Induction of Hsp70 by the Herbicide Oxyfluorfen (Goal) in the Egyptian Nile Fish, Oreochromis niloticus

This paper deals with the expression of the biomarker hsp70 in the liver and kidney of the freshwater fish Oreochromis niloticus following exposure to the herbicide oxyfluorfen (Goal). Fishes were exposed to three concentrations, the 96-h LC50 (3 mg/L), the 96-h (1/2)LC50 (1.5 mg/L), and the 96-h (1/4)LC50 (0.75 mg/L) of oxyfluorfen for 6, 15, and 24 days, respectively, and samples were taken at three different time periods for each concentration. The livers responded to the herbicide by an induction of the expression of both the constitutive (hsp75; Mr 75 kDa) and the inducible (hsp73; Mr 73 kDa) hsp70 proteins. In kidney, the herbicide induced a time-dependent increase in the expression o…

Multifunctional polymer-derivatized γ-Fe2O3 nanocrystals as a methodology for the biomagnetic separation of recombinant His-tagged proteins

Abstract Multifunctional polymer-derivatized superparamagnetic iron oxide (γ-Fe2O3) nanoparticles were prepared for biomagnetic separation of histidine-tagged recombinant proteins building up a faster and efficient method for protein separation by making use of their intrinsic magnetic properties. Using polymer bound γ-Fe2O3 nanocrystals, a 6× histidine-tagged recombinant protein (silicatein) with a molecular weight of 24 kDa has been isolated and purified. The supermagnetic iron oxide nanocrystals were characterized by transmission electron microscopy (TEM), high-resolution TEM (HRTEM), SQUID and Mossbauer and the polymer functionalization of the γ-Fe2O3 nanocrystals was monitored by UV–vi…

Synergistic effect of peplomycin in combination with bleomycin on L5178y mouse lymphoma cells in vivo.

Studying the treatment of NMRI mice with ip injections of bleomycin (BLM) for 5 days we found an approximate LD50 of 35 mg/kg; the toxicity of peplomycin (PEP) was slightly higher (LD50: approximately 25 mg/kg). The effect of the two drugs on growth of L5178y mouse lymphoma cells in NMRI mice was examined. BLM alone caused at a concentration of 2.5 mg/kg an almost complete inhibition of tumor cell growth; the same effect was determined with 1 mg PEP/kg. At these concentrations the drugs caused an increase of the survival time of 110% (BLM) or 104% (PEP). Given in combination, one-sixth of the optimal doses yielded an 100% increase of the median survival time. These results indicate a signif…

Morphogenetically active scaffold for osteochondral repair (Polyphosphate/alginate/N,O-carboxymethyl chitosan)

Here we describe a novel bioinspired hydrogel material that can be hardened with calcium ions to yield a scaffold material with viscoelastic properties matching those of cartilage. This material consists of a negatively charged biopolymer triplet, composed of morphogenetically active natural inorganic polyphosphate (polyP), along with the likewise biocompatible natural polymers N,O-carboxymethyl chitosan (N,O-CMC) and alginate. The porosity of the hardened scaffold material obtained after calcium exposure can be adjusted by varying the pre-processing conditions. Various compression tests were applied to determine the local (nanoindentation) and bulk mechanical properties (tensile/compressio…

Functional Polymer-Opals from Core-Shell Colloids

Colloidal photonic crystals were prepared from monodisperse core-shell particles. The shell is hereby formed from a functional monomer, such as glycidylmethacrylate or different reactive ester monomers, which can perform chemical reactions and the core from a standard monomer, which yields highly monodisperse colloids. It was possible to crystallize the core-shell particles into artificial opals with excellent optical properties. Reactions on the functional surface of the colloids were carried out, which lead to a dramatic rise in the mechanical stability or to a functionalization of His-tagged silicatein, which acts as nanoreactor to synthesize and immobilize gold nanoparticles from auric …

Activation of MAP kinase signaling pathway in the mussel Mytilus galloprovincialis as biomarker of environmental pollution

Stimulation of MAP kinase signal transduction pathway by various stressful stimuli was investigated in the marine bivalve Mytilus galloprovincialis. Analyses were performed in animals exposed in laboratory to selected pollutants and in mussels collected in winter and summer along the eastern Adriatic coast (Croatia). Effects of oxidative stress, induced by tributyltin, hydrogen peroxide and water soluble fraction of diesel fuel on the activation/phosphorylation of the three Mitogen-Activated Protein Kinases (MAPKs) p38, JNK and ERK using a newly developed ELISA procedure were evaluated. MAP kinase activation was analyzed 1 h after exposure of mussels to chemical agents, and after recovery p…

Differential effect of insulin and epidermal growth factor on the mRNA translocation system and transport of specific poly(A+) mRNA and poly(A-) mRNA in isolated nuclei.

The efficiency of efflux of rapidly labeled poly(A)-containing mRNA from isolated rat liver nuclei was found to be modulated by insulin and epidermal growth factor (EGF) in a biphasic but opposite way. At physiological concentrations (10 pM insulin and 1 pM EGF), maximal stimulation of the transport rate by insulin (to 137%) and maximal inhibition by EGF (to 69%) were obtained; at higher concentrations (greater than 100 pM and greater than 10 pM, respectively), the amount of poly(A)-containing mRNA released into the postnuclear supernatant was nearly identical with the level found in untreated nuclei (= 100%). Using mRNA entrapped into closed nuclear envelope (NE) vesicles as a model system…

Synthesis of the neurotoxin quinolinic acid in apoptotic tissue from Suberites domuncula: cell biological, molecular biological and chemical analyses

Sessile marine animals, such as sponges, are prone to infection by prokaryotic as well as by eukaryotic attacking organisms. In the present study we document for the first time that in tissue from sponges which underwent apoptosis, a toxic compound is produced which very likely controls the elimination of the dying tissue. The marine sponge Suberites domuncula develops in the field occasionally apoptotic tissue areas which are rapidly eliminated. In the present study apoptosis was induced in S. domuncula by exposing the specimens in aquaria to 5 µg/ml Dip or by maintaining the sponges for 3 - 5 days under non-aeration conditions. After that treatment only one eukaryotic epibiont, the mollus…

Molecular/chemical ecology in sponges. Evidence for an adaptive antibacterial response in Suberites domuncola

Sponges (Porifera) represent the evolutionary oldest metazoan phylum still extant today. They have developed a complex Bauplan, based on the existence of structural and regulatory molecules; many of these have been cloned and analyzed in the past years. The demosponge Suberites domuncula has been used as a suitable model to demonstrate that these animals not only possess an adaptive immune response on the level of cytokines, but also, as pointed out here, on the level of synthesis of bioactive alkyl-lipid derivatives. From specimens of S. domuncula the two lyso-PAF (platelet-activating factor) compounds, 1-O-hexadecyl-sn-glycero-3-phosphocholine and 1-O-octadecyl-sn-glycero-3-phosphocholine…

A new polyphosphate calcium material with morphogenetic activity

Abstract Polyphosphate [polyP] has been proven to elicit morphogenetic activity on bone cells. By applying mild reaction conditions, a Ca-polyP material that displays a hardness of ≈1.3 GPa has been fabricated. The Ca-polyP granules are prone to hydrolytic degradation during in vitro incubation of the cells, suggesting that this property is associated with the observed bioactivity.

Silicate modulates the cross-talk between osteoblasts (SaOS-2) and osteoclasts (RAW 264.7 cells): inhibition of osteoclast growth and differentiation

It has been shown that inorganic monomeric and polymeric silica/silicate, in the presence of the biomineralization cocktail, increases the expression of osteoprotegerin (OPG) in osteogenic SaOS-2 sarcoma cells in vitro. In contrast, silicate does not affect the steady-state gene expression level of the osteoclastogenic ligand receptor activator of NF-κB ligand (RANKL). In turn it can be expected that the concentration ratio of the mediators OPG/RANKL increases in the presence of silicate. In addition, silicate enhances the growth potential of SaOS-2 cells in vitro, while it causes no effect on RAW 264.7 cells within a concentration range of 10-100 µM. Applying a co-cultivation assay system,…

Cordycepin analogues of 2',5'-oligoadenylate inhibit human immunodeficiency virus infection via inhibition of reverse transcriptase.

Analogues of 2',5'-oligoadenylates (2-5A), the cordycepin (3'-deoxyadenosine) core trimer (Co3) and its 5'-monophosphate derivative (pCo3), were shown to display pronounced anti-human immunodeficiency virus type 1 (HIV-1) activity in vitro. Treatment of HIV-1 infected H9 cells with 1 microM Co3 or pCo3 resulted in an almost 100% inhibition of virus production. The compounds were encapsulated in liposomes targeted by antibodies specific for the T-cell receptor molecule CD3. Substitution of one or two cordycepin units in Co3 or pCo3 decreased the antiviral activity of the compounds. pCo3 did not stimulate 2-5A-dependent ribonuclease L activity and displayed no effect on the amount of cellular…

Biomineral Amorphous Lasers through Light-Scattering Surfaces Assembled by Electrospun Fiber Templates

New materials aim at exploiting the great control of living organisms over molecular architectures and minerals. Optical biomimetics has been widely developed by microengineering, leading to photonic components with order resembling those found in plants and animals. These systems, however, are realized by complicated and adverse processes. Here we show how biomineralization might enable the one-step generation of components for amorphous photonics, in which light is made to travel through disordered scattering systems, and particularly of active devices such as random lasers, by using electrospun fiber templates. The amount of bio-enzymatically produced silica is related to light-scatterin…

Characterization and osteogenic activity of a silicatein/biosilica-coated chitosan-graft-polycaprolactone.

Several attempts have been made in the past to fabricate hybrid materials that display the complementary properties of the polyester polycaprolactone (PCL) and the polysaccharide chitosan (CHS) for application in the field of bone regeneration and tissue engineering. However, such composites generally have no osteogenic activity per se. Here we report the synthesis of a chitosan-graft-polycaprolactone (CHS-g-PCL) and its subsequent characterization, including crystallinity, chemical structure and thermal stability. Upon surface-functionalization of CHS-g-PCL with osteogenic biosilica via the surface-immobilized enzyme silicatein, protein adsorption, surface morphology and wettability were a…

Inhibition of expression of human immunodeficiency virus-1 in vitro by antibody-targeted liposomes containing antisense RNA to the env region.

Previous studies revealed that antisense oligodeoxynucleotides to specific regions of the human immunodeficiency virus-1 (HIV-1) are potent inhibitors of replication of HIV-1 in vitro (Zamecnik, P. C., Goodchild, J., Taguchi, Y., and Sarin, P. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4143-4146). We now report that antisense RNA, synthesized in vitro using T7 and SP6 RNA polymerase, displayed an anti-HIV-1 effect in the HTLV-IIIB/H9 system in vitro. Treatment of HIV-1-infected H9 cells with viral env region antisense RNA encapsulated in liposomes targeted by antibodies specific for the T cell receptor molecule CD3 almost completely inhibited HIV-1 production. The viral env segment cover…

Modulation of the Antiviral 2-5A System in Human Immunodeficiency Virus-1-Infected CEM Cells by Propentofylline

2′,5′-OIigoadenylates (2-5A) play an essential role in the establishment of the antiviral state of cells exposed to virus infection. However, - after an initial increase observed in some cell lines - the activity of the interferon (IFN)-inducible, 2-5A-forming 2′,5′-oligoadenylate synthetase (2-5A synthetase) strongly decreases soon after infection of cells with the human immunodeficiency virus-1 (HIV-1). In the present report, we show that in IFN-treated human T lymphoblastoid CEM cells, the decrease in 2-5A synthetase activity had already occurred at day 1 post infection (p.i.)- At days 3 and 5 p.i., the 2-5A synthetase activity in the IFN-treated infected cells amounted to only 10-12% of…

Enzyme-Mediated Deposition of a TiO2Coating onto Biofunctionalized WS2 Chalcogenide Nanotubes

A chemically specific and facile method for the biofunctionalization of WS2 nanotubes (NT-WS2) is reported. The covalent modification strategy is based on the affinity of the nitrilotriacetic acid (NTA) side chain, which serves as a ligand for the surface binding to NT-WS2 and simultaneously as an anchor group for the binding of His-tagged proteins to the polymer backbone. The polymer functionalized WS2 nanotubes can be solubilized either in water or organic solvents; they are stable for at least one week. The probes were characterized by FT-IR and UV-vis spectroscopy. The immobilization of silicatein, a hydrolytic protein encountered in marine sponges, was visualized by scanning force micr…

DNA Damage and Developmental Defects After Exposure to UV and Heavy Metals in Sea Urchin Cells and Embryos Compared to Other Invertebrates

The depletion of the stratospheric ozone layer and the resulting increase in hazardous ultraviolet-B (UV-B) radiation reaching the Earth are of major concern not only for terrestrial but also for aquatic organisms. UV-B is able to penetrate clear water to ecologically significant depths. This chapter deals with the effects of UV radiation on DNA integrity in marine benthic organisms, in particular sea urchins in comparison to other marine invertebrates (sponges and corals). These animals cannot escape the damaging effects of UV-B radiation and may be additionally exposed to pollution from natural or anthropogenic sources. Besides eggs and larvae that lack a protective epidermal layer and ar…

17β-Estradiol-dependent regulation of chaperone expression and telomerase activity in the marine sponge Geodia cydonium

It is known that species belonging to the lowest metazoan phylum, the Porifera (sponges), do not develop tumors. Sponge cells share with tumor cells of higher animals at least one characteristic; they contain high levels of telomerase activity, suggesting that they possess a high proliferation capacity. This assumption, however, has not been substantiated experimentally. In addition, sponges show a specific bauplan, leading us to postulate that they undergo apoptosis to replace a given set of cells at a given time. In the present study, 17β-estradiol (βE2) was used as a defined agent to assess its effect on both the telomerase activity and the process of apoptosis in the marine sponge Geodi…

Monitoring chemical and physical stress using sea urchin immune cells.

Coelomocytes are the cells freely circulating in the body fluid contained in echinoderm coelom and constitute the defence system, which, in response to injuries, host invasion, and adverse conditions, is capable of chemotaxis, phagocytosis, and production of cytotoxic metabolites. Red and colourless amoebocytes, petaloid and philopodial phagocytes, and vibratile cells are the cell types that, in different proportions, constitute the mixed coelomocyte cell population found in sea urchins. Advances in cellular and molecular biology have made it possible to identify a number of specific proteins expressed in coelomocytes under resting conditions or when activated by experimentally induced stre…

Demonstration of an endocrine signaling circuit for insulin in the sponge Geodia cydonium.

Abstract The existence of an insulin-mediated cell-to-cell signaling in the sponge Geodia cydonium is demonstrated in this study by molecular biological and immunological techniques. The sequence of a sponge cDNA clone encoding preproinsulin was analyzed for the first time and determined to comprise a high homology to human preproinsulin (60-80% homology). The predicted polypeptide of preproinsulin from sponge contains two disulfide bridges which link the A- to the B-chain. The intra-A chain disulfide bridge is absent. Applying immunological and electron microscopical techniques it is shown that insulin is produced in specialized cells (spherulous cells). Experimental evidence is presented …

The nucleocytoplasmic shuttling of the La antigen in CV-I cells

Homologies Between Different Forms of 2-5A Synthetases

(2′-5′) Oligoadenylate synthetases (2-5A synthetases; EC 2.7.7.19) are present in mammalian cells and tissues and synthesize from ATP a series of oligomers termed 2-5A [general formula: ppp(A2′p)nA; with 1 ≤ n < 18 and usually 1 ≤ n < 6] (Hovanessian 1991). For full enzymic activity of the 2-5A synthetases, binding of double-stranded RNA is required (Sen 1982). Three principal 2-5A synthetase isoenzymes have been described with Mr’s of 40–46, 69, and 100 kDa (Chebath et al. 1987; Hovanessian et al. 1987, 1988). In the following they are classified as 2-5A synthetase I [Mr 40–46 000], II [Mr 69 000] and III [Mr 100 000]. All three isoforms are induced in cells by interferon (Cohen et al. 198…

Sustainable production of bioactive compounds from sponges: primmorphs as bioreactors

Sponges [phylum Porifera] are a rich source for the isolation of biologically active and pharmacologically valuable compounds with a high potential to become effective drugs for therapeutic use. However, until now, only one compound has been introduced into clinics because of the limited amounts of starting material available for extraction. To overcome this serious problem in line with the rules for a sustainable use of marine resources, the following routes can be pursued; first, chemical synthesis, second, cultivation of sponges in the sea (mariculture), third, growth of sponge specimens in a bioreactor, and fourth, cultivation of sponge cells in vitro in a bioreactor.

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

Interaction of prion protein mRNA with CBP35 and other cellular proteins: possible implications for prion replication and age-dependent changes.

Abstract A study of the intracellular distribution of prion protein (PrP) in N2a neuroblastoma cells which had been infected with prions (ScN2a cells) revealed that most PrP is present in the cytoplasm. However, a significant amount of PrP is also present in the nucleus (predominantly in the nucleoli) of these cells, as analyzed by confocal laser scanning microscopy. By contrast, no PrP could be detected in the nucleus of uninfected N2a cells. The steady-state level of PrP mRNA did not markedly differ between the two cell strains. Likewise, no changes were found in the rate of transcription and in the half-life of PrP mRNA. A number of cellular proteins, among them the nuclear lectin CBP35,…

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…

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.

Effects of a phycotoxin, okadaic acid, on oyster heart cell survival

Okadaic acid (OA) is a dinoflagellate toxin which accumulates in shellfish producing diarrhetic shellfish poisoning (DSP) in humans. It was found that OA is a highly selective inhibitor of protein phosphatase types 1 (PP1) and 2A (PP2A) which produces a marked increase in phosphorylation of several proteins, including p38 mitogen-activated protein (MAP) kinase. The cytotoxicity attributed to OA and the effects on p38 MAP kinase and calcium current were examined in the oyster Crassostrea gigas in this study. Data showed that p38 MAP kinase is strongly expressed in oyster heart and that OA bioaccumulated in cultured heart cells. Hence the effects of OA was tested in vitro and in vivo on oyste…

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 …

Alterations inras-gene expression and intracellular distribution of protein kinase C in the spongeGeodia cydonium in response to marine pollution

The siliceous spongeGeodia cydonium Jameson was used to study the influence of pollution in marine environments on selected parameters of the intracellular signal transduction pathway. The parameters chosen were: intracellular distribution of protein kinase C (PK-C),ras-gene expression and DNA polymerasealpha (DNA Polalpha) activity. Both PK-C andras-gene product (ras-protein) have previously been established to be key molecules in the intracellular signalling pathway in sponges; increased level ofras-protein mediates events following sponge cell-cell contact. Three unpolluted and three polluted sites in the off-shore seawater around Rovinj (Yugoslavia) were selected for the study in 1989. …

Modulation of Nuclear Matrix-associated 2′,5′-Oligoadenylate Metabolism and Ribonuclease L Activity in H9 Cells by Human Immunodeficiency Virus

Human T cells (H9), infected with the HTLV-IIIB strain of the human immunodeficiency virus (HIV-1), have been used to study the alteration of 2',5'-oligoadenylate [2'-5')A) metabolism in relation to virus production. The synthesis of (2'-5')A was determined to proceed in close association with the nuclear matrix. After HIV infection the (2'-5')A synthetase activity increased from 1.1 to 1.5 pmol of (2'-5')A synthesized/100 micrograms of nuclear matrix protein (during a 3-h in vitro incubation period) to 8.2 pmol at day 3 after infection. Then the activity dropped to the initial values. In non-infected H9 cells the (2'-5')A synthetase activity remained unchanged. Simultaneously with the decr…

Growth of fibrous aggregates of silica nanoparticles: Fibre growth by mimicking the biogenic silica patterning processes

We describe the self-assembly of discrete SiO2 nanofibers via grafting of silicatein side chains to a polymer backbone. The covalent binding of silicatein to the backbone of the polymer is based on the affinity of the nitrilotriacetic acid (NTA) side chain, which serves as a ligand for the immobilization of His-tagged silicatein. The surface charge and the bulkiness of the protein moieties prevent the entropically favoured coil formation of the polymer and force it to adopt an open chain structure after hydrolysis of the silica precursors. The probes were characterized by scanning force microscopy (SFM) and optical light microscopy. Surface complexation of the resulting silica nanoparticles…

The 2-5A System and HIV Infection

The human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of acquired immunodeficiency syndrome (AIDS). The progression of this retro viral disease is associated with various clinical manifestations, including the acquisition of an immunodeficient state, the frequent presence of neurological disorders, and some malignancies (reviewed in Barre-Sinoussi et al. 1983; Wong-Staal and Gallo 1985; Fauci 1988). Immunologic dysfunctions caused by HIV-1 infection include disorders in the production of cytokines (Murray et al. 1984; Abb et al. 1986). For example, a significant decrease in the production of interferon-α (IFN-α) by cultured peripheral blood mononuclear cells (PBMC) from pat…

Inhibition of Formation of Rev-RRE Complex by Pyronin Y

The interaction of pyronin Y, an RNA intercalating drug, with the binding of Rev protein from human immunodeficiency virus type 1 (HIV-1) to Rev-responsive element (RRE)-containing env RNA was studied. In gel retardation assays, recombinant Rev protein tightly bound to in vitro transcribed RRE RNA. Nitrocellulose-filter-binding studies revealed a dissociation constant of ≈(1–2) = 10−10M (Pfeifer et al., 1991). Pyronin Y efficiently suppressed formation of the Rev-RRE complex. At a concentration of 1 μg ml−1, complex formation was almost completely inhibited. Electron microscopy showed that Rev oligomerizes in the presence of RRE-containing RNA with the formation of short rod-like structures…

Cell-cell recognition system in gorgonians: description of the basic mechanism

The dissociation of the gorgonian Eunicella cavolinii (Koch) into single cells was successfully accomplished. These cells readily formed aggregates of a size of 2 100 μm during incubation in roller tubes; no aggregate formation was observed in non-rotating Petri dishes. The formation of aggregates was not influenced by Ca++, urea or trypsin; it was also independent of temperature (4° to 30°C) and pH (5.5–9.0). The intercellular material of the gorgonian contains a galactose-specific lectin, as determined by double diffusion experiments and haemagglutination inhibition experiments using a series of galactoglycoconjugates. This lectin converted the aggregation-susceptible cells to aggregation…

Inorganic Polyphosphate in Eukaryotes: Enzymes, Metabolism and Function

Inorganic polyphosphates (polyP) are linear polymers of orthophosphate (Pi) residues linked by high-energy phosphoanhydride bonds. These polymers are widely distributed in nature, from archaebacteria, eubacteria, fungi, algae, and protozoa to higher plants and animals (for reviews, see Kulaev 1979; Wood and Clark 1988; Kornberg 1994, 1995). PolyP molecules are stable in neutral aqueous solutions, but are hydrolyzed by heat-treatment, and under acidic or alkaline conditions. The chain length of polyP may range from 3 to more than 1000 Pi residues; it can be analyzed on urea/polyacrylamide gels (Clark and Wood 1987; Lorenz et al. 1994a). In contrast to the linear polymer, branched inorganic p…

Purification and characterization of an exopolyphosphatase from Saccharomyces cerevisiae.

An exopolyphosphatase (polyphosphate phosphohydrolase; EC 3.6.1.11) activity that cleaves inorganic polyphosphates to orthophosphate has been purified to apparent homogeneity (> 95% pure) from Saccharomyces cerevisiae. The exopolyphosphatase is a monomeric protein with a polypeptide molecular mass of 28 kDa. The enzyme, which can be stabilized in the presence of Triton X-100, has a pH optimum of 7.5 and requires, for maximal activity, Co2+ or Mg2+ ions. In the absence of these ions, the exopolyphosphatase binds to polyphosphate but does not degrade it, allowing affinity purification of the enzyme on a polyphosphate-modified zirconia support. o-Vanadate, Cu2+, and Ca2+ are effective inhibito…

Synergistic Anti-Human Immunodeficiency Viral (HIV-1) Effect of the Immunomodulator Ampligen (Mismatched Double-Stranded RNA) with Inhibitors of Reverse Transcriptase and HIV-1 Regulatory Proteins

The potent antiviral effect of double stranded RNA, such as the mismatched poly(l)·poly(C12U) [Ampligen], 2′,3′-dideoxy-3′-fluorothymidine (FddThd) and antisense oligodeoxynucleotides (ODN) has been established in in vitro systems using cells infected with the human immunodeficiency virus type 1 (HIV-1). We report here that the immunomodulator poly(l)·poly(C12U) interacts synergistically with (1) the reverse transcriptase inhibitor FddThd (FIC value: 0.43), (2) the modified (5′- and 3′-end capped thioates) antisense ODN-4 directed against the splice acceptor site of the HIV-1/ tat gene (FIC value: 0.66) and (3) also with pyronin Y, a compound which prevents binding of HIV-1 Rev protein to t…

Mechanism of the Antiretroviral Effect of dsRNA

The development of AIDS seems to be linked to an impairment of processes which are induced or activated by double-stranded RNA (dsRNA), such as the biosynthesis of interferon (IFN), production of 2′,5′-oligoadenylate (2-5A), ribonuclease L (RNase L) activity and different cell-mediated immune functions. A restriction of available bioactive dsRNA (or of dsRNA-dependent enzymes) may play an important role in the disease progression. The results summarized in this review show that defects in dsRNA-dependent pathways exhibited by AIDS patients can be reversed, at least in part, by exogenously supplied dsRNA.

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…

Human conglutinin-like protein inhibits infection by the human immunodeficiency virus-1 in vitro.

In summary the lectin-like protein analogous to bovine conglutinin was purified from human serum. Using a lectin-based ELISA system, it was demonstrated that conglutinin-like protein binds to human immunodeficiency virus-1 (HIV1) glycoprotein 120 (gp 120) via its carbohydrate binding site. In vitro experiments with T-lymphoblastoid CEM cells revealed that conglutinin-like protein abolishes infection by HIV1; a 50 % cytoprotective concentration of 23.9 μg/ml was measured.

The Chemokine Networks in Sponges: Potential Roles in Morphogenesis, Immunity and Stem Cell Formation

Porifera (sponges) are now well accepted as the phylum which branched off first from the common ancestor of all metazoans, the Urmetazoa. The transition to the Metazoa became possible because during this phase, cell-cell as well as cell-matrix adhesion molecules evolved which allowed the formation of a colonial stage of animals. The next prerequisite for the evolution to the Urmetazoa was the establishment of an effective immune system which, flanked by apoptosis, allowed the formation of a first level of individuation.

Studies on protein kinases involved in regulation of nucleocytoplasmic mRNA transport

The rate of energy-dependent nucleoside triphosphatase (NTPase)-mediated nucleocytoplasmic translocation of poly(A)-containing mRNA [poly(A)+mRNA] across the nuclear envelope is thought to be regulated by poly(A)-sensitive phosphorylation and dephosphorylation of nuclear-envelope protein. Studying the phosphorylation-related inhibition of the NTPase, we found that phosphorylation of one polypeptide of rat liver envelopes by endogenous NI- and NII-like protein kinase was particularly sensitive to poly(A). This polypeptide (106 kDa) was also phosphorylated by nuclear-envelope-bound Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C). Activation of kinase C by tumour-pr…

V2O5 nanowires with an intrinsic peroxidase-like activity

V2O5 nanowires exhibit an intrinsic catalytic activity towards classical peroxidase substrates such as 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 3,3,5,5,-tetramethylbenzdine (TMB) in the presence of H2O2. These V2O5 nanowires show an optimum reactivity at a pH of 4.0 and the catalytic activity is dependent on the concentration. The Michaelis-Menten kinetics of the ABTS oxidation over these nanowires reveals a behavior similar to that of their natural vanadium-dependent haloperoxidase (V-HPO) counterparts. The V2O5 nanowires mediate the oxidation of ABTS in the presence of H2O2 with a turnover frequency (k(cat)) of 2.5 x 10(3) s(-1). The K-M values of the V2O5 nanowire…

Retrovirus infection and aging: Increase in UAG suppressor tRNA expression in aged mice.

Summary The effect of aging on expression of a natural glutamine suppressor tRNA (tRNA Gln(UmUG) ) was studied in different tissues of mice; this tRNA recognizes UAG and inserts glutamine at the site of the termination codon. The level of tRNA Gln(UmUG) was found to be strongly increased in aged mice, compared to newborn and mature animals. An elevated expression of tRNA Gln(UmUG) has also been found in retrovirus-infected cells; in Moloney virus-infected cells the suppressor tRNA allows to read-through the UAG codon within the retroviral protease gene. We suggest that the increase in the level of tRNA Gln(UmUG) may influence retroviral gene expression with age.

Molecular phylogeny of the freshwater sponges in Lake Baikal

The phylogenetic relationship of the freshwater sponges (Porifera) in Lake Baikal is not well understood. A polyphyletic and/or monophyletic origin have been proposed. The (endemic) Baikalian sponges have been subdivided into two families: endemic Lubomirskiidae and cosmopolitan Spongillidae. In the present study, two new approaches have been made to resolve the phylogenetic relationship of Baikalian sponges; analysis of (1) nucleotide sequences from one mitochondrial gene, the cytochrome oxidase subunit I (COI) and of (2) one selected intron from the tubulin gene. Specimens from the following endemic Baikalian sponge species have been studied; Lubomirskia baicalensis, Baikalospongia interm…