Attenuating effects of melatonin on pilocarpine-induced seizures in rats

Daily melatonin (10-50 mg/kg, i.p.) treatment at 08.30 h or 17.00 h for 1 week of female rats (2-months-old) increased the latency to the appearance of the first convulsion in the pilocarpine-induced seizure model. Other behavior parameters remained unaltered. The anticonvulsant effect of melatonin seemed to be more intense at the light-dark transition. Moreover, the effect of repeated melatonin treatment was also age-related, since it showed a lower threshold in 2-month-old than in 21-day-old rats, and the acute treatment was not efficient. [3H]N-methylscopolamine binding was unaltered in the hippocampus and striatum of adult rats after the association of melatonin and pilocarpine. While muscarinic binding was unaltered in adult rats, it increased in the hippocampus of young rats in the presence of melatonin (50 mg/kg) and pilocarpine, and did not change in the striatum. Melatonin partially recovered [3H]GABA binding in the hippocampus in the presence of pilocarpine-induced seizures, and intensified pilocarpine effects in the striatum of adult rats.     

Localization of VIP36 in the post-Golgi secretory pathway also of rat parotid acinar cells.

VIP36 (36-kD vesicular integral membrane protein), originally purified from Madin-Darby canine kidney (MDCK) epithelial cells, belongs to a family of animal lectins and may act as a cargo receptor. To understand its role in secretory processes, we performed morphological analysis of the rat parotid gland. Immunoelectron microscopy provided evidence that endogenous VIP36 is localized in the trans-Golgi network, on immature granules, and on mature secretory granules in acinar cells. Double-staining immunofluorescence experiments confirmed that VIP36 and amylase co-localized in the apical regions of the acinar cells. This is the first study to demonstrate that endogenous VIP36 is involved in the post-Golgi secretory pathway, suggesting that VIP36 plays a role in trafficking and sorting of secretory and/or membrane proteins during granule formation.     

Downregulation of citrin, a mitochondrial AGC, is associated with apoptosis of hepatocytes

Citrin is a mitochondrial aspartate–glutamate carrier primarily expressed in liver. Adult-onset type II citrullinemia is caused by mutations in the SLC25A13 gene that encodes for citrin, and patients with this condition do not express citrin. We found apoptotic hepatocytes in one such patient. This finding prompted us to investigate the role of citrin in hepatocyte survival. Knockdown of citrin by a vector-based short-hairpin RNA technique reduced cell viability and induced apoptosis of a hepatocellular carcinoma cell line, Hep3B cells. Caspase-3/7 and caspase-9 were activated, and PARP was cleaved. Citrin knockdown also increased the expression of Bax and Bak, and reduced expression of Bcl-xL and Bcl-2. These alterations resulted in the release of cytochrome c from the mitochondria. Our results indicated that citrin downregulation induces apoptosis of hepatocytes through the mitochondrial death pathway, highlighting the importance of citrin in survival of hepatocytes and maintenance of liver function.     

IL-17-mediated regulation of innate and acquired immune response against pulmonary Mycobacterium bovis bacille Calmette-Guerin infection

IL-17 is a cytokine that induces neutrophil-mediated inflammation, but its role in protective immunity against intracellular bacterial infection remains unclear. In the present study, we demonstrate that IL-17 is an important cytokine not only in the early neutrophil-mediated inflammatory response, but also in T cell-mediated IFN-gamma production and granuloma formation in response to pulmonary infection by Mycobacterium bovis bacille Calmette-Guérin (BCG). IL-17 expression in the BCG-infected lung was detected from the first day after infection and the expression depended on IL-23. Our observations indicated that gammadelta T cells are a primary source of IL-17. Lung-infiltrating T cells of IL-17-deficient mice produced less IFN-gamma in comparison to those from wild-type mice 4 wk after BCG infection. Impaired granuloma formation was also observed in the infected lungs of IL-17-deficient mice, which is consistent with the decreased delayed-type hypersensitivity response of the infected mice against mycobacterial Ag. These data suggest that IL-17 is an important cytokine in the induction of optimal Th1 response and protective immunity against mycobacterial infection.     

Molecular analysis of the Escherichia coli hns gene encoding a DNA-binding protein, which preferentially recognizes curved DNA sequences.

Summary We previously demonstrated that the E. coli protein, H-NS (or Hla), encoded by the gene hns (or osmZ or bglY preferentially recognizes curved DNA sequences in vitro. In order to gain further insight into the complex function of H-NS and the significance of DNA curvature, we constructed a structurally defined hns deletion mutant on the E. coli chromosome. The hns deletion mutant thus obtained showed a variety of phenotypes previously for other lesions in hns. It was further demonstrated that, in this hns deletion background, numerous E. coli cellular proteins were either strongly expressed or remarkably repressed, as compared to their expression levels in wild-type cells.     

Interferon γ but not tumor necrosis factor α decreases susceptibility of human renal cell cancer cell lines to lymphokine-activated killer cells

Summary Human renal cell cancer (RCC) cell lines, ACHN and KRC/Y, with or without exposure to cytokines, were examined for their susceptibility to lymphokine-activated killer (LAK) cells. Flow-cytometric analysis demonstrated constitutional expression of class I antigen on both cell lines, which was enhanced by interferon (IFN), IFN and tumor necrosis factor (TNF). A 4-h⁵¹Cr-release cytotoxicity assay demonstrated that pretreatment of both cell lines with IFN or IFN, but not with TNF, decreased their susceptibility to LAK cells. IFN also decreased susceptibility to natural killer cells in a 16-h⁵¹Cr-release cytotoxicity assay. IFN treatment decreased the susceptibility of ACHN cells in a dose-dependent manner. Cold-target competition assay clearly showed that IFN- but not TNF-pretreated cells compete less effectively than do untreated target cells. Pretreatment with IFN, however, increased expression of intercellular adhesion molecule-1 (ICAM-1) to a degree comparable to that with TNF. Northern blot analyses using a 520-base-pair ICAM-1 cDNA as a probe demonstrated that more 3.3-kb mRNA is expressed in IFN- and TNF-pretreated cells. These results suggest that IFN-treated RCC cell lines may reduce their ability to be recognized by LAK cells, and that IFN-induced protection of RCC cell lines against LAK cells may depend upon a mechanism independent of the expression of class I antigens or ICAM-1 on tumor cells.     

Characterization of multicopy suppressor genes that complement a defect in the Wis1-Sty1 MAP kinase cascade involved in stress responses in Schizosaccharomyces pombe

The Wis1-Sty1 mitogen-activated protein (MAP) kinase cascade is one of the major signaling systems involved in a wide range of stress responses in Schizosaccharomyces pombe. It is known that Deltawis1 and Deltasty1 mutants exhibit highly pleiotropic phenotypes, including a phenotype of temperature sensitivity for growth. In this study, we screened multicopy suppressor genes that allow both the Deltawis1 and Deltasty1 mutants to grow simultaneously at a non-permissive temperature, 37 degrees C. Two such multicopy suppressors were cloned and characterized as sds23(+) and hxk2(+) genes. The former is known to specify a protein that functions as a multicopy suppressor for mutations of the PP1 protein phosphatase and the 20S cyclosome/anaphase-promoting complex (APC), and the latter encodes hexokinase 2. It was revealed that the multicopy sds231 gene restored a defect in the mating efficiency caused by the Deltawis1 and Deltasty1 mutations, whereas the multicopy hxk2(+) gene suppressed a phenotype of heat-shock sensitivity for growth of these mutant cells. These findings are discussed with special reference to the Wis1-Sty1 MAP kinase signaling pathway in S. pombe.     

Osmoregulation of fission yeast: cloning of two distinct genes encoding glycerol-3-phosphate dehydrogenase, one of which is responsible for osmotolerance for growth

Many types of microorganisms, including both prokaryotes and eukaryotes, have developed mechanisms to adapt to severe osmotic stress. In this study, we isolated multicopy suppressor genes for a Schizosaccharomyces pombe mutant, which exhibited the clear phenotype of being osmosensitive for growth (Osm^s) on agar plates containing high concentrations of either non-ionic or ionic osmotic solutes. Two genes were thus identified, and each was suggested to encode an NADH-dependent glycerol-3-phosphate dehydrogenase (GPD), which is required for glycerol synthesis. The nucleotide sequences, determined for these genes (named gpd1 ⁺ and gpd2 ⁺, respectively), revealed that S. pombe has two distinct GPD isozymes. They are only 60% identical to each other in their amino acid sequences. One such isozyme, GPD1, was shown to be directly involved in osmoregulation, based on the following observations. (i) Expression of gpd1 ⁺ was regulated at the mRNA level in response to osmotic upshift, (ii) It was demonstrated that wild-type cells markedly accumulated internal glycerol under high-osmolarity growth conditions. (iii) Δ gpd1 mutants, however, failed to do so even in a high-osmolarity medium, and thus exhibited an Osm^s phenotype. On the other hand, the gpd2 ⁺ gene was constitutively expressed at a particular low level, regardless of the osmolarity of the medium.     

Isolation and characterization of two outer membrane preparations from Escherichia coli

A method was developed for releasing specifically a part of outer membrane during spheroplast formation. A highly purified outer membrane (outer membrane I) was obtained from the spheroplast medium by isopycnic sucrose gradient centrifugation. The remaining outer membrane (outer membrane II) and cytoplasmic membrane was also isolated from the spheroplasts by the isopycnic centrifugation.Two outer membrane preparations were different from the cytoplasmic membrane in protein composition, enzyme localization, phospholipid composition, lipopolysaccharide content and electron micrographs. Although outer membranes I and II were almost the same in various respects, they seemed to be different from each other under electron microscope and in cardiolipin content. It is suggested that the outer membrane I and the outer membrane II, at least a part of the outer membrane II, are integrated in a different fashion in the outer-most layer of Escherichia coli cell surface.     

IL-17A produced by gammadelta T cells plays a critical role in innate immunity against listeria monocytogenes infection in the liver

IL-17A is originally identified as a proinflammatory cytokine that induces neutrophils. Although IL-17A production by CD4(+) Th17 T cells is well documented, it is not clear whether IL-17A is produced and participates in the innate immune response against infections. In the present report, we demonstrate that IL-17A is expressed in the liver of mice infected with Listeria monocytogenes from an early stage of infection. IL-17A is important in protective immunity at an early stage of listerial infection in the liver because IL-17A-deficient mice showed aggravation of the protective response. The major IL-17A-producing cells at the early stage were TCR gammadelta T cells expressing TCR Vgamma4 or Vgamma6. Interestingly, TCR gammadelta T cells expressing both IFN-gamma and IL-17A were hardly detected, indicating that the IL-17A-producing TCR gammadelta T cells are distinct from IFN-gamma-producing gammadelta T cells, similar to the distinction between Th17 and Th1 in CD4(+) T cells. All the results suggest that IL-17A is a newly discovered effector molecule produced by TCR gammadelta T cells, which is important in innate immunity in the liver.