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Biomedical subjects

K Hirai

Publications and source records attributed to K Hirai.

At least 19 recordsLinked to original sources
PubMed1992

Cloning and nucleotide sequence of the Pseudomonas aeruginosa nfxB gene, conferring resistance to new quinolones.

The gene nfxB is one of the genes which affect the cell membrane permeability of quinolones in Pseudomonas aeruginosa PAO. Both wild-type nfxB and a mutant nfxB (nfx13E) were cloned and the DNA sequences were determined. The wild-type gene was dominant in PAO strains. The nfxB mutation was a point mutation (cytosine----guanine) which generates an amino acid exchange (arginine----glycine) in the putative nfxB product. The amino acid sequence of the wild-type NfxB protein revealed that it has a helix-turn-helix motif which may be responsible for the ability to bind in a sequence-specific manner to DNA. This finding indicated that the NfxB protein may regulate the expression of genes that are associated with cell permeability of drugs in P. aeruginosa. The position of the amino acid substitution between the NfxB protein and the Nfx13E protein was located within a possible DNA-binding domain, suggesting that the mutant protein (Nfx13E) may have lost DNA binding ability and regulator activity.

Amino Acid Sequence↗
PubMed1992

Behavior of axons, Schwann cells and perineurial cells in nerve regeneration within transplanted nerve grafts: effects of anti-laminin and anti-fibronectin antisera.

Wistar rats (close cloned strain) were used to investigate the effect of endogenous laminin and fibronectin on axons, Schwann cells and perineurial cells in the regenerating peripheral nervous system (PNS). Sciatic nerve grafts obtained from donor rats were frozen, thawed and treated with rabbit anti-rat laminin or anti-fibronectin antiserum. Control grafts were treated with normal rabbit serum alone. One cm long portions of the sciatic nerve of the recipient rats were replaced with grafts. At 15 days after transplantation the number of regenerated axons in the laminin- and fibronectin-depleted grafts was half of that in the control. The growing axons in the laminin-depleted grafts did not recognize the basal lamina scaffolds (BLS) remaining in the basal lamina tubes, while in the control and fibronectin-depleted grafts 90% or more of axons grew inside the BLS. Elongation of axons always preceded migration of Schwann cells with the latter subsequently adhering to and wrapping around the former. Perineurium-forming fibroblastic cells recognized the combination of axons and Schwann cells and formed perineurial fasciculi around them. These fibroblastic cells did not recognize empty BLS but responded to them only when fibronectin was depleted. Macrophages sometimes closely faced the naked axons which elongated outside the BLS. These results suggest that in the early stages of nerve regeneration endogenous laminin and fibronectin not only regulate the growth of regenerating nerve fibers, but also exert a positive influence on perineurial cells and macrophages, both of which play important roles in nerve tissue injury and repair.

Animals↗
PubMed1992

Identification of thyroiditogenic epitope on porcine thyroid peroxidase for C57BL/6 mice.

C57BL/6 mice show thyroid lesions when immunized with porcine thyroid peroxidase (pTPO) emulsified in CFA. We attempted to clarify a thyroiditogenic epitope on pTPO. Thyroid peroxidase treated with cyanogen bromide was fractionated by reverse phase chromatography, and six fractions (A to F) were obtained. Two of these fractions (D and E) stimulated lymph node cells (LNC) primed with pTPO in vitro and induced thyroiditis in vivo. Tricine-SDS-PAGE and rechromatography showed that fraction D consisted solely of a fragment of Mr 9500 Da and that fraction E contained mainly fragments of Mr of 5400 and 9500 Da. The fragment of fraction D was rechromatographed and 20 NH2-terminal amino acids were analyzed. This segment was found to correspond to residue 726-745 of pTPO deduced from cDNA at a probability of 80%. Four peptides ranging from residue 746-827 were first synthesized and tested for their thyroiditogenicity. Only Pep-2 (29 amino acids) could stimulate LNC primed with pTPO and induce thyroiditis. Pep-2 was divided into two smaller peptides (Pep-2-1 and -2-2) and their thyroiditogenicity was tested again. Pep-2-1 corresponding to residue 774-788, GPA-QITCTPRGWDSP, had thyroiditogenicity as well as the ability to stimulate LNC. It was thought that this segment was at least one of the thyroiditogenic epitopes on porcine thyroid peroxidase for C57BL/6 mice.

Amino Acid Sequence↗
PubMed1992

Stem cell factor has histamine releasing activity in rat connective tissue-type mast cells.

Stem cell factor (SCF) was documented to be involved in the growth of mast cells controlled by fibroblasts. We tested the effect of recombinant rat SCF on degranulation from rat peritoneal mast cells (connective tissue-type mast cells: CTMC). SCF induced histamine release (approximately 20% of total histamine content) in a dose-dependent fashion. The release response was relatively rapid and reached a maximum within 5 min. The release showed total dependence on the presence of extracellular phosphatidylserine (PTS). These results reveal that SCF has histamine releasing activity in CTMC.

Animals↗
PubMed1992

The cell adhesion molecule CD31 is phosphorylated after cell activation. Down-regulation of CD31 in activated T lymphocytes.

We report the independent cloning of the cDNA for CD31, a recently described cell adhesion molecule of the immunoglobulin gene superfamily present on platelets, granulocytes, monocytes, lymphocytes, and endothelial cells. Northern analysis revealed three major mRNA transcripts in Jurkat (a human T cell line) and K562 and HEL (leukemia cell lines) cells with an additional 5.3-kilobase transcript seen in cultured human umbilical vein endothelial cells. Following T cell activation, CD31 mRNA was down-regulated by Northern analysis, and decreased CD31 protein expression was confirmed by immunoblots. The down-regulation of CD31 was partially mediated by decreased transcription as demonstrated by nuclear run-on studies. CD31 became rapidly phosphorylated in platelets, Jurkat cells, and endothelial cells after cell activation. We were unable to demonstrate the presence of a phosphotyrosine in CD31 using monoclonal and polyclonal phosphotyrosine antibodies. In addition, CD31 phosphorylation in platelets was induced by phorbol ester and was blocked by staurosporin, a protein kinase C inhibitor, suggesting that CD31 phosphorylation is mediated by protein kinase C and involves serine and/or threonine residues. The phosphorylation of CD31 following cell activation may modulate its cellular adhesiveness, and the down-regulation of its expression may serve to impart target specificity and to localize effector lymphocytes to areas of inflammation.

Antibodies, Monoclonal↗
PubMed1992

The role of laminin, a component of Schwann cell basal lamina, in rat sciatic nerve regeneration within antiserum-treated nerve grafts.

Regeneration of the sciatic nerve in transplanted nerve grafts in which laminin was inactivated was examined electron microscopically. Nerve grafts for transplantation were obtained from close cloned donor Wistar rats; 1-cm nerve segments of the sciatic nerve were frozen and thawed to kill the Schwann cells. Control recipient rats received grafts treated with normal rabbit serum to repair the artificially-made complete defect of the right sciatic nerve, and the experimental group of rats received grafts doubly treated with normal serum and rabbit anti-laminin antiserum. In the control grafts regenerating axons grew almost completely through the inside of the basal lamina scaffolds (92%) and adhered to the structure, while in the anti-laminin antiserum treated grafts the axons were present outside (52%) and inside (48%) the scaffolds simultaneously. In this case, the adhesion of axons to the scaffolds was obscure. Axons were associated with and without Schwann cells both inside and outside the basal lamina scaffolds. No unassociated Schwann cells were observed. The maximal number of axons in a 2 mm portion of the antiserum-treated grafts was approximately 250 axons per 100 x 100 microns square and 520 in the control at 15 days. At 30 days, almost the same number of axons was found at the distal (8 mm) portion of both groups. The growth in the former was delayed for 3 days. These results indicate that regenerating peripheral nerve axons may enter the basal lamina scaffolds and grow well because of the neurotrophic function of laminin present at the inner side of Schwann cell basal lamina.

Animals↗
PubMed1992

Application of immunocryoultramicrotomy to free cells: ultrastructural localization of myeloperoxidase in neutrophils of human peripheral blood.

Immunocryoultramicrotomy is generally considered the most suitable method for observing the immunostainings by monoclonal antibodies to intracellular antigens by electron microscopy. However, this procedure with the free cells such as peripheral blood cells or cultured cells is not widely used because of the difficulty in preparing ultrathin frozen sections. We report here a new technique for making ultrathin frozen sections of peripheral blood cells by positioning the cell in a semigelatinized medium for ultrasectioning. Monoclonal antibody against myeloperoxidase (MPO) was reacted with the ultrathin sections made with this new technique to observe the ultrastructural localization of intracellular MPO. MPO was observed in the primary granules of the neutrophils.

Antibodies, Monoclonal↗
PubMed1992

Diagnostic usefulness of dipeptidyl aminopeptidase IV monoclonal antibody in paraffin-embedded thyroid follicular tumours.

Monoclonal antibodies to dipeptidyl aminopeptidase IV (DAP IV, EC 3.4.14.5) were raised and selectively applied to paraffin-embedded sections of thyroid carcinoma. Five monoclonal antibodies were found to stain paraffin sections of thyroid carcinomas. Using one of these antibodies (44-4), we studied retrospectively aberrant expression of DAP IV in thyroid carcinoma to determine whether immunohistochemical staining with DAP IV antibody is useful in pathological diagnosis. In almost all cases of thyroid follicular and papillary carcinoma, tumour cells were positive (99.0 per cent) with DAP IV, whereas the cases of follicular adenoma showed a low incidence (27.1 per cent) of positive staining. Follicular adenoma with incomplete capsular invasion had a higher positive incidence (50 per cent) than follicular adenoma without incomplete capsular invasion (9.6 per cent). In positive staining cases previously diagnosed as benign tumours, 11 benign cases reacting positively with DAP IV were rediagnosed as carcinoma after re-examination of more thyroid paraffin block sections or serial sections. These findings suggest that DAP IV monoclonal antibody is very useful in distinguishing thyroid follicular carcinoma from follicular adenoma.

Adenoma↗
PubMed1992

Sequence determination and genetic content of an 8.9-kb restriction fragment in the short unique region and the internal inverted repeat of Marek's disease virus type 1 DNA.

The DNA sequence (8.9 kb) covering about 70% of the short unique region (Us) and part of the short inverted repeat of the Marek's disease virus type 1 GA strain was determined. Computer analysis of the sequence showed the presence of nine potential open reading frames (ORFs), consisting of more than 300 nucleotides in the Us region. Of these ORFs, four were found to be homologous to US10 (minor virion protein), US3 (protein kinase), US2, and US6 (gD) in the Us region of alpha-herpesvirus herpes simplex virus type 1. The protein kinase homologue is especially well conserved in alpha-herpesviruses. No counterpart of the nine MDV1 ORFs was found in the beta-herpes virus human cytomegalovirus and gamma-herpesvirus Epstein-Barr virus, suggesting that MDV1 is more similar to the alpha-herpesviruses. The junction of the Us region and the short inverted repeat was also determined by comparison between the sequences of the DNA fragments, including the terminal and internal repeats. Northern blot analysis showed that the Us region within the 8.9 kb sequence was transcriptionally active in MDV1-infected cells.

Amino Acid Sequence↗
PubMed1992

The restriction endonuclease map of Marek's disease virus (MDV) serotype 2 and collinear relationship among three serotypes of MDV.

A BamHI, EcoRI, and XhoI restriction endonuclease map of Marek's disease virus (MDV) serotype 2 (MDV2) DNA was constructed by double-digest analyses of 28 cloned BamHI and 11 cloned EcoRI fragments of MDV2 DNA, followed by hybridization tests of these cloned BamHI DNA fragments with electrophoretically separated digests of MDV2-infected cell DNA. On this map, MDV2 genome consisted of two segments which have unique regions inserted between two inverted repeat regions as observed in MDV serotype 1 and 3 genomes. Further, the DNA homology among three serotypes of MDV was examined by hybridization under less stringent conditions using cloned BamHI fragments of MDV2 DNA. Most of the MDV2 fragments located within the unique regions hybridized with MDV serotype 1 and 3 DNAs, indicating the presence of the collinear relationship among three serotypes. In addition, MDV2 DNA fragments which hybridized with the DNA fragments encoding MDV1 gp57-65 (or A antigen) or MDV1 gp100, gp60, gp49 (or B antigen) were identified and these fragments of serotypes 1 and 2 found to be collinear.

Animals↗
PubMed1992

Sequence determination of cDNA clones of transcripts from the tumor-associated region of the Marek's disease virus genome.

The number of 132-bp tandem direct repeats within the long inverted repeat region of the Marek's disease virus type 1 (MDV1) genome increases concomitantly with the loss of oncogenicity during serial passages in cultured cells. Twelve clones carrying the 132-bp sequence were isolated from a cDNA library constructed from chicken embryo fibroblasts infected with the MDV1 Md5 strain. Through sequence analysis of a cDNA clone and primer extension analysis, the corresponding mRNA was found to be a linear transcript which included the two 132-bp tandem direct repeats. Two open reading frames were found in this transcript. One had a week homology with v-fms. The other should increase its size concomitantly with expansion of the 132-bp tandem direct repeat. PCR analysis of both cDNA clones and RNA gave amplified products which were as large as that produced from the genomic clone, indicating that a majority of mRNA from this region is composed of unspliced transcripts.

Amino Acid Sequence↗
PubMed1992

Increased sensitivity for detection of human cytomegalovirus in urine by removal of inhibitors for the polymerase chain reaction.

The presence of inhibitors in urine interferes with the enzymatic reaction of the polymerase chain reaction (PCR) for detection of human cytomegalovirus (HCMV). To remove inhibitors, HCMV virions in urine were precipitated with polyethylene glycol, or DNA was extracted from urine by the use of glass powder and subjected to PCR followed by Southern blot hybridization with alkaline phosphatase-linked oligonucleotide probes. These simple, rapid methods increased significantly the sensitivity of PCR for detection of HCMV in urine.

Base Sequence↗
PubMed1992

Paraquat damage of rat liver mitochondria by superoxide production depends on extramitochondrial NADH.

Pure rat liver heavy mitochondrial fractions, in which the absence of significant microsomal contamination was confirmed by electron microscopy and by the lack of glucose-6-phosphatase activity, were used to demonstrate the effect of paraquat on mitochondrial ultrastructure in the presence of external NADH. Starved mitochondria (orthodox conformation) did not show O2 uptake or structural injury from either paraquat alone or NADH alone. Marked O2 uptake and structural breakage occurred only when paraquat and NADH were added in combination. These alterations were resistant to rotenone and malate plus glutamate or NADPH could not substitute for NADH. Paraquat was reduced anaerobically by the mitochondria in the presence of NADH, but not of NADPH. The addition of superoxide dismutase, ferricytochrome c or p-benzoquinone protected against the breakage of mitochondria caused by paraquat plus NADH. These results demonstrate that mitochondria may produce paraquat radicals in the presence of extramitochondrial NADH and thus generate superoxide anion radicals, resulting in structural injury to the mitochondria, by mechanisms that may involve the mitochondrial outer membrane rather than the electron transfer chain. These mitochondrial mechanisms in paraquat toxicity seemed to be more probable in vivo than are microsomal mechanisms; the latter are postulated to function in detoxication because phenobarbital diminished paraquat toxicity and SKF 525-A or cobaltous ions enhanced the toxicity.

Animals↗
PubMed1992

Functional correlation of fetal and adult forms of glycine receptors with developmental changes in inhibitory synaptic receptor channels.

Functional maturation of the nicotinic acetylcholine receptor is executed by its gamma-to-epsilon subunit switching. The glycine receptor also has fetal (alpha 2) and adult (alpha 1) isoforms. However, whether subunit switching is responsible for developmental changes in glycine receptor function is not known. We recorded single-channel currents from homomeric glycine receptors expressed in Xenopus oocytes with cRNAs encoding the alpha 2 or alpha 1 subunits and compared them with those recorded from native glycine receptors in rat spinal neurons at various ontogenic periods. The mean channel life times of the alpha 1 and mature glycine receptors were equally short, whereas both the alpha 2 and fetal receptors showed a significantly longer open time. Consistent with these results, the decay time of the glycinergic inhibitory postsynaptic currents (IPSCs) in spinal neurons became shorter during postnatal development. We conclude that developmental switching of alpha subunits may accelerate the kinetics of IPSCs.

Aging↗