The role of virulence antigens (VW) in the protection of mice againstYersinia pestis infection

Subcutaneous infection withYersinia enterocolitica harboring plasmid responsible for Ca²⁺ dependence at 37°C induced cell-mediated protective immunity against a lethal challenge withYersinia pestis; the isogenic derivative strain cured from this plasmid subverted the immunity in mice. This is the first identification of the antigen(s) responsible for the induction of cell-mediated protective immunity against the facultatively intracellular bacteria.     

Requirement of the core structure of a complex-type glycopeptide for the binding to immobilized lentil- and pea-lectins

Structural requirements for the binding of oligosaccharides and glycopeptides to immobilized lentil- and pea-lectins were investigated by use of radioactively-labeled glycopeptides and oligosaccharides. The results indicate that an intact 2- acetamido-2-deoxy-β-d-glucopyranosyl residue at the reducing end of a complex-type oligosaccharide is essential for high-affinity binding to lentil lectin-Sepharose but not to concanavalin A-Sepharose and that an asparagine residue is required for the binding of a complex-type glycopeptide to pea lectin-Sepharose. In addition, interaction of a complex-type oligosaccharide with lentil lectin-Sepharose was enhanced by exposure of nonreducing, terminal 2-acetamido-2-deoxy-β-d-glucopyranosyl groups, whereas interaction with pea lectin-Sepharose was enhanced only after exposure of nonreducing, terminal α-d-mannopyranosyl groups.     

Immunochemical approach to characterize advanced glycation end products of the Maillard reaction. Evidence for the presence of a common structure

Reaction of protein amino groups with glucose (the Maillard reaction) leads from early stage products such as Schiff base and Amadori products to advanced glycation end products (AGE), structures implicated in diabetic complications and the aging process. We have prepared the polyclonal anti-AGE antibody and the monoclonal anti-AGE antibody against AGE-bovine serum albumin and made an immunochemical approach to characterize AGE structures. Both polyclonal and monoclonal antibodies reacted with AGE-proteins such as AGE-bovine serum albumin, AGE-human serum albumin, and AGE-hemoglobin but not with unmodified counterparts. Treatments of these AGE-proteins with borohydride had no effect on the immunoreactivity. Moreover, fructosyl-epsilon-caproic acid, a synthetic Amadori compound, did not serve as an antigen, indicating that these antibodies were specific for AGE products but not for early stage products of the Maillard reaction. In addition, these antibodies were also able to recognize AGE products prepared either from alpha-tosyl-1-lysine, alpha-tosyl-1-lysine methyl ester, monoaminocarboxylic acid such as epsilon-aminocaproic acid, gamma-amino-n-butyric acid, and beta-alanine. Thus, these results strongly suggest the presence of a common structure in AGE preparations, regardless of whether AGE products are generated from proteins, amino acids, or monoaminocarboxylic acids.     

Structure of triphosphonoglycosphingolipid containing N-acetylgalactosamine 6-O-2-aminoethylphosphonate in the nervous system of Aplysia kurodai

A phosphonoglycosphingolipid, named F-21, was found in the nervous system of Aplysia kurodai by two-dimensional thin-layer chromatography (Abe, S., Araki, S., and Satake, M. (1986) Biomed. Res. (Tokyo) 7, 47-51). F-21 was isolated from the nervous tissue of Aplysia in this study, and its chemical structure was characterized as follows, where 2-AEP is 2-aminoethylphosphonate. (Formula; see text) The major aliphatic components of the ceramide portion were palmitic acid (75%), stearic acid (22%), octadeca-4-sphingenine (43%), and anteisononadeca-4-sphingenine (54%). Some information on the steric interactions in the sugar moiety was obtained by NMR spectroscopy. The ring protons of the internal galactose, H1, H3, and H4 and the H3 of the side chain galactose were shifted, as compared to the corresponding protons of dephosphonylated F-21. This may indicate the interactions between the 2-AEP residue of N-acetylgalactosamine and the internal galactose and between the N-acetyl group of N-acetylgalactosamine and the side chain galactose, implying a sterically restricted and unique structure that may relate to some biological functions of F-21.     

Novel phosphonoglycosphingolipids containing pyruvylated galactose from the nervous system of Aplysia kurodai

We have reported the existence of a phosphonoglycosphingolipid containing a pyruvylated galactose, FGL-IIb, in nerve fibers of Aplysia kurodai (Araki, S., Abe, S., Ando, S., Kon, K., Fujiwara, N. & Satake, M. (1989) J. Biol. Chem. 264, 19922-19927). We have now isolated two other pyruvylated galactose-containing phosphonoglycosphingolipids, named FGL-V and FGL-IIa, from the nervous tissue of Aplysia, and characterized them as [3,4-O-(S-1-carboxyethylidene)]Gal beta 1----3GalNAc alpha 1----3[6'-O-(2- aminoethylphosphonyl)Gal alpha 1----2] (2-aminoethylphosphonyl----6) Gal beta 1----4Glc beta 1----1 ceramide and [3,4,O-(S-1-carboxyethylidene)] Gal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2]Gal beta 1----4Glc beta 11----ceramide, respectively. Their major aliphatic components are palmitic acid, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. Thus, the nervous system of Aplysia contains several pyruvylated phosphonoglycolipids.     

Conformational changes of recombinant human granulocyte-colony stimulating factor induced bypH and guanidine hydrochloride

Fluorescence and circular dichroism were used to follow thepH-dependent conformational changes of granulocyte colony stimulating factor (G-CSF). Tryptophan fluorescence of the spectra monitored at 344 nm, or after deconvolution of the emission spectra, at 345 nm, showed a decrease in intensity on going frompH 7 to 4, with a midtransitionpH of 5.8. On the other hand, tyrosine fluorescence measured either by the ratio of intensity at 308 nm to that at 344 nm, or by the fluorescence intensity at 303 nm after deconvolution of the spectra, increased in intensity as thepH was changed from 6 to 2.5, with a midtransitionpH of 4.5. Near UV circular dichroic spectra also showed changes betweenpH 7.5 and 4.5, which correlated with the transition monitored by the tryptophan fluorescence. The guanidine hydrochloride-induced conformational changes of G-CSF at fivepH values from 2.5 to 7.5 were also studied. Circular dichroic and fluorescence spectra revealed minor conformational changes by the addition of 1 or 2 M guanidine HCl at allpH values examined, while the major conformational transition occurred between 2 and 4 M guanidine hydrochloride. The secondary structure of the protein was most stable betweenpH 3.3 and 4.5. The guanidine HCl-induced denaturation of G-CSF involved more than a two-state transition, with detectable intermediate(s) present, and the structure of the intermediate(s) appeared to depend on thepH used. These results are consistent with thepH dependence of the structure described above, and demonstrate the complex conformational properties of G-CSF.     

The complete amino acid sequence of yam (Dioscorea japonica) chitinase. A newly identified acidic class I chitinase.

The complete amino acid sequence of acidic chitinase from yam (Dioscorea japonica) aerial tubers was determined. The protein is composed of a single polypeptide chain of 250 amino acid residues and has a calculated molecular mass of 27,890 Da. There is an NH2-terminal domain, a hinge region, and a main structure, typical for class I chitinases (Shinshi, H., Neuhaus, J.-M., Ryals, J., and Meins, F., Jr. (1990) Plant Mol. Biol. 14, 357-368). We have obtained the first evidence for an acidic class I chitinase. Comparison with sequences of other class I chitinases revealed approximately 40% sequence similarity, a value lower than that for other class I chitinases (70-80%). We assume that there is a local conformational change in the molecule; cysteine residues that probably form disulfide bonds are completely conserved, with the exception of Cys-178. The difference in structure between this chitinase and other basic class I chitinases suggests that acidic and basic isoforms should be grouped into subclasses; this protein is an ethylene- or a pathogen-independent chitinase produced by a gene that is inherent in the tuber.     

An analysis of the effect of changes in growth temperature on proteolysis in vivo in the psychrophilic bacterium Vibrio sp. strain ANT-300.

In the psychrophilic bacterium Vibrio sp. strain ANT-300, the rate of protein degradation in vivo, measured at fixed temperatures, increased with elevation of the growth temperature. A shift in growth temperature induced a marked increase in this rate. Dialysed cell-free extracts hydrolysed exogenous insulin, globin and casein (in decreasing order of activity) but did not hydrolyse exogenous cytochrome c. Cells contained at least seven protease separated by DEAE-Sephacel chromatography, one of which was an ATP-dependent serine protease. The ATP-dependent proteolytic activity in extracts of cells incubated for 3 h at 16 degrees C after a shift-up from 0 degrees C increased to a level 36% and 17% higher than that of cells grown at 0 degrees C and 13 degrees C, respectively. A shift-down to 0 degrees C from 13 degrees C induced only a slight increase in the proteolytic activity. Extracts of all cells, whether exposed to temperature shifts or not, showed the same temperature dependence with respect to both ATP-dependent and ATP-independent protease activity. In all the extracts these proteases also exhibited the same heat lability. The ATP-dependent protease was inactivated by incubation at temperatures above 25 degrees C. There was an increase in ATP-independent protease activity during incubation at temperatures between 25 and 30 degrees C, but a decrease at 35 degrees C and higher. These results suggest that the marked increases in proteolysis in vivo, caused by a shift in temperature, may result not only from increases in levels of ATP-dependent serine protease(s) but also from increases in the susceptibility of proteins to degradation.     

Half-site recombinations mediated by yeast site-specific recombinases Flp and R.

The Flp recombinase of Saccharomyces cerevisae and the related R recombinase of Zygosaccharomyces rouxii can efficiently catalyze strand cleavage and strand exchange reactions in half recombination sites. A half-site consists of one recombinase binding element, a recombinase cleavage site on one strand and a 5' spacer hydroxyl group on the other that can initiate the strand exchange reaction. We have studied the various types of strand exchanges that half-sites can participate in. Reaction between a left half-site and a right half-site generates a full recombination site. Strand transfer between two left half-sites or between two right half-sites produces pseudo-full-sites. Strand transfer within a half-site results in a stem-loop or hairpin product. The half-site strand transfer reaction is fairly indifferent to the spacer sequence of the substrate per se and is less sensitive to variations in spacer lengths than a full-site recombination reaction. The optimal spacer length of eight to ten nucleotides observed for the Flp half-site reaction likely permits the most productive catalytic interactions between two Flp monomers bound to each of two partner half-sites. When reacted with a full-site, the half-site can give rise to a normal or reverse recombinant, corresponding to homologous or non-homologous alignments of the spacer sequences during substrate synapsis. The contrary recombination (resulting from non-homologous spacer alignment), whose level is low relative to normal recombination, is partly suppressed when the half-site spacer ends in a 5'-phosphate rather than a 5'-hydroxyl group. Thus, the early steps of recombination, namely synapsis and initial stand transfer, are not dependent on complete spacer homology between the two recombining substrates. The selection of properly aligned substrate partners must occur at the homology dependent branch migration step. In reactions containing a mixture of Flp and R half-sites, Flp and R catalyze strand transfer, almost exclusively, within or between their respective cognate substrates. However, under conditions where self-crosses are inhibited, strand exchange between a Flp half-site and an R half-site appears to be stimulated by a combination of R and Flp.