Immunogenicity of the Shigella flexneri Serotype Y (SFL124) Vaccine Strain Expressing Cloned Glucosyl Transferase Gene of Converting Bacteriophage SfX

The glucosyl transferase gene (gtr) from bacteriophage phage X (SfX) caused partial conversion of serotype Y (group antigen 3, 4) to X (group antigen 7, 8) when introduced into a candidate vaccine strain of Shigella flexneri serotype Y (SFL124). The gtr gene caused conversion of O-antigens but did not eliminate the adsorption of the corresponding phage SfX. The hybrid strain expressing both group antigens 7, 8 and 3, 4 showed 75% protection when immunized guinea pigs were challenged with a wild-type S. flexneri serotype X strain. No protection was observed against serotype Y challenge, although group antigen 3, 4 was detected in the LPS of the hybrid strain. This suggests the importance of O-antigen immunity in the host defense against shigellosis.     

Physical characterization of a glucose-dehydrogenase-bearing plasmid from ketoacid-producingErwinia herbicola

Erwinia herbicola (ATCC 21998), a facultative anaerobe, has two plasmids: pVQ1 and pVQ2. Curing with mitomycin C indicated that pVQ2 was cryptic but pVQ1, a 7.4-kb plasmid, bears a 4.3SacI fragment which strongly hybridized to the C-terminal region of the glucose dehydrogenase gene ofAcinetobacter calcoaceticus. A restriction map of plasmid pVQ1 is presented.The authors are with the Department of Biotechnology, Regional Research Laboratory, Canal Road, Jammu Tawi-180 001, India;     

Collagenase of a new streptomycete: Its induction and purification

Rifampin and chloramphenicol inhibited the synthesis of collagenase of Streptomyces sp. A₈, suggesting de novo synthesis. The collagenase was induced by insoluble collagen, its macromolecular fragments, gelatin, peptone, hide powder and yeast extract. Growth as well as collagenase synthesis were dependent on substrate availability. Purification of collagenase by DEAE-cellulose chromatography resulted in approximately 25-fold increase in activity (268.6 μmol glycine equivalents min^?1 mg^?1 protein) relative to the activity of the culture filtrate (10.5 μmol glycine equivalents min^?1 mg^?1 protein).     

Theoretical studies on the modes of binding of some of the derivatives of d-mannose to concanavalin A

The possible modes of binding for $\text{methyl}\text{-α-}\text{d}\text{-mannopyranoside}$, $\text{methyl}\text{-β-}\text{d}\text{-mannopyranoside}$, $\text{2-O-}\text{methyl}\text{-α-}\text{d}\text{-mannopyranoside}$, $\text{methyl-2-O-methyl}\text{-α-}\text{d}\text{-mannopyranoside}$ and $\text{methyl}\text{-α-}\text{d}\text{-N-}\text{acetylmannosamine}$ to concanavalin A have been investigated using theoretical methods. All these sugars, except $\text{methyl}\text{-α-}\text{d}\text{-N-}\text{acetylmannosamine}$, reach the active site of concanavalin A with a highly restricted number of binding orientations. Present investigations suggest that the failure of $\text{methyl}\text{-α-}\text{d}\text{-N-}\text{acetylmannosamine}$ to bind to concanavalin A is not so much due to steric factors as to repulsive electrostatic interactions. $\text{Methyl}\text{-2-O-}\text{methyl}\text{-α-}\text{d}\text{-mannopyranoside}$ can bind to concanavalin A in one mode whereas the other sugars can bind in more than one mode. The high potency of $\text{methyl}\text{-α-}\text{d}\text{-mannopyranoside}$ over $\text{methyl}\text{-β-}\text{d}\text{-mannopyranoside}$ is mainly due to the possibility of hydrophobic interactions of the α-methoxy group with Leu(99) or Tyr(100) and also due to the possibility of formation of better and more hydrogen bonds with the protein. A comparison of these data with those for the d-glucopyranosides suggests that the change of the hydroxyl at the C-2 atom from equatorial to axial orientation increases the stereochemically allowed region as well as the possible binding modes. From these studies it is also suggested that the overall shape of the oligosaccharides rather than the terminal or internal mannose alone affects the binding potency of saccharides to concanavalin A.     

Theoretical studies on the modes of binding of some of the derivatives of d-mannose to concanavalin A

The possible modes of binding for methyl-α-d-mannopyranoside, methyl-β-d-mannopyranoside, 2-O-methyl-α-d-mannopyranoside, methyl-2-O-methyl-α-d-mannopyranoside and methyl-α-d-N-acetylmannosamine to concanavalin A have been investigated using theoretical methods. All these sugars, except methyl-α-d-N-acetylmannosamine, reach the active site of concanavalin A with a highly restricted number of binding orientations. Present investigations suggest that the failure of methyl-α-d-N-acetylmannosamine to bind to concanavalin A is not so much due to steric factors as to repulsive electrostatic interactions. Methyl-2-O-methyl-α-d-mannopyranoside can bind to concanavalin A in one mode whereas the other sugars can bind in more than one mode. The high potency of methyl-α-d-mannopyranoside over methyl-β-d-mannopyranoside is mainly due to the possibility of hydrophobic interactions of the α-methoxy group with Leu(99) or Tyr(100) and also due to the possibility of formation of better and more hydrogen bonds with the protein. A comparison of these data with those for the d-glucopyranosides suggests that the change of the hydroxyl at the C-2 atom from equatorial to axial orientation increases the stereochemically allowed region as well as the possible binding modes. From these studies it is also suggested that the overall shape of the oligosaccharides rather than the terminal or internal mannose alone affects the binding potency of saccharides to concanavalin A.     

Structure and chromosomal arrangement of leghemoglobin genes in kidney bean suggest divergence in soybean leghemoglobin gene loci following tetraploidization

We have determined the structure of one of the leghemoglobin (Lb) genes of Phaseolus vulgaris (kidney bean) and deduced the chromosomal arrangement of leghemoglobin genes by genomic hybridizations with Lb and two other sequences, each specific to the 5' or 3' region of the soybean leghemoglobin loci. By comparing this organization with two other species of legumes, Glycine max (soybean) and G. soja (wild soybean), a phylogeny of leghemoglobin gene loci was traced. The intragenic structure of the kidney bean leghemoglobin gene shows the same intron/exon arrangement as that of soybean leghemoglobin genes and extensive sequence homologies in both coding as well as 5' and 3' non-coding regions. The presence in the kidney bean genome of four leghemoglobin genes suggests that tandem duplications of a single primordial plant globin gene had occurred to generate four leghemoglobin genes in an `Lb-locus' before Glycine and Phaseolus species diverged. Chromosome duplication by tetraploidization in Glycine generated two loci containing four genes each. A large deletion in one of the two four-gene loci in soybean resulted in the generation of the Lbc₂ locus containing two leghemoglobin genes, one functional and another pseudo (LbΨ₂). This pseudogene, unlike that present on the main locus, is represented by only two and a half exons and appears to be truncated. The two other truncated genes (LbT₁ and LbT₂) were probably generated similarly in the genome of Glycine spp. following tetraploidization before the divergence of G. max and G. soja.     

Two Ca2+-requiring p-nitrophenylphosphatase activities of the highly purified Ca2+-pumping adenosinetriphosphatase of human erythrocyte membranes, one requiring calmodulin and the other ATP

The highly purified Ca2+-pumping ATPase from human erythrocyte membranes displays two p-nitrophenylphosphatase (NPPase) activities: one of these requires calmodulin and low concentrations of Ca2+, while the other requires ATP and higher Ca2+ concentrations. The free Ca2+ concentrations required for the expression of the two NPPase activities differed very substantially. Both activities required high free Mg2+ concentrations and displayed simple hyperbolic kinetics toward p-nitrophenyl phosphate (NPP) with a Km in the range of 5-20 mM. Study of the dependence of the calmodulin-stimulated NPPase on Mg2+ and NPP indicated that the Mg-NPP complex is not the substrate of the enzyme. Under conditions optimal for ATP-requiring NPPase (1 mM free Ca2+), the Ca2+-ATPase displayed simple hyperbolic kinetics with a low Km for ATP. NPP competitively inhibited this activity, and the apparent Ki for NPP was less than 1 mM, much lower than the Km for NPP as a substrate. If NPP were inhibiting the ATPase by binding at the same site at which NPP is hydrolyzed, the apparent Ki for NPP as inhibitor would be the same as the Km for NPP as substrate. (Under these circumstances, the apparent Ki and the Km can be directly compared, since NPP was being hydrolyzed under both circumstances.) Since Ki was much lower than Km, NPP must have been inhibiting at another site; thus, these data show the existence of two types of NPP sites on the enzyme, one at which NPP is hydrolyzed and the other at which it inhibits ATP hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance Raman spectra of beta-carotene in native and modified low-density lipoprotein

Low-density lipoproteins isolated between density 1.02 and 1.063 g/cm3 from normal fasting human plasma, show strong resonance Raman spectra due to the presence of beta-carotene. Three intense bands, at 1010, 1160 and 1530 cm-1, are assigned to the stretching vibrations of -C-CH3, = C-C = and -C = C- bonds, respectively, of beta-carotene. High-resolution spectra of the 1500-1600 cm-1 region reveal multiple features, suggesting the coexistence of several structural populations of beta-carotene. The modifications of lipoproteins with pH and temperature (30 degrees-42 degrees) change the resonance Raman spectra of beta-carotene. The specific binding of LDL at pH 7.0 by fibroblast cells is suppressed. Our experiments thus suggest that physical and chemical perturbations of plasma lipoproteins modify the lipid-protein interactions and thereby alter the configurational distribution of beta-carotene molecules within these particles.     

Characterization of enterotoxin produced by four Yersinia enterocolitica strains of pig origin

All four isolates of Yersinia enterocolitica and one isolate of Y. frederiksenii from pigs were found to be enterotoxigenic. Whole-cell preparations of Y. enterocolitica isolates did not induce any change in the rabbit ligated gut test after 6 and 18 h of inoculation, but Y. frederiksenii on the other hand showed a positive gut response at 18 h. Cell-free supernatant (CFS) of all five isolates induced dilatation in the rabbit gut up to 6 h, after which Y. enterocolitica became negative, while Y. frederiksenii continued to show a reaction up to 18 h. CFS of all five isolates were also found positive with the infant mouse test. Of the five isolates of Yersinia, three gave a positive reaction for the permeability factor on rabbit skin. Yersinia enterotoxin could be concentrated by methanol extraction. It was stable at 100°C for 20 min and at 120°C for 15 min. However, its activity was lost at low (2.0) and high pH (10.0). Enterotoxic preparations of Y. enterocolitica lost part of their enterotoxic activity upon dialysis.