The effect of vitamin A depletion on antigen-stimulated trapping of peripheral lymphocytes in local lymph nodes of rats

The effect of vitamin A depletion on antigen-stimulated trapping of peripheral lymphocytes in lymphatic organs was studied in rats. Distribution of [3H]-uridine-labelled syngenic peripheral lymphocytes was quantified by assaying radioactive content of brachial and axillary lymph nodes, spleen and liver of normal and vitamin A-depleted F344/Ducrj rats immunized with sheep red blood cells. Localization of labelled cells in the ipsilateral brachial lymph nodes of the normal rats was stimulated by three times upon immunization with sheep erythrocytes as compared with the contralateral nodes. Recruitment of cells in axillary lymph nodes, spleen and liver was not significantly different from non-immunized values. The vitamin A-depleted rats exhibited marked deterioration in antigen-stimulated trapping of labelled cells in the draining brachial lymph nodes. These results suggest that this effect of vitamin A depletion is due to derangement of integrity of lymphocyte-trapping mechanism in the draining lymph nodes and not to any change in nature of lymphocytes per se.     

Rapid screening of point mutations of the Neisseria gonorrhoeae parC gene associated with resistance to quinolones.

To detect quinolone resistance-associated mutations within the Asp-86, Ser-87, Ser-88, and Glu-91 codons of the Neisseria gonorrhoeae parC gene, we developed a rapid and simple assay based on amplification of the regions of the parC gene containing the mutations sites by PCR and digestion of the PCR products with restriction enzymes. By using the method of primer-specified restriction site modification, artificial SalI, PstI, EcoRI, and HinfI restriction sites were created in the regions containing the Asp-86, Ser-87, Ser-88, and Glu-91 codons, respectively. The mutations generating alterations at Asp-86, Ser-87, Ser-88, and Glu-91 were detected as failures of SalI, PstI, EcoRI, and HinfI to digest the respective PCR products. Fifty-five clinical strains of N. gonorrhoeae were examined for mutations in the parC gene by this assay. Appropriate mutations at either the Asp-86, Ser-87, Ser-88, or Glu-91 codon were detected in each of 11 strains in which a mutation had previously been observed by DNA sequencing. This rapid and simple assay could be a useful device for screening genetic alterations in the parC gene associated with resistance to quinolones in N. gonorrhoeae.     

Vesicles in the subacrosomal space and partial diaphragms in the subacrosomal nuclear envelope of round spermatids of a rat injected intravenously with gold labeled-testosterone-bovine serum albumin conjugate: vesicular trafficking from acrosome to nucleus

Colloidal gold labeled-testosterone-bovine serum albumin conjugate (testosterone-BSA-gold) injected into the vascular system of rats is taken up by endocytosis into round spermatids. Based on observation of silver deposits indicating testosterone-BSA-gold with silver enhancement, we have suggested that testosterone-BSA-gold enters the nuclei through not only the postacrosomal nuclear envelope but also the subacrosomal nuclear envelope (SNE) via the acrosome (Nishimura and Nakano, 1997). However, it was unclear how testosterone-BSA-gold in the acrosome entered the nucleoplasm. Spermatids showing silver deposits on the subacrosomal space were observed under electron microscope without silver enhancement, to clarify the courses of translocation. In the spermatids, vesicles with the gold particles were seen in the subacrosomal space. Some of the vesicles were in contact with the SNE. A part of the outer nuclear membrane projected into the space. Furthermore, local single-bilayer nuclear membranes, which seemed to partially lack nuclear lamina, were present in the SNE. These results indicate the possibility that the vesicles mediate the transport of testosterone-BSA-gold from acrosome to nucleus, and that the vesicle membrane fuses with not only the outer nuclear membrane but also a shared bilayer in the SNE.     

Serotonin-immunoreactive axons in the cell column of sympathetic preganglionic neurons in the spinal cord of the filefish Stephanolepis cirrhifer

Serotonin-immunoreactive axonal components were observed in the central autonomic nucleus (CAN), a cell column of sympathetic preganglionic neurons in the rostral spinal cord of the filefish Stephanolepis cirrhifer. Serotonin-positive axonal varicosities were seen around neuronal perikarya through the whole rostrocaudal extent of the CAN, although their distribution pattern in the rostral CAN was different from that in the caudal CAN. Electron microscopically, serotonin-positive axonal varicosities were found to make axodendritic and axosomatic synapses on CAN neurons. Many serotonin-positive neuronal cell bodies were seen in the raphe nuclei in the lower brainstem, whereas only a few were found in the spinal cord. Thus most of serotoninergic axons within the CAN were considered to originate from the raphe nuclei in the lower brainstem.     

Detection of circulating intercellular adhesion molecule-1 antigen in malignant diseases.

A new monoclonal antibody (MoAb) HA58 (IgG1) was prepared, which recognizes the binding site on the intercellular adhesion molecule-1 (ICAM-1) antigen to the lymphocyte function-associated antigen-1 (LFA-1). The double-determinant immunoassay (DDIA) was established with use of MoAb HA58 and another anti-ICAM-1, MoAb CL207, to detect the soluble, shedding ICAM-1 antigen. Human recombinant interferon-gamma (IFN-gamma) induced not only the expression of cell surface ICAM-1, but also the shedding ICAM-1 antigen in an IFN-gamma concentration-dependent and incubation-time-dependent manner. DDIA was applied to detect the shedding ICAM-1 antigen in the sera of patients with malignant or benign diseases. The incidence of positivity for ICAM-1 antigen in malignant diseases was higher than that in benign diseases or in healthy controls. Furthermore, the sera of cancer patients with liver metastasis showed higher levels of the shedding ICAM-1 antigen. These findings suggest that serum ICAM-1 antigen may be a useful marker to monitor tumor burden in cancer patients.     

Significance of antibody to hepatitis C virus in Japanese patients with viral hepatitis: relationship between anti-HCV antibody and the prognosis of non-A, non-B post-transfusion hepatitis.

In a retrospective study, antibody to hepatitis C virus (anti-HCV antibody) was measured in 80 patients with acute viral hepatitis (type A, 18; type B, 21; type non-A,non-B, 41). Anti-HCV antibody was found in 12 of 20 patients (60%) with non-A,non-B post-transfusion hepatitis (NANB-PTH) and in 9 of 21 patients (43%) with sporadic NANB hepatitis (NANB-SPO). Patients with acute hepatitis type A or type B did not have anti-HCV antibody. The number of patients who developed chronic hepatitis was greater in the group with anti-HCV antibody than in the anti-HCV negative group in both NANB-PTH and NANB-SPO. The difference was significant in those with NANB-PTH (P less than 0.05). To investigate the relationship between the long-term prognosis of NANB-PTH and the course of anti-HCV, we studied anti-HCV antibody in 12 patients who developed chronic type C hepatitis (C-CH) after PTH and followed them for more than 5 years after the development of PTH. One year after the development of PTH, all 12 had anti-HCV antibody. Five lost anti-HCV antibody (group 1) while 7 remained positive (group 2) at the final examination. Four of the 5 patients in group 1 had normal serum transaminases; however, abnormal transaminase persisted in all 7 patients in group 2 until the end of follow-up (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C-dependent inhibition of K+ currents in noradrenaline-induced depolarization of smooth muscle of guinea-pig vas deferens

Ionic mechanisms and signal transduction underlying noradrenaline (NA)-induced depolarization in single smooth muscle cells of guinea-pig vas deferens were studied. NA caused depolarization followed by action potentials through activation of 1-adrenoceptors. In the presence of nifedipine, no action potential was generated, and the magnitude of the depolarization depended on the concentration of NA (0.1-100 micrometer). NA, through 1-adrenoceptor activation, reduced the magnitude of membrane currents in response to voltage ramp pulses from -90 to -30 mV in a concentration-dependent manner. The reversal potential of the current inhibited by NA changed proportionally to the change in the equilibrium potential of K+, suggesting that NA inhibited K+ channel activity. Treatment of cells with GDPS, an inhibitor of G proteins, or bisindolylmaleimide (BIM), a selective protein kinase C (PKC) inhibitor, prevented the NA inhibition of the currents. Application of 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, mimicked the effect of NA. It is suggested that in the smooth muscle of guinea-pig vas deferens, activation of 1-adrenoceptors and the subsequent activation of PKC led to inhibition of K+ currents, which is responsible for the depolarization induced by NA.     

Multiple downstream signalling pathways from ROCK, a target molecule of Rho small G protein, in reorganization of the actin cytoskeleton in Madin-Darby canine kidney cells

BACKGROUND: In Madin-Darby canine kidney cells, Rho small G protein regulates formation of stress fibres, focal adhesions, and peripheral bundles through reorganization of the actin cytoskeleton. There are two morphologically distinguishable types of Rho-regulated stress fibres: parallel and stellate. Of these, effects of Rho small G protein, mDia1 regulates the formation of parallel stress fibres, whereas ROCK regulates the formation of stellate stress fibres, peripheral bundles and focal adhesions. Both mDia1 and ROCK are direct downstream targets of Rho small G protein. RESULTS: The ROCK-induced formation of stellate stress fibres is regulated mainly through the myosin light chain kinase-dependent phosphorylation of myosin light chain and the LIM-kinase-dependent phosphorylation of cofilin. The ROCK-induced formation of focal adhesions is mainly regulated through a downstream pathway of ROCK other than myosin light chain and cofilin. The ROCK-induced formation of peripheral bundles is regulated at least through ERM proteins, but not through the myosin light chain or cofilin. CONCLUSION: Our present and previous findings suggest the presence of multiple downstream signalling pathways from ROCK to reorganization of the actin cytoskeleton in Madin-Darby canine kidney cells.     

Effect of Pseudomonas aeruginosa exotoxin A on endotoxin-induced tumour necrosis factor production in murine lung

The ability of several Pseudomonas aeruginosa exo-enzymes, including exotoxin A (ETA), to induce inflammation and their influence on endotoxin-induced tumour necrosis factor (TNF) production in murine lung were evaluated. Intratracheal administration of lipopolysaccharide (LPS; 0.1-10 microg/mouse), 2(-1) LD50 of P. aeruginosa alkaline protease (7.5 microg/mouse) and elastase (1.2 microg/mouse) elevated total cell number and the percentage of neutrophils in broncho-alveolar lavage fluid (BALF), whereas ETA (0.1 microg/mouse) did not. LPS induced TNF production in BALF in a dose-dependent manner, whereas the P. aeruginosa exo-enzymes did not. When ETA was inoculated into the respiratory tract before LPS, production of TNF in BALF was significantly suppressed in a dose-dependent manner. ETA also suppressed TNF production by alveolar macrophages (AMs) stimulated with LPS in vitro. Flow cytometric analysis showed that ETA markedly reduced the expression of CD14 and CD11c/CD18 on the surface of AMs. ETA also depressed partially the expression of TNF-alpha mRNA in AMs. These findings suggest that ETA regulates TNF production in murine lung by suppressing LPS receptor expression, mRNA expression and protein synthesis and/or secretion of TNF.