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 .     

Effect of the dietary alpha-linolenate/linoleate balance on leukotriene production and histamine release in rats

Rats were fed semi-purified diets supplemented either with safflower seed oil rich in linoleate (18:2n-6) or with perilla seed oil rich in alpha-linolenate (18:3n-3) through two generations. In the major phospholipids of polymorphonuclear leukocytes (PMNs), the proportions of n-6 polyunsaturated fatty acids (18:2, 20:4, 22:4 and 22:5) were higher but those of n-3 acids (20:5, 22:5 and 22:6) were lower in the safflower group than in the perilla group. When stimulated with a calcium ionophore, the PMNs from the safflower group produced 27% more leukotriene (LT)B4 than those from the perilla group. The formation of LTB5 which has biological activities less than 1/10 those of LTB4, was negligible in the safflower group but was 40 ng/10(7) PMN cells in the perilla group. The amount of the total LTB formed in the perilla group tended to be more than in the safflower group. The formation of SRS-A (slow-reacting substances of anaphylaxis) by PMNs was determined by measuring the spasmogenic activities of LTs on guinea pig ileum. SRS-A activity was 59% higher in the safflower group than in the perilla group. In contrast, histamine release from rat peritoneal mast cells was not significantly different between the two groups. Thus, the increasing the alpha-linolenate/linoleate ratio of diets results in the decreased formation of LTs derived from 20:4n-6 in PMNs. This may be beneficial in lowering the severity of allergic and inflammatory responses caused by LTs, and thereby shifting the pathological symptoms to normal self-defense mechanism.     

Taq I-generated HLA-DQ αpolymorphism in Japanese patients with narcolepsy

Taq I-generated HLA-DQrestriction fragment length polymorphism was examined in Japanese patients with narcolepsy. All patients were DR2 positive and shared a 6.0 kb fragment, although this fragment was found only in 54 % of the healthy DR2-positive Japanese. This finding added the DQ gene to the list of candidates for the possible narcolepsy-susceptibility gene. In contrast, there was no complete association between narcolepsy and DXrestriction fragment length polymorphism. These findings suggest that a narcolepsy-susceptibility gene is located closer to the DQ locus than to the DX locus.     

Differential regulation by Ly-5 and Lyb-2 of IgG production induced by lipopolysaccharide and B cell stimulatory factor-1 (IL-4)

We have previously shown that mAb Ly-5 which on B cells recognizes a 220,000-Da (B220) molecule, inhibits LPS-induced IgG responses without affecting IgM or proliferative responses, whereas mAb Lyb-2 which modulates B cell activation processes induced by B cell stimulatory factor-1 (BSF-1) or IL-4, has no effect on LPS-induced B cell responses. In this report we further examined the cellular mechanisms of Ly-5 antibody action and the effect of Lyb-2 antibody in IgG responses induced by LPS and BSF-1. The results presented demonstrated that the inhibitory effect of Ly-5 antibody seems to be restricted to the IgG class and is observed in all IgG subclasses induced by LPS. Limiting dilution analysis showed that the Ly-5 antibody reduces primarily the precursor frequency of IgG-secreting cells and that the effect on the clone size is partial. Lyb-2 antibody, on the other hand, greatly inhibited IgG1 induction initiated by LPS and BSF-1 by the action on processes triggered by BSF-1, although it could not reverse the reduced IgG2b or IgG3 responses. Limiting dilution analysis revealed that Lyb-2 antibody reduces the precursor frequency but not the clone size of BSF-1-induced IgG1-producing cells, supporting our previous proposition that Lyb-2 plays a critical role in the B cell differentiation mediated by BSF-1. Taken together, these results indicate that both Ly-5 and Lyb-2 are important molecules in IgG subclass regulation, each acting on a distinct activation step.     

Molecular cloning and sequence analysis of full-length cDNA for rabbit liver NADPH-cytochrome P-450 reductase mRNA

The nucleotide sequence of the mRNA for NADPH-cytochrome P-450 reductase from rabbit liver was determined from a full-length cDNA clone (pFP105). The clone contains 2,269 nucleotides complementary to rabbit liver reductase mRNA. The single open reading frame of 2,037 nucleotides codes for a 679-amino acid polypeptide with a calculated molecular weight of 76,583 daltons. The cloned cDNA contains the complete 3'-noncoding region of 193 nucleotides, including 68 nucleotides of poly(A), and 39 nucleotides of the 5'-noncoding region. The nucleotide sequence in the coding region of cDNA of rabbit reductase (pFP105) showed 85% homology to that of rat reductase (Porter, T.D. & Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U.S. 82, 973-977, and Murakami, H. et al. (1986) DNA 5, 1-10). Rabbit reductase has one more amino acid residue than the rat enzyme, and the amino acid compositions of the two enzymes are similar. The amino acid sequence of the rabbit enzyme showed 91% identity with that of the rat enzyme. The segment related to binding of FMN and FAD was well conserved among rabbit, rat, and pig reductases. The sequence related to AMP moiety-binding was also conserved among these species, and was found in the amino acid sequence of NADH-cytochrome b5 reductase, another flavoenzyme in the microsomal electron transport system.     

Multiple catalytic properties of the purified and reconstituted cytochrome P-450 (P-450sccII) system of pig testis microsomes

The catalytic properties of the testis microsomal P-450, termed P-450sccII, have been studied in a refined assay system which consists of P-450sccII (13 nmol of P-450 heme/mg of protein) and its reductase has been purified extensively from pig testis. The results indicated that P-450sccII was highly active in catalyzing hydroxylation of 11 beta-hydroxyprogesterone at the 17 alpha-position to give 21-deoxycortisol and cleavage of 17 alpha-hydroxyprogesterone at the 17-20 bond to give androstenedione with turnover numbers of 25 and 30 mol/min X mol of P-450, respectively. In contrast, many physiologically important corticosteroids we tested were found to be poor substrates for both the hydroxylase and lyase reactions. The possible reason for the importance of these substrate specificity of P-450sccII in production of both corticosteroids and androgens in the endocrine tissues is discussed. P-450sccII also catalyzed conversion of testosterone to androstenedione, but 18O experiments failed to show incorporation of atmospheric oxygen into the androstenedione formed. However, this does not preclude the possibility that the P-450-bound intermediate gem-diol stereoselectively dehydrates to give the nonlabeled ketosteroid. In addition to these steroid-oxidizing activities, P-450sccII revealed considerable specificities toward various xenobiotics, suggesting that P-450sccII and liver microsomal P-450 are basically similar as regards enzymatic functions and activities.     

Aplysia oxymyoglobin with an unusual stability property

Native oxymyoglobin was isolated directly from the radular muscle of Aplysia kurodai with complete separation from metmyoglobin on a DEAE-cellulose column. It was examined for its spectral and stability properties. The spectrum of Aplysia MbO2 , which lacks the distal histidine, is very similar to those of mammalian oxymyoglobins , the alpha-peak being higher than the beta-peak and the absorbance ratio being 1.03. Its stability, however, is quite different from those of the mammalian oxymyoglobins , and Aplysia MbO2 is found to be extremely susceptible to autoxidation. Its rate is one-hundred times higher at pH 9.0, and its pH dependence is unusual and much less steep, when compared with sperm whale MbO2 as reference.     

Differential subcellular localization of ACTH and α-MSH in corticotropes of the rat anterior pituitary

Summary Specific antisera to -melanotropin (-MSH) and corticotropin (ACTH 1-39) were used to obtain immunocytochemical evidence for the differential localization of -MSH and ACTH in the secretory granules of corticotropes of rat anterior pituitary. The specificity of the antisera was established by binding ¹³¹I-labeled -MSH and ACTH 1-39 to their respective antisera. Double-labeling immunocytochemistry (for -MSH, ferritin; for ACTH, colloidal gold) was performed. Some secretory granules were labeled with ferritin particles (-MSH), whereas others contained gold particles (ACTH). Only a few granules showed both ACTH and -MSH. In typical corticotropes (stellate in form with a small number of secretory granules aligned along the cell periphery) only some of the secretory granules that were labeled with anti-ACTH serum were also immunoreactive to anti--MSH. In atypical corticotropes (polygonal in shape and containing a large number of secretory granules) almost all of the immunoreactive ACTH secretory granules were also positive to anti--MSH serum. An intermediate type of corticotrope was observed containing a small number of secretory granules, almost all of which were labeled with anti--MSH. Thus, rat anterior pituitary corticotropes may be classified into three types according to the distribution and content of -MSH. The light-microscopic immuncytochemistry provided similar results.