Differential polyadenylation pattern of ovalbumin precursor RNAs during development.

The expression of the ovalbumin gene encoding for the major hen oviduct protein slows down with age. Analysis of Northern blots of electrophoretically separated total and poly(A) + RNA from oviducts of hens of different age with an ovalbumin-specific probe (nick-translated 9.5 kb ovalbumin gene DNA cloned into pBR322) revealed that the largest high molecular weight ovalbumin RNA precursor (7.9 kb band, representing the putative primary transcript of the ovalbumin gene) was most intense if total RNA from non-egg-laying old hen oviduct was checked as compared to that from egg-laying mature animals. On the other side, the 7.9 kb RNA precursor band was readily detected in the poly(A) + RNA from…

FORMATION OF A SMALL RIBONUCLEOPROTEIN PARTICLE BETWEEN TAT PROTEIN AND TRANS-ACTING RESPONSE ELEMENT IN HUMAN IMMUNODEFICIENCY VIRUS-INFECTED CELLS

The trans-acting response element (TAR) within the long terminal repeat of human immunodeficiency virus (HIV) is present in all 5' termini of HIV mRNAs and is recognized by the viral Tat protein. Now we describe that the 59-nucleotide-long TAR-RNA exists as a ribonucleoprotein particle in polysomal and heterogeneous nuclear RNP fractions of HIV-1-infected HeLa-T4+ cells. Applying an immunoprecipitation technique this Tat.TAR complex could be isolated from total cell extracts as well as from polysomal or heterogeneous nuclear RNP fractions. The chain length and the identity of the TAR-RNA were established by RNase protection assays while the Tat protein was confirmed by Western blotting tech…

A novel galactose- and arabinose-specific lectin from the sponge Pellina semitubulosa: isolation, characterization and immunobiological properties.

A new lectin from the sponge Pellina semitubulosa is derived which was extracted and purified to homogeneity. The purified lectin is probably a hexamer of polypeptide chains (each M(r) 34,000) which are covalently linked via disulfide linkages; the isoelectric point is 6.1. The lectin displays the following specificities: D-galactose (50% inhibition of hemagglutination at 0.2 mM) = L-arabinose (0.2 mM) greater than D-fucose (1.5 mM) greater than D-glucose (3.0 mM). It precipitates human erythrocytes (A1, A2, A1B, B, and O) with a titer between 2(8) and 2(11) and erythrocytes from sheep and rabbits with a titer between 2(5) and 2(10). The Pellina lectin displays a strong mitogenic effect on …

Enhancement of Wound Healing in Normal and Diabetic Mice by Topical Application of Amorphous Polyphosphate. Superior Effect of a Host⁻Guest Composite Material Composed of Collagen (Host) and Polyphosphate (Guest).

The effect of polyphosphate (polyP) microparticles on wound healing was tested both in vitro and in a mice model in vivo. Two approaches were used: pure salts of polyphosphate, fabricated as amorphous microparticles (MPs, consisting of calcium and magnesium salts of polyP, “Ca–polyp-MPs” and “Mg–polyp-MPs”), and host–guest composite particles, prepared from amorphous collagen (host) and polyphosphate (guest), termed “col/polyp-MPs”. Animal experiments with polyP on healing of excisional wounds were performed using both normal mice and diabetic mice. After a healing period of 7 days “Ca–polyp-MP” significantly improved re-epithelialization in normal mice from 31% (control) to 72% (polyP micr…

Intracellular distribution of the La antigen in CV-1 cells after herpes simplex virus type 1 infection compared with the localization of U small nuclear ribonucleoprotein particles.

The La antigen is known to associate, at least transiently, with a series of small nuclear and cytoplasmic ribonucleoprotein particles (snRNPs and scRNPs), e.g. U1 and U6 snRNPs. In CV-1 cells a monoclonal antibody (MAb), directed against the La protein (La1B5), immunostained intranuclear speckles. These speckles were found to co-localize with speckles that were stained by MAbs directed against either all U snRNPs or only against U1 snRNPs. Two h after infection of CV-1 cells with herpes simplex virus type 1 (HSV-1) (strain HFEM) the staining of nuclear speckles with the anti-La MAb disappeared and the La protein was found quantitatively in the cytoplasm. In contrast nuclear speckles remain…

Cover Picture: NanoSIMS: Insights into the Organization of the Proteinaceous Scaffold within Hexactinellid Sponge Spicules (ChemBioChem 8/2010)

Arginine kinase in the demosponge Suberites domuncula:regulation of its expression and catalytic activity by silicic acid

SUMMARY In Demospongiae (phylum Porifera) the formation of the siliceous skeleton,composed of spicules, is an energetically expensive reaction. The present study demonstrates that primmorphs from the demosponge Suberites domuncula express the gene for arginine kinase after exposure to exogenous silicic acid. The deduced sponge arginine kinase sequence displays the two characteristic domains of the ATP:guanido phosphotransferases; it can be grouped to the `usual' mono-domain 40 kDa guanidino kinases (arginine kinases). Phylogenetic studies indicate that the metazoan guanidino kinases evolved from this ancestral sponge enzyme; among them are also the `unusual'two-domain 80 kDa guanidino kinas…

Induction of DNA strand breaks and expression of HSP70 and GRP78 homolog by cadmium in the marine sponge Suberites domuncula

The marine sponge Suberites domuncula was used as a bioindicator to study the effects of cadmium on the occurrence of DNA strand breakage and on the induction of the expression of the stress biomarkers, heat shock protein 70 (HSP70) and glucose-regulated protein 78 (GRP78) homolog. The cDNA encoding GRP78 homolog from S. domuncula was isolated and characterized. The GRP78 cDNA has a length of 2.1 kb and displays characteristic features of the HSP70 family ; it encodes an aa sequence of M-r 72, 000. Exposure of S. domuncula to 1 mg/L of cadmium chloride for 24 h caused a strong(16.6-fold) increase in cadmium content to 7.7 mu g/g wet weight of sponge tissue ; after an incubation period of 6 …

Specific phosphorylation of proteins in pore complex-laminae from the sponge Geodia cydonium by the homologous aggregation factor and phorbol ester. Role of protein kinase C in the phosphorylation of DNA topoisomerase II.

We have recently shown that the aggregation factor (AF) from the sponge Geodia cydonium stimulates DNA synthesis in quiescent, dissociated cells from the same organism; this event was correlated with the release of the two second messengers: inositol trisphosphate and diacylglycerol. Here we describe that after binding of the AF to the plasma membrane-bound aggregation receptor, a rapid and drastic increase in the incorporation of 32Pi into a series of proteins in the pore complex-lamina fraction occurs. Addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, to quiescent cells resulted in a similar stimulation of phosphorylation of nuclear proteins. Among them we have selecte…

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.

Generation of TCR-Engineered T Cells and Their Use To Control the Performance of T Cell Assays

Abstract The systematic assessment of the human immune system bears huge potential to guide rational development of novel immunotherapies and clinical decision making. Multiple assays to monitor the quantity, phenotype, and function of Ag-specific T cells are commonly used to unravel patients’ immune signatures in various disease settings and during therapeutic interventions. When compared with tests measuring soluble analytes, cellular immune assays have a higher variation, which is a major technical factor limiting their broad adoption in clinical immunology. The key solution may arise from continuous control of assay performance using TCR-engineered reference samples. We developed a simp…

Correlation between the level of the potential biomarker, heat-shock protein, and the occurrence of DNA damage in the dab, Limanda limanda: a field study in the North Sea and the English Channel.

In the present study, heat-shock protein of M-r 70 kDa (HSP70), a marker of cellular stress response, was validated as a potential biomarker under field conditions. The dab, Limanda limanda (female, size greater than or equal to 25 cm, spawning maturity stage 2) was used as the indicator organism. The data on HSP level were correlated with the occurrence of DNA damage, measured in the same specimens of L. limanda, to prove the usefulness of the method. The area under investigation was the North Sea. Four locations were selected: station N01, close to Heligoland, in the North Sea; station N04 at the Dogger Bank; station N06 at the Firth of Forth; and station G08 in the English Channel. Ten a…

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…

The Galactose-Specific Lectin from the Sponge Chondrilla Nucula Displays Anti-Human Immunodeficiency Virus Activity in vitro via Stimulation of the (2′-5′)Oligoadenylate Metabolism

A new lectin has been isolated from the sponge Chondrilla nucula. The purified CN lectin is a protein composed of four polypeptide chains with a molecular weight (MW) of 15600. The isoelectric point is 4.5 and the amino acid composition is rich in aspartic and glutamic acid. The lectin precipitates erythrocytes from humans (A, B, O) with a titre between 25 and 210. The CN lectin is d-galactose-specific and displays a moderate mitogenic effect on spleen lymphocytes from mice and on CD4-positive human H9 cells. An interesting feature of this lectin is its ability to stimulate the (2′-5′)oligoriboadenylate [(2′-5′)A] metabolic pathway in non-infected and human immunodeficiency virus (HIV-1)-i…

A monoclonal Ro-antibody and the serum of a Ro-positive patient with subacute cutaneous lupus erythematosus (SCLE) react with basal layers of human epidermis.

Skin lesions, especially at areas exposed to sunlight, prove to be a major form of manifestation of diseases related to Ro-antibodies and neonatal-, 'ANA-negative-', and cutaneous types of lupus erythe- matosus. A monoclonal Ro-antibody established by our group reacts with a 60 kD polypeptide in extracts from human spleen, whereas in extracts from human epidermis the monoclonal Ro-antibody and a purified Ro-antibody from a monospecific serum of a patient with subacute cutaneous lupus erythematosus reacted with a 60 kD and a 48 kD protein. Performing immunofluorescence microscopy on HEp2-cells both antibodies showed a nuclear speckled staining pattern and a reaction with cytokeratin filament…

Interaction of polyribosomal components and polyribonucleotides with microtubule proteins

To demonstrate the affinity of RNA-containing polyribosomal components (isolated from L5178y cells) to microtubules, microtubule protein was attached to an insoluble matrix. In contrast to ribosomes, poly(A) (+) mRNA and poly(A)-RNP were found to bind to the matrix. Using synthetic polyribonucleotides, no significant differences in the binding properties of single- and double stranded polymers of different base composition to microtubule protein were observed. However, binding is dependent on the size of the polymer; a minimal chain length of 12 nucleotide units is required.

In vivo and in vitro induction of natural killer cells by cloned human tumor necrosis factor

The natural killer (NK) cell activity of mice in the peritoneal cavity is very low or undetectable and testing peritoneal NK cells is a useful model for studying the influence of activating substances upon local injection. Injection of tumor necrosis factor (TNF) at doses of 10-200 ng caused a marked activation of NK cell activity which was maximal after 24 h and declined rapidly on day 2. A similar effect was observed when interferons alpha and beta were injected, and there were additive results when interferon was injected together with TNF. The NK cell nature of the effector cells activated by TNF was substantiated by the finding that previous injection with anti-asialo GM 1 antibody pre…

Functionalized Magnetic Nano-particles for Selective Targeting and Sensing of Cells

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…

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.…

Influence of the xyloadenosine analogue of 2?,5?-oligoriboadenylate on poly(A)-specific, 2?,5?-oligoriboadenylate degrading 2?,3?-exoribonuclease and further enzymes involved in poly(A)(+)mRNA metabolism

The homogeneous poly(A)-specific 2′,3′-exoribonuclease from calf thymus gland, which cleaves both 3′,5′-and 2′,5′-linked oligoriboadenylates, does not degrade (xyloA2'p)2 xyloA, the xylofuranosyladenosine analogue of the 2-5A core. This oligonucleotide, which is supposed to enter intact cells rapidly, was found to possess an increased stability and an enhanced antiherpesvirus activity compared to the natural (A2'p)2A (Eppstein, D. A., Barnett, J. W., Marsh, Y. V., Gosselin, G. and Imbach, J.-L. (1983) Nature 302, 723–724). The poly(A) anabolic enzyme, poly(A) polymerase (Mn2+-dependent), from the same source, which is initiated by (A3'p)2A and its higher oligomers, does not accept 2–5A core…

The unique skeleton of siliceous sponges (Porifera; Hexactinellida and Demospongiae) that evolved first from the Urmetazoa during the Proterozoic: a review

Abstract. Sponges (phylum Porifera) had been considered as an enigmatic phylum, prior to the analysis of their genetic repertoire/tool kit. Already with the isolation of the first adhesion molecule, galectin, it became clear that the sequences of the sponge cell surface receptors and those of the molecules forming the intracellular signal transduction pathways, triggered by them, share high similarity to those identified in other metazoan phyla. These studies demonstrated that all metazoan phyla, including the Porifera, originate from one common ancestor, the Urmetazoa. The sponges evolved during a time prior to the Ediacaran-Cambrian boundary (542 million years ago (myr)). They appeared du…

Cytostatic Activity of Aeroplysinin-1 against Lymphoma and Epithelioma Cells

(±)-Aeroplysinin-1, an optically active 1.2-dihydroarene-1.2-diol. was isolated from the marine sponges Verongia aerophoba (+-isomer) and lanthella ardis (--isomer). For the experiments presented we used the +-isomer from Verongia aerophoba. Here we describe the hitherto unknown biological and pharmacological property of this compound to display pronounced anticancer activity against L5178y mouse lymphoma cells (ED50: 0.5 μm). Friend erythroleukemia cells (ED50: 0.7μm) , human mamma carcinoma cells (ED50: 0.3μm) and human colon carcinoma cells (ED50: 3.0 μm) in vitro. Furthermore, aeroplysinin caused a preferential inhibition of [3H]thymidine (dThd) incorporation rates in L5178y mouse lymph…

Speciation of sponges in Baikal-Tuva region: an outline

Lake Baikal is known for its high percentage of endemic fauna and flora. The most abundant sessile animal taxa in the littoral zone of Baikal are the photosymbiotic sponges. These endemic sponges are grouped to the family Lubomirskiidae and are separated, based on molecular data, from the cosmopolitan family Spongillidae Gray 1867. In the present review, recent data on the potential driving forces of the rapid speciation in Lake Baikal have been unified. Current data suggest that the genetic repertoire of the sponges was sufficiently large to cope with the major cold events, occurring 2.8–2.5 and 1.8–1.5 Ma. It is proposed that during those periods of climatic incisions founder populations …