0000000000020917
AUTHOR
Branko Kurelec
Immunological and biological identification of tumour necrosis-like factor in sponges: Endotoxin that mediates necrosis formation in xenografts
Xenografts of the sponge Geodia cydonium in its closely related species G. rovinjensis resulted in a rapid rejection of the graft within a period of 5 days. We identified an immunoreactive tumour necrosis factor (TNF)-like activity in the xenograft (Mr of 30,000) two days after grafting. In-vivo injection of 5 micrograms human recombinant TNF-alpha induced cytotoxicity in sponge cells in the same pattern and time course as during natural xenograft rejection. Anti-TNF-alpha polyclonals were found to react with xenograft extracts, by Western blot analysis, as from day 2 after grafting. Using ELISA we detected the TNF-like activity from day 2 after grafting with peak levels at days 4 and 5, wh…
Determination of pollutants with multixenobiotic -resistance inhibiting properties
Abstract Recent evidence indicates that inherent presence of multixenobiotic resistance (MXR) mechanism represents a general biological defence mechanism for protection of organisms against both endogenous and environmental toxins. In aquatic organisms exposed to polluted waters, this P-170 glycoprotein pump also removes ‘new’ man-made toxic chemicals out of cells. Many chemical agents may alter the function of this fragile mechanism. A new class of compounds, referred to as ‘chemosensitizers’, deserve a top rank among environmentally-hazardous chemicals, since they block this basic natural defence mechanism. The authors measured the concentration of such MXR-inhibiting substances by two me…
Characterization of the trimeric, self-recognizing Geodia cydonium lectin I.
A D-galactose-specific lectin I was extracted from the sponge Geodia cydonium and purified by affinity chromatography. The molecular weight of lectin I as determined by high-pressure liquid gel chromatography, was found to be 36500 +/- 1300. Disc gel electrophoresis in the presence and in the absence of sodium dodecyl sulfate showed that lectin I is a trimer composed of three different subunits (Mr: 13800, 13000 and 12200); two of the three subunits are linked by one disulfide bond. Isoelectric focusing gave a pI of 5.6 for the native molecule and a pI of 4.4 and of 7.4 for the subunits. The three subunits carry carbohydrate side chains, composed of D-galactose (94%) and of arabinose (5%). …
Aggregation of sponge cells. Isolation and characterization of an inhibitor of aggregation receptor from the cell surface.
From the cell membranes of the sponge Geodia cydonium a component was isolated and purified which inhibits the aggregation factor isolated from the same source; the component was termed anti-aggregation receptor. This molecule was characterized as a glycoprotein (54% neutral carbohydrate) and its molecular weight is in the range of 180,000 One biological site of the anti-aggregation receptor was determined to be D-galactose. Indirect evidence presented seems to indicate that this molecule is present in an active form in aggregation-deficient cells and absent in aggregation-susceptible cells.
Potential multidrug resistance genePOHL: An ecologically relevant indicator in marine sponges
Sponges are sessile filter feeders found in all aquatic habitats from the tropics to the arctic. Against potential environmental hazards, they are provided with efficient defense systems, e.g., protecting chaperones and/or the P-170/multidrug resistance pump system. Here we report on a further multidrug resistance pathway that is related to the pad one homologue (POH1) mechanism recently identified in humans. It is suggested that proteolysis is involved in the inactivation of xenobiotics by the POH1 system. Two cDNAs were cloned, one from the demosponge Geodia cydoniumand a second from the hexactinellid sponge Aphrocallistes vastus. The cDNA from G. cydonium, termed GCPOHL, encodes a deduce…
The multixenobiotic resistance mechanism in the marine sponge Suberites domuncula: its potential applicability for the evaluation of environmental pollution by toxic compounds
Experiments were carried out with the marine sponge Suberites domuncala to determine whether sponges may express - like mammalian tumor cells a multidrug-like transporter system. The results demonstrate that sponge cells possess such a protective system termed multixenobiotic resistance (MXR) pump or P-glycoprotein-like pump, The protein was identified by antisera for the mammalian P170 multidrug resistance protein as a 130 kDa molecule, Binding studies were performed with H-3-vincristine (H-3-VCR) and membrane vesicles ; this process is ATP-dependent and inhibited by verapamil, which is known to reverse the multidrug-resistance phenotype in mammalian systems, Accumulation experiments were …
Species-Specific Aggregation Factor in Sponges
An aggregation receptor (AR) from the siliceous sponge Suberites domuncula has been isolated and purified by chromatography to about 55% purity. The AR consists primarily of neutral carbohydrate and is characterized by a buoyant density of 1.59 g/ml and by an apparent molecular weight of 42,500. The average density of the AR on Suberite cells is about 3.8 × 10 5 per μm 2 . The AR contains considerable amounts of hexuronic acid. The isolated AR can bind not only to receptor-depleted Suberites cells but also to receptor depleted cells from another siliceous species (Geodia cydontum) . After being charged with Suberites ARs, Geodia cells form aggregates in the presence of the species-specific …
Inhibitory effects of extracts from the marine alga Caulerpa taxifolia and of toxin from Caulerpa racemosa on multixenobiotic resistance in the marine sponge Geodia cydonium
The invasive growth of the introduced green alga Caulerpa taxifolia, already affecting the richness and diversity of the littoral ecosystems, has become a major ecological problem in the Mediterranean Sea. Previously, we demonstrated that the water pollutant tributyltin induces apoptosis in tissue of the marine sponge Geodia cydonium at concentrations of 3 μM and higher. Here we show that exposure of G. cydonium to low (non-toxic) concentrations of Caulerpa extract or purified caulerpin (10 μg/ml) together with low doses of tributyltin (1 μM; non-toxic), results in a strong apoptotic effect. Evidence is presented that the enhancement of toxicity of tributyltin by Caulerpa extract is at leas…
Species-Specific Aggregation Factor In Sponges.
In dissociated single cells from the sponge Geodia cydonium, DNA synthesis is initiated after incubation with a homologous, soluble aggregation factor. During the DNA-initiation phase the cyclic AMP- and cyclic GMP levels vary drastically; the cyclic AMP content drops from 2.2 pmol/10(6) cells to 0.3 pmol/10(6) cells while the cyclic GMP content increases from 0.6 pmol to 3.7 pmol/10(6) cells. The activity of neither the adenylate cyclase nor of the guanylate cyclase isolated from cells which have been incubated for different periods of time with the aggregation factor, is changed. The soluble as well as the particulate enzyme activities were checked in vitro. The cyclic nucleotide receptor…
Determination of lectin-cell-binding parameters by a new agglutination technique.
We applied a recently described technique which is based on a light transmission/scattering method to determine the association characteristics of the Geodia lectin to sheep erythrocytes. The agglutination assays were performed in a total volume of 3 ml with 5.4 x 10(6) erythrocytes/ml. At a concentration of 360 ng/ml 50% of the lectin molecules were bound to the cells within the first 10 s of incubation. Scatchard analyses revealed an association constant (K(a)) of 0.9 +/- 0.1 x 10(8) M-1 and a number of 3.8 +/- 0.6 x 10(6) lectin binding sites on one erythrocyte. The method was also successfully applied to determine quantitatively the inhibitory potential of sugars competing with cell sur…
Combinatory effects of temperature stress and nonionic organic pollutants on stress protein (hsp70) gene expression in the freshwater spongeEphydatia fluviatilis
This is the first documentation of a heat shock protein (hsp) response in sponges. Subjecting the freshwater sponge Ephydatia fluviatilis to temperature stress (18 to 33 C; 2 h) resulted in an increased expression (>10 times) of the M{sub r}70,000 (hsp70). The induction of hsp70 could be demonstrated on the level of gene expression and by quantification of the hsp70 protein. Temperature stress also resulted in a 25% reduction of sponge cell proliferation. A mixture of nonionic organic compound was extracted from water from the polluted Schwarzbach River (S. Hesse, Germany) by adsorption onto XAD-7 resin. Concentrations of this Schwarzbach River water extract at two and four items ambient le…
Thymine content of sea water as a measure of biosynthetic potential
A hydrolysis procedure along with a high-pressure liquid chromatographic procedure is given enabling simple and reliable thymine determinations in the nanogram range in different fractions of sea-water samples taken from three different locations in the Northern Adriatic Sea. The levels corresponded to 1–3 μg DNA per liter. From total polyanionic thymine, which had been precipitated as the cetyltrimethylammonium salt, the highest percentage was linked to the particulate fraction, with a definite subsurface minimum at 10 to 15 m. There was a corresponding maximum of a high molecular “non-particulate” thymine-containing fraction at the corresponding depth. From the bottom at 30 m upwards to a…
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…
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…
Species-Specific Aggregation Factor in Sponges
An aggregation factor (AF) from the siliceous sponge Suberites domuncula has been isolated and purified by the following steps: Sepharose 2 B gel chromatography, sucrose gradient, Nonidet treatment, Sephadex G-100 gel chromatography and DEAE-Sephadex ion-exchange chromatography. By this procedure the AF was purified 1340-fold with a 63% yield nearly to homogeneity. The AF is originally associated with large particles, characterized by a sedimentation of 2200 S. These particles have been visualized electron microscopically; they are characterized by a filament-like shape of a length of 3400 A and a cross-sectional diameter of 230 A. The purified, low-molecular weight AF has a buoyant density…
Identification and isolation of the primary aggregation factor from the cell membrane of the sponge Geodia cydonium
The primary aggregation factor (pAF) of sponge cells is a glycoprotein that is firmly associated with the cell membrane. Polyspecific antibodies (anti-GM) prepared from sera raised against membranes of cells from the siliceous sponge Geodia cydonium were found to inhibit initial aggregation of homologous cells. The inhibition of aggregation, caused by anti-GM was neutralized by pAF. The pAF had been successfully solubilized and enriched by affinity chromatography, gel filtration and density gradient centrifugation, if checked by polyacrylamide gel electrophoresis in the presence of urea. The Mr of the native pAF was approximately 40 000 as estimated by gel filtration; under denaturing condi…
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. …
Regulated expression and phosphorylation of the 23-26-kDa ras protein in the sponge Geodia cydonium.
We have cloned, sequenced and examined the sponge Geodia cydonium cDNA encoding a protein homologous to ras proteins. The sponge ras protein has a more conserved N-terminal region and a less conserved C-terminal region, especially in comparison to Dictyostelium discoideum; the similarity to human c-Ha-ras-1 and to Saccharomyces cerevisiae is less pronounced. The sponge ras cDNA comprises five TAG triplets; at the translational level these UAG termination codons are suppressed by a Gln-tRNA. The sponge ras protein was isolated and partially purified (23-26 kDa) and found to undergo phosphorylation at a threonine moiety, when dissociated cells were incubated in the presence of a homologous ag…
Xenograft rejection in marine sponges. Isolation and purification of an inhibitory aggregation factor from Geodia cydonium.
In sponges there exists a graft rejection mechanism in which an inhibitory aggregation factor is involved. The inhibitory aggregation factor has been isolated from a culture medium containing dissociated cells of the sponge Geodia cydonium. Using ion-exchange and gel fractionation the factor was purified and shown to be electrophoretically pure. The factor has a molecular weight of 27000 and was characterized as a glycoprotein. The activity of the inhibitory aggregation factor was not affected by heat treatment, but treatment with trichloroacetic acid resulted in the irreversible loss of activity. The inhibitory aggregation factor affects the aggregation-factor-mediated reaggregation of dis…