Structural organization of cholera toxin gene and its expression in an environmental non-pathogenic strain ofVibrio cholerae

Non-pathogenic, environmental strain ofVibrio cholerae, ELTOR Ogawa EW6 carries a copy of the cholera toxin gene in its chromosome. Restriction enzyme digestion followed by Southern blot analysis revealed that the structure of the cholera toxin gene in this organism is different from that found in the virulent strains. The xbaI site which has been found to be conserved in the cholera toxin of the virulent strains examined so far, is absent here. Results of the RNA dot blot analysis indicated that the cholera toxin gene in EW6 is transcribed much less efficiently compared to the cholera toxin gene present in the virulent strainVibrio cholerae classical Inaba 569B.     

Chemical modification studies on a lectin from Saccharomyces cerevisiae (baker''s yeast).

The effect of chemical modification on a galactose-specific lectin isolated from a fatty acid auxotroph of Saccharomyces cerevisiae was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of 50 free amino groups with succinic anhydride or citraconic anhydride led to an almost complete loss of activity. This could not be protected by the inhibitory sugar methyl alpha-D-galactopyranoside. Treatment with N-bromosuccinimide and N-acetylimidazole, for the modification of tryptophan and tyrosine residues, did not affect lectin activity. Modification of carboxy groups with glycine ethyl ester greatly affected lectin activity, although sugars afford partial protection. Modification of four thiol groups with N-ethylmaleimide was accompanied by a loss of 85% of the agglutinating activity, and two thiol groups were found to be present at the sugar-binding site of the lectin. Modification of 18 arginine residues with cyclohexane-1,2-dione and 26 histidine residues with ethoxyformic anhydride led to a loss of lectin activity. However, in these cases, modification was not protected by the abovementioned inhibitory sugar, suggesting the absence of these groups at the sugar-binding site. In all the cases, immunodiffusion studies with modified lectin showed no gross structural changes which could disrupt antigenic sites of the lectin.     

Cloning and DNA sequence of plasmid determinant iss, coding for increased serum survival and surface exclusion, which has homology with lambda DNA

Summary Escherichia coli K12 cells carrying a cloned 1.4 kb HindIII fragment from plasmid ColV2-K94, showed increased survival in guinea pig serum. The recombinant plasmid also conferred group II surface exclusion, i.e. the cells were reduced in recipient ability towards the incoming plasmid R538drd in conjugation experiments. Southern blotting suggested homology with bacteriophage lambda DNA and to the insertion element IS2. Determination of the DNA sequence of the fragment demonstrated the presence of a truncated IS2 (165 bp), separated by 250 bp from a 900 bp stretch of homology with lambda DNA, beginning within the Rz gene and continuing in the rightward direction on the lambda map. A 97 amino acid open reading frame (ORF) adjacent to Rz and on the opposite strand, remained intact in iss, with several amino acid changes. The ORF in iss is preceded by sequences resembling prokaryotic ribosome binding sites and promoters.     

Longitudinal age changes in a fishermen population of West Bengal

The longitudinal anthropological age changes in a sample of coastal fishermen in West Bengal are discussed. The results show that some traits follow the trends observed in ageing of Western populations, for instance nasal length and breadth continue to increase throughout life in both sexes. Stature follows also the trend generally observed: a decrease occurs indeed from 35 years of age in both sexes, the decline is becoming higher with increasing age. The typical ecological conditions of our population influence the rate of change in other traits, such as weight and biacromial diameter. Weight is known to be influenced by the nutritional status and the way of life. It explains that in our study of males an early increase of weight and biacromial diameter is observed first and a decrease afterwards. In females, however, a decrease is observed already from 35 years of age. This kind of analysis was only possible with the help of longitudinal data and in a well defined population: only longitudinal data is free from the effect of secular changes and of selective death.     

Abnormalities of the erythrocyte membrane in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a hematologic malignancy characterized by excessive growth of myeloid cells and their progenitors. The proportion of spectrin dimers compared to tetramers extracted from membranes at 4 degrees C, under low ionic strength conditions, increased in CML erythrocytes. These also displayed abnormal thermal sensitivity (between 45 and 46 instead of 49 degrees C). Crosslinking with the bifunctional reagent, dimethyl adipimidate (8.6 A) showed significant organizational modification of not only spectrin, but other cytoskeletal components such as ankyrin, bands 4.2 and 5. Enhanced concanavalin A (Con-A) agglutinability of CML erythrocytes also suggests altered topographic distribution of a functionally important membrane protein, band 3. The anion transport activities of erythrocytes from patients with CML and normal donors were comparable. In CML erythrocytes, significant reduction in the number of ankyrin-binding sites, present in the cytoplasmic domain of band 3, may lead to partial loss of cytoskeletal anchorage to the bilayer and account for their increased Con-A agglutinability and heat sensitivity and may lead to their premature removal from the circulation.     

Biological activity and conformational isomerism in position 9 analogues of the des-1-tryptophan,3-beta-cyclohexylalanine-alpha-factor from Saccharomyces cerevisiae

Analogues of the des-1-tryptophan,3-beta-cyclohexylalanine-alpha-factor of Saccharomyces cerevisiae, where the glycyl residue of position 9 was replaced by D-Ala, L-Ala, D-Leu, and L-Leu, were synthesized and evaluated by morphogenesis assays and circular dichroism spectroscopy. Synthesis was accomplished in solution phase with mixed anhydrides and p-nitrophenyl active esters as the coupling agents. All crude dodecapeptides were purified to greater than 98% homogeneity by preparative high-performance liquid chromatography on a reversed-phase column. The Gly9, D-Ala9, and D-Leu9 analogues elicited morphogenic alterations in MATa strains of S. cerevisiae at concentrations of 1-2 micrograms/mL and exhibited similar CD patterns in both trifluoroethanol and tris(hydroxymethyl)aminomethane buffer, pH 7.4. In contrast, the L-Ala9 and L-Leu9 analogues were more than 200 times less active in the morphogenesis assay and had markedly different CD spectra. These results demonstrate that the position 9 residue plays an important role in determining the biological activity and solution conformation of alpha-factor. We suggest the presence of a type II beta-turn in the Lys7-Gln10 region when the alpha-factor assumes its biologically active conformation.     

Evidence that a system similar to the recA system of Escherichia coli exists in Vibrio cholerae

Summary Two lines of evidence suggest that a gene analogous to the recA gene of Escherichia coli exists in Vibrio cholerae and that its product serves a proteolytic function in the SOS response. Firstly, Southern blot hybridization using the recA gene of E. coli as a probe revealed a genomic sequence in V. cholerae which hybridized with the probe. Secondly, the SOS-like response in V. cholerae (as measured by beta phage induction) triggered by DNA damaging agents like Furazolidone could be blocked by Antipain, a protease inhibitor known to inhibit RecA protease action in E. coli. Maximal blocking effect of Antipain on beta phage induction occurred at 1 mM. At this concentration neither the viability of the host bacterium nor the lytic growth of a clear plaque mutant of the phage was affected by Antipain.     

Proton translocation during denitrification in Rhodopseudomonas sphaeroides f. denitrificans

Proton translocation during the reduction of NO ₃ ^- , NO ₂ ^- , N₂O and O₂, with endogenous substrates, in washed cells of Rhodopseudomonas sphaeroides f. denitrificans was investigated by an oxidant pulse method. On adding NO ₂ ^- to washed cells, anaerobically in the dark, an alkalinization occurred in the reaction mixture followed by acidification. When NO ₃ ^- , N₂O or O₂ was added to cells in the dark or with these compounds and NO ₂ ^- in light an acidification only was observed. Proton translocation was inhibited by carbonyl cyanide-m-chlorophenyl hydrazone.Valinomycin treated cells produced acid in response to the addition of either NO ₃ ^- , NO ₂ ^- , N₂O or O₂. The proton extrusion stoichiometry ( ratios) in illuminated cells were as follows: NO ₃ ^- 0.5N₂, 4.82; NO ₂ ^- 0.5N₂, 5.43; N₂ON₂, 6.20; and O₂H₂O, 6.43. In the dark the comparable values were 3.99, 4.10, 4.17 and 3.95. Thus, illuminated cells produced higher values than those in the dark, indicating a close link between photosynthesis and denitrification in the generation of proton gradients across the bacterial cell membranes.When reduced benzyl viologen was the electron donor in the presence of 1 mM N-ethylmaleimide and 0.5 mM 2-n-heptyl-4-hydroxyquinoline-N-oxide in the dark, the addition of either NO ₃ ^- , NO ₂ ^- or N₂O to washed cells resulted in a rapid alkalinization of the reaction mixture. The stoichiometries for proton consumption, ratios without a permeant ion were NO ₃ ^- NO ₂ ^- ,-1.95; NO ₂ ^- 0.5 N₂O,-3.03 and N₂ON₂,-2.02. The data indicate that these reductions occur on the periplasmic side of the cytoplasmic membrane.Abbreviations BVH reduced benzyl viologen - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DIECA N, N-diethyl-dithiocarbamate - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - NEM N-ethylmaleimide