The effect of cholera toxin on the inhibition of vasopressin-stimulated inositol phospholipid hydrolysis is a cyclic AMP-mediated event at the level of receptor binding.

Incubation of L6 skeletal myoblasts for 16 h with cholera toxin but not with pertussis toxin, led to the inhibition of inositol phosphate generation induced by subsequent exposure to vasopressin. The effects of the toxin on inositol lipid metabolism were accompanied by the total ADP-ribosylation of the available cholera-toxin substrates within the cells. Immunological analysis demonstrated that the two polypeptides modified in vivo by cholera toxin were different forms of Gs alpha (alpha subunit of Gs). No novel cholera-toxin substrate(s) were detected. The cholera-toxin-mediated inhibition of vasopressin-stimulated inositol phosphate generation could be mimicked by both forskolin and dibutyryl cyclic AMP, but not by the separated subunits of the toxin. Receptor-binding studies demonstrated that the inhibition of agonist-stimulated inositol phosphate generation was accompanied by a decrease in cell-surface vasopressin-binding sites, with no effect on the affinity of these for the hormone. We suggest that the effect of cholera toxin and agents which increase intracellular cyclic AMP on vasopressin-stimulated inositol lipid hydrolysis is an effect on receptor number, and that there is no requirement to postulate a role for a novel G-protein, which is a substrate for cholera toxin, in the regulation of inositol phospholipid metabolism.     

Synthesis of an affinity ligand ('UPHIT') for in vivo acylation of the kappa-opioid receptor

The isothiocyanate analog (1S,2S-trans-2-isothiocyanato-4,5-dichloro-N- methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide, 3a) of the highly selective kappa-opioid receptor agonist, U50,488, was prepared as a potential site-directed affinity ligand for acylation of kappa-opioid receptors in vivo. The isothiocyanate (3a) which we have designated UPHIT and its enantiomer (3b) were synthesized in 3 steps starting from optically pure (1S,2S)-(+)-trans-2-pyrrolidinyl-N-methyl-cyclohexylamine (4a) and its enantiomer (4b), respectively, thus defining their absolute stereochemistry. Binding in vitro of the 1S,2S enantiomer 3a to kappa receptors labelled by [3H]U69,593 was shown to occur with an IC50 value of 25.92 +/- 0.36 nM, whereas 827.42 +/- 5.88 and 115.10 +/- 1.23 nM were obtained for the IC50 value of the 1R,2R enantiomer (3b) and (+/-)-3 respectively. Intracerebroventricular (ICV) injection of 100 micrograms of (+/-)-3 into guinea-pig brain followed by analysis of remaining kappa-binding sites 24 h later revealed that (+/-)-3 depleted 98% of the kappa receptors that bind [3H]U69,593 and 40% of those that bind [3H]bremazocine. These preliminary data suggest exciting uses for these compounds in furthering our knowledge of the kappa-opioid receptor.     

Nonrandom structural features in the heparin polymer

Computer simulation studies were used to prepare an ensemble of heparin number chains. The polydispersity of these chains was simulated by introducing a specific "fraction of terminators", and it closely resembled the experimentally observed polydispersity of a porcine mucosal, glycosaminoglycan heparin. The same percentage of simulated chains contained antithrombin III (ATIII) binding site sequences as are typically found to contain ATIII binding sites using affinity chromatography. Heparin lyase action was then simulated by using Michaelis-Menten kinetics. In one model, heparin chains were constructed from the random assembly of monosaccharide units using the observed mole percentage of each. After simulated depolymerization, the final oligosaccharides formed were compared to the observed oligosaccharide products. The simulation which assumed a random distribution of monosaccharide units in heparin did not agree with experimental observations. In particular, no ATIII binding site sequences were found in the simulated number chains. The results of this simulation indicate that heparin is not simply a random assembly of monosaccharide units. These results are consistent with the known, ordered biosynthesis of heparin. In a second model, heparin chains were constructed from randomly assembled oligosaccharides at the mole percentage in which each is found in the final product mixture. The action of heparin lyase was then simulated, and the distribution of the oligosaccharide products was measured throughout the simulated time course of the depolymerization reaction. The simulated rate of formation and final concentration of a particular oligosaccharide which contains a portion of heparin's ATIII binding site were similar to those observed experimentally. These results are consistent with the random distribution of ATIII binding sites within glycosaminoglycan heparin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstituted skin in culture:a simple method with optimal differentiation

Human skin is a unique organ, which can be reconstituted in vitro and represents an interesting system for studying cell proliferation and differentiation. A simple technique for producing reconstituted skin with optimal epidermal differentiation is described and characterized. A 4-mm punch biopsy of normal human skin is deposited on the epidermal side of mortified de-epidermized human dermis maintained at the air-liquid interface with a metallic support. The culture medium contains insulin, epidermal growth factor (EGF), cholera toxin, hydrocortisone, penicillin/streptomycin and fungizone. A well-differentiated epidermis develops within 15 days. Morphological and ultrastructural studies show a neoepidermis resembling normal skin. Differentiation markers such as involucrin, filaggrin, and various cytokeratins detected with pancytokeratin antibody are present and confirm this resemblance. The keratin profile is comparable to that observed in other skin culture models. A basement-membrane-like structure is reconstituted with hemidesmosomes and anchoring-filament formation. Bullous pemphigoid (BP) antigen is observed at the dermo-epidermal junction after 21 days of culture. Moreover, both dermal substrates and punch biopsies can be kept frozen for long-term storage, with little or no loss of epidermal growth kinetics and morphology. This skin culture technique is rapid, simple, economical and reproducible. Characterization has here shown high-quality epidermal differentiation. Scientists interested in epidermal in vitro studies should take interest in all these advantages.     

New services of the EMBL Data Library.

The existing services of the EMBL Data Library for external users have been improved and extended in several ways. The EMBL File Server has been reorganised, and many new databases and other information relevant to biologists are now accessible via global computer networks. A broad range of software for molecular biology is freely available for different popular computer systems, including the EMBL enhancements to the Wisconsin (GCG) Package. The new Mail-Quicksearch and Mail-FastA services give access to the latest sequence data for database searches by ordinary electronic mail.     

High-resolution dynamic and morphological G-bandings (GBG and GTG): a comparative study

Summary A high-resolution replication banding technique, dynamic GBG banding (G-bands after 5-bromodeoxyuridine [BrdUrd] and Giemsa), showed that, at a resolution of 850 bands/genome, GBG banding and GTG banding (G-bands after trypsin and Giemsa) produce almost identical patterns. RBG band (R-bands after BrdUrd and Giemsa) and RHG band (R-bands after heat denaturation and Giemsa) patterns were previously shown to be only 75%–85% coincident; thus GTG banding more accurately reflects replication patterns than does RHG banding. BrdUrd synchronization uses high concentrations of BrdUrd both to substitute early replicating DNA and to arrest cells before the late bands replicate. Release from the block is via a low thymidine concentration. The banding is revealed by the fluorochrome-photolysis-Giemsa (FPG) technique and produces the GBG banding that includes concomitant staining of constitutive heterochromatin. As opposed to other replication G-banding procedures, BrdUrd synchronization and GBG banding produces a reproducible replication band pattern. The discordance between homologs after GBG banding is similar to that after GTG banding and no lateral asymmetry of the constitutive heterochromatin has been observed. Also, BrdUrd synchronization neither significantly depresses the mitotic index, nor induces chromosome breaks. Thus, GBG banding seems as clinically useful as GTG banding and provides important information regarding replication time.     

Production,cross reactivity,and epitope analysis of monoclonal antibodies against rat kidney gamma glutamyltransferase

Eighteen IgGl monoclonal antibodies (blabs) have been produced against gamma-glutamyl transferase (GGT) from rat kidney. They were specific to the light subunit of the enzyme with affinity constants ranging from 0.3 to 7.5 10⁸ M^–1, while they did not react with GGT from other sources i.e. human and pig kidney, rat and guinea pig liver, suggesting species and organ specificity. Two of the blabs (N° 11 and 21) lost their immunoreactivities towards rat kidney GGT in the presence of N-acetyl-neuraminic acid, while immunoreactivities of the other blabs were unchanged. Furthermore, Mabs No 11 and 21 did not react with desialylated rat kidney GGT. These findings suggest that N-acetyl-neuraminic acid is involved in the epitopes recognized by these two Mabs.Abbreviations ELISA enzyme linked immunosorbent assay - GGT gamma-glutamyltransferase - Mab monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Characterization of benzodiazepine receptors with fluorescent ligands

Fluorescein conjugates of the high-affinity benzodiazepine receptor ligands Ro 15-1788 and Ro 7-1986 were synthesized. The binding of these fluorescent ligands (BD 621 and BD 607) to benzodiazepine receptors was characterized by direct fluorescence measurement. Both the equilibrium dissociation constants (KD) of BD 621 and BD 607 and the maximum number of binding sites (Bmax) estimated by fluorescence monitoring were consistent with values obtained by using radioligand binding techniques. The binding of BD 621 and BD 607 assessed by fluorescence measurement was reversible, abolished by photoaffinity labeling with Ro 15-4513, and unaffected by a variety of substances that do not bind to benzodiazepine receptors. The potencies of chemically diverse benzodiazepine receptor compounds to inhibit fluorescent ligand binding were highly correlated (r = 0.94, P less than 0.001), with potencies obtained from radioligand binding techniques. These findings demonstrate the feasibility of using direct fluorescence measurement techniques to quantitate ligand-receptor interactions.     

Seed size variation: magnitude,distribution, and ecological correlates

Summary We examined seed-mass variation in 39 species (46 populations) of plants in eastern-central Illinois, USA. The coefficient of variation of seed mass commonly exceeded 20%. Significant variation in mean seed mass occurred among conspecific plants in most species sampled (by hierarchical ANOVA), averaging 38% of total variance. For most species, within-plant variation was the larger component of total variance, averaging 62% of total variance. Variation in seed mass among fruits within crops was significant in most species tested.We conclude that variation in seed mass among and within plants is widespread and common. There was little evidence of trade-offs between number of seeds and mean or variance of seed mass, and little correlational evidence of local competition for maternal resources. No consistent ecological (dispersal mode and growth form) correlates of variance of seed mass were evident.     

Purification of an antitrypanosomal factor fromPseudomonas fluorescens by high-pressure liquid chromatography

Studies on the purification of an antitrypanosomal factor (ATF-II) obtained fromPseudomonas fluorescens disclosed that high-pressure liquid chromatography (HPLC) with a Rad-pak porasil (10 silica) column and a GPC-60 Å column was an efficient procedure for the separation of the active components. Extraction of the factor with absolute ethanol prior to elution significantly enhanced the lytic activity of the HPLC eluates, as shown by marked pathologic changes followed by lysis in bioassays performed withTrypanosoma equiperdum. HPLC provided an increase of purification 30 times that obtained with gel filtration of the crude bacterial product. The lipopolysaccharide content of the purified fractions was markedly reduced and indicated an additional advantage for further in vivo tests in experimental infections withT. cruzi.