PubMed Health⌕ Search

Biomedical subjects

Y Ohba

Publications and source records attributed to Y Ohba.

At least 19 recordsLinked to original sources
PubMed1996

A novel silent posttranslational mechanism converts methionine to aspartate in hemoglobin Bristol (beta 67[E11] Val-Met->Asp).

The first reported case of congenital Heinz body hemolytic anemia was subsequently shown to be caused by an unstable hemoglobin, Hb Bristol [beta 67(E11) Val-Asp]. This has become one of the classic models of an unstable hemoglobin, the hydrophilic aspartate disrupting the hydrophobic heme pocket. We have restudied this original case, who remains clinically well after nearly 50 years of severe hemolysis with a hemoglobin level of about 7 g/dL and two unrelated Japanese cases. Surprisingly, all three cases show the same DNA changes, predicting a valine to methionine change at beta 67, rather than the expected aspartate. Further analysis with electrospray ionization mass spectrometry and globin chain biosynthesis strongly suggests that this anomaly is because of a novel posttranslational mechanism, with slow conversion of the translated methionine into an aspartate residue. The proximity of the heme and oxygen may be important in facilitating the reaction. These findings show the importance of complete characterization of variant hemoglobins using protein, DNA, and biosynthetic analyses.

Adolescent↗
PubMed1996

Inhibitory mechanisms of H(+)-ATPase inhibitor bafilomycin A1 and carbonic anhydrase II inhibitor acetazolamide on experimental bone resorption.

The effects of the vacuolar-type H(+)-ATPase inhibitor bafilomycin A1 (baf.A1) and the carbonic anhydrase II inhibitor acetazolamide (AZ) on bone resorption and procathepsin L secretion of rat osteoclasts were investigated using the bone slice assay method, pit formation test. Baf.A1 completely suppressed osteoclastic bone resorption stimulated by parathyroid hormone (PTH), but did not affect procathepsin L secretion, while AZ suppressed both bone resorption and procathepsin L secretion. These findings suggest that bone resorption by procathepsin L secretion and its processing are regulated by proton production and proton secretion.

Acetazolamide↗
PubMed1996

Suppressive effect of N-(benzyloxycarbonyl)-L-phenylalanyl-L-tyrosinal on bone resorption in vitro and in vivo.

The suppressive effect of N-(benzyloxycarbonyl)-L-phenylalanyl-L-tyrosinal on bone resorption was examined in vitro and in vivo. This synthetic peptidyl aldehyde was found to be a potent and selective cathepsin L inhibitor in our screening for cysteine protease inhibitors. In the pit formation assay with unfractionated rat bone cells, 1.5 nM of this compound markedly inhibited parathyroid hormone-stimulated osteoclastic bone resorption. In addition, intraperitoneal administration of this peptidyl aldehyde (2.5-10 mg/kg) for 4 weeks suppressed bone weight loss dose dependently in the ovariectomized mouse, experimental model of osteoporosis. Hydroxyproline measurement of the decalcified femurs from these ovariectomized mice suggested that this compound acts as a bone resorption suppressor through the inhibition of collagen degradation.

Animals↗
PubMed1996

In vitro and in vivo analysis of human leukocyte binding by the antitumor polysaccharide, lentinan.

Lentinan (LNT), a (1-->3)-beta-D-glucan with (1--6)-beta-D-glucopyranoside branches, has marked antitumor effects in syngeneic and autochtonous hosts. Clinically, LNT has proved effective with chemotherapeutic agents in patients with recurrent gastric and colorectal cancer. However, the mechanism that triggers subsequent immunologic reactions remains obscure. We hypothesized that LNT must first bind to the host cells. Accordingly, we analyzed LNT binding to host cells in healthy volunteers after incubating their cells under a variety of conditions as well as intravenously injecting LNT then subjecting them to flow cytometry and immunofluorescent staining using monoclonal antibody (anti-LNT mAb). LNT bound to monocytes and neutrophils, but not to lymphocytes in vitro. The most avid LNT binding was to monocytes. The percentage of LNT-bound monocytes after 60 min incubation at 4 degrees C was greater than that at 37 degrees C. The binding of LNT to monocytes was inhibited slightly by anti-iC3b receptor (anti-CR3) mAb, strongly by anti-C3b receptor (anti-CR1) mAb, and completely by anti-CR1 and anti-CR3 mAb together. The percentages of LNT-binding monocytes in the peripheral blood increased significantly 3 and 4 after 2 mg LNT-injection and returned to low levels 5 h later. However, no increase in LNT-binding neutrophils and lymphocytes was observed. We concluded that binding of LNT to human monocytes may initiate the influence of this compound on the immune system and differ between individuals. Its binding site may be similar to the C3b receptor.

Adult↗
PubMed1996

The existence of thyroid hormone responsive element, TRE, was confirmed in the first intron of rat alpha 1-acid glycoprotein gene.

A set of acute-phase proteins was found to have been induced in rat after triiodothyronine (T3) treatment. To study the mechanism of gene activation by T3, the rat gene of alpha 1-acid glycoprotein (AGP), one of the major acute-phase proteins, was cloned and analyzed. CAT assay identified a thyroid hormone responsive element (TRE) in the first intron of the AGP gene and showed that mutations introduced into the TRE region destroyed the TRE activity. Gel shift assay and methylation interference assay showed that the thyroid hormone receptor bound to the TRE. The TRE identified here had a palindromic structure and was highly homologous to other known TREs.

Acute-Phase Proteins↗
PubMed1996

Improved production of phenomycin by a genetically engineered Escherichia coli.

We established an improved production of an antitumor polypeptide anti biotic, phenomycin (PHM), by using a genetically engineered Escherichia coli. Phenomycin consists of 89 natural amino acids without intramolecular disulfide bridge. PHM gene was synthesized as a fusion gene in which PHM at the C-terminus and the residues 1 approximately 20 of Hirudin variant 1 (HV1) at the N-terminus connected by a factor Xa recognition sequence (Ile-Glu-Gly-Arg). E coli JM 109 transformed with a plasmid containing the synthesized gene expressed a fusion protein and the trypsinization of the fusion protein purified by ultrafiltration and ion-exchange chromatography gave efficiently recombinant PHM at a final yield of 50 mg/liter of culture. This PHM yield was six times higher than that obtained by a natural PHM producing strain of Streptoverticillium baldacci. Recombinant PHM was not distinguishable from natural PHM in all aspects observed.

Anti-Bacterial Agents↗
PubMed1996

Major histocompatibility complex expression in muscle of rats with graft-versus-host disease.

Immunohistochemical examination of rat skeletal muscle during graft-versus-host disease (GVHD), a systemic immune reaction, was performed to investigate specific immune reactivities focusing on major histocompatibility complex (MHC) expression and inflammatory cell infiltration of skeletal muscle during a systemic immune reaction. MHC class II expression and inflammatory cell infiltration did not increase. MHC class I was expressed along the contour of muscle fibres, and most strongly expressed by the cells which were distributed throughout the endomysium and perimysium. Seventy-six percent of these MHC class I+ cells carried endothelial cell-markers, while 24% of them did not. The latter cells were revealed not to be inflammatory cells such as lymphocytes, granulocytes or macrophages when examined by immunostaining using several exudate-cell markers. Neither were they myosatellite cells because they were located outside the basement membrane. These results may be useful for considering animal models of inflammatory myopathies such as polymyositis and dermatomyositis.

Animals↗
PubMed1996

[A case of leiomyomatosis in pelvic lymph nodes].

We report a rare case of leiomyomatosis in iliac lymph nodes, which was found in a woman operated with a diagnosis as keratinizing epidermoid carcinoma of the cervix. A 39-year-old Japanese female, married, who had received hormonal therapy in her past history, visited the Department of Obstetrics and Gynecology at Kinki University Hospital, with a chief complaint of bloody discharge. Colposcopy and biopsy suggested a diagnosis of keratinizing epidermoid carcinoma of the cervix. A radical hysterectomy and bilateral salpingo-oophorectomy with pelvic lymph nodes dissection was performed. Histopathological examination showed a keratinizing epidermoid carcinoma of the cervix. An intramural leiomyoma nodule (0.5cm in diameter) was detected in the fundus of the uterus. Histopathologically, this was a typical benign leiomyoma. The lymph nodes were free of neoplasms. But bilateral iliac lymph nodes were enlarged up to soybean size. Microscopically, the iliac lymph nodes contained a large amount of well differentiated smooth muscle tissue (11/30). Immunohistochemical investigation showed a positive reaction for smooth muscle actin and desmins in the spindle cells proliferated in the lymph nodes; no cytokeratin positivity was detected. Leiomyomatosis of lymph node may rise through metaplasia of intranodal decidua or endometriosis by myofibroblasts or smooth muscle cells, reflecting the multipotentiality of the pelvic subcoelomic mesenchyme that can be found in the peripheral sinus of lymph nodes.

Adenocarcinoma, Papillary↗
PubMed1996

[Homozygous beta(+)-thalassemia due to -31 A to G mutation].

A 20-year-old Japanese woman was admitted to our hospital with anemia and mild splenomegaly. Peripheral blood examination revealed Hb 9.4 g/dl, Ht 29.3%, RBC 4.74 x 10(6)/microliters, reticulocytes 2.4%, WBC 5,200/microliters, platelets 24.9 x 10(4)/microliters, MCV 61.7 fl, and MCH 19.9 pg. Poikilocytosis with target cells was recognized on the peripheral blood smear. A bone marrow aspirate revealed erythroid hyperplasia. Serum iron and ferritin were in the normal range. beta-thalassemia was suggested by the increase in HbA2 (6.5%) and HbF (7.5%). Analysis of beta globin DNA by single strand conformation polymorphism (SSCP) and amplification refractory mutation system (ARMS) confirmed a diagnosis of homozygous beta(+)-thalassemia due to -31 A to G mutation. A familial study revealed that her parents were heterozygous for this allele. This is the 8th case of homozygous beta(+)-thalassemia due to -31 A to G mutation in Japan.

Adult↗
PubMed1995

Pyruvate kinase deficiency of mice associated with nonspherocytic hemolytic anemia and cure of the anemia by marrow transplantation without host irradiation.

Mutant mice with splenomegaly and nonspherocytic hemolytic anemia were found in an inbred colony of the CBA/N (hereafter CBA) strain maintained in the Japan SLC Haruno farm (Shuchi-gun, Shizuoka, Japan). The activity of pyruvate kinase (PK) in red blood cells (RBCs) of the anemic mutants decreased to 16.2% of normal (+/+) CBA mice. Because the mutant CBA mice showed a remarkable reticulocytosis (41.6%) and because the PK activity of reticulocytes is much higher than that of mature RBCs, the PK activity in mature RBCs of the mutant CBA mice was calculated to be 2.8% that of mature RBCs of CBA-(+/+) mice. Because RBC type PK is encoded by the Pk-1 locus of the mouse (chromosome 3), we designated the mutant locus as Pk-1slc. The anemia and PK deficiency of CBA-Pk-1slc/Pk-1slc mice were cured by bone marrow transplantation (BMT) from CBA-(+/+) mice. Prior irradiation was not necessary for the curative BMT. On the other hand, the BMT from CBA-Pk-1slc/Pk-1slc mice to nonirradiated CBA-(+/+) mice did not result in the decrease of RBCs and the reduction of PK activity. The present results indicate that CBA-Pk-1slc/Pk-1slc mice are a potentially useful animal model for studying pathophysiology of PK deficiency and for developing new therapeutic methods to correct PK deficiency.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1995

Purification of an angiogenesis inhibitor from culture medium conditioned by a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8.

We previously reported that a novel human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, produced an anti-tumor angiogenesis factor and secreted it into the culture medium [Takigawa et al.: Anticancer Res., 10, 311-316, 1990]. In the present study, we purified the inhibitor by monitoring gelatinase inhibitory activity from the conditioned medium (CM) of HCS-2/8 cells. By a simple three-step procedure, gel filtration chromatography, ion-exchange chromtography, and reverse-phase HPLC, 200 micrograms of the inhibitor was obtained from 6 liters of CM with 239-fold enrichment. Purified inhibitor, named hCHIAMP (human chondrocyte-derived inhibitor of angiogenesis and metalloproteinase activity), showed a single protein band with a molecular mass (M(r)) of 24,000 (24K) under reducing conditions and M(r) 22K under nonreducing conditions on SDS-PAGE. On reverse-zymography, purified hCHIAMP showed a single band of 22K M(r) under nonreducing conditions. Its NH2-terminal amino acid sequence determined up to the 11th amino acid residue was identical with that of the tissue inhibitor of metalloproteinases-2 (TIMP-2). On Western blotting, anti-human TIMP-2 antibody cross-reacted with hCHIAMP, hCHIAMP at a dose of as little as 0.45 microgram significantly inhibited angiogenesis in the yolk sac of chick embryos induced by 0.25 mumol of spermine. Because HCS-2/8 is a clonal cell line, these findings clearly showed for the first time that chondrocytes themselves produce a potent inhibitor of angiogenesis, which is also an inhibitor of gelatinase. The findings also indicate that hCHIAMP is a TIMP-2-like molecule. Because the HCS-2/8 cells are an immortal cell line and of human origin, hCHIAMP could be useful for therapy of angiogenic diseases including solid tumors.

Chondrosarcoma↗
PubMed1995

Secretion and processing mechanisms of procathepsin L in bone resorption.

Secretion of procathepsin L into the culture medium from a bone cell mixture was markedly enhanced by addition of parathyroid hormone (PTH), 1 alpha,25-(OH)2D3 or tumor necrosis factor alpha (TNF alpha). These stimulators of secretion of procathepsin L enhanced bone pit formation, which was inhibited by E-64, but not by CA-074, a specific inhibitor of cathepsin B. Procathepsin L may thus participate in the process of bone collagenolysis during bone resorption. Procathepsin L partially purified from rat long bones under cold conditions was rapidly converted to the mature form under acidic conditions at room temperature. This conversion was inhibited by E-64, suggesting that the procathepsin L secreted into lacunae is catalytically converted to the mature enzyme by cysteine proteinase(s).

Animals↗
PubMed1995

Transcription stimulation of the Fas-encoding gene by nuclear factor for interleukin-6 expression upon influenza virus infection.

We previously found that the level of Fas, a cell surface receptor for an apoptosis signal, increases at the mRNA level in influenza virus-infected HeLa cells prior to their death by apoptosis. Here we investigated the mechanism of activation of the Fas-encoding gene expression upon influenza virus infection. Nucleotide sequences for the binding of nuclear factor for interleukin-6 expression (NF-IL6), also known as CCAAT/enhancer-binding protein beta, were repeated 8 times in the 5'-end region of the human FAS gene, spanning from -1360 to +320. This region directed the expression of a downstream marker gene when introduced into HeLa cells and the activity of the FAS gene promoter was stimulated about 2-fold upon influenza virus infection. Gene expression driven by the FAS promoter was activated when human NF-IL6 was overproduced in a DNA when human NF-IL6 was overproduced in a DNA co-transfection study. Moreover, the DNA-binding activity of NF-IL6 increased after infection with the virus, whereas the amount of NF-IL6 seemed unchanged. The results suggest that NF-IL6 is activated upon influenza virus infection through post-translational modification and that the modified factor stimulates the transcription of the human FAS gene.

Antigens, Surface↗
PubMed1995

Ultraviolet resonance Raman studies of quaternary structure of hemoglobin using a tryptophan beta 37 mutant.

Environmental changes of tyrosine and tryptophan residues of hemoglobin (Hb) upon its T to R transition of quaternary structure were investigated with ultraviolet resonance Raman (UVRR) spectroscopy excited at 235 nm. DeoxyHb A (T-form) showed a UVRR spectrum distinctly different from those of the ligated Hbs (R-form) including oxyHb, COHb, and metHb A, whereas the ligated Hbs exhibited similar UVRR spectra irrespective of the ligand species and the oxidation state of the heme. To characterize the spectral change of Trp-beta 37 at the alpha 1 beta 2 interface due to the quaternary structure transition, the UVRR spectra of Hb A were compared with the corresponding spectra of Hb Hirose (Trp-beta 37-->Ser). A difference spectrum between deoxyHb A and deoxyHb Hirose showed only Trp resonance Raman (RR) bands, which were reasonably ascribed to Trp-beta 37 in deoxyHb A. RR bands at 873 cm-1 (W17) and at 1360 and 1343 cm-1 (W7, Fermi doublet) indicated that the indole ring of Trp-beta 37 in deoxyHb A formed a strong hydrogen bond at the N1H site in hydrophobic environments. Tyr residues in deoxyHb Hirose seemed to be in the same environments as those of deoxyHb A. In contrast, the difference spectrum between Hb A and Hb Hirose in the ligated state displayed peaks for RR bands of both Trp and Tyr. The difference spectra were unaltered by the addition of 5 mM inositol hexaphosphate. This means that the differences were not caused by the tetramer to dimer dissociation but by a conformation change within a tetramer. Comparison of the Hb A-Hb Hirose difference spectra in the oxy and deoxy states revealed that the oxygenation-induced changes of Trp RR bands arose mostly from Trp-beta 37 with the small portion of remaining changes coming from Trp-beta 15, demonstrating that Trp-beta 37 plays a pivotal role in the quaternary structural change in Hb A.

Amino Acid Sequence↗
PubMed1995

Multi-enzyme reference material from established human cell lines and human sources.

A multi-enzyme reference material was prepared from seven enzymes of asparatate aminotransferase (AST, EC 2.6.1.1), alanine aminotransferase (ALT, EC 2.6.1.2), alkaline phosphatase (ALP, EC 3.1.3.1), lactate dehydrogenase (LD, EC 1.1.1.27), creatine kinase (CK, EC 2.7.2.2), gamma-glutamyltranspeptidase (gamma-GT, EC 2.3.2.2) and amylase (AMY, EC 3.2.1.1) which were purified from human sources including established human cell lines. The enzymatic properties of the material closely resembled those of human serum. In lyophilized form the preparation was stable for at least 200 days when stored at 40 degrees C. Intermethod comparisons of the enzyme activities in 80 clinical specimens were done by correcting the mean values with calibration constants for different assay methods resulting from use of a human serum, the multi-enzyme reference and a commercial control serum. The results from the comparison for the six enzymes of AST, ALT, LD, CK, gamma-GT and AMY in use of the multi-enzyme reference were almost the same as those with use of a human serum as a calibrator, but were not satisfactory for ALP. Even though further search for more reliable material for ALP is required the multi-enzyme reference material can be used for standardization in clinical chemistry.

Alanine Transaminase↗
PubMed1995

Mouse p87wee1 kinase is regulated by M-phase specific phosphorylation.

We have cloned a mouse wee1 kinase cDNA (mwee1). The clone is 2258 bp in length and its open reading frame corresponds to 646 amino acid residues. The molecular weight of this kinase is 87 kDa in SDS-PAGE, which is about 1.7-fold larger than the human p50wee1 kinase reported previously. In a cell cycle, the mouse wee1 kinase is phosphorylated at M-phase, and an in vitro study using a mitotic extract revealed that phosphorylation occurs in the N-terminal domain, which is absent from the human wee1 kinase, resulting in inactivation of the kinase activity. The N-terminal domain or entire molecule is extensively phosphorylated by cdc2-cyclin B kinase. Furthermore, the activity of the wee1 kinase was reduced by phosphorylation with the mitotic extract which contained cdc2-cyclin B kinase.

Amino Acid Sequence↗