Analysis of common antigen of flagella in Bacillus cereus and Bacillus thuringiensis

Abstract Flagellar antigen of Bacillus cereus H.1 was purified and tested for serodiagnostic antigen by ELISA. The antibody against the flagellar antigen of B. cereus H.1 reacted not only with the homologous specific antigen but also reacted with the flagellar antigens of 23 strains of B. cereus . This common flagellar antigen of B. cereus was found to be due to 61-kDa protein by SDS-PAGE and immunoblot assay. Monoclonal antibody H15A5 against common antigenic epitope of B. cereus also reacted with flagellar antigens of 21 strains of Bacillus thuringiensis by ELISA. This monoclonal antibody reacted with the 61-kDa protein of the flagella of B. cereus H.1 and H.2 and B. thuringiensis Kurstaki HD1, Alesti and Aizawai juroi by immunoblot analysis. These results indicated that the common antigenic epitope of the 61-kDa protein existed in the flagella both of B. cereus and B. thuringiensis .     

The specificity of fumarate as a switching factor of the bacterial flagellar motor

Fumarate restores to flagella of cytoplasm-free, CheY- containing envelopes of Escherichia coli and Salmonella typhimurium the ability to switch from one direction of rotation to another. To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues. Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate. These observations were made both with wild-type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching-promoting activity of the analogues was not caused by their conversion to fumarate. Aspartate and lactate did not promote switching. Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemo-taxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate). All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching. Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour.     

Microstructural changes in the basal ganglia in restless legs syndrome and migraine

Sleep and Biological Rhythms -     

Immunochemical properties of mannan-protein complex isolated from viable cells of Saccharomyces cerevisiae 4484-24D-1 mutant strain by the action of zymolyase

Viable cells of Saccharomyces cerevisiae 4484-24D-1 mutant strain were treated with an Arthrobacter sp. beta-1,3-glucanase, Zymolyase-60,000, in the presence of a serine protease inhibitor, phenylmethylsulfonyl fluoride. Fractionation of the solubilized materials with Cetavlon (cetyltrimethylammonium bromide) yielded a purified mannan-protein complex, which had a molecular weight of ca. 150,000, approximately three times higher than that of the mannan isolated from the same cells by the hot-water extraction method at 135 C. The amino acid composition of the mannan-protein complex was found to be very similar to that of the mannan-protein complexes of S. cerevisiae X2180-1A wild and S. cerevisiae X2180-1A-5 mutant strains, indicating the presence of large amounts of serine and threonine. It was unexpected that the antibody-precipitating activity of this complex against the homologous anti-whole cell serum was about twice as great as that of the mannan isolated by hot-water extraction. Treatment of this complex with 100 mM NaOH, hot water at 135 C, and pronase, respectively, gave degradation products having the same molecular weight and antibody-precipitating activity as those of the hot-water extracted mannan, allowing the assumption that the protein moiety participated in a large part of this activity.     

Multimeric bivalent immunogens from recombinant tetanus toxin H<Subscript>C</Subscript> fragment,synthetic hexasaccharides,and a glycopeptide adjuvant

Using recombinant tetanus toxin H_C fragment (rTT-H_C) as carrier, we prepared multimeric bivalent immunogens featuring the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Ogawa, in combination with either the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Inaba, or a synthetic disaccharide tetrapeptide peptidoglycan fragment as adjuvant. The conjugation reaction was effected by squaric acid chemistry and monitored in virtually real time by SELDI-TOF MS. In this way, we could prepare well-defined immunogens with predictable carbohydrate–carrier ratio, whose molecular mass and the amount of each saccharide attached could be independently determined. The ability to prepare such neoglycoconjugates opens unprecedented possibilities for preparation of conjugate vaccines for bacterial diseases from synthetic carbohydrates.     

A novel magnesium-independent neutral sphingomyelinase associated with rat central nervous system meylin.

Purified myelin fractions prepared from young adult rat brain contain a novel sphingomyelinase which has a pH optimum of 7.0 and does not require divalent cations. This sphingomyelinase is different from the two previously known sphingomyelinases in the brain--the acidic sphingomyelinase and the magnesium-dependent neutral sphingomyelinase. When the distributions of the sphingomyelinases among the purified myelin, the total subcellular fractions heavier than myelin (greater than 0.85 M sucrose), and the microsomes were examined, the magnesium-independent sphingomyelinase was detected only in myelin, while the magnesium-dependent sphingomyelinase was present in the other two fractions but not in myelin. Therefore, this new sphingomyelinase appears to be specifically localized in the myelin sheath.     

Enzymatic synthesis of two ginsenoside Re-beta-xylosides.

Two new beta-xylosyl derivatives of ginsenoside Re, 20(S)-protopanaxatriol 6-O-alpha-L-rhamnopyranosyl-(1 --> 2)-[beta-D-xylopyranosyl-(1 --> 4)]-beta-D-glucopyranosyl-20-O-beta-D-glucopyranoside and 20(S)-protopanaxatriol 6-O-alpha-L-rhamnopyranosyl-(1 --> 2)-[beta-D-xylopyranosyl-(1 --> 6)]-beta-D-glucopyranosyl-20-O-beta-D-glucopyranoside, were respectively synthesized from p-nitrophenyl beta-D-xylopyranoside and phenyl beta-D-xylopyranoside as donors and ginsenoside Re as the acceptor in 25% acetone and acetonitrile by a cellulase preparation from Trichoderma viride and a beta-galactosidase preparation from Aspergillus oryzae. The latter enzyme preparation also catalyzed the hydrolysis of ginsenoside Re to the minor saponin, ginsenoside Rg2.     

Mutations in the hepatocyte nuclear factor-4alpha gene in Japanese with non-insulin-dependent diabetes: a nucleotide substitution in the polypyrimidine tract of intron 1b.

Mutations of the hepatocyte nuclear factor 4 alpha (HNF-4alpha) gene have been demonstrated in maturity-onset diabetes of the young (MODY) 1 families. To investigate the possibility that the HNF-4alpha gene contributes to the onset of non-insulin-dependent diabetes mellitus (NIDDM) in Japanese patients, we screened all exons and flanking introns of this gene for mutations in 100 patients with NIDDM diagnosed after 25 years of age. We identified two missense mutations: M49V in exon 1c and T1301 in exon 4; and two nucleotide substitutions in introns: cytosine to thymidine at -5 nt in intron 1b and adenine to thymidine at -21 nt in intron 5. We screened an additional 220 diabetic subjects for the polymorphism in intron 1b. The c/t substitution in intron 1b was associated with NIDDM. This substitution in the polypyrimidine tract, an important cis-acting element directing intron removal, is likely to influence pre-mRNA splicing of this gene. T1301 in exon 4 was observed in only two diabetic subjects. This mutation could influence the conformation of this peptide, resulting in changes in ligand binding domain function. M49V in exon 1c was found in both diabetic and non-diabetic subjects; isoforms HNF-4alpha 4, 5, and 6 with this mutation may impair glucose metabolism in tissue. In contrast to the primary cause of nonsense and missense mutations of the HNF-4alpha gene in MODY1, the nucleotide substitution in intron 1b may partially contribute to development of NIDDM in combination with other genetic and environmental factors.