Ca<Superscript>2+</Superscript>, Mg<Superscript>2+</Superscript>-dependent DNase Involvement in Apoptotic Effects in Spermatozoa of Sea Urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> Induced by Two-Headed Sphingolipid Rhizochalin

Previously, we have purified three distinct DNases from spermatozoa of sea urchin Strongylocentrotus intermedius and we suppose the role of Ca²⁺, Mg²⁺-dependent DNase (Ca, Mg-DNase) in apoptosis of spermatozoa. Two-headed sphingolipid rhizochalin (Rhz) induced characteristic apoptotic nuclear chromatin changes, internucleosomal DNA cleavage, and activation of caspase-9, caspase-8, and caspase-3 in spermatozoa as was shown by fluorescence Hoechst 33342/PI/FDA analysis, DNA fragmentation assay, and fluorescence caspase inhibitors FAM-LEHD-fmk, FAM-IETD-fmk, and FAM-DEVD-fmk, respectively. Inhibitor of caspase-3 z-DEVD-fmk subdued Rhz-induced internucleosomal ladder formation, which confirmed the major role of caspase-3 in apoptotic DNA cleavage probably through Ca, Mg-DNase activation. Participation of sea urchin Ca, Mg-DNase in apoptosis of spermatozoa was demonstrated by ions Zn²⁺ blocking of Rhz-induced DNA fragmentation due to direct inhibition of the Ca, Mg-DNase and internucleosomal cleavage of HeLa S and Vero E6 cell nuclei chromatin by highly purified Ca, Mg-DNase.     

Phosphatases and phosphodiesterases isolated from the red king crab (Paralithodes camtschatica) hepatopancreas

Five enzymes have been isolated from the hepatopancreas of the red king crab Paralithodes camtschatica by means of ion exchange and gel chromatography: two acid (AcP) and one alkaline (AlkP) phosphomonoesterases, one alkaline phosphodiesterase (AlkPI), and one acid phosphodiesterase (AcPD). The pH optimum values of these enzymes are: AlkPs and AlkPD, 7.5; AcP, 5.5; and AcPD, 5.0. The activity of AlkP and AlkPD demands Mg²⁺ ions. The molecular weights of the enzymes (kDa) are the following: AlkP, 80; AcPs, 80 and 82; AlkPD, 51; and AcPD, 57. The enzymes are relatively thermostable (ID 50 from 47 to 62°C). AlkP is inhibited by NaCl (IC 50 at 0.4 M). The AcP, AcPD, and AlkPD activities are tolerant of high ionic strength.     

Biologically Active Polypeptides and Hydrolytic Enzymes in Sea Anemones of Northern Temperate Waters

The content of biologically active polypeptides in aqueous and ethanol extracts of seven sea anemone species collected near Sakhalin Island (Sea of Okhotsk) and in Posyet Bay (Sea of Japan) was analyzed. Water extracts of the sea anemone Cribrinopsis similis showed the highest hemolytic activity, while ethanol extracts proved to have toxic properties. The levels of toxic and hemolytic activity of extracts of sea anemones inhabiting northern temperate waters were 2 to 3 orders of magnitude lower, compared to tropic species. The reason for this is likely to be the differences in the habitat conditions and biological traits of these animals. The water extracts of all species possessed proteolytic, phospholipase A2, and low DNAase activities, except Actinostola sp., whose aqueous extract contained a high activity alkaline DNAase. The species studied contained a wide range of proteinase inhibitors, O-glycosyl hydrolases (glycosidases and polysaccharide hydrolases). Water extracts of C. similis and Stomphia coccinea possessed the highest laminarinase activity. High activity of N-galactopyranosidase was found in water extracts of S. coccinea and Oulactis orientalis.     

Application of different test systems and biochemical indicators for environmental monitoring of the Troitsa Bay,Sea of Japan

The DNase express test system and the SF-bioassay (sea urchin sperm fertilization bioassay) were used to assess the quality of sea and bottom sediment water sampled at 8 stations of the Troitsa Bay (Possyet Bay, Sea of Japan). The specific activities of 8 enzymes (alkaline and acid DNases, RNases, phosphatases and phosphodiesterases) were determined for the liver and gill tissues of the mussel Crenomytilus grayanus sampled at the same stations. The activities of all enzymes were higher in the liver than in the gills, and the activity of acid nucleases in both tissues was significantly higher than that of alkaline nucleases. The specific activities of some nucleases in the mussel tissues correlated to the level of seawater pollution. The activities of acid RNases and phosphodiesterases decreased in both tissues upon an increase in water pollution, up to 1.5–3 times in the gills, while the activities of alkaline and acid phosphatases and phosphodiesterases increased 1.5–4 times. Thus, the specific activities of acid RNases, phosphatases and phosphodiesterases from mussel liver can be a useful indicator of marine pollution. This approach can be used for biotesting of ocean water and for assessment of the biological condition of invertebrates adapting to various ecological and anthropogenic effects.     

Trofosides A and B and other cytostatic steroid-derived compounds from the far east starfish <Emphasis Type="Italic">Trofodiscus über</Emphasis>

Three new polar steroids identified as trofoside A, 20R,24S)-24-O-(3-O-methyl-β-D-xylopyranosyl)-3β,6α,8,15β,24-pentahydroxy-5α-cholestane, its 22(23)-dehydro derivative (trofoside B), and 15-sulfooxy-(20R,24S)-5α-cholestane-3β,6β,8,15α,24-pentaol sodium salt, were isolated fromTrofodiscus über starfish extracts collected in the Sea of Ohotsk. Two known compounds, trofoside A aglycone, (20R,24S)-3β,6α,8,15β,24-pentahydroxy-5α-cholestane, and triseramide, (20R,24R,25S,22E)-24-methyl-3β6α,8,15β-tetrahydroxy-5α-cholest-22-en-27-oic acid (2-sulfoethyl)amide sodium salt, were also found. The structures of the isolated polyoxysteroids were established from their spectra. Minimal concentrations causing degradation of unfertilized egg-cells of the sea-urchin Strongylocentrotus intermedius(C _min) and terminating the cell division at the stage of the first division (C _min embr.), as well as the concentrations causing 50% immobilization of sperm cells (OC₅₀) and inhibiting their ability to fertilize egg-cells by 50% (IC₅₀) were determined for the isolated compounds. Of three compounds highly toxic in embryos and sea-urchin sperm cells, the polyol with a sulfo group in the steroid core was the most active; two glycosides with monosaccharide chains located at C3 and C24 atoms were less toxic. Note that all the compounds with the spermiotoxic activities differently affected the embryo development. The positions of monosaccharide residues in the core considerably influence the compound activity. For example, both mono-and double chained glycosides with the monosaccharide fragment at C3 and fragments at C3 and C4 atoms are active against sea-urchin sperm cells and embryos, whereas the C24 glycosylated trofoside A does not affect embryos and displays a poor spermiotoxicity.     

Novel salt-resistant alkaline phosphatase from eggs of the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis>

It was shown that the alkaline phosphatase (AP) isolated from eggs of the sea urchin Strongylocentrotus intermedius was a homodimer with a molecular mass of 150 kDa and that it exhibited maximal activity at a рН of 8.1–8.5 and a temperature of 45°С. Mg²⁺, Ca²⁺, and Mn²⁺ ions, as well as DTT, are activators of AP activity, while Zn²⁺, Cu²⁺, Cd²⁺, Pb²⁺, and Ni²⁺ ions, as well as EDTA, N-EMI, and p-CMB, inhibit its activity. The enzyme demonstrates unique salt resistance and can hydrolyze the substrate in seawater. It was shown by fluorescence methods and circular dichroism that an increase of the NaCl concentration above 1.0М caused noticeable changes in the secondary and tertiary structures of the protein and a decrease in enzyme activity. Analysis of the molecular masses of tryptic peptides conducted by mass spectrometry with the MASCOT program (which is based on the NCBI and SWISS-PROT databases) did not allow identification of the investigated protein. The uniqueness of the source and of the properties of the investigated AP, which were not characteristic for nonspecific AP, as well as the difficulties in primary structure identification with the fingerprinting technique, suggested that this enzyme was a nontypical AP with a novel structure.