Genetic studies on premating isolation in Drosophila simulans. II. A D. simulans line highly crossable to males of D. yakuba and D. teissieri

Females of the S1 line of Drosophila simulans, an isofemale line from a natural population at Mishima, Japan, were found to show high crossability to males of D. yakuba and D. teissieri. With D. yakuba males, 24.0-58.5% of the S1 females were inseminated, while the rate was only 0.0-6.3% for the control lines. The high crossability was ascribed to the genes on the X and third chromosomes. With D. teissieri males, 38.0-51.5% of the S1 females were inseminated, while the rate was only 0.0-6.2% for the control lines. The crossability was ascribed to the genes on the third chromosome, and possibly the second chromosome genes acted additively. The crossabilities of hybrid females between the S1 and the control lines were intermediate between the parental lines both to D. yakuba males and to D. teissieri males.     

Estrogen enhances depolarization-induced glutamate release through activation of phosphatidylinositol 3-kinase and mitogen-activated protein kinase in cultured hippocampal neurons

Changes in synaptic efficacy are considered necessary for learning and memory. Recently, it has been suggested that estrogen controls synaptic function in the central nervous system. However, it is unclear how estrogen regulates synaptic function in central nervous system neurons. We found that estrogen potentiated presynaptic function in cultured hippocampal neurons. Chronic treatment with estradiol (1 or 10 nm) for 24 h significantly increased a high potassium-induced glutamate release. The estrogen-potentiated glutamate release required the activation of both phosphatidylinositol 3-kinase and MAPK.The high potassium-evoked release with or without estradiol pretreatment was blocked by tetanus neurotoxin, which is an inhibitor of exocytosis. In addition, the reduction in intensity of FM1-43 fluorescence, which labeled presynaptic vesicles, was enhanced by estradiol, suggesting that estradiol potentiated the exocytotic mechanism. Furthermore, protein levels of synaptophysin, syntaxin, and synaptotagmin (synaptic proteins, respectively) were up-regulated by estradiol. We confirmed that the up-regulation of synaptophysin was blocked by the MAPK pathway inhibitor, U0126. These results suggested that estrogen enhanced presynaptic function through the up-regulated exocytotic system. In this study, we propose that estrogen reinforced excitatory synaptic transmission via potentiated-glutamate release from presynaptic sites.     

Genetic analysis and characterization of poly(aspartic acid) hydrolase-1 from Sphingomonas sp. KT-1

Sphingomonas sp. KT-1 hydrolyzes poly(aspartic acid) (PAA) containing alpha- and beta-amide units and has at least two different types of PAA hydrolases. The PAA hydrolase-1 hydrolyzes selectively beta-beta amide units in PAA. Molecular cloning of PAA hydrolase-1 from Sphingomonas sp. KT-1 has been carried out to characterize its gene products. Genetic analysis shows that the deduced amino acid sequence of PAA hydrolase-1 has a similarity with those of the catalytic domain of poly(3-hydroxybutyric acid) (PHB) depolymerases from Alcaligenes faecalis AE122 and Pseudomonas lemoignei. Site-specific mutation analysis indicates that (176)Ser is a part of a strictly conserved pentapeptide sequence (Gly-Xaa-Ser-Xaa-Gly), which is the lipase box, and plays as an active residue.     

MpFAE3, a beta-ketoacyl-CoA synthase gene in the liverwort Marchantia polymorpha L., is preferentially involved in elongation of palmitic acid to stearic acid

Fatty acid chain elongation is a crucial step in the biosynthesis of long chain fatty acids. An essential reaction in the elongation process is condensation of malonyl-CoA with acyl-CoA, which is catalyzed by beta-ketoacyl-CoA synthase (KCS) in plants. We have isolated and characterized the MpFAE3 gene, one of the KCS gene family in the liverwort Marchantia polymorpha. Transgenic M. polymorpha plants overexpressing MpFAE3 accumulate fatty acids 18:0, 20:0, and 22:0. In these plants, the amount of 16:0 is reduced to 50% of wild type. In a heterologous assay, transgenic methylotrophic yeast expressing the MpFAE3 gene accumulates fatty acid 18:0 and generates several longer fatty acids which are not detectable in the control, accompanied by a decrease of 16:0. These observations indicate that the MpFAE3 protein is preferentially involved in the elongation of 16:0 to 18:0 and also in the subsequent steps of 18:0 to 20:0 and 20:0 to 22:0 in M. polymorpha.     

Cleavage of Host Cytokeratin-6 by Lysine-Specific Gingipain Induces Gingival Inflammation in Periodontitis Patients

Background/PurposeLysine-specific gingipain (Kgp) is a virulence factor secreted from Porphyromonas gingivalis (P. gingivalis), a major etiological bacterium of periodontal disease. Keratin intermediate filaments maintain the structural integrity of gingival epithelial cells, but are targeted by Kgp to produce a novel cytokeratin 6 fragment (K6F). We investigated the release of K6F and its induction of cytokine secretion.MethodsK6F present in the gingival crevicular fluid of periodontal disease patients and in gingipain-treated rat gingival epithelial cell culture supernatants was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer-based rapid quantitative peptide analysis using BLOTCHIP. K6F in gingival tissues was immunostained, and cytokeratin 6 protein was analyzed by immunofluorescence staining and flow cytometry. Activation of MAPK in gingival epithelial cells was evaluated by immunoblotting. ELISA was used to measure K6F and the cytokines release induced by K6F. Human gingival fibroblast migration was assessed using a Matrigel invasion chamber assay.ResultsWe identified K6F, corresponding to the C-terminus region of human cytokeratin 6 (amino acids 359–378), in the gingival crevicular fluid of periodontal disease patients and in the supernatant from gingival epithelial cells cultured with Kgp. K6F antigen was distributed from the basal to the spinous epithelial layers in gingivae from periodontal disease patients. Cytokeratin 6 on gingival epithelial cells was degraded by Kgp, but not by Arg-gingipain, P. gingivalis lipopolysaccharide or Actinobacillus actinomycetemcomitans lipopolysaccharide. K6F, but not a scrambled K6F peptide, induced human gingival fibroblast migration and secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1. These effects of K6F were mediated by activation of p38 MAPK and Jun N-terminal kinase, but not p42/44 MAPK or p-Akt.ConclusionKgp degrades gingival epithelial cell cytokeratin 6 to K6F that, on release, induces invasion and cytokine secretion by human gingival fibroblasts. Thus, Kgp may contribute to the development of periodontal disease.