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Biomedical subjects

K Asai

Publications and source records attributed to K Asai.

At least 55 records · Page 3Linked to original sources
PubMed2001

Acid stimulates E-cadherin surface expression on gastric epithelial cells to stabilize barrier functions via influx of calcium.

BACKGROUND AND AIMS: E-cadherin, which is a [Ca2+]-dependent, homotypic cell-cell adhesion molecule, is expressed in gastrointestinal epithelial cells. Much has been learned about the down-regulation of E-cadherin expression in gastrointestinal tumours, Barrett's oesophageal dysplasia, and Crohn's disease, but the functions of this molecule in normal gastrointestinal mucosa are less known. METHODS: In this study, we investigated the relationship between E-cadherin expression and permeability using rat cultured gastric and intestinal epithelial cells following a 30-min exposure to various pH solutions. We also investigated the participation of [Ca2+] in these events. RESULTS: E-cadherin expression increased under acid (pH 4) but not alkali (pH 10 or 11) exposure only for gastric epithelial cells. Gastric epithelial permeability was maintained only against acid exposure while intestinal permeability increased under both conditions. Transient influx of [Ca2+] was only observed for gastric epithelial cells just after acid exposure. CONCLUSIONS: These findings suggest that E-cadherin expression on gastric epithelium stabilizes the epithelial barrier against acid, probably through influx of [Ca2+]. This event is thought to be one of the protective mechanisms in gastric mucosa against acid back-diffusion, which is one of the causes of peptic ulcer formation.

Acids↗
PubMed2001

Calcium oxalate crystal attachment to cultured rat kidney epithelial cell, NRK-52E.

Madin-Darby canine kidney (MDCK) cells have been used in research on crystal adhesion to epithelial cells. Recently, matrix proteins were identified, and studies of the genes and proteins expressed in renal epithelial cells have become active. The present study confirms the usefulness of the NRK-52E cell line, derived from the rat, in the study of attachment with calcium oxalate crystals. The calcium oxalate crystal suspension was distributed on top of the cells. After incubation, the monolayers were rinsed to remove non-associated crystals. After fixation, the association of crystals and NRK-52E cells was visualized using scanning electron microscopy. Calcium oxalate crystals were attached to the surface of NRK-52E cells. Under high magnification, many of the microvilli of the cells had elongated towards the crystals, and microvilli projections appeared to catch the crystals. The NRK-52E cell line is useful in the study of attachment between crystals and urinary epithelial cells in the kidney, especially for the regulation and analysis of genes and proteins.

Animals↗
PubMed2001

[Review of the research of glia maturation factor and cloning of human and rat glia maturation factor-gamma (GMFG) cDNA].

Glia maturation factor-beta (GMFB) is a 17-kDa protein that was initially identified as a growth and differentiation factor acting on neurons as well as glia in the vertebrate brain. We isolated human and rat glia maturation factor-gamma (GMFG) cDNA and examined the tissue distribution of GMFG in human and rat by Northern blots and Western blots. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein of 142 amino acid residues. The deduced amino acid sequence of its putative product is highly homologous to GMFB. Northern blot analysis indicated that a 0.9 kb mRNA is predominantly expressed in rat thymus, testis, and spleen. In comparison with GMFB, the current study demonstrated that the tissue distribution of GMFG is not the same as that of GMFB, and GMFG is predominantly in proliferative and differentiative organs.

Animals↗
PubMed2001

Biosynthetic response of cultured articular chondrocytes to mechanical vibration.

The objective of this study was to determine the effects of mechanical vibration loading on DNA and proteoglycan syntheses in cultured rabbit articular chondrocytes. Chondrocyte culture plates were placed in a vibratory apparatus and subjected to a mechanical vibratory load at various frequencies and periods in culture. Mechanical vibration was applied at a sinusoidal waveform of 1.4 g acceleration with frequencies of 200, 300, 400, 800, and 1600 Hz. 3H-Thymidine and H2(35)SO4 incorporation were used to detect radiolabeled DNA and proteoglycan syntheses, respectively. A frequency of 300 Hz showed a time-dependent augmentation of DNA synthesis and gave a maximal increase at day 3 with periodic vibration (8 h per day) and at 72 h or longer with continuous vibration. It also promoted proteoglycan synthesis in long-term culture (from 3 to 15 days) by periodic vibration. However. frequencies above 400 Hz suppressed biosynthesis. These results suggest that mechanical vibration at certain frequencies may modulate biosynthetic response of articular chondrocytes.

Animals↗
PubMed2001

Dopamine D2 receptor inhibition of adenylyl cyclase is abolished by acute ethanol but restored after chronic ethanol exposure (tolerance).

Dopamine D2 (D2) receptors seem to mediate reinforcing responses to addicting drugs. A stably transfected NG108-15 cell line expressing the long form of the rat brain D2 receptor (D2L) was used to determine how ethanol modifies D2 receptor coupling to adenylyl cyclase. Activation of D2L receptors by the D2 receptor-specific agonist R-(-)-2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) inhibits both basal and receptor-stimulated cAMP production in these cells. Ethanol added acutely prevents D2L receptor inhibition of cAMP production. After chronic exposure to ethanol, however, D2L receptor coupling to adenylyl cyclase becomes tolerant to rechallenge with ethanol, i.e., ethanol no longer inhibits D2L receptor coupling and NPA inhibition of cAMP production is restored. Acute ethanol does not change NPA binding to D2 receptor in cell membranes but abolishes guanosine-5'-O-(3-thio)triphosphate induction of a lower-affinity state; chronic ethanol is without effect. The protein kinase A (PKA) inhibitor adenosine 3',5' cyclic monophosphorothioate, Rp-isomer, prevents acute ethanol inhibition of D2L receptor coupling. In contrast, the PKA activator adenosine 3',5' cyclic monophosphorothioate, Sp-isomer, reverses chronic ethanol-induced tolerance of D2L receptor coupling, restoring coupling to an ethanol-sensitive state. These results suggest that D2L receptor coupling to adenylyl cyclase via G(i) develops tolerance to ethanol inhibition, which appears to be influenced by PKA activity.

Adenylyl Cyclase Inhibitors↗
PubMed2000

Suppression of temperature-sensitive sporulation mutation in the Bacillus subtilis sigA gene by rpoB mutation.

We isolated a temperature-sensitive sporulation defective mutant of the sigA gene, encoding a major sigma factor, sigma(A) protein, in Bacillus subtilis, and designated it as sigA21. The sigA21 mutation caused a single-amino acid substitution, E314K, in region 4 of the sigma(A) protein. In this mutant, expression of the spoIIG gene, whose transcription depends on both sigma(A) and the phosphorylated Spo0A protein, Spo0A approximately P, a major transcription factor during early stages of sporulation, was greatly reduced at 43 degrees C. To obtain further information on the mechanism of sigma(A) function during the early spore development, we isolated a spontaneous sporulation-proficient suppressor mutant at 43 degrees C. This extragenic suppressor mutation was mapped within the rpoB gene, encoding the beta subunit of RNA polymerase, and was found to have a single-amino acid substitution, A863G. In this mutant, the expression of the spoIIG is partially restored at 43 degrees C.

Amino Acid Substitution↗
PubMed2000

The yabG gene of Bacillus subtilis encodes a sporulation specific protease which is involved in the processing of several spore coat proteins.

The synthesis and proteolysis of the spore coat proteins, SpoIVA and YrbA, of Bacillus subtilis were analyzed using antisera. Almost no intact full-length proteins of either type were extracted from wild-type spores, while yabG mutant spores contained intact SpoIVA and YrbA proteins. We purified recombinant YrbA and YabG proteins from Escherichia coli transformants and found that YrbA was cleaved to the smaller moiety in the presence of YabG in vitro. These observations indicate that YabG is a protease involved in the proteolysis and maturation of SpoIVA and YrbA proteins, conserved with the cortex and/or coat assembly by B. subtilis.

Bacillus subtilis↗
PubMed2000

Akt-dependent cytokine production in mast cells.

Cross-linking of FcepsilonRI induces the activation of three protein tyrosine kinases, Lyn, Syk, and Bruton's tyrosine kinase (Btk), leading to the secretion of a panel of proinflammatory mediators from mast cells. This study showed phosphorylation at Ser-473 and enzymatic activation of Akt/protein kinase B, the crucial survival kinase, upon FcepsilonRI stimulation in mouse mast cells. Phosphorylation of Akt is regulated positively by Btk and Syk and negatively by Lyn. Akt in turn can regulate positively the transcriptional activity of interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha promoters. Transcription from the nuclear factor kappaB (NF-kappaB), nuclear factor of activated T cells (NF-AT), and activator protein 1 (AP-1) sites within these promoters is under the control of Akt activity. Accordingly, the signaling pathway involving IkappaB-alpha, a cytoplasmic protein that binds NF-kappaB and inhibits its nuclear translocation, appears to be regulated by Akt in mast cells. Catalytic activity of glycogen synthase kinase (GSK)-3beta, a serine/threonine kinase that phosphorylates NF-AT and promotes its nuclear export, seems to be inhibited by Akt. Importantly, Akt regulates the production and secretion of IL-2 and TNF-alpha in FcepsilonRI-stimulated mast cells. Altogether, these results revealed a novel function of Akt in transcriptional activation of cytokine genes via NF-kappaB, NF-AT, and AP-1 that contributes to the production of cytokines.

Animals↗
PubMed2000

Redundant and opposing functions of two tyrosine kinases, Btk and Lyn, in mast cell activation.

Protein-tyrosine kinases play crucial roles in mast cell activation through the high-affinity IgE receptor (FcepsilonRI). In this study, we have made the following observations on growth properties and FcepsilonRI-mediated signal transduction of primary cultured mast cells from Btk-, Lyn-, and Btk/Lyn-deficient mice. First, Lyn deficiency partially reversed the survival effect of Btk deficiency. Second, FcepsilonRI-induced degranulation and leukotriene release were almost abrogated in Btk/Lyn doubly deficient mast cells while singly deficient cells exhibited normal responses. Tyrosine phosphorylation of cellular proteins including phospholipases C-gamma1 and C-gamma2 was reduced in Btk/Lyn-deficient mast cells. Accordingly, FcepsilonRI-induced elevation of intracellular Ca2+ concentrations and activation of protein kinase Cs were blunted in the doubly deficient cells. Third, in contrast, Btk and Lyn demonstrated opposing roles in cytokine secretion and mitogen-activated protein kinase activation. Lyn-deficient cells exhibited enhanced secretion of TNF-alpha and IL-2 apparently through the prolonged activation of extracellular signal-related kinases and c-Jun N-terminal kinase. Potentially accounting for this phenomenon and robust degranulation in Lyn-deficient cells, the activities of protein kinase Calpha and protein kinase CbetaII, low at basal levels, were enhanced in these cells. Fourth, cytokine secretion was severely reduced and c-Jun N-terminal kinase activation was completely abrogated in Btk/Lyn-deficient mast cells. The data together demonstrate that Btk and Lyn are involved in mast cell signaling pathways in distinctly different ways, emphasizing that multiple signal outcomes must be evaluated to fully understand the functional interactions of individual signaling components.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2000

Determinants of the cardiomyopathic phenotype in chimeric mice overexpressing cardiac Gsalpha.

Mice with overexpressed cardiac Gsalpha develop cardiomyopathy, characterized by myocyte hypertrophy and extensive myocardial fibrosis. The cardiomyopathy likely involves chronically enhanced beta-adrenergic signaling, because it can be blocked with long-term propranolol treatment. It remains unknown whether the genotype of the myocyte is solely responsible for the progressive pathological changes. A chimeric population in the heart should answer this question. Accordingly, we developed a chimeric animal, which combined cells from a transgenic overexpressed Gsalpha parent and a Rosa mouse containing the LacZ reporter gene, facilitating identification of the non-Gsalpha cells, which express a blue color with exposure to beta-galactosidase. We studied these animals at 14 to 17 months of age (when cardiomyopathy should have been present), with the proportion of Gsalpha cells in the myocardium ranging from 5% to 88%. beta-Galactosidase staining of the hearts demonstrated Gsalpha and Rosa cells, exhibiting a mosaic pattern. The fibrosis and hypertrophy, characteristic of the cardiomyopathy, were not distributed randomly. There was a direct correlation (r=0.85) between the extent of myocyte hypertrophy (determined by computer imaging) and the quantity of Gsalpha cells. The fibrosis, determined by picric acid Sirius red, was also more prominent in areas with the greatest Gsalpha cell density, with a correlation of r=0.88. Thus, the overexpressed Gsalpha can exert its action over the life of the animal, resulting in a local picture of cardiomyopathic damage in discrete regions of the heart, where clusters of the overexpressed Gsalpha cells reside, sparing the clusters of normal cells derived from the normal Rosa parent.

Animals↗
PubMed2000

Predominant subpopulations of T lymphocytes in the mammary gland secretions during lactation and intraepithelial T lymphocytes in the intestine of dairy cows.

Lymphocytes obtained from mammary gland secretions (MGS) during lactation or the dry period of dairy cows were simultaneously analyzed and compared to ileal intraepithelial lymphocytes (IEL) and peripheral blood lymphocytes (PBL) using monoclonal antibodies (mAb) specific for bovine leukocyte differentiation antigens. The T-lymphocytes of MGS during lactation and those in IEL were predominantly CD8(+), while T-cells in MGS during the dry period were predominantly CD4(+). In addition, the proportion of gamma delta T-cells in MGS during lactation and IEL was fairly high. A large percentage of CD8(+) cells and T-cells coexpressed the activation molecule, ACT2, yielding a high proportion of ACT2(+) CD8 T-cells and ACT2(+) gamma delta T-cells, in MGS during lactation and IEL. However, both types of cells were found at an extremely low level in MGS during the dry period and in PBL. Thus, the predominant T-cell populations in MGS during lactation are phenotypically similar to those in IEL in the intestine.

Animals↗
PubMed2000

Genetic analysis of SecA-SecY interaction required for spore development in Bacillus subtilis.

All spontaneous suppressor mutations obtained from a secA12 sporulation-defective mutant in Bacillus subtilis were localized in highly conserved membrane-spanning regions of SecY. The expression of early sporulation genes, kinA and spo0A encoding a histidine kinase and a transcription regulator for several sporulation genes, respectively, was restored in these suppressor mutants. These results indicate that the secretion function of translocase combined with Sec proteins is required for sporulation in B. subtilis.

ATP-Binding Cassette Transporters↗
PubMed2000

AT motif binding factor 1-A (ATBF1-A) negatively regulates transcription of the aminopeptidase N gene in the crypt-villus axis of small intestine.

This is the first study to demonstrate that the AT motif binding factor 1-A (ATBF1-A) is expressed in the crypts and the bases of villi of the small intestine and negatively regulates transcription of brush-border enzyme gene, aminopeptidase-N (APN). In situ hybridization visualized a limited ATBF1-A mRNA expression in the crypts and the bases of villi. Transient transfection and dual luciferase-reporter assay demonstrated that ATBF1-A suppressed the activity of APN promoter, but did not that of AT motif deleted promoter. These results imply that ATBF1-A inhibits the transcription of APN gene through its direct binding to the AT motif element. Furthermore, butyrate-induced differentiation of Caco-2 cells, retaining the enterocytic phenotypes such as a villus structure and the expression of brush-border enzymes, leads to a reduced expression of ATBF1-A mRNA. We proposed that ATBF1-A regulating APN gene expression in the crypt-villus axis of the small intestine is a landmark of enterocyte differentiation and maturation.

Animals↗
PubMed2000

Synovial inflammation and hyperplasia induced by gliostatin/platelet-derived endothelial cell growth factor in rabbit knees.

Neovascularization, proliferation of synovial cells, and mononuclear cell influx and activation are characteristic events observed in synovial joints in the pathohistology of rheumatoid arthritis (RA). The objective of this study was to examine synovial inflammation in rabbit knees induced by intra-articular administration of human gliostatin/platelet-derived endothelial cell growth factor (GLS/PD-ECGF), which shares a high degree of chemical homology with thymidine phosphorylase (dThdPase) and is known to have angiogenic activity. Purified recombinant human gliostatin (rHuGLS) and its mutant protein, which was prepared by site-directed mutagenesis and which lacks dThdPase activity, were administered at various doses to rabbit knee joints. The effects of rHuGLS and the mutant were examined histologically. Intra-articular injection of rHuGLS resulted in the development of diffuse synovitis resembling RA. The mutant protein also brought about the same effect. These findings suggest that human GLS can cause RA-like synovitis in rabbit knee joints via a mechanism other than its dThdPase activity.

Animals↗