Stereospecific induction of starfish oocyte maturation by (8R)-hydroxyeicosatetraenoic acid

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine. This induction of meiotic divisions can be triggered also by four fatty acids: 5,8,11-20:3; 5,8,11,14-20:4 (arachidonic acid); 6,9,12,15-20:4; 5,8,11,14,17-20:5, all other fatty acids being completely inactive. This maturation triggered by eicosanoids occurs in the micromolar range and is facilitated by the presence of calcium. A variety of arachidonic acid derivatives (esters, epoxides, etc.) and metabolites (cyclooxygenase and lipoxygenase products) has been tested; the biological activity is restricted to 8-hydroxyeicosatetraenoic acid (8-HETE), other mono- and poly-HETEs being completely inactive. Maturation triggered by 8-HETE occurs around 10 nM and is insensitive to the presence of calcium. 8-HETE methyl ester and 8-hydroperoxyeicosatetraenoic acid are able to induce maturation at higher concentrations. Both (8S) and (8R) stereoisomers have been tested; the biological activity is strictly restricted to the (8R) isomer. 8-HETE triggers a complete maturation, i.e. maturation-promoting factor appearance, germinal vesicle breakdown, emission of the polar bodies, and formation of a female pronucleus. (8R)-HETE, but not (8S)-HETE, triggers the typical decrease in cyclic AMP concentration induced by 1-methyladenine and the burst of protein phosphorylation associated with maturation. Starfish oocytes oxidize exogenous arachidonic acid into 8-HETE and other HETEs. 8-HETE was identified, after high pressure liquid chromatography purification, by gas chromatography mass spectrometry. Furthermore, it was found that the starfish oocytes only produce the (8R)-HETE isomer. This highly stereospecific induction of oocyte maturation by (8R)-HETE suggests that this fatty acid, or a very closely related fatty acid, may play a role in the transduction of the 1-methyladenine message at the plasma membrane level.     

Production of native, correctly folded bovine pancreatic trypsin inhibitor by Escherichia coli

A gene for bovine pancreatic trypsin inhibitor (BPTI) was fused to the coding sequence for the Escherichia coli alkaline phosphatase signal peptide and expressed in E. coli under the control of the alkaline phosphatase promoter. When induced in phosphate-depleted medium such cells produced a trypsin inhibitor that was indistinguishable from native, properly folded BPTI. In particular, the BPTI produced by E. coli had three disulfide bonds that appeared to be identical to those found in native BPTI, as assayed by sensitivity to iodoacetate, dithiothreitol, and urea. This expression/secretion system will make possible the production of variant BPTI molecules, thus allowing the perturbing effects of amino acid substitutions on BPTI folding, structure, and function to be assessed.     

Structure of an antifreeze polypeptide precursor from the sea raven, Hemitripterus americanus

The cystine-rich antifreeze polypeptides (AFP) from sea raven were fractionated by reverse-phase high performance liquid chromatography into several components, with SR2 (Mr 17,000) as the major AFP. Sea raven AFP cDNA clones were isolated from a liver cDNA library using a synthetic oligonucleotide, and the identity of one of the clones, C2-1, was confirmed by hybridization selection and cell-free translation. C2-1 encodes a pre-AFP of 195 amino acids with no evidence of any profragments. Comparison of the deduced amino acid sequence with partial peptide sequences from SR2 showed substitutions in at least four amino acid positions, suggesting that C2-1 cDNA codes for a minor component. Both the primary and the predicted secondary structures of sea raven AFP are completely different from those of other fish AFP. This further confirms that sea raven AFP belongs to a different class of antifreezes. The high frequency of reverse turns and the presence of paired hydrophilic amino acids in these structures are striking features of the protein and may contribute to their antifreeze action.     

Structural investigation of the capsular polysaccharide produced by a novel Klebsiella serotype (SK1). Location of O-acetyl substituents using NMR and MS techniques

The capsular polysaccharide of Klebsiella SK1 was investigated by methylation analysis, Smith degradation, and ¹H NMR spectroscopy. The oligosaccharides (P1 and P2) obtained by bacteriophage ΦSK1 degradation of the polymer were studied by methylation analysis, and 1D- and 2D-NMR spectroscopy. The resulting data showed that the patent repeating unit is a branched pentasaccharide having a structure identical to the revised structure recently proposed for Klebsiella serotype K8 capsular polysaccharide.

The 2D-NMR data showed that one third of the glucuronic acid residues in the SK1 polymer are acetylated at O-2, O-3, or O-4. FABMS studies confirmed the presence of monoacetylated glucuronic acid residues. Thus, the relationship between the Klebsiella K8 and SK1 polymers is akin to that found for Klebsiella polysaccharides K30 and K33, which have been typed as serologically distinct yet their structures differ only in the degree of acetylation.     

Reaction on alternating GC oligonucleotide templates

Summary The condensation reactions of activated nucleotides, ImpN or 2-MeImpN, with the selfcomplementary ribo-octanucleotide GCGCGCGC or with the partially self-complementary heptanucleotide GCGCGCG were studied. The templatedirected reaction of 2-meImpC with the heptamer yields the 3–5 octamer as the main product. All other reactions yield 2–5-linked octamers and pyrophosphates as major products. Surprisingly, 2-MeImpG facilitates the reaction of 2-MeImpC with the heptamer.Procedures for the analysis by gel electrophoresis of the oligomeric products obtained in reactions of this kind are described.     

The extracellular processing and catabolism of hyaluronan in cultured adult articular cartilage explants.

Hyaluronan was shown to have the same turnover time as aggrecan in explant cultures of adult bovine articular cartilage. Inclusion of fetal calf serum in the culture medium resulted in a similar decrease in the rate of catabolism of both hyaluronan and proteoglycan. Less than 9% of the hyaluronan lost from the explants in the course of the experiment was recovered from the culture medium as hyaluronan, suggesting that the catabolism of hyaluronan involves the uptake of this glycosaminoglycan by the chondrocytes. Analysis of the molecular size of the newly synthesized hyaluronan in these cultures showed that the hyaluronan was initially synthesized as large macromolecules that were gradually depolymerized with time within the extracellular matrix. The resulting size distribution of newly synthesized hyaluronan molecules after 12 days in culture was similar to that determined for the endogenous hyaluronan. The kinetics of depolymerization of the newly synthesized hyaluronan was consistent with a random fragmentation of the macromolecule. The rate constants for the depolymerization of hyaluronan indicate that oxygen-derived radicals may be involved in the fragmentation of this macromolecule. Inclusion of either cycloheximide or proteinase inhibitors in the medium of the explant cultures resulted in a marked decrease in the rate of loss of hyaluronan from the tissue and in the inhibition of the depolymerization of the newly synthesized macromolecule. This suggests that both the catabolism and the depolymerization of hyaluronan are cell mediated and depend on metabolically active cells.     

Specificity of binding of beta-glucoside activators of ryegrass (1-->3)-beta-glucan synthase and the synthesis of some potential photoaffinity activators.

Structure-activity relationships among glycoside activators of ryegrass (Lolium multiflorum) (1-->3)-beta-glucan synthase were investigated using a number of natural and synthetic glycosides, including some carrying photoaffinity functions. There is an absolute requirement for a beta-D-glycosyl moiety in the activator, both S- and N-glucosides are active, and the position of the glucosidic linkage in beta-glucose disaccharides has a significant effect on the affinity of binding. However, the binding requirement does not extend beyond a single beta-D-glucosyl residue, and beta-D-oligoglucosides are less effective than disaccharides. The nature of the aglycon has a major influence on the binding affinity. Hydrophobic aglycons lower the concentration required for half-maximal stimulation of the enzyme obtained from an Eadie-Hofstee plot of kinetic data (Ka) for activation, but charge aglycons increase Ka. Relative to methyl-beta-D-glucoside and cellobiose (Ka 1.1 mM), the most potent compounds tested were N-[4-(benzoyl)benzoyl]-beta-D-glucosylamine and 2'-[4-azidosalicylamino]ethyl-1-thio-beta-D-glucoside with K(a)s of approximately 30 microM. The latter also was tested for its potential to specifically label the beta-glucoside-binding site on the synthase, but under the conditions used the binding was found to be nonspecific.     

Diversity and conservation of blackwater fishes in Peninsular Malaysia,particularly in the North Selangor peat swamp forest

One of the most extreme freshwater habitats in Peninsular Malaysia is the peat swamp forest, with dark-coloured and highly acidic waters. Surprisingly, little is known about blackwater fishes in Peninsular Malaysia. Until 1968, only 26 fish species were known from blackwaters throughout Peninsular Malaysia, of which only one can be regarded as stenotopic. A recent intensive survey of part of the North Selangor peat swamp forest yielded 47 species, of which 14 are probably stenotopic taxa. These include four undescribed species and several new records for western Peninsular Malaysia. These discoveries are significant in that they include the family Chaudhuriidae which until 1985, was not reported from Sundaic Southeast Asia, and the rare genus Encheloclarias which had not been encountered for over 50 years. The rapid rate of destruction of the peat swamp forest owing to development, forestry and agricultural activities must be halted or slowed significantly to enable the proper zoological surveys and studies to be conducted. Conservation plans and environmental impact assessments based on inadequate sampling and knowledge of species present is acutely dangerous. There are no longer substantial undisturbed blackwater peat swamp forests left in most of Peninsular Malaysia. Conservation of the remaining blackwater biotopes is critically important if extinction of many species, here regarded as economically valuable renewable resources, is to be prevented.