The effect on enterotoxicity of protease purified from Vibrio cholerae O1

Abstract The effect on enterotoxicity of protease purified from Vibrio cholerae O1 was investigated by the inoculation of live vibrio cells into the protease-treated loops of the ileal loop model. Fluid accumulation ratios in the protease-treated loops were elevated in a dose-dependent manner by challenge with live vibrio cells byt not that with toxin. An enhancement effect of protease on enterotoxicity was observed in both serotypes of V. cholerae O1 and V. cholerae non-O1. It is suggested, therefore, that the enterotoxicity was enhanced by treatment with protease when live vibrio cells were inoculated into the ileal loops of rabbits.     

Quantitative and qualitative differences in the in vivo response of NKT cells to distinct alpha- and beta-anomeric glycolipids

NKT cells represent a unique subset of immunoregulatory T cells that recognize glycolipid Ags presented by the MHC class I-like molecule CD1d. Because of their immunoregulatory properties, NKT cells are attractive targets for the development of immunotherapies. The prototypical NKT cell ligand alpha-galactosylceramide (alpha-GalCer), originally isolated from a marine sponge, has potent immunomodulatory activities in mice, demonstrating therapeutic efficacy against metastatic tumors, infections, and autoimmune diseases, but also has a number of adverse side effects. In vivo administration of alpha-GalCer to mice results in the rapid activation of NKT cells, which is characterized by cytokine secretion, surface receptor down-regulation, expansion, and secondary activation of a variety of innate and adaptive immune system cells. In this study, we have evaluated the in vivo immune response of mice to a set of structural analogues of alpha-GalCer. Our results show that, contrary to current thinking, beta-anomeric GalCer can induce CD1d-dependent biological activities in mice, albeit at lower potency than alpha-anomeric GalCer. In addition, we show that the response of NKT cells to distinct GalCer differs not only quantitatively, but also qualitatively. These findings indicate that NKT cells can fine-tune their immune responses to distinct glycolipid Ags in vivo, a property that may be exploited for the development of effective and safe NKT cell-based immunotherapies.     

Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by alpha-galactosylceramide in mice

The role of mouse liver NK1.1 Ag(+) T (NKT) cells in the antitumor effect of alpha-galactosylceramide (alpha-GalCer) has been unclear. We now show that, whereas alpha-GalCer increased the serum IFN-gamma concentration and alanine aminotransferase activity in NK cell-depleted C57BL/6 (B6) mice and B6-beige/beige mice similarly to its effects in control B6 mice, its enhancement of the antitumor cytotoxicity of liver mononuclear cells (MNCs) was abrogated. Depletion of both NK and NKT cells in B6 mice reduced all these effects of alpha-GALCER: Injection of Abs to IFN-gamma also inhibited the alpha-GalCer-induced increase in antitumor cytotoxicity of MNCS: alpha-GalCer induced the expression of Fas ligand on NKT cells in the liver of B6 mice. Whereas alpha-GalCer did not increase serum alanine aminotransferase activity in B6-lpr/lpr mice and B6-gld/gld mice, it increased the antitumor cytotoxicity of liver MNCS: The alpha-GalCer-induced increase in survival rate apparent in B6 mice injected intrasplenically with B16 tumor cells was abrogated in beige/beige mice, NK cell-depleted B6 mice, and B6 mice treated with Abs to IFN-gamma. Depletion of CD8(+) T cells did not affect the alpha-GalCer-induced antitumor cytotoxicity of liver MNCs but reduced the effect of alpha-GalCer on the survival of B6 mice. Thus, IFN-gamma produced by alpha-GalCer-activated NKT cells increases both the innate antitumor cytotoxicity of NK cells and the adaptive antitumor response of CD8(+) T cells, with consequent inhibition of tumor metastasis to the liver. Moreover, NKT cells mediate alpha-GalCer-induced hepatocyte injury through Fas-Fas ligand signaling.     

Cloning, overexpression and mutagenesis of cDNA encoding dihydrolipoamide succinyltransferase component of the porcine 2-oxoglutarate dehydrogenase complex.

Dihydrolipoamide succinyltransferase (E2o) is the structural and catalytic core of the 2-oxoglutarate dehydrogenase (OGDH) complex. The cDNA encoding porcine E2o (PE2o) has been cloned. The PE2o cDNA spans 2547 bases encoding a presequence (68 amino-acid residues) and a mature protein (387 residues, Mr = 41 534). Recombinant porcine E2o (rPE2o) (residues 1-387), C- and N-terminal truncated PE2os, and site-directed mutant PE2os were overexpressed in Escherichia coli via the expression vector pET-11d and purified. The succinyltransferase activity of the rPE2o was about 2.2-fold higher than that of the native PE2o. Electron micrographs of the rPE2o negatively stained showed a cube-like structure very similar to that of the native PE2o. Deletion of five amino-acid residues from the C-terminus resulted in a complete loss of both enzymatic activity and formation of the cube-like structure, but the deletion of only the last two residues had no effect on either function, suggesting the important roles of the C-terminal leucine triplet (Leu383-384-385). Substitution of Ser306 with Ala, and Asp362 with Asn, Glu or Ala in the putative active site, and Leu383-384-385 with Ala or Asp abolished both functions. Substitution of His358 with Cys resulted in an 8.5-fold reduction in kcat, with little change in Km values for dihydrolipoamide and succinyl-CoA. However, self-assembly was not affected. These data indicate that Ser306, Asp362 and the Leu383-384-385 triplet are important residues in both the self-assembly and catalytic mechanism of PE2o.     

Interactions between the Nucleus and Cytoplasmic Organelles during the Cell Cycle of Euglena gracilis in Synchronized Cultures III. Association between the Nucleus and Chloroplasts at an Early Stage in the Cell Cycle under Photoorganotrophic Conditions (Part II)

Three-dimensional models of chloroplasts before and during theirassociation with the nucleus in Euglena gracilis Z were constructedbased on serial sections of cells taken during an early phaseof the cell cycle in synchronized cultures under photoorganotrophiccondition. Before association, the cell contained two to threechloroplasts which were composed of elongated tubular bodiesextending in different directions toward the cell periphery.These tubular arms of chloroplasts were drawn toward the nucleus,and folded, with concurrent coalescence, into a single giantbody surrounding the nucleus. A DAPI-fluorescence photomicrographof a giant chloroplast revealed that the chloroplast-nucleoidswere in the form of a continuous strand lying throughout thechloroplast body, and that some protuberances from the nucleoidstrand were in especially close proximity to the nuclear periphery. A new mode of the chloroplast-nucleus connection was observed.The nucleus had conspicuous protrusions, whose distal ends wereconnected with the chloroplast. The outer membrane of the nuclearenvelope was continuous with the outer chloroplast membrane,and at some sites, an open space was formed between the innernuclear membrane and the inner chloroplast membrane. ¹ This work was reported at the 48th Annual Meeting of the BotanicalSociety of Japan, held in Kyoto, in October, 1983. For PartI see Ehara et al. (1984).     

Filamentous vibriophage fs2 encoding the rstC gene integrates into the same chromosomal region as cholera toxin X

The genome of the filamentous phage of Vibrio cholerae fs2 was found to contain rst C and rst B1 (truncated) genes downstream of ORF500. att -fs2-dir and att- fs2-rev sequences homologous to that of att -CTXφ were found between orf 500 and rst C of the fs2 genome. This prompted us to search for the integration site of fs2 in the genomes of V. cholerae O1 and O139. The genome of fs2 was found to integrate downstream of att RS of the CTXφ phage, which integrated into chromosome I of V. cholerae O1 and O139. When infected with fs2, a fimbriate strain of V. cholerae O1 appeared to reduce fimbrial production in an adult rabbit ileal loop assay.     

Minor pilin subunits are conserved in Vibrio cholerae type IV pili

The nucleotide sequences of five open reading frames within the Vibrio cholerae NAGV14 type IV pilus gene cluster were determined. The genes showed high homology to the mannose-sensitive hemagglutinin (MSHA) pilus genes mshB, mshC, mshD, mshO and mshP. PCR analysis showed that a MSHA-like gene cluster is highly conserved among different V. cholerae strains, with the exception of the previously reported major pilin subunit. Recombinant MshB and MshO proteins were purified and specific antiserum was raised to each of them. Western blotting analyses showed that these antisera reacted with purified NAGV14 and MSHA pili. The results suggested that MshB and MshO are minor components of the pilus fiber. Although there was no cross-reaction between the major pilin subunits of NAGV14 and MSHA pili, minor components seemed to be highly homologous and immunologically cross-reactive.     

Side chain effect on ion channel characters of Aib rich peptides.

As models of ion channel proteins and naturally occurring pore-forming peptides, we designed a series of Aib rich peptides [Ac-(Aib-Xxx-Aib-Ala)(5)-NH(2) (Xxx = Lys, Glu, Ser, and Gly: BXBA-20)] to investigate the effects of the side chains of the amino acid residues Lys, Glu, Ser, and Gly on the conformation and electrophysiological properties of ion channels. The conformation of peptides and their affinity for phospholipid membranes were evaluated by CD spectroscopy. Patch-clamp experiments revealed that all BXBA-20 peptides form ion channels in DPhPC bilayers exhibiting clearly resolved transitions between the open and closed states. The channel forming frequency was in the order BKBA-20>BEBA-20>BSBA-20>BGBA-20. In the case of BKBA-20 and BEBA-20, the self-assembled conductive oligomers expressed homogeneous and voltage-independent single channel conductances. In contrast, heterogeneous conductance was observed in BSBA-20 and BGBA-20 ion channels under similar experimental conditions. From these results, we conclude that peptides with a high degree of helical conformation, high amphipathicity, high affinity for lipid membranes, and self-associating characters in vesicles are most suitable for inducing ion channels with a high frequency of occurrence. Moreover, BEBA-20, BSBA-20, and BGBA-20 channels were cation-selective, whereas the BKBA-20 channel was non-selective.     

CXCL12 chemokine and CXCR4 receptor: association with susceptibility and prognostic markers in triple negative breast cancer

CXCL12/CXCR4 signaling has been implicated in breast carcinogenesis, and genetic polymorphisms in these molecules have been associated with different types of cancer. The present study analyzed genetic polymorphisms in CXCL12 (rs1801157, G?>?A) and CXCR4 (rs2228014, C?>?T) and CXCR4 immunostaining in tumor tissues from patients with triple negative breast cancer (TNBC) aiming to evaluate their possible role in its’ susceptibility and prognosis. Genetic polymorphisms were analyzed in 59 TNBC patients and 150 control women; age-adjusted logistic regression showed no association when variants were considered in isolation; however, a statistically significant interaction was noted for heterozygosis for both allelic variants increasing the odds for TNBC (CXCL12-GA by CXCR4-CT: OR 7.23; 95% CI 1.15–45.41; p?=?0.035). CXCL12 polymorphism was correlated negatively with proliferation index (Ki67) (Tau-b?=???0.406; p?=?0.006). CXCR4 immunostaining was evaluated in 37 TNBC patients (22 with paired tumor-normal adjacent tissue). CXCR4 was detected more intensely in cell cytoplasm than in membrane, and was more expressed in tumor than in normal adjacent tissues, although not statistically significant. CXCR4 expression on the membrane of tumor cells was correlated positively with histopathological grade (Tau-b?=?0.271; p?=?0.036) and negatively with lymph node metastasis (Tau-b?=???0.478; p?=?0.036). The present study indicates that CXCL12 and CXCR4 polymorphisms and CXCR4 immunostaining might have susceptibility and prognostic roles in TNBC pathogenesis.