (mediated by ALPL and AK1 enzymes). Both enzymes translocate to the cell membrane in response to polyP. To distinguish the process(es) of AMP and ADP formation during ALP hydrolysis from the ATP generated via the AK reaction, inhibition studies with the AK inhibitor A(5')P5(5')A were performed. We found that ADP formation in the extracellular …

Molecular phylogeny of Metazoa (animals): monophyletic origin.

The phylogenetic relationships within the kingdom Animalia (Metazoa) have long been questioned. Focusing on the lowest eukaryotic multicellular organisms, the metazoan phylum Porifera (sponges), it remained unsolved if they evolved multicellularity independently from a separate protist lineage (polyphyly of animals) of derived from the same protist group as the other animal phyla (monophyly). After having analyzed genes typical for multicellularity (adhesion molecules/receptors and a nuclear receptor), we present evidence that Porifera should be placed in the kingdom Animalia. We therefore suggest a monophyletic origin for all animals.

Polyketides from the marine-derived fungus Aspergillus falconensis: In silico and in vitro cytotoxicity studies.

Abstract Fermentation of the marine-derived fungus Aspergillus falconensis, isolated from sediment collected from the Red Sea, Egypt on solid rice medium containing 3.5% NaCl yielded a new dibenzoxepin derivative (1) and a new natural isocoumarin (2) along with six known compounds (3–8). Changes in the metabolic profile of the fungus were induced by replacing NaCl with 3.5% (NH4)2SO4 that resulted in the accumulation of three further known compounds (9–11), which were not detected when the fungus was cultivated in the presence of NaCl. The structures of the new compounds were elucidated by HRESIMS and 1D/2D NMR as well as by comparison with the literature. Molecular docking was conducted fo…

Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro

Inorganic polymeric phosphate/polyphosphate (polyP) is a natural polymer existing in both pro- and eukaryotic systems. In the present study the effect of polyP as well as of polyP supplied in a stoichiometric ratio of 2 m polyP:1 m CaCl2 [polyP (Ca2+ complex)] on the osteoblast-like SaOS-2 cells and the osteoclast-like RAW 264.7 cells was determined. Both polymers are non-toxic for these cells up to a concentration of 100 µm. In contrast to polyP, polyP (Ca2+ complex) significantly induced hydroxyapatite formation at a concentration > 10 µm, as documented by alizarin red S staining and scanning electron microscopic (SEM) inspection. Furthermore, polyP (Ca2+ complex) triggered in SaOS-2 cell…

Modelling genetic regulation of growth and form in a branching sponge

We present a mathematical model of the genetic regulation controlling skeletogenesis and the influence of the physical environment on a branching sponge with accretive growth (e.g.Haliclona oculataorLubomirskia baikalensis). From previous work, it is known that high concentrations of silicate induce spicule formation and upregulate thesilicateingene. The upregulation of this gene activates locally the production of spicules in the sponge and the deposition of the skeleton. Furthermore, it is known that the expression of the geneIroquoisinduces the formation of an aquiferous system, consisting of exhalant and inhalant pores. We propose a model of the regulatory network controlling the separa…

Interaction of the retinoic acid signaling pathway with spicule formation in the marine sponge Suberites domuncula through activation of bone morphogenetic protein-1

Abstract Background The formation of the spicules in siliceous sponges involves the formation of cylinder-like structures in the extraspicular space, composed of the enzyme silicatein and the calcium-dependent lectin. Scope of review Molecular cloning of the cDNAs (carotene dioxygenase, retinal dehydrogenase, and BMB-1 [bone morphogenic protein-1]) from the demosponge Suberites domuncula was performed. These tools were used to understand the retinoid metabolism in the animal by qRT-PCR, immunoblotting and TEM. Major conclusions We demonstrate that silintaphin-2, a silicatein-interacting protein, is processed from a longer-sized 15-kDa precursor to a truncated, shorter-sized 13 kDa calcium-b…

NanoSIMS: insights into the organization of the proteinaceous scaffold within Hexactinellid sponge spicules.

The giant basal spicules (GBS) from Monorhaphis chuni (Porifera [sponges], Hexactinellida) represent the largest biosilica structures on Earth and can reach lengths of 300 cm (diameter of 1.1 cm). The amorphous silica of the inorganic matrix is formed enzymatically by silicatein. During this process, the enzyme remains trapped inside the lamellar-organized spicules. In order to localize the organic silicatein scaffold, the inside of a lamella has been analyzed by nano-secondary ion mass spectrometry (NanoSIMS). It is shown that the GBSs are composed of around 245 concentrically arranged individual siliceous lamellae. These surround an internal siliceous axial cylinder. The lamellae adjacent…

Tanzawaic acid derivatives from freshwater sediment-derived fungus Penicillium sp.

Abstract Chemical investigation of a freshwater sediment-derived fungus, Penicillium sp. (S1a1), led to the isolation of three new tanzawaic acid derivatives, including penitanzchroman (1), tanzawaic acids Y (2) and Z (3), along with six known tanzawaic acid analogues (4-9), three known isochromans (10-12) and two known benzoquinones (13 and 14). The structures of the new compounds were established based on high-resolution mass spectrometry, and detailed analysis of one- and two-dimensional NMR spectroscopy. The relative configuration of the new compounds was assigned on the basis of NMR spectroscopic data including ROESY spectra. The absolute configuration was determined based on the speci…

Co-expression and Functional Interaction of Silicatein with Galectin

Sponges (phylum Porifera) of the class of Demospongiae are stabilized by a siliceous skeleton. It is composed of silica needles (spicules), which provide the morphogenetic scaffold of these metazoans. In the center of the spicules there is an axial filament that consists predominantly of silicatein, an enzyme that catalyzes the synthesis of biosilica. By differential display of transcripts we identified additional proteins involved in silica formation. Two genes were isolated from the marine demosponge Suberites domuncula; one codes for a galectin and the other for a fibrillar collagen. The galectin forms aggregates to which silicatein molecules bind. The extent of the silicatein-mediated s…

Human immunodeficiency virus infection in cells of myeloid-monocytic lineage.

We established persistent infection with a strain of human immunodeficiency virus type 1, HTLV-IIIB, in a promyelomonocytic cell line, ML-1 (CD4 antigen nearly negative and CD4 mRNA negative), and a promonocytic cell line, THP-1 (CD4 antigen positive). Different reaction of giant cell formation was found after co-cultivation of infected and uninfected cells of ML-1, HL-60, THP-1 and U-937 cell lines with uninfected and infected MOLT4 (a T-lymphoma cell line).

Origin of metazoan adhesion molecules and adhesion receptors as deduced from cDNA analyses in the marine sponge Geodia cydonium: a review.

The phylogenetic relationships of the kingdom Animalia (Metazoa) have long been questioned. Whether the lowest eukaryotic multicellular organisms, the metazoan phylum Porifera (sponges), independently evolved multicellularity from a separate protist lineage (polyphyly of animals) or whether they were derived from the same protist group as the other animal phyla (monophyly) remains unclear. Analyses of the genes that are typical for multicellularity, e.g. those coding for adhesion molecules (galectin) and adhesion receptors (receptor tyrosine kinase, integrin receptor, receptors featuring scavenger receptor cysteine-rich domains) or elements involved in signal transduction pathways (G-protei…

Identification of sapovirus infection among Japanese infants in a day care center.

A total of 921 fecal specimens collected from 44 infants in a day care center in Tokyo, Japan during June 1999 to July 2000 were tested for the presence of sapovirus by reverse transcription-polymerase chain reaction (RT-PCR). Of 88 fecal specimens from infants with acute gastroenteritis, 2.3% (2) were found to be positive for sapovirus. Twenty-two of 833 (2.6%) fecal specimens collected from asymptomatic infants were also infected with this virus. Another interesting feature was the demonstration of high incidence of sapovirus infection (95.5%, 21 of 22) identified in a single day care center, which was not due to viral shedding after the latest acute gastroenteritis. Sapovirus was subject…

Marine sponge collagen: isolation, characterization and effects on the skin parameters surface-pH, moisture and sebum

A previously described isolation procedure for collagen of the marine sponge Chondrosia reniformis Nardo was modified for scaling-up reasons yielding 30% of collagen (freeze-dried collagen in relation to freeze-dried sponge). Light microscope observations showed fibrous structures. Transmission electron microscopy studies proved the collagenous nature of this material: high magnifications showed the typical periodic banding-pattern of collagen fibres. However, the results of the amino acid analysis differed from most publications, presumably due to impurities that still were present. In agreement with earlier studies, sponge collagen was insoluble in dilute acid mediums and all solvents inv…

Axial growth of hexactinellid spicules: Formation of cone-like structural units in the giant basal spicules of the hexactinellid Monorhaphis

The glass sponge Monorhaphis chuni (Porifera: Hexactinellida) forms the largest bio-silica structures on Earth; their giant basal spicules reach sizes of up to 3 m and diameters of 8.5 mm. Previously, it had been shown that the thickness growth proceeds by appositional layering of individual lamellae; however, the mechanism for the longitudinal growth remained unstudied. Now we show, that the surface of the spicules have towards the tip serrated relief structures that are consistent in size and form with the protrusions on the surface of the spicules. These protrusions fit into the collagen net that surrounds the spicules. The widths of the individual lamellae do not show a pronounced size …

Morphogenetic (Mucin Expression) as Well as Potential Anti-Corona Viral Activity of the Marine Secondary Metabolite Polyphosphate on A549 Cells

The mucus layer of the nasopharynx and bronchial epithelium has a barrier function against inhaled pathogens such as the coronavirus SARS-CoV-2. We recently found that inorganic polyphosphate (polyP), a physiological, metabolic energy (ATP)-providing polymer released from blood platelets, blocks the binding of the receptor binding domain (RBD) to the cellular ACE2 receptor in vitro. PolyP is a marine natural product and is abundantly present in marine bacteria. Now, we have approached the in vivo situation by studying the effect of polyP on the human alveolar basal epithelial A549 cells in a mucus-like mucin environment. These cells express mucins as well as the ectoenzymes alkaline phospha…

Transformation of Amorphous Polyphosphate Nanoparticles into Coacervate Complexes: An Approach for the Encapsulation of Mesenchymal Stem Cells.

Inorganic polyphosphate [polyP] has proven to be a promising physiological biopolymer for potential use in regenerative medicine because of its morphogenetic activity and function as an extracellular energy-donating system. Amorphous Ca2+ -polyP nanoparticles [Ca-polyP-NPs] are characterized by a high zeta potential with -34 mV (at pH 7.4). This should contribute to the stability of suspensions of the spherical nanoparticles (radius 94 nm), but make them less biocompatible. The zeta potential decreases to near zero after exposure of the Ca-polyP-NPs to protein/peptide-containing serum or medium plus serum. Electron microscopy analysis reveals that the particles rapidly change into a coacerv…

Collagen-inducing biologization of prosthetic material for hernia repair: Polypropylene meshes coated with polyP/collagen

Prostethic mesh material such as nonabsorbable polypropylene used in open and laparoscopic hernia repair are characterized by controllable mechanical properties but may elicit undesirable physiological reactions due to the nonphysiological inert polymer material. We succeeded in developing a biocompatible coating for these meshes, based on a physiological inorganic polymer, polyphosphate (polyP) that is morphogenetically active and used as a metabolic energy source, and a collagen matrix. The polyP/collagen hydrogel material was prepared by a freeze-extraction method, with amorphous Ca-polyP microparticles. Electron microscopy (SEM and REM) studies revealed that the polyP/collagen coats are…

Polyphosphate: A Morphogenetically Active Implant Material Serving as Metabolic Fuel for Bone Regeneration

The initial mineralization centers during human bone formation onto osteoblasts are composed of CaCO3 . Those bioseeds are enzymatically formed via carbonic anhydrase(s) in close association with the cell surface of the osteoblasts. Subsequently, the bicarbonate/carbonate anions are exchanged non-enzymatically by inorganic phosphate [Pi ]. One source for the supply of Pi is polyphosphate [polyP] which is a physiological polymer, formed in the osteoblasts as well as in the platelets. The energy-rich acid anhydride bonds within the polyP chain are cleaved by phosphatase(s); during this reaction free-energy might be released that could be re-used, as metabolic fuel, for the maintenance of the …

Pharmacological intervention in age-associated brain disorders by Flupirtine: Alzheimer’s and Prion diseases

Alzheimer's disease, a major form of dementia in the elderly has become an increasingly important health problem in developed countries. In vitro studies on primary neurons demonstrate that Flupirtine (Katadolon) at a concentration of 1 microg/ml, significantly reduces the neurotoxic (apoptotic) effect displayed by A beta25-35, a segment of the amyloid beta-protein precursor the etiologic agent of Alzheimer's disease. Flupirtine, which has been in clinical use since 10 years ago, prevents the toxic effect of PrP, the presumed etiologic agent of the Creutzfeldt-Jakob disease as well as the excitatory amino acid glutamate on cortical neurons. Flupirtine displays a bimodal activity. Its strong…

Au@MnO nanoflowers: hybrid nanocomposites for selective dual functionalization and imaging.

Recently, the development of hybrid nanostructures consisting of various materials has attracted considerable interest. The assembly of different nanomaterials with specific optical, magnetic, or electronic properties to multicomponent composites can change and even enhance the properties of the individual constituents. Specifically tuning the structure and interface interactions within the nanocomposites has resulted in novel platforms of materials that may lead the way to various future technologies, such as synchronous biolabeling, protein separation and detection, heterogeneous catalysis, and multimodal imaging in biomedicine. Of the various kinds of nanomaterials, gold nanorods show an…

The enzyme carbonic anhydrase as an integral component of biogenic Ca-carbonate formation in sponge spicules

The inorganic scaffold of the spicules, the skeletal elements of the calcareous sponges, is formed of calcium carbonate (CaCO3). The growth of the approximately 300-μm large spicules, such as those of the calcareous sponge Sycon raphanus used in the present study, is a rapid process with a rate of about 65 μm/h. The formation of CaCO3 is predominantly carried out by the enzyme carbonic anhydrase (CA). The enzyme from the sponge S. raphanus was isolated and prepared by recombination. The CA-driven deposition of CaCO3 crystallites is dependent on temperature (optimal at 52 °C), the pH value of the reaction assay (7.5/8.0), and the substrate concentration (CO2 and Ca2+). During the initial pha…

Axial (apical-basal) expression of pro-apoptotic and pro-survival genes in the lake baikal demosponge Lubomirskia baicalensis.

Like in all other Metazoa, also in sponges (Porifera) proliferation, differentiation, and death of cells are controlled by apoptotic processes, thus allowing the establishment of a Bauplan (body plan). The demosponge Lubomirskia baicalensis from the Lake Baikal is especially suitable to assess the role of the apoptotic molecules, since its grade of construction is highly elaborated into an encrusting base and branches composed of modules lined up along the apical-basal axis. The four cDNAs, ALG-2, BAK, MA-3, and Bcl-2, were isolated from this sponge species. The expression levels of these genes follow characteristic gradients. While the proapoptotic genes are highly expressed at the base of…

Corynebacterium parvum (Propionibacterium acnes): an inducer of tumor necrosis factor-alpha in human peripheral blood mononuclear cells and monocytes in vitro.

The present study investigates the potential capacity of the immunostimulant Corynebacterium parvum (C.p.) to induce tumor necrosis factor-alpha (TNF-alpha) in human peripheral blood mononuclear cells (PBMC) and blood monocytes (BMo) in vitro. Both at the mRNA and protein level, stimulation of PBMC and BMo upon C.p. induces TNF-alpha. Compared to the hitherto used TNF-alpha inducers in vitro such as Sendai virus, phytohemagglutinin or lipopolysaccharide the C.p. stimulus displayed a threefold stronger induction of TNF-alpha production (p less than 0.001). Using C.p. as an inducer it was possible to demonstrate that TNF-alpha production is regulated by prostaglandin E2; preincubation of the …

Enzymatically Synthesized Inorganic Polymers as Morphogenetically Active Bone Scaffolds

In recent years a paradigm shift in understanding of human bone formation has occurred that starts to change current concepts in tissue engineering of bone and cartilage. New discoveries revealed that fundamental steps in biomineralization are enzyme driven, not only during hydroxyapatite deposition, but also during initial bioseed formation, involving the transient deposition and subsequent transformation of calcium carbonate to calcium phosphate mineral. The principal enzymes mediating these reactions, carbonic anhydrase and alkaline phosphatase, open novel targets for pharmacological intervention of bone diseases like osteoporosis, by applying compounds acting as potential activators of …

Determination of 14-3-3 protein levels in cerebrospinal fluid from Creutzfeldt-Jakob patients by a highly sensitive capture assay.

The level of 14-3-3gamma protein was determined in the cerebrospinal fluid (CSF) from patients with Creutzfeldt-Jakob disease (CJD) and non-CJD patients applying a new and fast microplate assay (14-3-3 protein capture assay), based on the binding to a peptide comprising a phosphorylated recognition motif of 14-3-3 protein. The levels of the gamma-isoform of 14-3-3 protein in CSF samples from CJD patients (n=41) were significantly higher than those observed in patients with non-CJD dementias (n=36) suggesting that this capture assay is a reliable method in the diagnosis of CJD. Since this assay allows the direct measurement of 14-3-3 protein in the CSF without prior concentration it is an ea…

Dispacamide E and other bioactive bromopyrrole alkaloids from two Indonesian marine sponges of the genus Stylissa.

Chemical investigation of methanolic extracts of the two Indonesian marine sponges Stylissa massa and Stylissa flabelliformis yielded 25 bromopyrrole alkaloids including 2 new metabolites. The structures of all isolated compounds were unambiguously elucidated based on extensive 1D and 2D NMR, LR-MS and HR-MS analyses. All isolated compounds were assayed for their antiproliferative and protein kinase inhibitory activities. Several of the tested compounds revealed selective activity(ies) which suggested preliminary SARs of the isolated bromopyrrole alkaloids.

Alteration of DNA topoisomerase II activity during infection of H9 cells by human immunodeficiency virus type 1 in vitro: a target for potential therapeutic agents.

Infection of H9 cells with human immunodeficiency virus type 1 (HIV-1) was found to decrease the phosphorylation of DNA topoisomerase II during the initial phase of infection. Simultaneously, with a later overshoot of phosphorylation and the subsequent activation of DNA topoisomerase II, the production of HIV-1 started. Applying three new protein kinase C inhibitors from the class of O-alkylglycerophospholipids we demonstrated that inhibition of protein kinase C-mediated phosphorylation of DNA topoisomerase II resulted in an inhibition of HIV-1 production. Based on the differential effect of the two protein kinase C activators, phorbol ester and bryostatin, we conclude that phosphorylation …

Identification of highly conserved genes: SNZ and SNO in the marine sponge Suberites domuncula: their gene structure and promoter activity in mammalian cells

Abstract Recently, we reported that cells from the sponge Suberites domuncula respond to ethylene with an increase in intracellular Ca 2+ level [Ca 2+ ] i , and with an upregulation of the expression of (at least) two genes, a Ca 2+ /calmodulin-dependent protein kinase and the potential ethylene-responsive gene, termed SDSNZERR (A. Krasko, H.C. Schroder, S. Perovic, R. Steffen, M. Kruse, W. Reichert, I.M. Muller, W.E.G. Muller, J. Biol. Chem. 274 (1999)). Here, we describe for the first time that also mammalian (3T3) cells respond to ethylene, generated by ethephon, with an immediate and transient, strong increase in [Ca 2+ ] i . Next, the promoter for the sponge SDSNZERR gene was isolated …

Genome size and chromosomes in marine sponges [Suberites domuncula, Geodia cydonium]

The genome size of the marine sponges Suberites domuncula and Geodia cydonium has been determined by flow cytofluorometric analysis using diamidino-phenylindole [DAPI]. Using human lymphocytes as reference the amount of DNA in cells from S. domuncula has been determined to be 3.7 pg and that of G. cydonium 3.3 pg. While no chromosomes could be identified in G. cydonium, the karyotype of the Suberites domuncula is 32 chromosomes in the diploid state. The size of the chromosomes was between 0.25 and 1.0 micron. No pronounced banding pattern was visible.

Cloning and expression of the putative aggregation factor from the marine sponge Geodia cydonium.

Sponges (phylum Porifera) have extensively been used as a model system to study cell-cell interaction on molecular level. Recently, we identified and cloned the putative aggregation receptor (AR) of the sponge Geodia cydonium, which interacts in a heterophilic way with the aggregation factor (AF) complex. In the present study, antibodies against this complex have been raised that abolish the adhesion function of the enriched sponge AF, the AF-Fraction 6B. Using this antibody as a tool, a complete 1.7 kb long cDNA, GEOCYAF, could be isolated from a cDNA library that encodes the putative AF. Its deduced aa sequence in the N-terminal section comprises high similarity to amphiphysin/BIN1 sequen…

Triple-target stimuli-responsive anti-COVID-19 face mask with physiological virus-inactivating agents

Conventional face masks to prevent SARS-CoV-2 transmission are mostly based on a passive filtration principle. Ideally, anti-COVID-19 masks should protect the carrier not only by size exclusion of virus aerosol particles, but also be able to capture and destroy or inactivate the virus. Here we present the proof-of-concept of a filter mat for such a mask, which actively attracts aerosol droplets and kills the virus. The electrospun mats are made of polycaprolactone (PCL) a hydrophilic, functionalizable and biodegradable polyester, into which inorganic polyphosphate (polyP) a physiological biocompatible, biodegradable and antivirally active polymer (chain length, ∼40 Pi units) has been integr…

Enzyme-based biosilica and biocalcite: biomaterials for the future in regenerative medicine

The oldest animals on Earth, sponges, form both the calcareous and the siliceous matrices of their spicules enzymatically. Until recently, it has been neglected that enzymes play crucial roles during formation of these biominerals. This paradigm shift occurred after the discovery that the enzyme silicatein, which catalyzes the polycondensation of silica, and the enzyme carbonic anhydrase (CA), which catalyzes the formation of bicarbonate (HCO3(-)/CaCO3), produce solid amorphous bioglass or biocalcite. This suggests that in mammals, biosilica and biocalcite can act anabolically during hydroxyapatite (HA) synthesis and bone formation. Biosilica and biocalcite are thus promising candidates for…

Hierarchical structure and cytocompatibility of fish scales from Carassius auratus

Abstract To study the structure and the cytocompatibility of fish scales from Carassius auratus , scanning electron microscopy (SEM) was used to observe the morphology of fish scales treated with different processing methods. Based on varying morphologies and components, the fish scales can be divided into three regions on the surface and three layers in vertical. The functions of these three individual layers were analyzed. SEM results show that the primary inorganic components are spherical or cubic hydroxyapatite (HA) nanoparticles. The fish scales have an ~ 60° overlapped plywood structure of lamellas in the fibrillary plate. The plywood structure consists of co-aligned type I collagen …

Cloning and expression of the sponge longevity gene SDLAGL.

Porifera show a characteristic Bauplan in spite of the fact that (almost) all cells are telomerase-positive and presumably provided with an unlimited potency for cell proliferation. One gene, SDLAGL, was identified in the marine sponge Suberites domuncula whose deduced polypeptide showed high sequence similarity to the longevity assurance genes from other Metazoa. While in single cells no transcripts of SDLAGL could be identified, high expression was seen after re-aggregation of single cells and in proliferating cells of primmorphs.

Differentiation capacity of epithelial cells in the sponge Suberites domuncula.

Sponges (phylum Porifera) represent the oldest metazoans. Their characteristic metazoan adhesion molecules and transcription factors enable them to establish a complex "Bauplan" ; three major differentiated cell types (epithelial cells, skeletal cells/sclerocytes, and contractile cells) can be distinguished. Since no molecular markers are as yet available to distinguish these somatic cells or the corresponding embryonic cells from which they originate, we have selected the following three genes for their characterization: noggin (a signaling molecule in development), a caspase that encodes an apoptotic molecule, and silicatein. Silicatein is an enzyme that is involved in the synthesis of si…

Siliceous spicules in marine demosponges (example Suberites domuncula)

All metazoan animals comprise a body plan of different complexity. Since-especially based on molecular and cell biological data-it is well established that all metazoan phyla, including the Porifera (sponges), evolved from a common ancestor the search for common, basic principles of pattern formation (body plan) in all phyla began. Common to all metazoan body plans is the formation of at least one axis that runs from the apical to the basal region; examples for this type of organization are the Porifera and the Cnidaria (diploblastic animals). It seems conceivable that the basis for the formation of the Bauplan in sponges is the construction of their skeleton by spicules. In Demospongiae (w…

Poly(silicate)-metabolizing silicatein in siliceous spicules and silicasomes of demosponges comprises dual enzymatic activities (silica polymerase and silica esterase)

Siliceous sponges can synthesize poly(silicate) for their spicules enzymatically using silicatein. We found that silicatein exists in silica-filled cell organelles (silicasomes) that transport the enzyme to the spicules. We show for the first time that recombinant silicatein acts as a silica polymerase and also as a silica esterase. The enzymatic polymerization/polycondensation of silicic acid follows a distinct course. In addition, we show that silicatein cleaves the ester-like bond in bis(p-aminophenoxy)-dimethylsilane. Enzymatic parameters for silica esterase activity are given. The reaction is completely blocked by sodium hexafluorosilicate and E-64. We consider that the dual function o…

Endoribonuclease IV. A poly(A)-specific ribonuclease from chick oviduct. 1. Purification of the enzyme.

A new endoribonuclease, termed endoribonuclease IV, has been described. This enzyme has been isolated from chick oviducts and purified 15 000-fold in a 25% yield nearly to homogeneity. The nuclease, which specifically degrades poly(A), forms oligonucleotides of an average chain length of 10. These (A)-10 fragments are terminated by 3'-hydroxyl and 5'-phosphate groups. The enzyme has a pH optimum at 8.7, requires Mn2+ or Mg2+ as a cofactor, and has a molecular weight of about 45 000.

A novel method for determination of inorganic polyphosphates using the fluorescent dye fura-2.

A method for determining inorganic polyphosphate, which is based on the Mn2+-induced quenching of the fluorescence of the calcium indicator fura-2, is described. The effect of Mn2+ ions on fura-2 fluorescence is gradually abolished in the presence of increasing concentrations of polyphosphate; this allows the quantification both of synthetic polyphosphates and of the naturally occurring polymer isolated from tissues or cells. The described method has some advantages compared to conventional procedures for detection of polyphosphates based on the metachromatic effect on toluidine blue. It can be applied for the determination of pyrophosphate, tripolyphosphate and other short-chain polyphosph…

Okadaic Acid, an Apoptogenic Toxin for Symbiotic/Parasitic Annelids in the Demosponge Suberites domuncula

ABSTRACT The role of okadaic acid (OA) in the defense system of the marine demosponge Suberites domuncula against symbiotic/parasitic annelids was examined. Bacteria within the mesohyl produced okadaic acid at concentrations between 32 ng/g and 58 ng/g of tissue (wet weight). By immunocytochemical methods and by use of antibodies against OA, we showed that the toxin was intracellularly stored in vesicles. Western blotting experiments demonstrated that OA also existed bound to a protein with a molecular weight of 35,000 which was tentatively identified as a galectin (by application of antigalectin antibodies). Annelids that are found in S. domuncula undergo apoptotic cell death. OA is one ca…

Functional characterization of Tat protein from human immunodeficiency virus. Evidence that Tat links viral RNAs to nuclear matrix.

The processes of transcription and posttranscription are assumed to proceed in close association with the nuclear matrix. In this study we demonstrated that Tat, the trans-activating protein from human immunodeficiency virus type 1 (HIV-1), binds both to the TAR region of the nascent HIV mRNAs and the nuclear matrix with high affinity. Both North/Western blotting experiments and nitrocellulose binding studies revealed that Tat binds with an association constant (K alpha) of approximately 1 x 10(9) M-1 to the TAR segment of HIV RNA; binding of Tat to this sequence which is present between position 32 and 82 downstream from the TATA box was also confirmed by gel retardation assays. Binding of…

Interaction of 68–kDa TAR RNA-binding protein and other cellular proteins with rpion protein-RNA stem-loop

The RNA stem-loop structure of the trans-activating region TAR sequence of human immunodeficiency virus-1 mRNA is the binding site for a number of host cell proteins. A virtually identical set of proteins from HeLa nuclear extracts was found to bind to the predicted RNA hairpin element of prion protein (PrP) mRNA, as demonstrated in UV cross-linking/RNase protection and Northwestern assays. We show that the cellular TAR loop-binding protein, p68, is among those proteins which associate with PrP RNA. Competition experiments with various TAR RNA mutants revealed that binding of partially purified p68 to PrP RNA stem-loop occurs sequence-specifically. The 100-kDa 2-5A synthetase which is invol…

The stem cell concept in sponges (Porifera): Metazoan traits.

Sponges are considered the oldest living animal group and provide important insights into the earliest evolutionary processes in the Metazoa. This paper reviews the evidence that sponge stem cells have essential roles in cellular specialization, embryogenesis and Bauplan formation. Data indicate that sponge archaeocytes not only represent germ cells but also totipotent stem cells. Marker genes have been identified which are expressed in totipotent stem cells and gemmule cells. Furthermore, genes are described for the three main cell lineages in sponge, which share a common origin from archaeocytes and result in the differentiation of skeletal, epithelial, and contractile cells.

In vitroandin vivoenhancement of osteogenic capacity in a synthetic BMP-2 derived peptide-coated mineralized collagen composite

Enhancement of osteogenic capacity was achieved in a mineralized collagen composite, nano-hydroxyapatite/collagen (nHAC), by loading with synthetic peptides derived from BMP-2 residues 32-48 (P17-BMP-2). Rabbit marrow stromal cells (MSCs) were used in vitro to study cell biocompatibility, attachment and differentiation on the mineralized collagen composite by a cell counting kit, scanning electron microscopy (SEM) and real-time reversed transcriptase-polymerase chain reaction analysis (RT-PCR). Optimal peptide dosage (1.0 µg/mL) was obtained by RT-PCR analysis in vitro. In addition, the relative expression level of OPN and OCN was significantly upregulated on P17-BMP-2/nHAC compared with nH…

Flashing light signaling circuit in sponges: Endogenous light generation after tissue ablation in Suberites domuncula

The skeleton of siliceous sponges (phylum Porifera: classes Demospongiae and Hexactinellida), composed of tightly interacting spicules that assemble to a genetically fixed scaffold, is formed of bio-silica. This inorganic framework with the quality of quartz glass has been shown to operate as light waveguide in vitro and very likely has a similar function in vivo. Furthermore, the molecular toolkit for endogenous light generation (luciferase) and light/photon harvesting (cryptochrome) has been identified in the demosponge Suberites domuncula. These three components of a light signaling system, spicules—luciferase—cryptochrome, are concentrated in the surface layers (cortex) of the poriferan…

Induction of heat-shock (stress) protein gene expression by selected natural and anthropogenic disturbances in the octocoral Dendronephthya klunzingeri

Previously it was found that the expression of selected heat-shock proteins is upregulated in corals after exposure to elevated temperature. We published that HSPs are suitable markers in sponges to monitor the degree of environmental stress on these animals. In the present study the heat-shock proteins (HSPs) with a molecular weight of 90 kDa have been selected to prove their potential usefulness as biomarkers under controlled laboratory conditions and in the field. The studies have been performed with the octocoral Dendronephthya klunzingeri from which the cDNA coding for HSP90 was cloned first. The expression of the HSP90 gene is upregulated by thermal stress; treatment of the animals fo…

Oxygen-Controlled Bacterial Growth in the Sponge Suberites domuncula: toward a Molecular Understanding of the Symbiotic Relationships between Sponge and Bacteria†

ABSTRACT Sponges (phylum Porifera), known to be the richest producers among the metazoans of bioactive secondary metabolites, are assumed to live in a symbiotic relationship with microorganisms, especially bacteria. Until now, the molecular basis of the mutual symbiosis, the exchange of metabolites for the benefit of the other partner, has not been understood. We show with the demosponge Suberites domuncula as a model that the sponge expresses under optimal aeration conditions the enzyme tyrosinase, which synthesizes diphenols from monophenolic compounds. The cDNA isolated was used as a probe to determine the steady-state level of gene expression. The gene expression level parallels the lev…

Inorganic Polyphosphates: Biologically Active Biopolymers for Biomedical Applications

Inorganic polyphosphate (polyP) is a widely occurring but only rarely investigated biopolymer which exists in both prokaryotic and eukaryotic organisms. Only in the last few years, this polymer has been identified to cause morphogenetic activity on cells involved in human bone formation. The calcium complex of polyP was found to display a dual effect on bone-forming osteoblasts and bone-resorbing osteoclasts. Exposure of these cells to polyP (Ca2+ complex) elicits the expression of cytokines that promote the mineralization process by osteoblasts and suppress the differentiation of osteoclast precursor cells to the functionally active mature osteoclasts dissolving bone minerals. The effect o…

Self-healing, an intrinsic property of biomineralization processes

The sponge siliceous spicules are formed enzymatically via silicatein, in contrast to other siliceous biominerals. Originally, silicatein had been described as a major structural protein of the spicules that has the property to allow a specific deposition of silica onto their surface. More recently, it had been unequivocally demonstrated that silicatein displays a genuine enzyme activity, initiating and maintaining silica biopolycondensation at low precursor concentrations (<2 mM). Even more, as silicatein becomes embedded into the biosilica polymer, formed by the enzyme, it retains its functionality to enable a controlled biosilica deposition. The protection of silicatein through the biosi…

Matrix-mediated canal formation in primmorphs from the sponge Suberites domuncula involves the expression of a CD36 receptor-ligand system.

Sponges (Porifera), represent the phylogenetically oldest metazoan phylum still extant today. Recently, molecular biological studies provided compelling evidence that these animals share basic receptor/ligand systems, especially those involved in bodyplan formation and in immune recognition, with the higher metazoan phyla. An in vitro cell/organ-like culture system, the primmorphs, has been established that consists of proliferating and differentiating cells, but no canals of the aquiferous system. We show that after the transfer of primmorphs from the demosponge Suberites domuncula to a homologous matrix (galectin), canal-like structures are formed in these 3D-cell aggregates. In parallel …

Self-Healing Properties of Bioinspired Amorphous CaCO3/Polyphosphate-Supplemented Cement

There is a strong interest in cement additives that are able to prevent or mitigate the adverse effects of cracks in concrete that cause corrosion of the reinforcement. Inorganic polyphosphate (polyP), a natural polymer that is synthesized by bacteria, even those on cement/concrete, can increase the resistance of concrete to progressive damage from micro-cracking. Here we use a novel bioinspired strategy based on polyP-stabilized amorphous calcium carbonate (ACC) to give this material self-healing properties. Portland cement was supplemented with ACC nanoparticles which were stabilized with 10% (w/w) Na&ndash

Indole alkaloids from the coprophilous fungus Aphanoascus fulvescens

Abstract The Ascomycete fungus Aphanoascus fulvescens isolated from goose dung was investigated for its secondary metabolites, yielding five new indole alkaloids okaramines V–Z (1–5) and eleven known derivatives (6–16). Their structures were determined by 1D, 2D NMR spectra and HRMS data. Compounds 6, 8, 11 and 12 showed significant to moderate cytotoxicity against the mouse lymphoma cell line L5178Y with IC50 values ranging from 4.0 to 14.7 μM. Preliminary structure-activity relationships are discussed.

Changes in metabolism of inorganic polyphosphate in rat tissues and human cells during development and apoptosis

Age-dependent studies show that the amount of inorganic polyphosphate in rat brain strongly increases after birth. Maximal levels were found in 12-months old animals. Thereafter, the concentration of total polyphosphate decreases to about 50%. This decrease in the concentration of total polyphosphate is due to a decrease in the amount of insoluble, long-chain polyphosphates. The amount of soluble, long-chain polyphosphates does not change significantly in the course of ageing. In rat embryos and newborns, mainly soluble polyphosphates could be detected. In rat liver, the age-dependent changes are less pronounced. The changes in polyphosphate level are accompanied by changes in exopolyphosph…

Mineralization of SaOS-2 cells on enzymatically (silicatein) modified bioactive osteoblast-stimulating surfaces.

There is a demand for novel bioactive supports in surgery, orthopedics, and tissue engineering. The availability of recombinant silica-synthesizing enzyme (silicatein) opens new possibilities for the synthesis of silica-containing bioactive surfaces under ambient conditions that do not damage biomolecules like proteins. Here it is shown that growth of human osteosarcoma SaOS-2 cells on cluster plates precoated with Type 1 collagen is not affected by additional coating of the plates with the recombinant silicatein and incubation with its enzymatic substrate, tetraethoxysilane (TEOS). However, the enzymatic modification of the plates by biosilica deposition on the protein-coated surface cause…

Effect of bioglass on growth and biomineralization of SaOS-2 cells in hydrogel after 3D cell bioprinting.

We investigated the effect of bioglass (bioactive glass) on growth and mineralization of bone-related SaOS-2 cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP), administered as polyP • Ca2+-complex, or silica, or as biosilica that had been enzymatically prepared from ortho-silicate by silicatein. These hydrogels, together with SaOS-2 cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, with a size of 55 nm and a molar ratio of SiO2 : CaO : P2O5 of 55 : 40 : 5, did not affect the growth of the encapsulated cells. If silica, biosilica, or polyP …

Cladosporinone, a new viriditoxin derivative from the hypersaline lake derived fungus Cladosporium cladosporioides

A new cytotoxic viriditoxin derivative, cladosporinone (1), along with the known viriditoxin (2) and two viriditoxin derivatives (3 and 4) were obtained from the fungus C ladosporium cladosporioides isolated from the sediment of a hypersaline lake in Egypt. The structure of the new compound (1) was determined by 1D and 2D NMR measurements as well as by high-resolution ESIMS and electronic circular dichroism spectroscopy. All isolated compounds were studied for their cytotoxicity against the murine lymphoma cell line L5187Y and for their antibiotic activity against several pathogenic bacteria. Viriditoxin (2) was the most active compound in both bioassays. Compound 1 also exhibited strong cy…

Molecular Evolution of Apoptotic Pathways: Cloning of Key Domains from Sponges (Bcl-2 Homology Domains and Death Domains) and Their Phylogenetic Relationships

Cells from metazoan organisms are eliminated in a variety of physiological and pathophysiological processes by apoptosis. In this report, we describe the cloning and characterization of molecules from the marine sponges Geodia cydonium and Suberites domuncula, whose domains show a high similarity to those that are found in molecules of the vertebrate Bcl-2 superfamily and of the death receptors. The Bcl-2 proteins contain up to four Bcl-2 homology regions (BH). Two Bcl-2-related molecules have been identified from sponges that are provided with two of those regions, BH1 and BH2, and are termed Bcl-2 homology proteins (BHP). The G. cydonium molecule, BHP1_GC, has a putative size of 28,164, w…

3D printing of hybrid biomaterials for bone tissue engineering: Calcium-polyphosphate microparticles encapsulated by polycaprolactone.

Abstract Here we describe the formulation of a morphogenetically active bio-ink consisting of amorphous microparticles (MP) prepared from Ca 2+ and the physiological inorganic polymer, polyphosphate (polyP). Those MP had been fortified by mixing with poly-e-caprolactone (PCL) to allow 3D-bioprinting. The resulting granular PCL/Ca-polyP-MP hybrid material, liquefied by short-time heating to 100 °C, was used for the 3D-printing of tissue-like scaffolds formed by strands with a thickness of 400 µm and a stacked architecture leaving ≈0.5 mm 2 -sized open holes enabling cell migration. The printed composite scaffold turned out to combine suitable biomechanical properties (Young’s modulus of 1.60…

Bio-sintering processes in hexactinellid sponges: Fusion of bio-silica in giant basal spicules from Monorhaphis chuni☆

The two sponge classes, Hexactinellida and Demospongiae, comprise a skeleton that is composed of siliceous skeletal elements (spicules). Spicule growth proceeds by appositional layering of lamellae that consist of silica nanoparticles, which are synthesized via the sponge-specific enzyme silicatein. While in demosponges during maturation the lamellae consolidate to a solid rod, the lamellar organization of hexactinellid spicules largely persists. However, the innermost lamellae, near the spicule core, can also fuse to a solid axial cylinder. Similar to the fusion of siliceous nanoparticles and lamella, in several hexactinellid species individual spicules unify during sintering-like processe…

Caged Dexamethasone/Quercetin Nanoparticles, Formed of the Morphogenetic Active Inorganic Polyphosphate, are Strong Inducers of MUC5AC

Inorganic polyphosphate (polyP) is a widely distributed polymer found from bacteria to animals, including marine species. This polymer exhibits morphogenetic as well as antiviral activity and releases metabolic energy after enzymatic hydrolysis also in human cells. In the pathogenesis of the coronavirus disease 2019 (COVID-19), the platelets are at the frontline of this syndrome. Platelets release a set of molecules, among them polyP. In addition, the production of airway mucus, the first line of body defense, is impaired in those patients. Therefore, in this study, amorphous nanoparticles of the magnesium salt of polyP (Mg-polyP-NP), matching the size of the coronavirus SARS-CoV-2, were pr…

Damipipecolin and damituricin, novel bioactive bromopyrrole alkaloids from the Mediterranean sponge Axinella damicornis

Two new bromopyrrole alkaloids, damipipecolin (1) and damituricin (2), have been isolated from the Mediterranean sponge Axinella damicornis, and their structures established through spectroscopic methods. Compounds 1 and 2 extend the structural variety of the so far known pyrrole alkaloids; in these compounds, the 4-bromopyrrole 2-carboxylic acid is directly condensed with a non-protein cyclic alpha-amino acid, the (2R, 4R)-trans-4-hydroxypipecolic acid and (2R, 4R)-cis-N,N'-dimethyl-4-hydroxyproline (D-turicine) in 1 and 2, respectively. Compounds 1 and 2 were found to display a modulating effect of the serotonin receptor activity in vitro.

A new printable and durable N,O-carboxymethyl chitosan–Ca2+–polyphosphate complex with morphogenetic activity

Biomimetic materials have been gaining increasing importance in tissue engineering since they may provide regenerative alternatives to the use of autologous tissues for transplantation. In the present study, we applied for bioprinting of a functionalized three-dimensional template, N,O-carboxymethyl chitosan (N,O-CMC), mimicking the physiological extracellular matrix. This polymer, widely used in tissue engineering, has been provided with functional activity by integration of polyphosphate (polyP), an osteogenically acting natural polymer. The two polymers, N,O-CMC and polyP, are linked together via Ca2+ bridges. This N,O-CMC + polyP material was proven to be printable and durable. The N,O-…

Molecular Mechanism of Spicule Formation in the Demosponge Suberites domuncula: Silicatein-Collagen-Myotrophin

In living organisms four major groups of biominerals exist: (1) iron compounds, which are restricted primarily to Prokaryota; (2) calcium phosphates, found in Metazoa; (3) calcium carbonates, used by Prokaryota, Protozoa, Plantae, Fungi and Metazoa and (4) silica (opal) present in sponges and diatoms (reviewed in: Bengtson 1994; Baeuerlein 2000). It is surprising that the occurrence of silica as a major skeletal element is restricted to some Protozoa and to sponges (Porifera). The element silicon (Si) contributes to 28% of the earth crust and is - after oxygen - the second most abundant element on earth (Windholz 1983).

Multicolor 3D Printing of Complex Intracranial Tumors in Neurosurgery.

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. The model can be used for planning and simulating critical steps of a surgical approach. Therefore, it is important that anatomical structures such as blood vessels inside a tumor can be printed to be colored not only on their surface, but throughout their whole volume. During simulation this allows for the removal of certain parts (e.g., with a high-speed drill) and revealing internally located structures of a different color. Thus, diagnostic information from various imaging modalities (e.g., CT, MRI) can be combined in a single compact …

Circumferential spicule growth by pericellular silica deposition in the hexactinellid sponge Monorhaphis chuni.

SUMMARY The giant basal spicule of the hexactinellid sponge Monorhaphis chuni represents the longest natural siliceous structure on Earth. This spicule is composed of concentrically arranged lamellae that are approximately 10 μm thick. In the present study, we investigated the formation of outer lamellae on a cellular level using microscopic and spectroscopic techniques. It is shown that the formation of an outermost lamella begins with the association of cell clusters with the surface of the thickening and/or growing spicule. The cells release silica for controlled formation of a lamella. The pericellular (silica) material fuses to a delimited and textured layer of silica with depressions …

Kinetics of expression of prion protein in uninfected and scrapie-infected N2a mouse neuroblastoma cells.

The scrapie prion protein, PrPSc, is formed from its isoform, the cellular PrPc. There is evidence available indicating that PrPSc is necessary component of the infectious prion particle to cause a series of transmissible spongiform encephalopathies. We have used immunocytochemistry and RNA blotting techniques to investigate if infection with prions results in an increased PrP gene expression. For the experiments we used N2a cells which had been infected with prions (ScN2a cells). We demonstrated by confocal laser scanning microscopy that PrP-protein was present in the nucleus (predominantly in the nucleoli) of ScN2a cells. Analysis of the PrP-mRNA levels both in N2a- and in ScN2a cells usi…

The morphogenetically active polymer, inorganic polyphosphate complexed with GdCl 3 , as an inducer of hydroxyapatite formation in vitro

Inorganic polyphosphate (polyP) is a physiological polymer composed of tens to hundreds of phosphate units linked together via phosphoanhydride bonds. Here we compared the biological activity of polyP (chain length of 40 phosphate units), complexed with Gd(3+) (polyP·Gd), with the one caused by polyP (as calcium salt) and by GdCl3 alone, regarding their potencies to induce hydroxyapatite (HA) formation in SaOS-2 cells in vitro. The three compounds, GdCl3, polyP and polyP·Gd were found to be non-toxic at concentrations up to at least 30μM. Selecting a low, 5μM, concentration it was found that polyP·Gd significantly induced HA formation, as determined by Alizarin Red S staining and by quantit…

Establishment of an HIV cell-cell fusion assay by using two genetically modified HeLa cell lines and reporter gene.

Infection of human cells with the human immunodeficiency virus type I (HIV-1) can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 together with the appropriate human chemokine receptors. In this study, a T-tropic HIV cell-cell fusion assay was established that utilized CD4, human CXCR4 and HIV NL4-3 gp160 as fusion components and a T7 polymerase-activated luciferase as a reporter system. The HeLa T4 cells used, expressed CD4 and CXCR4, and the applied HeLa KS386 cells expressed HIV NL4-3 gp160. By combining HeLa T4 cells with HeLa KS386 cells, an approximately about 100- to 300-fold increase in luciferase activity c…

A biomimetic approach to ameliorate dental hypersensitivity by amorphous polyphosphate microparticles.

Abstract Objective Dental hypersensitivity has become one of the most common and most costly diseases in the world, even though those maladies are very rarely life threatening. Using amorphous microparticles, fabricated from the natural polymer (polyphosphate), we intend to reseal the dentinal tubules exposed and reduce by that the hypersensitivity. Methods Amorphous microparticles (termed aCa-polyP-MP) were prepared from Na-polyphosphate (polyP) and CaCl 2 , then incubated with human teeth. The potential of the microparticles to plug the dentinal tubules was determined by microscopic and spectroscopic techniques. Results We demonstrate that, in contrast to polyP, the aCa-polyP-MP efficient…

Electrospun bioactive mats enriched with Ca-polyphosphate/retinol nanospheres as potential wound dressing

Background While electrospun materials have been frequently used in tissue engineering no wound dressings exist that significantly improved wound healing effectively. Methods We succeeded to fabricate three-dimensional (3D) electrospun poly(D,l-lactide) (PLA) fiber mats into which nanospheres, formed from amorphous calcium polyphosphate (polyP) nanoparticles (NP) and encapsulated retinol (“retinol/aCa-polyP-NS” nanospheres [NS]), had been incorporated. Results Experiments with MC3T3-E1 cells revealed that co-incubation of the cells with Ca-polyP together with retinol (or incubation with retinol/aCa-polyP-NS) resulted in a significant synergistic effect on cell growth compared with particle-…

Bio-silica and bio-polyphosphate: applications in biomedicine (bone formation)

Bio-silica represents the main mineral component of the sponge skeletal elements (siliceous spicules), while bio-polyphosphate (bio-polyP), a multifunctional polymer existing in microorganisms and animals acts, among others, as reinforcement for pores in cell membranes. These natural inorganic bio-polymers, which can be readily prepared, either by recombinant enzymes (bio-silica and bio-polyP) or chemically (polyP), are promising materials/substances for the amelioration and/or treatment of human bone diseases and dysfunctions. It has been demonstrated that bio-silica causes in vitro a differential effect on the expression of the genes OPG and RANKL, encoding two mediators that control the …

The role of bestatin, an inhibitor of cell surface proteases, in the interaction of serum with untransformed cells in culture.

Bestatin is an inhibitor of cell surface-associated aminopeptidase B and leucine aminopeptidase. This microbial product simulates the role of serum as an activator of uridine uptake in quiescent BHK cells. The compound significantly stimulates the incorporation of labelled thymidine into the acid-insoluble fraction of serum-starved Nil 8 cells in the presence of low concentration of serum. The possible mechanisms of these interactions are discussed.

Protein synthesis of the sponge Geodia cydonium: characterization of the system.

Abstract The ribosomal population of the sponge Geodia cydonium has been examined. The monosomes have a sedimentation constant of 80 S, the sizes of the subunits are approximately 60 S and 45 S respectively. The polyribosomes contain up to 40 ribosomal units. Cell free protein synthesizing systems (cell homogenate as well as reconstituted system) have been prepared and characterized with respect to Mg2+, KCI and ATP concentrations, temperature, pH and time course of the reaction. In the cell-free system and in the cellular system the protein biosynthesis is inhibited by chloramphenicol. It is not affected by cycloheximide.

Isolation and characterization of five Fox (Forkhead) genes from the sponge Suberites domuncula.

Fox or Forkhead genes constitute a subgroup of the helix-turn-helix class of transcription factors with a characteristic and highly conserved DNA binding domain. To date, around 100 different Fox genes have been reported ranging from yeast to humans; these have been classified into 18 subclasses (A to P). Fox proteins are responsible for a wide range of functions and key roles in early developmental processes, during organogenesis and also for the function of the major organs and tissues in the adult. Here, we report the isolation and phylogenetic characterization of five members of the Fox family from the sponge Suberites domuncula. Four of them (Sd-FoxL2, Sd-FoxP, Sd-FoxD and Sd-FoxF) fal…

Preparation and karyotype analysis of mitotic chromosomes of the freshwater sponge Spongilla lacustris.

The present study documents for the first time the karyotype and mitotic chromosomes of a sponge. For the studies the freshwater sponge Spongilla lacustris (Lin. 1758) was used. Its karyotype comprises nine different chromosome pairs ranging in size from 2.1 to < or = 0.7 microns. Changes in size and shape of the chromosomes during the progression of mitosis are documented both light and electron microscopically. The data reveal that the lowest multicellular eukaryotes, the sponges, have already reached a high level of evolution of the mitotic mechanism.

&lt;p&gt;Cuprous oxide nanoparticles reduces hypertrophic scarring by inducing fibroblast apoptosis&lt;/p&gt;

Background Less apoptosis and excessive growth of fibroblasts contribute to the progression of hypertrophic scar formation. Cuprous oxide nanoparticles (CONPs) could have not only inhibited tumor by inducing apoptosis and inhibiting proliferation of tumor cells, but also promoted wound healing. The objective of this study was to further explore the therapeutic effects of CONPs on hypertrophic scar formation in vivo and in vitro. Methods In vivo, a rabbit ear scar model was established on New Zealand albino rabbits. Six full-thickness and circular wounds (10 mm diameter) were made to each ear. Following complete re-epithelization observed on postoperative day 14, an intralesional injection o…

Inducible ASABF-Type Antimicrobial Peptide from the Sponge Suberites domuncula: Microbicidal and Hemolytic Activity in Vitro and Toxic Effect on Molluscs in Vivo

Since sponges, as typical filter-feeders, are exposed to a high load of attacking prokaryotic and eukaryotic organisms, they are armed with a wide arsenal of antimicrobial/cytostatic low-molecular-weight, non-proteinaceous bioactive compounds. Here we present the first sponge agent belonging to the group of ASABF-type antimicrobial peptides. The ASABF gene was identified and cloned from the demospongeSuberites domuncula. The mature peptide, with a length of 64 aa residues has a predicted pI of 9.24, and comprises the characteristic CSαβ structural motif. Consequently, the S. domuncula ASABF shares high similarity with the nematode ASABFs ; it is distantly related to the defensins. The recom…

Artificial cartilage bio-matrix formed of hyaluronic acid and Mg2+-polyphosphate.

Here we show that inorganic polyphosphate (polyP), a polyanionic metabolic regulator consisting of multiple phosphate residues linked by energy-rich phosphoanhydride bonds, is present in the synovial fluid. In a biomimetic approach, to enhance cartilage synthesis and regeneration, we prepared amorphous polyP microparticles with Mg2+ as counterions. The particles were characterised by X-ray diffraction (XRD), energy-dispersive X-ray (EDX) and Fourier transformed infrared spectroscopic (FTIR) analyses. Similar particles were obtained after addition of Mg2+ ions to a solution containing hyaluronic acid, as a major component of the synovial fluid, and soluble Na-polyP. The viscous paste-like ma…

Induction of new metabolites from sponge-associated fungus Aspergillus carneus by OSMAC approach.

Abstract A comparative study on the metabolic profile of the sponge-associated fungus Aspergillus carneus using the OSMAC approach was conducted. The fungal strain was fermented on three different media including solid rice medium with or without sea salt and modified Czapek medium. Three new natural products, isopropylchaetominine (1), isoterrelumamide A (2) and 5′-epi-averufanin (3), together with fourteen known compounds (4–17) were isolated. The structures of the new compounds were established by 1D and 2D NMR spectroscopic analysis as well as by HRESIMS. Compound 2 was only found when the fungus was cultivated on modified Czapek medium, whereas compounds 4, 7, 11, 12, and 14 were only …

Innate Immune Defense of the Sponge Suberites domuncula against Bacteria Involves a MyD88-dependent Signaling Pathway

Sponges (phylum Porifera) are the phylogenetically oldest metazoa; as filter feeders, they are abundantly exposed to marine microorganisms. Here we present data indicating that the demosponge Suberites domuncula is provided with a recognition system for Gram-negative bacteria. The lipopolysaccharide (LPS)-interacting protein was identified as a receptor on the sponge cell surface, which recognizes the bacterial endotoxin LPS. The cDNA was isolated, and the protein (Mr 49,937) was expressed. During binding to LPS, the protein dimerizes and interacts with MyD88, which was also identified and cloned. The sponge MyD88 (Mr 28,441) is composed of two protein interaction domains, a Toll/interleuki…

Molecular Biomineralization: Toward an Understanding of the Biogenic Origin of Polymetallic Nodules, Seamount Crusts, and Hydrothermal Vents

Polymetallic nodules and crusts, hydrothermal vents from the Deep Sea are economically interesting, since they contain alloying components, e.g., manganese or cobalt, that are used in the production of special steels; in addition, they contain rare metals applied for plasma screens, for magnets in hard disks, or in hybrid car motors. While hydrothermal vents can regenerate in weeks, polymetallic nodules and seamount crusts grow slowly. Even though the geochemical basis for the growth of the nodules and crusts has been well studied, the contribution of microorganisms to the formation of these minerals remained obscure. Recent HR-SEM (high-resolution scanning electron microscopy) analyses of …

Cultural Heritage: Porifera (Sponges), A Taxon Successfully Progressing Paleontology, Biology, Biochemistry, Biotechnology and Biomedicine

In 1876, Campbell (Campbell, 1876 [p. 446]) wrote “those beautiful ‘glass-rope sponges’, Hyalonema etc., have been found by our researchers to be ‘the most characteristic inhabitants of the great depths all over the world, and with them ordinary siliceous sponges, some of which rival Hyalospongiae in beauty’ “. The admiration for the beauty of sponges is documented since Aristotle (cited in Camus 1783), however the nature of these organisms and their phylogenetic position remained enigmatic until less than 10 years ago. E.g., in 1988 Loomis (Loomis, 1988 [p. 186]) wrote “the sponge cells are unspecialized flagellates held together by a glycoprotein extracellular matrix... they are multicell…

14-3-3 in the cerebrospinal fluid of patients with variant and sporadic Creutzfeldt–Jakob disease measured using capture assay able to detect low levels of 14-3-3 protein

Abstract A protein capture assay was used to measure 14-3-3 (γ-isoform) in the cerebrospinal fluid (CSF) of patients with either variant or sporadic Creutzfeldt–Jakob disease (CJD). The results were compared with those obtained using Western blotting. Elevated levels of 14-3-3γ were found in 58% of variant CJD (vCJD) patients and 82% of sporadic CJD (spCJD) patients using the protein capture assay. Using a Western blotting technique, the presence of CSF 14-3-3γ was detected in 58% of vCJD patients and in 89% of spCJD patients. When the results from the protein capture assay and the Western blot were combined, 14-3-3γ was detected in 77% of vCJD patients and in 91% of spCJD patients. These r…

Kinetic models for nucleocytoplasmic transport of messenger RNA

Abstract Much is known about the mechanism by which mRNAs cross the nuclear envelope (the translocation stage of nucleocytoplasmic transport), but far less is known about the preceding (intranuclear migration/release) and succeeding (cytoplasmic binding) stages. Therefore, existing information suffices for articulating detailed kinetic models of translocation, but not models for the overall mRNA transport process. In this paper, we show that simple kinetic models of translocation can (i) accommodate date about nucleocytoplasmic distributions of endogenous transcripts; (ii) predict the overall effects on these distributions of effectors such as insulin and epidermal growth factor; (iii) thro…

Outbreak of sapovirus infection among infants and children with acute gastroenteritis in Osaka City, Japan during 2004–2005

One hundred and twenty five fecal specimens were collected from sporadic cases of acute gastroenteritis in a pediatric clinic in Osaka City, Japan from July 2004 to June 2005 and tested for the presence of rotavirus, norovirus, sapovirus, astrovirus, and adenovirus by RT-multiplex PCR. Among diarrheal viruses detected, norovirus was the most prevalent (19.2%, 24 of 125), followed by group A rotavirus (18.4%, 23 of 125), astrovirus (1.6%, 2 of 125), and adenovirus (0.8%, 1 of 125), respectively. Interestingly, sapovirus infection was identified with high incidence of 17.6% (22 of 125). Sapovirus was subjected to molecular genetic analysis by sequencing. It was found that sapovirus detected i…

Biochemistry and cell biology of silica formation in sponges

The main inorganic material forming the skeletal elements in Demospongiae as well as in Hexactinellida, the spicules, is amorphous silica. The spicules occur in the cytoplasm and the extracellular space and also in the nucleus (as silicate crystals) of some sponge cells; the function in the latter compartment is unknown. Recent evidence shows that the formation of spicules is mediated by the enzyme silicatein. The cDNA as well as the gene encoding this enzyme was cloned from Suberites domuncula. The recombinant silicatein catalyzes the syn- thesis of amorphous silicate using tetraethoxysilane as substrate. The enzyme is dependent on ferric iron. Silicatein also has proteolytic (cathepsin-li…

Strombine dehydrogenase in the demosponge Suberites domuncula: Characterization and kinetic properties of the enzyme crucial for anaerobic metabolism

Previously, the cDNA and the respective gene for a presumed tauropine dehydrogenase (TaDH) from Suberites domuncula (GenBank accession nos. AM712888, AM712889) had been annotated. The conclusion that the sequences encode a TaDH had been inferred from the 68% identity with the TaDH protein from the marine demosponge Halichondria japonica. However, subsequent enzymatic assays shown here indicate that the presumed S. domuncula opine dehydrogenase is in fact a strombine dehydrogenase (StDH). The enzyme StDH is highly specific for glycine and is inhibited by an excess of the substrate pyruvate. Besides kinetic data, we report in this study also on the predicted tertiary and quaternary structure …

Transition from Protozoa to Metazoa: An Experimental Approach

Until recently, stromatolites were thought to be the oldest fossils on earth that were very abundant 2000 to 3000 Ma (million years) ago (Walter 1994). Recently, the biological origin of these fossils has been questioned (Walter 1996). The universal phylogenetic tree exhibits a tripartite division of the living world into Bacteria (“eubacterial”), Archaea (“archebacterial”), and Eucarya [“eukaryotic” (Woese 1987; Woese et al. 1991)]. Based on comparisons of amino acid (aa) sequence data from enzymes, it has been proposed that the common ancestor of prokaryotes and eukaryotes lived about 2000 Ma ago (Doolittle et al. 1996). Phylogenetic analysis of the 70kDa heat-shock proteins suggested tha…

Binding of Tat Protein to TAR Region of Human Immunodeficiency Virus Type 1 Blocks TAR-Mediated Activation of (2′-5′)Oligoadenylate Synthetase

The TAR sequence of the 5' leader of HIV-1 long terminal repeat-directed mRNA was found to be able to bind to and to activate double-stranded RNA-dependent (2'-5')A synthetase. Binding of TAR to the purified synthetase in vitro was abolished by addition of HIV-1 Tat protein, which binds to this sequence with a high affinity. Inhibition of TAR-mediated activation of (2'-5')A synthetase by Tat was prevented in the presence of the Zn2+ and Cd2+ chelators o-phenanthroline and penicillamine, which did not impair TAR-synthetase interaction. Transient expression assays of bacterial chloramphenicol acetyltransferase (CAT) gene in HeLa cells revealed that the levels of both CAT mRNA and CAT protein …

Emergence and Disappearance of an Immune Molecule, an Antimicrobial Lectin, in Basal Metazoa

Sponges (phylum Porifera) represent the evolutionarily oldest metazoans that comprise already a complex immune system and are related to the crown taxa of the protostomians and the deuterostomians. Here, we demonstrate the existence of a tachylectin-related protein in the demosponge Suberites domuncula, termed Suberites lectin. The MAPK pathway was activated in response to lipopolysaccharide treatment of the three-dimensional cell aggregates, the primmorphs; this process was abolished by the monosaccharide D-GlcNAc. The cDNA encoding the S. domuncula lectin was identified and cloned; it comprises 238 amino acids (26 kDa) in the open reading frame. The deduced protein has one potential trans…

Differential mode of inhibition of terminal deoxynucleotidyl transferase by 3′-dATP, ATP, βaraATP and αaraATP

Intracellular signal transduction pathways in sponges.

Abstract Sponges are the lowest multicellular eukaryotic organisms. Due to the relatively low specialization, and concomitantly the high differentiation and dedifferentiation potency of their cells, the sponge cell system has proven to be a useful model to study the mechanism of cell-cell adhesion on molecular levels. Results of detailed biochemical and cell biological studies with the main cell adhesion molecules, the aggregation factor (AF) and the aggregation receptor, led to the formation of the modulation theory of cell adhesion. The events of cell adhesion are contigent on a multiplicity of precisely coordinated intracellular signal transduction pathways. Using the marine sponge Geodi…

Evolutionary relationships of Metazoa within the eukaryotes based on molecular data from Porifera

Recent molecular data provide strong support for the view that all metazoan phyla, including Porifera, are of monophyletic origin. The relationship of Metazoa, including the Porifera, to Plantae, Fungi and unicellular eukaryotes has only rarely been studied by using cDNAs coding for proteins. Sequence data from rDNA suggested a relationship of Porifera to unicellular eukaryotes (choanoflagellates). However, ultrastructural studies of choanocytes did not support these findings. In the present study, we compared amino acid sequences that are found in a variety of metazoans (including sponges) with those of Plantae, Fungi and unicellular eukaryotes, to obtain an answer to this question. We use…

A synthetic biology approach for the fabrication of functional (fluorescent magnetic) bioorganic–inorganic hybrid materials in sponge primmorphs

During evolution, sponges (Porifera) have honed the genetic toolbox and biosynthetic mechanisms for the fabrication of siliceous skeletal components (spicules). Spicules carry a protein scaffold embedded within biogenic silica (biosilica) and feature an amazing range of optical, structural, and mechanical properties. Thus, it is tempting to explore the low-energy synthetic pathways of spiculogenesis for the fabrication of innovative hybrid materials. In this synthetic biology approach, the uptake of multifunctional nonbiogenic nanoparticles (fluorescent, superparamagnetic) by spicule-forming cells of bioreactor-cultivated sponge primmorphs provides access to spiculogenesis. The ingested nan…

Assessing the root of bilaterian animals with scalable phylogenomic methods.

A clear picture of animal relationships is a prerequisite to understand how the morphological and ecological diversity of animals evolved over time. Among others, the placement of the acoelomorph flatworms, Acoela and Nemertodermatida, has fundamental implications for the origin and evolution of various animal organ systems. Their position, however, has been inconsistent in phylogenetic studies using one or several genes. Furthermore, Acoela has been among the least stable taxa in recent animal phylogenomic analyses, which simultaneously examine many genes from many species, while Nemertodermatida has not been sampled in any phylogenomic study. New sequence data are presented here from org…

Species-specific aggregation factor in sponges V. Influence on programmed syntheses

Isolated cells from the siliceous sponge Geodia cydonium as well as small primary aggregates (diameter: 70 mum) consisting of them show no increase in rates of programmed syntheses and mitotic activity with time. After addition of a highly purified aggregation factor to a culture with primary aggregates which subsequently form secondary aggregates (diameter: larger than 1000 mum), a dramatic increase of DNA, RNA and protein synthesis occurs. Together with this increase, the cells show a high mitotic activity. The values for the mitotic coefficient reach a first maximum 8 h after the beginning of the secondary aggregation process. The stimulation of the mitotic activity of cells during the a…

Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca2+ level in osteoblasts (SaOS-2 cells) in vitro

Inorganic polymeric phosphate is a physiological polymer that accumulates in bone cells. In the present study osteoblast-like SaOS-2 cells were exposed to this polymer, complexed in a 2:1 stoichiometric ratio with Ca(2+), polyP (Ca(2+) salt). At a concentration of 100 μM, polyP (Ca(2+) salt) caused a strong increase in the activity of the alkaline phosphatase and also an induction of the steady-state expression of the gene encoding this enzyme. Comparative experiments showed that polyP (Ca(2+) salt) can efficiently replace β-glycerophosphate in the in vitro hydroxyapatite (HA) biomineralization assay. In the presence of polyP (Ca(2+) salt) the cells extensively form HA crystallites, which r…

Influence of template inactivators on the binding of DNA polymerase to DNA.

The agents daunomycin, ethidium bromide, distamycin A and cytochrome c inhibit DNA dependent DNA polymerase I (E. coli) reaction competitively to DNA. The influence of these template inactivators on the binding of DNA polymerase to native as well as denatured DNA has been determined by affinity chromatography. Cytochrome c blocks the binding of the enzyme to double-stranded and to single-stranded DNA Sepharose. In contrast to these results daunomycin, ethidium bromide or distamycin A reduce the binding affinity only with denatured DNA Sepharose as matrix. These data are discussed with respect to the modification by template inactivators of the affinity of DNA to the different binding sites …

Formation of a micropatterned titania photocatalyst by microcontact printed silicatein on gold surfaces

The enzyme silicatein has been bioengineered to carry a thiol-bearing Au-affinity tag (Cys-tag) for direct immobilization on gold carriers in shortest time without the need for prior surface functionalization. Through microcontact printing, defined silicatein micropatterns were created on gold surfaces, facilitating the subsequent enzymatically controlled synthesis of photocatalytically active TiO(2).

Regional and modular expression of morphogenetic factors in the demosponge Lubomirskia baicalensis

Some sponges [phylum Porifera], e.g. the demosponges Lubomirskia baicalensis or Axinella polypoides, show an arborescent growth form. In the freshwater sponge L. baicalensis this morphotype is seen mostly in depths below 4 m while in more shallow regions it grows as a crust. The different growth forms are determined in nature very likely by water current and/or light. The branches of this species are composed of modules, arranged along the apical-basal axis. The modules are delimited by a precise architecture of the spicule bundles; longitudinal bundles originate from the apex of the earlier module, while at the basis of each module these bundles are cross-linked by traverse bundles under f…

A physiologically active interpenetrating collagen network that supports growth and migration of epidermal keratinocytes: zinc-polyP nanoparticles integrated into compressed collagen.

The distinguished property of the physiological polymer, inorganic polyphosphate (polyP), is to act as a bio-intelligent material which releases stimulus-dependent metabolic energy to accelerate wound healing. This characteristic is based on the bio-imitating feature of polyP to be converted, upon exposure to peptide-containing body fluids, from stable amorphous nanoparticles to a physiologically active and energy-delivering coacervate phase. This property of polyP has been utilized to fabricate a wound mat consisting of compressed collagen supplemented with amorphous polyP particles, formed from the inorganic polyanion with an over-stoichiometric ratio of zinc ions. The proliferation and t…

Metabolites from Combretum dolichopetalum and its associated endophytic fungus Nigrospora oryzae--Evidence for a metabolic partnership.

Abstract A new altersolanol derivative, 4-dehydroxyaltersolanol A ( 9 ), along with two known sesquiterpenoids, ( S )-7′-hydroxyabscisic acid ( 7 ) and ( S )-abscisic acid ( 8 ) were obtained from the endophytic fungus, Nigrospora oryzae , isolated from leaves of Combretum dolichopetalum . The host plant yielded six known compounds including ellagic acid ( 1 ), 3, 3′, 4-tri-O-methylellagic acid ( 2 ), arjunolic acid ( 3 ), 4′-dihydrophaseic acid ( 4 ), echinulin ( 5 ) and arestrictin B ( 6 ). Close structural similarities with regard to compounds 4 , 7 and 8 were observed between the metabolites from the host plant and those of the endophytic fungus. Furthermore compounds 5 and 6 are relate…

Alteration of nuclear (2'-5')oligoriboadenylate synthetase and nuclease activities preceding replication of human immunodeficiency virus in H9 cells.

After infection of the respective target cells with the human immunodeficiency virus (HIV-1) viral progeny is produced only after a short temporary delay of some days, depending on cell type. After this period of time a sudden onset of HIV-1 protein synthesis with a dramatic increase in virus release occurs. (2'-5')Oligoriboadenylates [(2'-5')A], capable to activate a latent ribonuclease (RNase L) degrading both mRNA and rRNA, are known mediators involved in the early response of cells to virus infection. Here we show that the (2'-5')A-synthesizing (2'-5')A synthetase, which is inducible by interferon and activated by double-stranded RNA, as well as a (2'-5')A nuclease (2',3'-exoribonucleas…

Identification of a cell surface-associated protein involved in mouse neural cell aggregation by means of antibodies against the sponge aggregation factor.

Polyclonal antibodies were raised against the purified aggregation factor (AF) from the sponge Geodia cydonium to elucidate possible immunological relationships between adhesion molecules of lower multicellular eukaryotic systems (sponges) and those of vertebrates. This anti-AF recognized a series of polypeptides associated with the AF, among them also a polypeptide with a Mr of 47,000 (p47). The formation of the antibody-p47 immunocomplexes could be prevented by adsorbing the anti-AF with a brain extract from DBA/2J mice. Moreover, this brain polypeptide inhibited the AF-mediated aggregation of sponge cells. Interestingly, the anti-AF recognized a p37 molecule in the brains of 2- to 3-day-…

Ethylene modulates gene expression in cells of the marine sponge Suberites domuncula and reduces the degree of apoptosis.

Sponges (phylum Porifera) live in an aqueous milieu that contains dissolved organic carbon. This is degraded photochemically by ultraviolet radiation to alkenes, particularly to ethylene. This study demonstrates that sponge cells (here the demosponge Suberites domuncula has been used), which have assembled to primmorphs, react to 5 microM ethylene with a significant up-regulation of intracellular Ca(2+) concentration and with a reduction of starvation-induced apoptosis. In primmorphs from S. domuncula the expression of two genes is up-regulated after exposure to ethylene. The cDNA of the first gene (SDERR) isolated from S. domuncula encodes a potential ethylene-responsive protein, termed ER…

S-type lectins occur also in invertebrates: high conservation of the carbohydrate recognition domain in the lectin genes from the marine sponge Geodia cydonium.

The marine sponge Geodia cydonium contains several lectins. The main component, called lectin-1, is composed of three to four identical subunits. The subunits of the lectins were cloned from a cDNA library; two clones were obtained. From the deduced aa sequence of one clone, LECT-1, a mol. wt of 15,313 Da is calculated; this value is in good agreement with mass spectrometric analysis of 15,453 +/- 25 Da. The sequence of another clone, LECT-2, was analysed and the aa sequence was deduced (15,433 Da). The two subunits have a framework sequence of 38 conserved aa which are characteristic for the carbohydrate-binding site of vertebrate S-type lectins. Clustering of lectin sequences of various s…

DNA-replication complex from cells infected with herpes virus.

Herpes simplex virus (HSV) DNA synthesis is initiated in an intact cell system by a 36-residue ribonucleotide stretch [W.E.G. Müller, R.K. Zahn, J. Arendes, and D. Falke (1979) Virology, 98, 200-210]. In the present study a nucleoplasmic fraction was isolated from rabbit kidney cells infected with HSV (type 1), which catalyzes DNA synthesis. By means of specific assays, containing single-stranded deoxyribopolymers, it was elucidated that the replication complex contains both an RNA-synthesizing and a DNA-synthesizing enzyme. These enzymes were characterized as host cell RNA polymerase II and HSV-induced DNA polymerase. The RNA polymerase II synthesizes an RNA initiator with an average chain…

Polymorphism in the immunoglobulin-like domains of the receptor tyrosine kinase from the sponge Geodia cydonium.

Sponges [Porifera] are the phylogenetically oldest phylum of the Metazoa. They are provided with both cellular and humoral allorecognition systems. The underlying molecules are not yet known. To study allorecognition in sponges we first determined the frequency of graft rejection in a natural population of the marine sponge Geodia cydonium. We then determined, for the first time at the molecular level, the degree of sequence polymorphism in segments of one molecule which may be related to sponge allorecognition and host defense: the Ig-like domains from the receptor tyrosine kinase [RTK]. Thirty six pairs of auto- and allografts were assayed, either by parabiotic attachment or insertion of …

Application of a MTT Assay for Screening Nutritional Factors in Growth Media of Primary Sponge Cell Culture

Marine sponges (Porifera) are producers of the largest variety of bioactive compounds among benthic marine organisms. In vitro culture of marine sponge cells has been proposed for the sustainable production of these pharmacologically interesting compounds from marine sponges but with limited success. The development of a suitable growth medium is an essential prerequisite for sponge cells grown in vitro. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was adapted to screen for potential nutritional factors in formulating a growth medium for primary cell culture of Suberites domuncula. In 96-well plates, the effects of nutritional factors including glutamine, pyr…

Isolation of the silicatein-α interactor silintaphin-2 by a novel solid-phase pull-down assay.

The skeleton of siliceous sponges consists of amorphous biogenous silica (biosilica). Biosilica formation is driven enzymatically by means of silicatein(s). During this unique process of enzymatic polycondensation, skeletal elements (spicules) that enfold a central proteinaceous structure (axial filament), mainly comprising silicatein, are formed. However, only the concerted action of silicatein and other proteins can explain the genetically controlled diversity of spicular morphotypes, from simple rods with pointed ends to intricate structures with up to six rays. With the scaffold protein silintaphin-1, a first silicatein interactor that facilitates the formation of the axial filament and…

Cyclic heptapeptides from the soil-derived fungus Clonostachys rosea

Abstract Three new cyclic heptapeptides (1–3) together with three known compounds (4–6) were isolated from a solid rice culture of the soil-derived fungus Clonostachys rosea. Fermentation of the fungus on white beans instead of rice afforded a new γ-lactam (7) and a known γ-lactone (8) that were not detected in the former extracts. The structures of the new compounds were elucidated on the basis of 1D and 2D NMR spectra as well as by HRESIMS data. Compounds 1 and 4 exhibited significant cytotoxicity against the L5178Y mouse lymphoma cell line with IC50 values of 4.1 and 0.1 µM, respectively. Compound 4 also displayed cytotoxicity against the A2780 human ovarian cancer cell line with an IC50…

A New Tetrahydrofuran Derivative from the Endophytic Fungus Chaetomium sp. Isolated from Otanthus maritimus

1 A hitherto unidentified endophytic strain of the genus Chaetomium, isolated from the medicinal plant Otanthus maritimus, yielded a new tetrahydrofuran derivative, aureonitolic acid (), along with 5 known natural products, 2 - 6. The structure of 1 was determined by extensive spectroscopic analysis and comparison with reported data. Extracts of the fungus, grown either in liquid culture or on solid rice media, exhibited considerable cytotoxic activity when tested in vitro against L5178Y mouse lymphoma cells. Compounds 2 and 6 showed significant growth inhibition against L5178Y cells with EC50 values of 7.0 and 2.7 μg/mL, respectively, whereas 1 was inactive

Novel mechanism for the radiation-induced bystander effect: nitric oxide and ethylene determine the response in sponge cells.

Until now the bystander effect had only been described in vertebrates. In the present study the existence of this effect has been demonstrated for the phylogenetically oldest metazoan phylum, the Porifera. We used the demosponge Suberites domuncula for the experiments in the two-chamber-system. The lower dish contained irradiated "donor" cells (single cells) and the upper dish the primmorphs ("recipient" primmorphs). The "donor" cells were treated with UV-B light (40 mJ/cm2) and 100 microM hydrogen peroxide (H2O2), factors that exist also in the natural marine aquatic environment of sponges; these factors caused a high level of DNA strand breaks followed by a reduced viability of the cells.…

Src proteins/src genes: from sponges to mammals

The genome of marine sponge Suberites domuncula, a member of the most ancient and most simple metazoan phylum Porifera, encodes at least five genes for Src-type proteins, more than, i.e., Caenorhabditis elegans or Drosophila melanogaster (two in each). Three proteins, SRC1SD, SRC2SD and SRC3SD, were fully characterized. The overall homology (identity+similarity) among the three S. domuncula Srcs (68-71%) is much lower than the sequence conservation between orthologous Src proteins from freshwater sponges (82-85%). It is therefore very likely that several src genes/proteins were already present in the genome of Urmetazoa, the hypothetical metazoan ancestor. We have identified in the S. domun…

Silicatein: from chemical through enzymatic silica formation, to synthesis of biomimetic nanomaterials

Silicateins are the enzymes that had been identified in sponges, then sequenced and expressed. They are not only the enzymes facilitating biosilica synthesis but also function as structure-guiding and structure-forming proteins. The three minireviews highlight the principles of silicatein-mediated biosilica formation and outline the bionic strategies which might be used for the design and fabrication of novel materials.

Experimental indication in favor of the introns-late theory: the receptor tyrosine kinase gene from the sponge Geodia cydonium.

Abstract We have analyzed the gene that encodes receptor tyrosine kinase (RTK) from the marine sponge Geodia cydonium, which belongs to the most ancient and simple metazoan groups, the Porifera. RTKs are enzymes found only in metazoa. The sponge gene contains two introns in the extracellular part of the protein. However, the rest of the protein (transmembrane and intracellular part), including the tyrosine kinase (TK)-domain, is encoded by a single exon. In contrast, all TK genes, so far known only from higher animals (vertebrates), contain several introns especially in the TK-domain. The TK-domain of G. cydonium shows similarity with numerous members of receptor as well as nonreceptor TKs.…

Selenium affects biosilica formation in the demosponge Suberites domuncula

Selenium is a trace element found in freshwater and the marine environment. We show that it plays a major role in spicule formation in the demosponge Suberites domuncula. If added to primmorphs, an in vitro sponge cell culture system, it stimulates the formation of siliceous spicules. Using differential display of transcripts, we demonstrate that, after a 72-h exposure of primmorphs to selenium, two genes are up-regulated; one codes for selenoprotein M and the other for a novel spicule-associated protein. The deduced protein sequence of selenoprotein M (14 kDa) shows characteristic features of metazoan selenoproteins. The spicule-associated protein (26 kDa) comprises six characteristic repe…

Bleomycin inhibition of DNA synthesis in isolated enzyme systems and in intact cell systems.

Abstract Blcomycin (BLM) inhibits DNA and RNA synthesis in different isolated enzyme systems. The inhibition effect can be reduced by adcling RNA to the reaction mixture. The activity of the RNA dependent DNA polymerase and of a cell-free protein synthesizing system is not affected by BLM. The antibiotic reduces cell proliferation (L5178y mouse lymphoma cells) in vitro at low concentrations by cytostatis and at higher concentrations by cytotoxicity. In BLM-treated L5178y cells DNA synthesis is strongly reduced, while RNA and protein synthesis are not affected. In vivo , using growing quail oviducts, cell proliferation and cytodifferentiation are markedly inhibited after BLM treatment. This …

Iron Induces Proliferation and Morphogenesis in Primmorphs from the Marine SpongeSuberites domuncula

Dissociated cells from marine demosponges retain their proliferation capacity if they are allowed to form special aggregates, the primmorphs. On the basis of incorporation studies and septin gene expression, we show that Fe3+ ions are required for the proliferation of cells in primmorphs from Suberites domuncula. In parallel, Fe3+ induced the expression of ferritin and strongly stimulated the synthesis of spicules. This result is supported by the finding that the enzymatic activity of silicatein, converting organosilicon to silicic acid, depends on Fe3+. Moreover, the expression of a scavenger receptor molecule, possibly involved in the morphology of spicules, depends on the presence of Fe3…

Electrospinning of Bioactive Wound-Healing Nets

The availability of appropriate dressings for treatment of wounds, in particular chronic wounds, is a task that still awaits better solutions than provided by currently applied materials. The method of electrospinning enables the fabrication of novel materials for wound dressings due to the high surface area and porosity of the electrospun meshes and the possibility to include bioactive ingredients. Recent results show that the incorporation of biologically active inorganic polyphosphate microparticles and microspheres and synergistically acting retinoids into electrospun polymer fibers yields biocompatible and antibacterial mats for potential dressings with improved wound-healing propertie…

Expression of silicatein and collagen genes in the marine sponge Suberites domuncula is controlled by silicate and myotrophin

The major skeletal elements in the (Porifera) sponges, are spicules formed from inorganic material. The spicules in the Demospongiae class are composed of hydrated, amorphous silica. Recently an enzyme, silicatein, which polymerizes alkoxide substrates to silica was described from the sponge Tethya aurantia. In the present study the cDNA encoding silicatein was isolated from the sponge Suberites domuncula. The deduced polypeptide comprises 331 amino acids and has a calculated size of Mr 36 306. This cDNA was used as a probe to study the potential role of silicate on the expression of the silicatein gene. For these studies, primmorphs, a special form of aggregates composed of proliferating c…

From anti-fouling to biofilm inhibition: New cytotoxic secondary metabolites from two Indonesian Agelas sponges

Chemical investigation of Indonesian marine sponges Agelas linnaei and A. nakamurai afforded 24 alkaloid derivatives representing either bromopyrrole or diterpene alkaloids. A. linnaei yielded 16 bromopyrrole alkaloids including 11 new natural products with the latter exhibiting unusual functionalities. The new compounds include the first iodinated tyramine-unit bearing pyrrole alkaloids, agelanesins A-D. These compounds exhibited cytotoxic activity against L5178Y mouse lymphoma cells with IC(50) values between 9.25 and 16.76 muM. Further new compounds include taurine acid substituted bromopyrrole alkaloids and a new dibromophakellin derivative. A. nakamurai yielded eight alkaloids among th…

The La antigen shuttles between the nucleus and the cytoplasm in CV-1 cells

Recently we established a monoclonal antibody against the La-protein (Bachmann et al., Proc. Natl. Acad. Sci. USA, 83, 7770, 1986). The antibody gives a nuclear speckled type staining and, in addition, a perinuclear cytoplasmic staining on cultured cells in immunofluorescence microscopy. After inhibition of RNA synthesis the La-protein is transported into the cytoplasm. After prolonged inhibition it returns into the nucleus forming large growing speckles. The transport into the nucleus apparently depends on glycosylation.

Bioactive polyketides and alkaloids from Penicillium citrinum , a fungal endophyte isolated from Ocimum tenuiflorum

Chemical investigation of the endophytic fungus Penicillium citrinum cultured on white beans or on rice led to the isolation of two new alkaloids (1 and 2), along with fourteen known polyketides (6-12, 14-20) and four known alkaloids (3-5, and 13). The structures of the isolated compounds were determined by extensive analysis of the 1D, 2D NMR, and MS data, and by comparison with the literature. Compound 13, which had been previously obtained only by chemical synthesis, was isolated as a natural product for the first time, while compound 6 was firstly reported as a fungal metabolite. A re-isolation of sclerotinin A (14) revealed it to be a diastereoisomeric mixture (14a and 14b), whose ster…

Biofabrication of biosilica-glass by living organisms

Biosilicification is an evolutionarily old and widespread type of biomineralization both in unicellular and multicellular organisms, including sponges, diatoms, radiolarians, choanoflagellates, and higher plants. In the last few years combined efforts in molecular biology, cell biology, and inorganic and analytical chemistry have allowed the first insight into the molecular mechanisms by which these organisms form an astonishing variety of siliceous structures that cannot be achieved by chemical methods. Here we report about the present stage of knowledge on structure, biochemical composition, and mechanisms of biosilica formation, focusing our attention particularly on sponges because of t…

Influence of Altered Microbes on Soil Organic Carbon Availability in Karst Agricultural Soils Contaminated by Pb-Zn Tailings.

Soil organic carbon (SOC) availability is determined via a complex bio-mediated process, and Pb-Zn tailings are toxic to the soil microbes that are involved in this process. Here, Pb-Zn-tailings- contaminated karst soils with different levels (paddy field &gt; corn field &gt; citrus field &gt; control group) were collected to explore the intrinsic relationship between Pb-Zn tailings and microbes due to the limited microbial abundance in these soils. The SOC concentration in the paddy fields is the highest. However, based on the soil microbial diversity and sole-carbon-source utilization profiles, the rate of SOC availability, McIntosh index, Shannon-Wiener diversity index, Simpson’s diversi…

Physicochemical and functional characterization of the polymerization process of the Geodia cydonium lectin

The extracellularly localized, galactose-specific lectin from the sponge Geodia cydonium binds at one class of sites, 40 mol Ca2+/mol lectin with an association constant (Ka) of 0.3 X 10(6)M-1. Stoichiometric calculations reveal that in the extracellular milieu 22 mol Ca2+ (maximum) are complexed per mol lectin. Binding of Ca2+ to the lectin increases its apparent Mr from 44000 to 56000 (electrophoretic determination) or from 36500 to 53500 (high-pressure liquid gel chromatographical determination); the s20, w increases from 4.3 S to 4.5 S if Ca2+ is added to the lectin. In the presence of Ca2+ the lectin undergoes a conformational change perhaps by expanding the carbohydrate side chains wh…

The therapeutic potential of inorganic polyphosphate: A versatile physiological polymer to control coronavirus disease (COVID-19).

Rationale: The pandemic caused by the novel coronavirus SARS-CoV-2 is advancing rapidly. In particular, the number of severe courses of the disease is still dramatically high. An efficient drug therapy that helps to improve significantly the fatal combination of damages in the airway epithelia, in the extensive pulmonary microvascularization and finally multiorgan failure, is missing. The physiological, inorganic polymer, polyphosphate (polyP) is a molecule which could prevent the initial phase of the virus life cycle, the attachment of the virus to the target cells, and improve the epithelial integrity as well as the mucus barrier. Results: Surprisingly, polyP matches perfectly with the ca…

Transformation of Construction Cement to a Self-Healing Hybrid Binder

A new biomimetic strategy to im prove the self-healing properties of Portland cement is presented that is based on the application of the biogenic inorganic polymer polyphosphate (polyP), which is used as a cement admixture. The data show that synthetic linear polyp, with an average chain length of 40, as well as natural long-chain polyP isolated from soil bacteria, has the ability to support self-healing of this construction material. Furthermore, polyP, used as a water-soluble Na-salt, is subject to Na+/Ca2+ exchange by the Ca2+ from the cement, resulting in the formation of a water-rich coacervate when added to the cement surface, especially to the surface of bacteria-containing cement/c…

Flupirtine increases the levels of glutathione and Bcl-2 in hNT (human ) neurons: mode of action of the drug-mediated anti-apoptotic effect

Flupirtine is a triaminopyridine analogue which has been successfully applied in clinics as a non-opiate analgesic drug. Previously we described that flupirtine acts like an N-methyl-D-aspartate (NMDA) receptor antagonist in neuronal cells both in vitro and in vivo. Here we show that flupirtine displays its anti-apoptotic effect also in hNT (human Ntera/D1) neurons. hNT neurons were induced to apoptosis applying glutamate (Glu; at concentrations > or = 1 mM) or NMDA (> or = 1 mM). During Glu/NMDA-mediated apoptosis the levels of the intracellular anti-apoptotic agents Bc1-2 and glutathione dropped by more than 50%. Flupirtine completely abolished this reduction of Bc1-2 and glutathione leve…

Oxygen as a morphogenic factor in sponges: expression of a tyrosinase gene in the sponge Suberites domuncula

Sponges live in a symbiotic relationship with microorganisms, especially bacteria. Here we show, using the demosponge Suberites domuncula as a model, that the sponge expresses the enzyme tyrosinase which synthesizes diphenols from monophenolic compounds. It is assumed that these products serve as carbon source for symbiotic bacteria to grow.

Mitochondrial genome of Suberites domuncula: palindromes and inverted repeats are abundant in non-coding regions.

The 26,300-nucleotide sequence of the mitochondrial DNA (mtDNA) molecule of the demosponge Suberites domuncula (Olivi, 1792), the largest in size yet found in Porifera, has been determined. We describe the second hadromerid sponge mitochondrial genome that contains the same set of 41 genes as the hadromerid sponge Tethya actinia, including trnMe(cau), trnI2(cau), trnR2(ucu), and atp9, all of which are transcribed in the same direction. Furthermore, rRNA genes for the small and large ribosomal subunit are very long, rns is indeed the longest among Metazoa (1833 bp). Intergenic regions (IGR) comprise about 25% of S. domuncula mtDNA and include numerous direct and inverted repeats, as well as …

Amorphous Polyphosphate and Ca‐Carbonate Nanoparticles Improve the Self‐Healing Properties of both Technical and Medical Cements

Cement is used both as a construction material and for medical applications. Previously, it has been shown that the physiological polymer inorganic polyphosphate (polyP) is morphogenetically active in regeneration of skin, bone, and cartilage. The present study investigates the question if this polymer is also a suitable additive to improve the self-healing capacity not only of construction cement but also of inorganic bone void fillers. For the application in the cement, two different polyP-based amorphous nanoparticles (NP) are prepared, amorphous Ca-polyP NP and amorphous Ca-carbonate (ACC) NP. The particles are integrated into poly(methyl methacrylate) in a concentration ratio of 1:10. …

Conservation of the positions of metazoan introns from sponges to humans

Abstract Sponges (phylum Porifera) are the phylogenetic oldest Metazoa still extant. They can be considered as reference animals (Urmetazoa) for the understanding of the evolutionary processes resulting in the creation of Metazoa in general and also for the metazoan gene organization in particular. In the marine sponge Suberites domuncula , genes encoding p38 and JNK kinases contain nine and twelve introns, respectively. Eight introns in both genes share the same positions and the identical phases. One p38 intron slipped for six bases and the JNK gene has three more introns. However, the sequences of the introns are not conserved and the introns in JNK gene are generally much longer. Intron…

Sponge homologue to human and yeast gene encoding the longevity assurance polypeptide: differential expression in telomerase-positive and telomerase-negative cells of Suberites domuncula.

Porifera show a characteristic Bauplan in spite of the fact that (almost) all cells are telomerase-positive and presumably provided with an unlimited potency for cell proliferation. Studies revealed that telomerase-positive cells can be triggered to telomerase-negative cells by dissociating them into single cells. Single cells from the demosponge Suberites domuncula, in contrast to cells present in primmorphs (a special form of cell aggregates), lack the property to proliferate and they undergo apoptosis. One gene, SDLAGL, was identified in primmorphs that showed high sequence similarity to the longevity assurance genes from other Metazoa. In single cells no transcripts of SDLAGL could be i…

Manganese/polymetallic nodules: Micro-structural characterization of exolithobiontic- and endolithobiontic microbial biofilms by scanning electron microscopy

Polymetallic/ferromanganese nodules (Mn-nodules) provide a rich source for manganese. It is not yet known if the nodules have a biogenic or an abiogenic origin. Here we applied the technique of high-resolution scanning electron microscopy, in combination with energy dispersive X-ray spectroscopical (EDX) analysis, to trace the existence of microbial biofilms. Two spatially separated assemblies exist, the exolithobiontic- and endolithobiontic colonizations. The exolithobiontic colonization is seen in the micro-canals, which traverse the outer surface layer of the nodules and are formed by elongated filamentous organisms, which show no signs of mineralization. In the center of the nodules thr…

Evidence for a biogenic, microorganismal origin of rock varnish from the Gangdese Belt of Tibet

In the present study we examined material from the Ashikule Basin of Tibet. Chemical analyses were performed by use of energy dispersive X-ray spectroscopy and electron probe microanalysis to clarify whether the varnish layers that had developed on the surface of the rhyolite are indeed composed of varnish bodies and silica glaze. Electron microscopic analyses revealed that the surface of the varnish is covered both by filamentous hyphae bacterial and cocci-shaped forms. Within the varnish mineral layer in those samples two forms of bacteria-like microorganisms exist; cocci as tightly packed bacterial aggregates [within varnish bodies], and bacillus-like microorganisms [within the varnish m…

Biosilica aging: From enzyme-driven gelation via syneresis to chemical/biochemical hardening

Abstract Background The distinguished property of the siliceous sponge spicules is their enzyme (silicatein)-catalyzed biosilica formation. The enzymatically formed, non-structured biosilica product undergoes a molding, syneresis, and hardening process to form the species-specifically shaped, hard structured skeletal spicules. Besides of silicatein, a silicatein-associated protein, silintaphin-2, is assumed to be involved in the process of biosilica formation in vivo. Methods Biosilica has been synthesized enzymatically and determined quantitatively. In addition, the subsequent hardening/aging steps have been followed by spectroscopic and electron microscopic analyses. Results The young spi…

Aged Mice Devoid of the M3 Muscarinic Acetylcholine Receptor Develop Mild Dry Eye Disease

The parasympathetic nervous system is critically involved in the regulation of tear secretion by activating muscarinic acetylcholine receptors. Hence, various animal models targeting parasympathetic signaling have been developed to induce dry eye disease (DED). However, the muscarinic receptor subtype (M1–M5) mediating tear secretion remains to be determined. This study was conducted to test the hypothesis that the M3 receptor subtype regulates tear secretion and to evaluate the ocular surface phenotype of mice with targeted disruption of the M3 receptor (M3R−/−). The experimental techniques included quantification of tear production, fluorescein staining of the ocular surface, environmenta…

Double-stranded RNA polyinosinic-polycytidylic acid immobilized onto gamma-Fe2O3 nanoparticles by using a multifunctional polymeric linker.

Cryptochrome in Sponges: A Key Molecule Linking Photoreception with Phototransduction

Sponges (phylum: Porifera) react to external light or mechanical signals with contractile or metabolic reactions and are devoid of any nervous or muscular system. Furthermore, elements of a photoreception/phototransduction system exist in those animals. Recently, a cryptochrome-based photoreceptor system has been discovered in the demosponge. The assumption that in sponges the siliceous skeleton acts as a substitution for the lack of a nervous system and allows light signals to be transmitted through its glass fiber network is supported by the findings that the first spicules are efficient light waveguides and the second sponges have the enzymatic machinery for the generation of light. Now…

Ubiquitins (polyubiquitin and ubiquitin extension protein) in marine sponges: cDNA sequence and phylogenetic analysis

The complete nucleotide sequences of twoSuberites domunculacDNAs and oneSycon raphanuscDNA, all encoding ubiquitin, have been determined. One cDNA fromS. domunculacodes for polyubiquitin with four tandemly repeated monomeric units and the second cDNA encodes ubiquitin fused to a ribosomal protein of 78 amino acids (aa).S. domunculapossesses at least one additional polyubiquitin gene, from which the last two monomers were also sequenced. All analysed genes fromS. domunculaencode identical ubiquitin proteins, with only one aa difference (Ala19) to the human/higher animals ubiquitin (Pro19). Ubiquitin inS. domunculais identical with the ubiquitin found in another Demospongia,Geodia cydonium. T…