Biomedical subjects

T Okazaki

Publications and source records attributed to T Okazaki.

At least 199 records · Page 11Linked to original sources

PubMed1995

Structural organization and chromosomal assignment of the human obese gene.

The obese (ob) gene has been identified through a positional cloning approach; the mutation of this gene causes marked hereditary obesity and diabetes mellitus in mice. We report here the isolation and characterization of the human ob gene. Southern blot analysis demonstrated a single copy of the ob gene in the human genome. The human ob gene spanned approximately 20 kilobases (kb) and contained three exons separated by two introns. The first intron, approximately 10.6 kb in size, occurred in the 5'-untranslated region, 29 base pair (bp) upstream of the ATG start codon. The second intron of 2.3 kb in size was located at glutamine +49. By rapid amplification of 5'-cDNA ends, the transcription initiation sites were mapped 54-57 bp upstream of the ATG start codon. The 172-bp 5'-flanking region of the human ob gene contained a TATA box-like sequence and several cis-acting regulatory elements (three copies of GC boxes, an AP-2-binding site, and a CCAAT/enhancer-binding protein-binding site). By the fluorescence in situ hybridization technique, the ob gene was assigned to human chromosome 7q31.3. This study should establish the genetic basis for ob gene research in humans, thereby leading to the better understanding of the molecular mechanisms underlying the ob gene.

Amino Acid Sequence↗

PubMed1995

Requirement of AP-1 for ceramide-induced apoptosis in human leukemia HL-60 cells.

Ceramide has emerged as a novel lipid mediator in cell proliferation, differentiation, and apoptosis. In this work, we demonstrate that the levels of c-jun mRNA, c-Jun protein, and DNA binding activity of a nuclear transcription factor AP-1 to 12-o-tetradecanoylphorbol 13-acetate responsive elements all increased following treatment with the cell-permeable ceramide, N-acetylsphingosine in human leukemia HL-60 cells. N-Acetylsphingosine (1-10 microM) increased the levels of c-jun mRNA in a dose-dependent manner, and maximal expression was achieved 1 h after treatment. Increase of c-jun expression treated with 5 microM N-acetyldihydrosphingosine, which could not induce apoptosis, was one third of that with 5 microM N-acetylsphingosine. Ceramide-induced growth inhibition and DNA fragmentation were both prevented by treatment with curcumin, 1,7-bis[4-hydroxy-3-methoxy-phenyl]-1,6-heptadiene-3,5-dione (an inhibitor of AP-1 activation), or antisense oligonucleotides for c-jun. These results suggest that the transcription factor AP-1 is critical for apoptosis in HL-60 cells and that an intracellular sphingolipid mediator, ceramide, modulates a signal transduction inducing apoptosis through AP-1 activation.

Apoptosis↗

PubMed1995

Cytosolic mercaptopyruvate sulfurtransferase is evolutionarily related to mitochondrial rhodanese. Striking similarity in active site amino acid sequence and the increase in the mercaptopyruvate sulfurtransferase activity of rhodanese by site-directed mutagenesis.

Rat liver mercaptopyruvate sulfurtransferase (MST) was purified to homogeneity. MST is very similar to rhodanese in physicochemical properties. Further, rhodanese cross-reacts with anti-MST antibody. Both purified authentic MST and expressed rhodanese possess MST and rhodanese activities, although the ratio of rhodanese to MST activity is low in MST and high in rhodanese. In order to compare the active site regions of MST and rhodanese, the primary structure of a possible active site region of MST was determined. The sequence showed 66% homology with that of rat liver rhodanese. An active site cysteine residue (Cys246; site of formation of persulfide in catalysis) and an arginine residue (Arg185; substrate binding site) in rhodanese were also conserved in MST. On the other hand, two other active site residues (Arg247 and Lys248) were replaced by Gly and Ser, respectively. Conversion of rhodanese to MST was tried by site-directed mutagenesis. After cloning of rat liver rhodanese, recombinant wild type and three mutants (Arg247-->Gly and/or Lys248-->Ser) were constructed. The enzymes were expressed in Escherichia coli strain BL21 (DE3) with a T7 promoter system. The mutation of these residues decreases rhodanese activity and increases MST activity.

Amino Acid Sequence↗

PubMed1995

Elevated levels of eukaryotic translation initiation factor eIF-4E, mRNA in a broad spectrum of transformed cell lines.

Translation initiation factor eIF-4E, which binds to the 5' cap structure of eukaryotic mRNAs, is believed to play an important role in the control of cell growth. Over-expression of eIF-4E in fibroblasts results in their malignant transformation. However, no information on eIF-4E expression in established transformed cell lines has been available. We report here that a variety of tumor cell lines, chemically, virally and oncogenically transformed, exhibit elevated levels of eIF-4E mRNA expression as compared to their normal counterparts. Overexpression of eIF-4E, which is normally rate-limiting in protein synthesis, may stimulate the translation of regulatory and oncogenic proteins involved in transformation.

Animals↗

PubMed1995

Expression of the Fas ligand in cells of T cell lineage.

Fas ligand (FasL) is a membrane-type cytokine belonging to the TNF family, and induces apoptosis through its cell-surface receptor, Fas. To determine the cell types that express FasL, various mouse tissues and cell lines were examined by Northern hybridization using a mouse FasL cDNA as a probe. Among tissues, lymphoid organs (thymus, lymph node, spleen), lung, and small intestine express low levels of FasL mRNA, suggesting the role of FasL in the general immune system and mucosal immunity. The testis expressed FasL mRNA most abundantly; however, the size of FasL mRNA in the testis was slightly shorter than those in other tissues. Distribution of FasL mRNA in a panel of cell lines indicated that the FasL expression is rather restricted to the cells of T cell lineage. Activation of the splenocytes with the T cell activators such as PMA and ionomycin, Con A, anti-CD3, or even IL-2 alone induced the expression of the FasL. CD8+ splenocytes expressed the FasL more abundantly than did the CD4+ splenocytes upon activation by Con A and IL-2. Among CD4+ CTL cell lines, the FasL was expressed in all Th1 and Th0, and some Th2 clones.

Animals↗

PubMed1995

High expression of c-kit in K562YO cells due to the prolonged half-life of its mRNA: the effects of modification with serine/threonine kinase signals.

We previously reported that the K562 cell line K562YO expressed a high level of the c-kit gene. In this study, we analyzed the mechanism of this expression and investigated the effects of the serine/threonine kinases such as protein kinase C (PKC) and cyclic adenosine 3',5'-monophosphate (cAMP)-dependent kinase (PKA) on it. The half-life of the c-kit mRNA in K562YO cells was greater than 10 hours, compared with 2 hours in the original K562 cells, which expressed a very low level of c-kit mRNA. This prolonged half-life can contribute to the high level of c-kit expression in K562YO cells. Cycloheximide (CHX), a protein synthesis inhibitor, caused increases in c-kit mRNA levels in K562YO cells. 12-O-tetradecanoylphorbol-13-acetate (TPA), by which PKC was activated at first and downregulated in a late phase, gradually decreased c-kit mRNA in K562YO cells until 9 hours and then returned to the control level 24 hours after treatment. TPA also rapidly decreased c-kit protein level on the membranes. In whole cells, c-kit protein was also decreased 6 hours after incubation with TPA. Calphostin C, a light-dependent PKC inhibitor, decreased c-kit mRNA levels within 30 minutes in a light-dependent manner. It also decreased c-kit protein in whole cells 2 hours after the addition. However, it increased the amount of c-kit protein on the cell surfaces. Dibutyryl cyclic AMP (dbc-AMP) increased c-kit mRNA as well as c-kit protein on membranes and in whole cells. Run-on transcriptional assay suggested that the agent (dbc-AMP) enhanced the transcription rate of the gene. These results suggest that c-kit protein on the membranes is downregulated by PKC activation and upregulated by PKC inhibition. In the whole cell lysate, c-kit proteins are decreased by PKC inhibition through downregulation of mRNA. On the other hand, the elevation of an intracellular cAMP level causes upregulation of both the mRNA and c-kit protein on membranes and in whole cells through enhanced transcription. Thus, c-kit gene expression is apparently modulated by PKC and PKA.

Bucladesine↗

PubMed1995

Microsatellite instability in primary and metastatic lung carcinomas.

Fifty-seven primary lung carcinomas and 35 metastatic lung carcinomas were analyzed for microsatellite instability at II different chromosomal loci. Although no instability was detected in 37 small cell lung carcinomas (SCLC), it was frequently detected in non-small cell lung carcinomas (NSCLC) (16/55, 29%). In NSCLC, the incidence of replication errors (RERs) in metastatic tumors (12/22, 55%) was significantly higher than that in primary tumors (4/33, 12%) (P = 0.0021). Among 10 pairs of primary tumors and corresponding metastases, there were 4 cases which manifested the identical RER phenotypes in both primary and metastatic tumors. In two cases, RER phenotypes were detected in metastatic but not in primary tumors. Never was an RER phenotype found only in a primary tumor but not in the metastases. RERs were detected more frequently in stage III or IV tumors (3/8, 38%) than stage I or II tumors (1/25, 4%) (P = 0.0359). Tumor cells with allelic losses on chromosome arm 3p or 18q tended to have RER phenotypes (P = 0.0432 and P = 0.0187, respectively). The data suggest that microsatellite instability is common in NSCLC but not in SCLC, and that genomic instability appears late in tumor progression and plays an important role in the acquisition of more malignant phenotypes in NSCLC.

Carcinoma, Non-Small-Cell Lung↗

PubMed1995

Long-term effects of intrasplenically transplanted adult hepatocytes and fetal liver in hyperbilirubinemic Gunn rats.

We performed adult hepatocyte transplantation (HCTx) and fetal liver transplantation (FLTx) into the spleens of hyperbilirubinemic Gunn rats in congenic combination and we compared the long-term effects of these procedures for as long as 12 months. Proliferative activity of intrasplenic hepatocytes was evaluated using antiproliferating cell nuclear antigen (PCNA) immunohistochemical staining. The serum total bilirubin levels (T. Bil) significantly decreased from 7.16 +/- 0.25 mg/dl to 4.38 +/- 0.60 mg/dl 2 months after HCTx and gradually decreased thereafter until 12 months after transplantation (3.23 +/- 0.37 mg/dl, P < 0.05 vs preoperative value). The T. Bil change after FLTx was similar to that of HCTx: 7.22 +/- 0.24 mg/dl before FLTx, and 4.92 +/- 0.24 and 3.06 +/- 0.47 mg/dl, 2 and 12 months after FLTx (P < 0.05), respectively. Bilirubin glucuronides, which were not detectable in the bile from untreated Gunn rats, appeared in considerable amounts 4 months after HCTx and FLTx (27.5% and 36.0% of total bile, respectively). PCNA labeling indices of intrasplenic hepatocytes (4.9% +/- 0.9% and 3.7% +/- 0.7%, 6 months after HCTx and FLTx, respectively) were slightly higher than those of normal hepatocytes (1.0% +/- 0.1%) in the host liver. In conclusion, both adult and fetal rat hepatocytes transplanted into the spleen in congenic combination functioned for at least a year in terms of bilirubin glucuronidation. The spleen is considered to be one of the optimal grafting sites for hepatocytes, with nearly lifelong significant function and proliferative activity.

Animals↗

PubMed1995

Postoperative flow characteristics of left internal thoracic artery grafts.

Twenty patients whose left internal thoracic artery (LITA) was anastomosed to the left anterior descending artery (LAD) underwent postoperative coronary angiography and Doppler ultrasound velocimetry. During angiography, the diameter of the LITA conduit was measured at three points: proximal, mid, and distal. The degree of left anterior descending artery stenosis proximal to the anastomotic site was evaluated by densitometry. The LITA flow velocity pattern was obtained at the three points to calculate the total, systolic, and diastolic flow volume. There were significant differences in the total LITA flow among the three points (proximal, 36.0 +/- 17.2 mL/min; mid, 29.9 +/- 15.2 mL/min; distal, 27.2 +/- 14.0 mL/min; p < 0.001 between the proximal and the mid or distal portions). The degree of left anterior descending artery stenosis affected the distal LITA flow and diameter (r = 0.823 and 0.811, respectively). There were significant differences in the systolic LITA flow among the three points (proximal, 13.2 +/- 6.5 mL/min; mid, 8.1 +/- 4.7 mL/min; distal, 5.6 +/- 3.4 mL/min; p < 0.001 between the proximal and the mid or distal portions). However, there was no statistically significant difference in the diastolic LITA flow among the three points (proximal, 22.9 +/- 11.0 mL/min; mid, 21.7 +/- 10.8 mL/min; distal, 21.6 +/- 10.8 mL/min). We conclude that a lower degree of LAD stenosis significantly reduces the LITA flow, inducing the string phenomenon. Additionally, during the diastolic phase, the LITA graft transports the blood primarily to the coronary artery but not to the side branches. Therefore, the steal phenomenon might not apply in the setting of an LITA graft.

Adult↗

PubMed1995

Quantitative analysis of neuronal innervation in the aganglionic bowel of patients with Hirschsprung's disease.

Although there is marked proliferation of nerve fibers in the aganglionic bowel of patients with Hirschsprung's disease (HD), controversy exists as to whether these fibers terminate in the cells of the bowel wall. This study quantitates biochemically the synaptic vesicle proteins and neurofilaments in the aganglionic bowel of HD patients. The bowel specimens obtained from 12 patients with HD (mean age, 4.6 months) and nine age-matched controls were analyzed by Western blot analysis using monoclonal antibody 171B5 against the synaptic vesicle 38,000-d protein (SVP38), and the specific band obtained was quantitated by densitometry. Similarly, using monoclonal antibody NF70K for detecting the 70,000-d component of neurofilament (NF70), nerve fibers were quantitated. In the aganglionic HD bowel, the quantity of SVP38, representing a proportion of synapses, was significantly lower than that in the normoganglionic bowel (P < .001). The quantity of NF70, representing a proportion of nerve fibers, was significantly greater than that in the normoganglionic bowel (P < .05). These findings suggest that the nerve fibers in the aganglionic bowel of HD patients have much less developed connections with the cells of the bowel wall, indicating overall poor innervation, despite marked proliferation of the nerve fibers.

Blotting, Western↗

PubMed1995

Differential regulation of catalytic and non-catalytic trkB messenger RNAs in the rat hippocampus following seizures induced by systemic administration of kainate.

Ribonuclease protection analysis and quantitative in situ hybridization histochemistry were used to investigate the coordination and regional expression of catalytic and non-catalytic trkB messenger RNAs in the adult rat hippocampus following systemic kainate-induced seizures. Changes in trkB expression were compared with the messenger RNA expression of its neurotrophic ligands, brain-derived neurotrophic factor and neurotrophin-3. TrkB messenger RNA expression was increased in the dentate granule cells at 1-4 h following the onset of seizures, and returned to control levels 16-24 h thereafter. In addition, seizures also induced expression of trkB messenger RNA in putative non-neuronal cells at four to seven days in the molecular layer of the dentate gyrus and the stratum lacunosum moleculare of the CA1 region. Hybridization with probes specific for the non-catalytic trkB receptor and the catalytic trkB receptor revealed that the increases at four and seven days in the molecular layers of the hippocampus reflected an up-regulation of only the non-catalytic form of the receptor. Furthermore, the neuronal increases observed 1-4 h were due to an up-regulation of both trkB TK- and trkB TK+ messenger RNAs. It was established that systemic administration of kainate increased brain-derived neurotrophic factor messenger RNA levels in the pyramidal and granule cell regions of the hippocampus 1-4 h following the onset of behaviorally manifested seizure activity. Early changes in neuronal expression of trkB TK- and trkB TK+ messenger RNA paralleled changes in brain-derived neurotrophic factor messenger RNA in the dentate granule cell and CA1 pyramidal cell layers, but not in the CA3 subregion. These data suggest that concomitant regulation of brain-derived neurotrophic factor and its cognate receptor may play a role in the selective vulnerability of hippocampal subregions to kainate-induced neuropathology. Furthermore, these data suggest a dual function for trkB receptor expression in the hippocampus following kainate-induced seizures, possibly related to both the plastic and degenerative consequences of seizure induction by kainate.

Animals↗

PubMed1995

[Quinolone-resistant mutations in Neisseria gonorrhoeae].

For 10 strains of Neisseria gonorrhoeae exhibiting decreased susceptibility to new quinolones (MIC, > or = 0.1 microgram/ml) isolated and preserved from patients with gonococcal urethritis during the period from February 1991 through January 1992. We investigated the mechanisms for development of resistance to new quinolones. After selecting 3 PCG-sensitive and new quinolone-resistant strains from among these strains, we first carried out transformation experiments of N. gonorrhoeae using a plasmid carrying the E. coli gyrA gene. We then determined the base sequence on the N. gonorrhoeae gyrA gene by PCR method. Of the 3 strains in which transformation experiments were carried out, 2 strains yielded transformants, one of which was 8 times more sensitive to norfloxacin (NFLX) than the original strain, and it was assumed that this strain has a mutation in the gyrA gene. In our study of the base sequence on the N. gonorrhoeae gyrA gene using the PCR method, both strains had the mutation of Ser 83 (TCC)-->Phe (TTC), and in addition to this mutation of Ser-->Phe, one other strain had the mutation of Asp-87 (GAC)-->Gly (GGC). This type of mutation of quinolone-resistant N. gonorrhoeae on the gyrA gene has high homology with the mutations reported for Escherichia coli and other strains and there appears to be a close correlation between the increasing frequency of use of the new quinolones in this country and the spread of such resistant strains of N. gonorrhoeae.

Drug Resistance, Microbial↗

PubMed1995

Analysis of protein-DNA and protein-protein interactions of centromere protein B (CENP-B) and properties of the DNA-CENP-B complex in the cell cycle.

We previously reported that centromere protein B (CENP-B) forms a stable complex (designated complex A) containing two alphoid DNAs in vitro. Domains in the CENP-B polypeptide involved in the formation of complex A were determined in the present study with truncated derivatives expressed in Escherichia coli and in rabbit reticulocyte lysates. It was revealed by gel mobility shift analyses that polypeptides containing the NH2-terminal DNA-binding domain bind a DNA molecule as a monomer, while dimerizing at a novel hydrophobic domain in the COOH-terminal region of 59 amino acid residues. This polypeptide dimerization activity at the COOH-terminal region was also confirmed with the two-hybrid system in Saccharomyces cerevisiae cells. The results thus proved that CENP-B polypeptides form a homodimer at the COOH-terminal hydrophobic domain, each binding a DNA strand at their NH2-terminal domains. The dimerization and DNA-binding domains fall into two of the three completely conserved sequences found in human and mouse CENP-B, and complex A-forming activity was also detected in nuclear extracts of mouse cells. Metaphase-specific phosphorylation of CENP-B was also detected, but this had no effect on its complex A-forming activity. On the basis of the present results, we propose that CENP-B plays an important role in the assembly of specific centromere structures by forming unique DNA-protein complexes at the sites of CENP-B boxes on the centromeric repetitive DNA both in interphase nuclei and on mitotic chromosomes.

Amino Acid Sequence↗

PubMed1995

Molecular cloning of rat obese cDNA and augmented gene expression in genetically obese Zucker fatty (fa/fa) rats.

The obese (ob) gene has recently been isolated through a positional cloning approach, the mutation of which causes a marked hereditary obesity and diabetes mellitus in mice. In the present study, we isolated rat ob cDNA and examined the tissue distribution of the ob gene expression in rats. We also studied the gene expression in genetically obese Zucker fatty (fa/fa) rats. The rat ob gene product, a 167 amino acid protein with a putative signal sequence, was 96 and 83% homologous to the mouse and human ob proteins, respectively. Northern blot analysis using the rat ob cDNA probe identified a single mRNA species of 4.5 kb in size in the adipose tissue, while no significant amount of ob mRNA was present in other tissues in rats. The ob gene was expressed in the adipose tissue with region specificities. The rank order of the ob mRNA level in the adipose tissue was epididymal, retroperitoneal, and pericardial white adipose tissue > mesenteric and subcutaneous white adipose tissue > or = interscapular brown adipose tissue. The ob gene expression occurred in mature adipocytes rather than in stromalvascular cells isolated from the rat adipose tissue. Expression of the ob gene was markedly augmented in all the adipose tissue examined in Zucker fatty (fa/fa) rats at the stage of established obesity. The present study leads to the better understanding of the physiologic and pathophysiologic roles of the ob gene.

Adipose Tissue↗

PubMed1995

Degradation of 2-ketoarginine by guanidinobutyrase in arginine aminotransferase pathway of Brevibacterium helvolum.

Guanidinobutyrase (EC 3.5.3.7) involved in the arginine oxygenase pathway of Brevibacterium helvolum IFO 12073 was found to catalyze also the hydrolysis of 2-ketoarginine (2-keto-5-guanidinovalerate) to 2-ketoornithine (2-keto-5-aminovalerate) and urea, the second step of the arginine aminotransferase pathway. No other enzyme that degraded 2-ketoarginine was found in cells grown on L-arginine. The enzyme hydrolyzed 2-ketoarginine with a relative rate of about 0.7% of that toward 4-guanidinobutyrate. The Km for 2-ketoarginine was 33 mM.

Arginine↗

PubMed1995

Human obese gene expression. Adipocyte-specific expression and regional differences in the adipose tissue.

The obese (ob) gene, the mutation of which results in severe hereditary obesity and diabetes in mice, has recently been isolated through positional cloning. In this study, we isolated a full-length human ob complementary DNA (cDNA) clone and examined the tissue distribution of ob gene expression in humans. The nucleotide sequences of the human ob cDNA coding region were 83% identical to those of the mouse and rat ob cDNA coding regions. Analysis of the deduced amino acid sequences revealed that the human ob protein is a 166-amino acid polypeptide with a putative signal sequence and is 84 and 83% homologous to the mouse and rat ob proteins, respectively. Northern blot analysis using the cloned human ob cDNA fragment as a probe identified a single messenger RNA (mRNA) species 4.5 kb in size found abundantly in the adipose tissues obtained from the subcutaneous, omental, retroperitoneal, perilymphatic, and mesenteric fat pads. However, no significant amount of ob mRNA was present in the brain, heart, lung, liver, stomach, pancreas, spleen, small intestine, kidney, prostate, testis, colon, or skeletal muscle. The ob mRNA level in the adipose tissue varied from region to region even in the same individual. Furthermore, in the human adipose tissue, ob gene expression occurred in mature adipocytes rather than in stromal-vascular cells. This study is the first report of the elucidation of ob gene expression in human tissues, thereby leading to better understanding of the physiological and clinical implications of the ob gene.

Adipocytes↗

PubMed1995

A novel bioactive delta lactone FD-211. Taxonomy, isolation and characterization.

During our screening program for natural product drugs effective against multidrug-resistant mammalian cells, we have discovered a new delta lactone FD-211 from the fermantation broth of Myceliophthora lutea TF-0409. FD-211 had a broad spectrum activity against cultured tumor cell lines, including adriamycin-resistant HL-60 cells.

Animals↗

PubMed1995

Biological response modifiers (BRM) as antigens. III. T cell lines specific for BRM kill tumor cells in a BRM-specific manner.

In order to investigate tumoricidal effector cells in therapy by biological response modifiers (BRM) such as Propionibacterium acnes, bacillus Calmette-Guérin (BCG), Streptococcus pyogenes and a protein-bound polysaccharide (PSK), we established T cell lines specific for each BRM from BALB/c mice immunized with the corresponding BRM. These T cell lines proliferated and produced interleukin-2 (IL-2) and/or IL-4, but only in the presence of the relevant BRM and BALB/c spleen cells as the antigen and antigen-presenting cells respectively. Cross-functional experiments indicated that each BRM acts as a nominal antigen, but not as a non-specific immunostimulator. In addition, the T cell lines killed Ia-positive syngeneic B lymphoma cells, but only in the presence of the relevant BRM. These experiments excluded the possibility of cytotoxic effects by each BRM. The T cell lines and clones also killed Ia-negative bystander target cells, but only in the presence of both a relevant antigen and antigen-presenting cells. The T cell clones specific for S. pyogenes or P. acnes tested were Thy1+, L3T4+ and Lyt2-. These results indicate that some BRM exert tumoricidal activity by inducing T cells that recognize them as an antigen and kill tumor cells in an antigen-specific manner. The T cells killed tumor targets in either a tumor-necrosis-factor(TNF)-dependent or a TNF-independent manner. The mediator of the latter pathway remains to be elucidated.

Animals↗