Histology of gonads and cephalic brood pouch of the marine gastropod Planaxis sulcatus of Karachi Coast (Pakistan)

This histological study of 141 specimens of adult Planaxis sulcatus from Karachi showed thepresence of only female gametes in gonadal follicles,supporting the conclusion of earlier workers that thespecies may be parthenogenetic. The gonadal folliclesof several snails were occupied by an unidentifiedtrematode parasite.     

Structure and biological evaluation of novel cytotoxic sterol glycosides from the marine red alga Peyssonnelia sp

Bioactivity-guided fractionation of the extract from a Fijian red alga Peyssonnelia sp. led to the isolation of two novel sterol glycosides 19-O-β-d-glucopyranosyl-19-hydroxy-cholest-4-en-3-one (1) and 19-O-β-d-N-acetyl-2-aminoglucopyranosyl-19-hydroxy-cholest-4-en-3-one (2), and two known alkaloids indole-3-carboxaldehyde (3) and 3-(hydroxyacetyl)indole (4). Their structures were characterized by 1D and 2D NMR and mass spectral analysis. The sterol glycosides inhibited cancer cell growth with mean IC₅₀ values (for 11 human cancer cell lines) of 1.63 and 1.41 μM for 1 and 2, respectively. The most sensitive cancer cell lines were MDA-MB-468 (breast) and A549 (lung), with IC₅₀’s in of 0.71–0.97 μM for 1 and 2. Modification of the sterol glycoside structures revealed that the α,β-unsaturated ketone at C-3 and oxygenation at C-19 of 1 and 2 are crucial for anticancer activity, whereas the glucosidic group was not essential but contributed to enhanced activity against the most sensitive cell lines.     

Unexpected fine-scale population structure in a broadcast-spawning Antarctic marine mollusc

Several recent empirical studies have challenged the prevailing dogma that broadcast-spawning species exhibit little or no population genetic structure by documenting genetic discontinuities associated with large-scale oceanographic features. However, relatively few studies have explored patterns of genetic differentiation over fine spatial scales. Consequently, we used a hierarchical sampling design to investigate the basis of a weak but significant genetic difference previously reported between Antarctic limpets (Nacella concinna) sampled from Adelaide and Galindez Islands near the base of the Antarctic Peninsula. Three sites within Ryder Bay, Adelaide Island (Rothera Point, Leonie and Anchorage Islands) were each sub-sampled three times, yielding a total of 405 samples that were genotyped at 155 informative Amplified Fragment Length Polymorphisms (AFLPs). Contrary to our initial expectations, limpets from Anchorage Island were found to be subtly, but significantly distinct from those sampled from the other sites. This suggests that local processes may play an important role in generating fine-scale population structure even in species with excellent dispersal capabilities, and highlights the importance of sampling at multiple spatial scales in population genetic surveys.     

Isolation and structure of axinastatin 5 from a republic of comoros marine sponge

The new cancer cell growth (human and murine) inhibitory (GI₅₀ 0.3 to 3.3 μg/ml) cyclo-octapeptide axinastatin 5 (2) was isolated in 3.8 x 10⁻⁷% yield from the Western Indian Ocean marine sponge Axinella cf. carteri. Structural elucidation was achieved by high field (400 and 500 MHz) 2 D-NMR techniques and the amino acid sequence was confirmed by results of NMR and high resolution mass spectral MS/MS interpretations. Other peptide cancer cell growth inhibitory constituents of this orange sponge were found to be the cyclic hepta- and octapeptides axinastatin 1 and hymenistatin 1 (1).     

Isolation and structure elucidation of new cytotoxic steroids from the gorgonian Leptogorgia sarmentosa

The gorgonian Leptogorgia sarmentosa contains three new steroids, (20S)-20-hydroxycholestane-3,16-dione (1), (16S, 20S)-16,20-dihydroxycholestan-3-one (2), and (20S)-20-hydroxycholest-1-ene-3,16-dione (3) together with a known related compound (4). Their structures were defined by spectroscopic analysis. The new steroids exhibited significant cytotoxicity against four tumor cell lines (ED50 = 1 microg/ml).     

Isolation and structure elucidation of cytotoxic polyacetylenes and polyenes from Echinacea pallida

Bioassay-guided fractionation of n-hexane extracts of Echinacea pallida (Asteraceae) roots led to the isolation and structure elucidation of two polyacetylenes (1, 3) and three polyenes (2, 4, 5). Two are known hydroxylated compounds, namely 8-hydroxy-pentadeca-(9E)-ene-11,13-diyn-2-one (1) and 8-hydroxy-pentadeca-(9E,13Z)-dien-11-yn-2-one (2). Two dicarbonylic constituents, namely pentadeca-(9E)-ene-11,13-diyne-2,8-dione (3) and pentadeca-(9E,13Z)-dien-11-yne-2,8-dione (4), were isolated and characterized for the first time. Furthermore, the structure elucidation of pentadeca-(8Z,13Z)-dien-11-yn-2-one (5) is described. The structure of the compounds isolated was determined on the basis of UV, IR, NMR (including 1D and 2D NMR experiments, such as 1H-1H gCOSY, gHSQC-DEPT, gHMBC, gNOESY) and MS spectroscopic data. The cytotoxic activity of the isolated constituents against MIA PaCa-2 human pancreatic adenocarcinoma cells was evaluated in the concentration range 1-100 microg/ml. Results show that the hydroxylated compounds (1, 2) have low cytotoxicity, while the more hydrophobic polyacetylenes (3) and polyenes (4, 5) displayed moderate activity.     

Cellulases of a marine mollusc, Dolabella sp

1. A crude cellulase extract was prepared from the hepatopancreas of a marine mollusc, Dolabella sp., and partially purified by ammonium sulphate fractionation. 2. The optimum pH values of the partially purified preparation were 6.5 and 8.0 for Walseth cellulose and CM-cellulose respectively. It was most stable at pH6.0 and showed moderate thermostability. 3. The partially purified preparation was subjected to starch-zone electrophoresis, and incompletely resolved into several fractions that contained one or more cellulase components of different substrate specificity. 4. Some of these cellulase fractions showed practically no aryl beta-glucosidase activity and hydrolysed aryl beta-cellobioside with difficulty. From substrates such as higher cello-oligosaccharides, cellodextrin, CM-cellulose, Walseth cellulose and cotton fibre, they produced cellobiose as the major and cellotriose as the minor end products, both of which were resistant to further attack by cellulase. 5. From the slope of the curves of viscosity-reducing power for CM-cellulose, the cellulase components from Dolabella were presumed to be of a ;more-random' or a ;less-random' type in the mode of action. 6. In the hepatopancreas of this mollusc, beta-glucosidases were also present, which hydrolysed cellobiose as well as aryl beta-glucosides. The optimum pH values of these enzymes were about 5.5.     

Isolation and structure of a novel peptide inhibitor of HIV-1 integrase from marine polychaetes

A homogeneous peptide with a mass of 683 Da which inhibits HIV-1 integrase with IC₅₀ 3 × 10^?5 M was separated from aqueous extracts of a marine worm Eunicidae sp. by multistage chromatography purification. The Asp-Leu-Hse-His-Ala-Gln structure was proposed for this peptide according to amino acid analysis, automated amino acid Edman sequences, and TLC with witness homoserine and MS/MS fragmentation. The proposed structure is the first example of a natural peptide containing an amino acid homoserine residue.     

Isolation of functional chloroplasts from the sacoglossan mollusc Elysia viridis Montague

A novel technique has been developed in which functional chloroplasts have been isolated and purified from the symbiotic sacoglossan mollusc Elysia viridis , Montague. This entailed the progressive filtration and gel-filtration of a hyaluronidase-incubated homogenate. Isopycnic centrifugation of the resultant crude chloroplast suspension reduced the cytoplasmic/mitochondrial and microbody contamination by approx. 70 and 90% respectively. Purified chloroplast suspensions were capable of linear rates of ¹⁴CO₂ fixation for at least 25 min at 50 μmol m² s⁻¹ (PPFD), even though approx. 54% of assimilate was being released into the bathing medium. Values for CO₂ fixation and assimilate release were similar to those obtained for the intact mollusc.     

Ophirasterol, a new C31 sterol from the marine sponge Topsentia ophiraphidites

Analysis of the sterol fraction obtained from the Colombian Caribbean sponge Topsentia ophiraphidites revealed that this sponge is a rich source of C30 and C31 sterols. Among them, a new C31 sterol, named ophirasterol, was isolated, and its structure was established as (22E,24R)-24-(1-buten-2-yl)cholesta-5,22-dien-3beta-ol (1) by spectral means and comparison with synthetic C-24 epimers with known configuration. Other isolated C30 and C31 sterols were the known 24-ethyl-24-methyl-22-dehydrocholesterol (2), 24-isopropyl-22-dehydrocholesterol (3), 24-isopropylcholesterol (4), 24-ethyl-24-methylcholesterol (5), 24-isopropenyl-25-methyl-22-dehydrocholesterol (6) and 24-isopropenyl-25-methylcholesterol (7), and 24-isopropenyl-22-dehydrocholesterol (8).     

Haemocyanin mRNA from arthropod and mollusc origin. Evidence for a multi-unit structure in mollusc haemocyanin mRNA.

Hepatocytes were preincubated with 10mM-glucagon and 100 microM-corticosterone to increase phosphatidate phosphohydrolase activity. Addition of 10 nM-glucagon or 100 microM-8-bromo cyclic GMP to a second incubation mixture that contained cycloheximide increased the half-life of the phosphohydrolase activity. Dexamethasone (100 nM) had no significant effect, but insulin (500 pM) or spermine (1 mM) decreased the half-life. None of these compounds altered the general rate of degradation of proteins labelled with [3H]leucine. There appears to be a specific control of the half-life of phosphatidate phosphohydrolase activity, which could contribute to its long-term regulation in the liver.     

Antineoplastic agents 390. Isolation and structure of phakellistatin 12 from a Chuuk archipelago marine sponge

A new cancer cell growth inhibitory (P388 lymphocytic leukemia ED(50) 2.8 microg/mL) cyclodecapeptide designated phakellistatin 12 (2) has been isolated as a trace (1.7 x 10(-6)% yield) constituent of the Western Pacific Ocean (Federated States of Micronesia-Chuuk) sponge Phakellia sp. Employing principally a combination of high resolution FAB with high field (500 MHz) 1H, 13C and 2-D NMR and chiral GC analyses the structure (all S chirality) cyclo-Ile-Phe-Thr-Leu-Pro-Pro-Tyr-Ile-Pro-Pro was assigned.     

Interactions of temperature and pH on the regulatory properties of pyruvate kinase from organs of a marine mollusc

The effects of low temperature assay (5 °C) on the properties of the aerobic (low phosphate) vs. anoxic (high phosphate) forms of pyruvate kinase (PK) from foot muscle and gill of the whelk Busycon canaliculatum (L.) were assessed at two pH values, pH 7.00 and 7.25, and compared to control conditions of 20 °C and pH 7.00 (all assayed in imidazole buffer). When pH was held constant at 7.00, the decrease in assay temperature to 5 °C had large effects on the measured kinetic parameters of all PK forms, as compared to 20 °C and pH 7.00. However, when assay pH was allowed to rise, from 7.00 to 7.25, with the temperature decrease to 5 °C there were fewer alterations of kinetic parameters and quantitatively smaller changes to enzyme properties. It appears, then, that when pH rises with decreasing temperature following alphastat predictions, kinetic properties of PK are largely conserved. Low temperature, at either pH value, had several significant effects on PK properties. For example, low temperature raised the S_0.5 for phosphoenolpyruvate of PK-anoxic from gill by 3–6 fold and decreased the I₅₀ Mg · ATP for PK-anoxic from foot by the same amount. Arrhenius plots of PK activity for the gill PK forms showed a distinct break at 10 °C; > 10 °C Q₁₀ was 2.5 whereas < 10 °C Q₁₀ was 8.4. Temperature-dependent changes in all cases affected enzyme properties in a manner that would restrict enzyme function at low temperature.     

Isolation and characterization of an alpha-macroglobulin from the gastropod mollusc Helix pomatia with tetrameric structure and preserved activity after methylamine treatment

A proteinase inhibitor with M(r) 697000 and 20.3% (w/w) carbohydrate was isolated from the haemolymph of the snail Helix pomatia and characterized. It was shown to have a tetrameric structure with subunits disulphide linked by two. It inhibited the activity of several types of proteinases against large substrates but not that of trypsin against N-alpha-benzoyl-DL-arginine-4-nitroanilide. This indicated a nonspecific and steric hindrance mode of inhibition. The ratio of trypsin molecules inactivated per inhibitor amounted to 1.5. This interaction led to a cleavage of the subunits into two equal fragments and to a slow to fast conformational change of the whole molecules. Experiments with 125I-labelled trypsin indicated that the proteinase had become covalently linked to one of the fragments. Heating of the inhibitor led to autolytic cleavage products but not when methylamine treated. Thiol titration after trypsin or methylamine treatment indicated the presence of one thiol ester bond per subunit. These facts are all indicative of an alpha-macroglobulin type of inhibitor. However, unlike for most of them the methylamine treatment did not induce a conformational change nor suppress its proteinase inhibitory activity. Moreover, invertebrate alpha-macroglobulins are mostly dimeric in structure but tetramers likewise do occur in Biomphalaria glabrata.     

Enkephalins increase dopamine levels in the CNS of a marine mollusc.

Intracardiac administration of methionine enkephalin and leucine enkephalin increased dopamine but not serotonin levels in the CNS of the marine mollusc $\text{Mytilus}$$\text{edulis}$. Naloxone blocked the effects of the enkephalins. These responses displayed a time dependent desensitization to methionine enkephalin. The study suggests the presence of an opiate receptor mechanism in this invertebrate species.     

Isolation of myxobacteria from the marine environment

In an attempt to isolate indigenous marine myxobacteria from coastal samples, we obtained two swarm forming bacteria. Both isolates formed cell aggregates which, at least in one isolate, developed to fruiting body-like structures consisting of a mass of myxospore-like cells. The optimum NaCl concentrations for their growth were between 2 and 3%, comparable to the NaCl concentration of seawater. This growth characteristic strongly suggests that the two isolates are specific marine bacteria. The 16S rDNA sequence studies indicated that the two isolates were related to the genus Nannocystis. Based on the phylogenetic distances between branches, we concluded that the isolates should be assigned to two new myxobacterial genera.     

Population dynamics of two snail species,Planaxis sulcatus and Cerithium moniliferum,and their trematode species at Heron Island,Great Barrier Reef

Summary The population dynamics of the prosobranch snail Planaxis sulcatus and its trematode parasites on 600 m of beachrock on the southern side of Heron Island, Great Barrier Reef, were studied. Populations of Planaxis show little mixing, due to lack of long-distance movements. From March 1973 to June 1975, an increase in the numbers of small and medium-sized snails occurred, but the biomass (dry tissue weight) of the snail population did not change. Snails were infected with one species of Aspidogastrea and six species of cerariae. Infections with cercariae did not significantly affect the relative weight of the snails. Multiple infections were random, i.e. neither negative nor positive interactions between trematode species could be demonstrated. Proportions of uninfected snails and snails infected with various trematode species remained more or less constant from July 1973 to February 1975.The population dynamics of the prosobranch snail Cerithium moniliferum and its 11 species of cercariae and one species of Aspidogastrea in a small area at Heron Island were studied. Numbers of large snails decreased and numbers of small snails increased from August 1973 to June 1975. The biomass of the Cerithium population increased only slightly. Prevalence of infection changed significantly only in large snails.Infections of Cerithium moniliferum and Peristernia australiensis with an aspidogastrid species decreased strongly from January 1971 to March/April 1972 and had not recovered by mid 1975.Seasonal fluctuations could not be demonstrated for any of the snails or parasites.The data for Planaxis suggest equilibrium conditions and saturation of the habitat.