Biomedical subjects

K Ohtani

Publications and source records attributed to K Ohtani.

At least 19 recordsLinked to original sources

PubMed1999

Human T-cell leukemia virus type 1 tax protein abrogates interleukin-2 dependence in a mouse T-cell line.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia. Tax, the viral protein, is thought to be crucial in the development of the disease, since it transforms healthy T cells in vitro and induces tumors in transgenic animals. We examined the effect of Tax activity on the growth of the interleukin-2 (IL-2)-dependent T-cell line CTLL-2. Stable expression of Tax in CTLL-2 transformed cell growth from being IL-2 dependent to IL-2 independent. Tax stimulated transcription through NF-kappaB and the cyclic AMP-responsive element-like sequence in the HTLV-1 promoter. The finding of Tax mutants segregating these two pathways suggested that the NF-kappaB pathway was essential for IL-2-independent growth of CTLL-2 cells while the CRE pathway was unnecessary. However, both pathways were necessary for another transformation-related activity (colony formation in soft agar) of CTLL-2/Tax. Our results show that Tax has at least two distinct activities on T cells, and suggest that Tax plays a crucial role in IL-2-independent T-cell transformation induced by HTLV-1, in addition to its well-known IL-2-dependent cell transformation.

Animals↗

PubMed1998

Regulation of cell growth-dependent expression of mammalian CDC6 gene by the cell cycle transcription factor E2F.

CDC6 of Saccharomyces cerevisiae regulates the DNA replication initiation through the origin recognition complex (ORC). Identification of a human homolog of the CDC6 gene (HsCdc6) suggests a universal role of the gene product in DNA replication. Expression of HsCdc6 is growth-regulated. We investigated the molecular basis of growth-regulated expression of mammalian Cdc6. The promoter activity of isolated HsCdc6 upstream region was activated at late G1 and G1/S boundary in the cell cycle of rat embryonic fibroblast REF52 cells by the addition of serum. The isolated promoter was activated by exogenous expression of E2F without serum stimulation. However a mutant promoter lacking the E2F recognition sites failed to respond to serum stimulation and exogenous expression of E2F. Expression of endogenous Cdc6 was induced by exogenous expression of E2F. Therefore, we concluded that the growth-regulated expression of mammalian Cdc6 was mediated by E2F. Moreover, we demonstrated that exogenous overexpression of either HsCdc6 or HsOrc1 failed to induce DNA synthesis unlike overexpression of E2F1, even though E2F1 induced both Cdc6 and Orc1, suggesting that E2F may regulate the expression of another gene(s), besides Cdc6 and Orc1, required for induction of cellular DNA synthesis in mammalian cells.

Animals↗

PubMed1998

High-performance liquid chromatographic determination of nemonapride and desmethylnemonapride in human plasma using an electrochemical detection.

A high-performance liquid chromatographic method using an electrochemical detector (HPLC-ED) was developed for the determination of nemonapride and its active metabolite, desmethylnemonapride in human plasma. Nemonapride, desmethylnemonapride and moperone chloride, which was used as the internal standard (I.S.) in plasma, were extracted by a rapid and simple procedure based on C18 bonded-phase extraction, and were separated by C8 reversed-phase HPLC column. Nemonapride and desmethylnemonapride were detected by high conversion efficiency amperometric detection at +0.84 V. Determination of both nemonapride and desmethylnemonapride were possible in the concentration range at 0.25-5.0 ng/ml, and the limit of detection for each was 0.1 ng/ml. The recoveries of nemonapride and desmethylnemonapride added to plasma were 97.0-98.2% and 96.7-98.8%, respectively, with coefficients of variation of less than 7.2% and 10.3%, respectively. This method is applicable to drug level monitoring in the plasma of schizophrenia patients treated with nemonapride and to the study of pharmacokinetics.

Antipsychotic Agents↗

PubMed1998

Molecular mechanisms of promoter regulation of the gp34 gene that is trans-activated by an oncoprotein Tax of human T cell leukemia virus type I.

We investigated the molecular mechanism of transcriptional activation of the gp34 gene by the Tax oncoprotein of human T cell leukemia virus type I (HTLV-I). gp34 is a type II transmembrane molecule belonging to the tumor necrosis factor family and is constitutively expressed on HTLV-I-producing cells but not normal resting T cells. The transcriptional regulatory region of the gp34 gene was activated by HTLV-I Tax in the human T cell line Jurkat, in which endogenous gp34 is induced by Tax. Sequence analysis demonstrated that two NF-kappaB-like elements (1 and 2) were present in the regulatory region. Both NF-kappaB-like elements were able to bind to NF-kappaB or its related factor(s) in a Tax-dependent manner. Chloramphenicol acetyltransferase assays indicated that NF-kappaB-like element 1 was Tax-responsive, although the activity was lower than that the native promoter. NF-kappaB-like element 2 elevated promoter activity when combined with NF-kappaB-like element 1, indicating cooperative function of the elements for maximum promoter function. Unlike typical NF-kappaB elements, the NF-kappaB-like elements in gp34 were not activated by treatment of Jurkat cells with phorbol ester despite induction of the NF-kappaB-like binding activity. Chloramphenicol acetyltransferase reporter assays using the region upstream of the NF-kappaB-like elements identified an upstream region that reduced transcription from cognate and noncognate core promoters in a Tax-independent manner. Our results imply complex regulation of expression of the gp34 gene and suggest implication of gp34 in proliferation of HTLV-I infected T cells.

Antigens, Surface↗

PubMed1998

Identification and characterization of a glucose-responsiveness region upstream of human insulin gene in transfected HIT-T 15 cells.

To determine possible regulation of full-length human insulin gene promoter activity by glucose, we examined a 2-kilobase pair (kbp) 5'-flanking region of the human insulin gene and characterized the DNA elements in transfected HIT-T 15 cells. The expression of the 2-kilobase pair 5'-flanking region human insulin gene fused to the luciferase reporter gene occurred by transfection. In 0.8 mM glucose of the F-12 K medium, the element mediating the negative regulatory region was localized from -1782 to -1295 base pairs (bp) and stimulatory element from -1295 to -1138 bp. The elements from -1138 to -880 bp and from -356 to +252 bp possessed the elements dose-dependently responsive to 0.8 mM, 7.0 and 22.2 mM glucose. In fragment D, cotransfection of oligonucleotide that confers RIPE3b1 activator decreased the glucose-stimulated promoter activity, but the other oligonucleotide that confers STF-1 did not. The present data indicated that 2 kbp possesses glucose-responsive region in the element from -1138 to -880 bp, in addition to the previously reported element from -356 to initiation site. There may exist a RIPE3b1 activator binding site in the glucose-responsive element from -1138 to -880 bp. In addition, negatively regulatory region may exist from -1782 to -1295 bp.

Binding Sites↗

PubMed1998

Oleanane saponins from Polyscias fruticosa.

From the leaves and roots of Polyscias fruticosa, eight new oleanolic acid saponins named polysciosides A to H were isolated together with three known saponins. The structures of the saponins were established by means of spectral data, particularly NMR, which included COSY, HSQC, HMBC, HOHAHA and ROESY techniques.

Carbohydrate Sequence↗

PubMed1998

Epidemiology of spina bifida in Tottori Prefecture, Japan, 1976-1995.

The authors studied the epidemiology of spina bifida in Tottori Prefecture, Japan, from 1976 to 1995. Thirty-four patients (16 men and 18 women) were registered in this study. Consanguineous marriages, familial occurrence, and abnormalities in prenatal history were not observed. The incidence rate in the entire prefecture and in the eastern, central, and western regions was 0.234, 0.148, 0.425, and 0.230 per 1,000 live births, respectively. The incidence rate in the central region was greater than that in the eastern region with statistical significance (P < 0.05), but the cause of the cluster is unknown. The incidence rate of 0.234 per 1,000 live births for 20 years is compatible with the previous two studies of 1922-1940 and 1948-1954 in Japan. Such apparently stable trends suggest that environmental factors have affected the Japanese less than genetic factors. Seasonal variations are not demonstrated.

Demography↗

PubMed1998

Molecular and biological characterization of rabbit mannan-binding protein.

Mannan-binding protein (MBP) is a member of the collectin family of protein. There are two types of MBP, MBP-A and MBP-C, which were found in rodent (rats and mice), rhesus monkey, and cynomolgus monkey, while chimpanzee and human have only one MBP. It was considered that the loss of one MBP gene occurred during hominoid evolution. In this article two rabbit MBP, a liver and serum MBP, were characterized biologically and genetically. Analyses by SDS-PAGE under reduced condition and their amino acid sequences of both MBPs showed that they have a same molecular weight of 32 kDa and their amino acid sequences were identical. A serum MBP has a higher ability to activate complement than does a liver MBP; however, a liver MBP inhibits hemagglutination by influenza virus as strongly as a serum MBP does. cDNA clones encoding the rabbit MBP were isolated from a rabbit cDNA liver library using whole cDNA of mouse MBP-C as a probe. The cDNA carried an insert of 744 bp coding for a protein of 247 acid residues with a signal peptide of 22 residues. The deduced amino acid sequence of the cDNA was identical to that of amino acid sequences of the 32 kDa proteins determined here. Northern blot analysis showed that mRNA transcripts of about 0. 9 and 3.0 kb were expressed only in the liver. The analysis of the phylogenetic tree of rabbit and bovine MBPs and other collectins indicates that the loss of MBP gene occurred not only during hominoid evolution but also at some points after the separation of birds and mammals.

Amino Acid Sequence↗

PubMed1998

Characterization of truncated human mannan-binding protein (MBP) expressed in Escherichia coli.

Mannan-binding protein (MBP) is a calcium-dependent mammalian serum lectin important in first-line host defense. MBP belongs to the collectin family, which is characterized by an NH2-terminal cysteine-rich domain, a collagen-like domain, a neck domain, and a carbohydrate recognition domain (CRD). We have expressed a recombinant human MBP, consisting of the short collagen region (two repeats of Gly-Xaa-Yaa amino acid sequences), the neck domain, and the CRD, in Escherichia coli. The truncated MBP was capable of forming trimers by association of the neck domain and could bind sugar with a specificity similar to that of the native form. Results of hemagglutination inhibition (HI) assay of influenza A virus showed that the truncated MBP inhibited hemagglutination less strongly, although the native MBP induced the HI phenomenon. These results suggest that an oligomeric structure is an advantage for MBP to have full biological activity against influenza A virus.

Carrier Proteins↗

PubMed1998

Hypothalamic corticotropin-releasing hormone is a mediator of the anorexigenic effect of leptin.

Leptin, ob gene product, inhibits feeding behavior and stimulates energy expenditure in rodents. Corticotropin-releasing hormone (CRH) and neuropeptide Y (NPY), which act in the hypothalamus to influence energy homeostasis, may mediate the anorexic effect of leptin. The present studies were undertaken to examine the possible involvement of hypothalamic CRH in the anorexigenic action of leptin in male Wistar rats. Recombinant leptin (2 microg/rat), microinjected into the third ventricle, inhibited food intake at 2 h by 33.3% (P < 0.01) in rats that were deprived of food for 18 h. The intracerebroventricular injection of 2 microg leptin also increased hypothalamic CRH content (P < 0.05) at 2 h after its administration. Simultaneous intracerebroventricular administration of 5 microg/rat alpha-helical CRH 9-41 (alpha-hCRH), a CRH antagonist, with 2 microg/rat leptin attenuated the anorexic effect of leptin by 2 h. In contrast, single intracerebroventricular injection of alpha-hCRH did not affect food consumption in food-deprived rats. These results implicate hypothalamic CRH as an important mediator of the anorexic effect of leptin in food-deprived rats.

Animals↗

PubMed1997

An evaluation of CA125 levels in 291 normal postmenopausal and 20 endometrial adenocarcinoma-bearing women before and after surgery.

Two hundred ninety-one normal postmenopausal women without hormone replacement therapy were studied for their blood levels of CA125. The levels ranged from 0.1 to 31.8 U/ml (mean+/-SEM 5.5+/-0.3 U/ml). Twenty patients with endometrial adenocarcinoma who had preoperative CA125 levels below 50 U/ml were also studied. The levels of CA125 in the latter group ranged from 2.1 to 43.0 U/ml (17.2+/-2.3 U/ml) and the difference of the mean value of the two groups was statistically significant (P < 0.001). The CA125 levels in normal postmenopausal women have a weak negative correlation with their age (CA125 = -0.025 x age + 6.9, P < 0.001), whereas in cancer patients, a positive correlation was seen (CA125 = 0.6 x age + 11.5, P < 0.05). Patients with endometrial cancer showed a decrease in their CA125 levels after a hysterectomy and salpingo-oophorectomy (6.5+/-1.2 U/ml, P < 0.0001). These results confirmed that the normal range of the CA125 of menopausal women is much lower than that of cycling women and each laboratory should establish its own normal range for the population. Also, it was suggested that CA125 levels which apparently fall within the normal range but are high, however, for the respective age, may indicate that an extensive search for an endometrial malignancy is necessary.

Adenocarcinoma↗

PubMed1997

Characterization of recombinant bovine conglutinin expressed in a mammalian cell.

We describe here the successful expression of recombinant bovine conglutinin in CHO cells as well as its physical and biological characteristics. Geneticin-resistant transformants harboring bovine conglutinin cDNA in the expression vector pNOW/CMV-A were screened by Western blot analysis for secretion into media of recombinant conglutinin. A four-day amplification of the transgene with increasing concentrations of methotrexate resulted in a dose-dependent increase in the production of recombinant conglutinin to a final concentration of 18.6 microg/ml of media. Recombinant conglutinin purified from this media by affinity column chromatography on mannan-agarose had a migration pattern similar to that of native conglutinin on polyacrylamide gel electrophoresis under reducing, nonreducing, and native conditions. The recombinant conglutinin exhibited sugar binding, conglutination, hemagglutination inhibition, and neutralization of influenza A virus, activities engaged in by the native conglutinin. This is the first report describing a high level of expression of a serum cruciform collectin with the full range of biological activity.

Animals↗

PubMed1997

Structure of a truncated human surfactant protein D is less effective in agglutinating bacteria than the native structure and fails to inhibit haemagglutination by influenza A virus.

Surfactant protein D (SP-D) is a lung-specific protein that is synthesized and secreted by lung epithelial cells and is believed to play an important role in lung host defence. This protein belongs to the C-type lectin family, which is characterized by an N-terminal cysteine-rich domain, a collagen-like domain, a neck domain and a carbohydrate recognition domain (CRD). To elucidate the biological actions of this animal lectin against such pathogens as micro-organisms, the biological activities of a recombinant partial SP-D lacking a collagen-like domain were examined. A recombinant human SP-D, consisting of a short collagen region (two repeats of Gly-Xaa-Yaa amino acid sequences), the neck domain and the CRD, was expressed in Escherichia coli. The recombinant SP-D was purified on a nickel column and then on a maltose-agarose column. This protein can form a trimeric structure owing to the neck domain and exhibits sugar-binding activity and specificity similar to those of native human SP-D. The recombinant SP-D caused dose-dependent and calcium-dependent agglutination of E. coli Y1088. The agglutination titre (the concentration required to achieve a 50% decrease in light transmission by agglutination) of recombinant SP-D was approx. 6-fold that of native SP-D. As for conglutination, the recombinant trimeric conglutinin required 8-16-fold higher concentrations than the native counterpart. In haemagglutination inhibition (HI) of influenza A virus, although native and recombinant conglutinin showed similar levels of HI activity, the recombinant SP-D was unable to inhibit haemagglutination, even at a concentration approx. 120-fold that of the native SP-D. The lectin precipitation and lectin blot assays showed that the truncated SP-D could bind to influenza A virus as well as native SP-D did. These results indicate that the agglutination activity of trimeric collectins can be largely retained, and furthermore that the oligomeric structure with several hands at opposite sites can enhance agglutination activity. The difference in HI activity against influenza A virus between native and recombinant SP-D suggests that SP-D uses a different mechanism from that of conglutinin to inhibit viral haemagglutination.

Agglutination↗

PubMed1997

Cloning and characterization of a cDNA encoding bovine mannan-binding protein.

To identify the bovine mannan-binding protein (MBP), a search for the cDNA homologue of human MBP was carried out. cDNA clones encoding bovine MBP were isolated from a bovine liver cDNA library using a cDNA fragment encoding a short collagen region, neck domain and carbohydrate recognition domain of human MBP. The cDNA carried an insert of 747 bp encoding a protein of 249 amino acid (aa) residues with a signal peptide of 19 aa. The mannan-binding protein fraction of bovine serum that eluted with 100 mM mannose from a mannan-Sepharose column was analyzed under reducing conditions by SDS-PAGE. The major band of 33 kDa obtained reacted with anti-human MBP rabbit serum. The partial aa sequence of the purified 33-kDa protein was identical to the aa sequence deduced from the obtained cDNA. Results of the passive hemolysis experiment using sheep erythrocytes coated with yeast mannan suggest that this MBP has the ability to activate complement. Northern blot analysis showed a 1.8-kb mRNA that was expressed only in the liver. Based on results of genomic analysis, this bovine MBP is likely to be a homologue of human MBP and to also have homology to rat and mouse MBP-C which are localized in liver cells rather than to rat and mouse MBP-A found in serum. Alignments of bovine collectins show that bovine MBP cannot be included among the other bovine collectins, such as bovine SP-D, conglutinin and CL-43. Finally, these genomic and biological analyses indicate that the cDNA obtained here encoded a bovine serum MBP.

Amino Acid Sequence↗

PubMed1997

Pulmonary metastatic lesion of endolymphatic stromal myosis expresses metastasis-related genes but not invasion-related matrix type metalloproteinase.

A case of endolymphatic stromal myosis (ELSM) with multiple metastasis to lungs was studied. A single biopsy specimen from the lung was analyzed for c-erbB-2, CD44E, and autocrine motility factor receptors (AMFR) mRNA expression, all putatively associated with metastasis. Estrogen and progestin receptors (ER, PR) expression were studied by reverse transcription polymerase chain reactions. Expression of an invasion-related gene, membrane type matrix metalloproteinase (MT-MMP) was also studied. The metastatic lesion showed positive expression of c-erbB-2, CD44E, AMFR, PR and ER expression, whereas no expression of MT-MMP was detected. These results correspond with the present clinical history that is early and multiple lung metastasis but essentially benign in nature and with excellent response to gestagen treatment.

Biopsy↗

PubMed1997

Collagenase gene (colA) is located in the 3'-flanking region of the perfringolysin O (pfoA) locus in Clostridium perfringens.

The 3'-flanking region of the beta-galactosidase gene (pbg), which is located downstream of the perfringolysin O gene (pfoA), and the 5'-flanking region of the collagenase gene (colA) of Clostridium perfringens strains NCTC8237 and 13, respectively, were analyzed. Southern analysis revealed that the colA gene is located 6.5 kb downstream of the pbg gene in the chromosome of C. perfringens. Sequence analysis showed that between the pbg and colA genes were the arcABDC and ahrC genes, whose putative products were quite similar to enzymes of the arginine deiminase pathway of Pseudomonas aeruginosa and the arginine repressor/activator of Bacillus subtilis, respectively. It is concluded that the genomic structure of the pfoA-colA region consists of pfoR-pfoA-ORF54-pbg-arcABDC-ahrC-colA.

Amino Acid Sequence↗

PubMed1997

Leptin stimulates insulin secretion and synthesis in HIT-T 15 cells.

Leptin, an ob gene product, corrects hyperinsulinemia in ob/ob mice. The leptin receptor may exist in pancreatic islets. The present studies were undertaken to determine the direct effect of 1-100 ng/ml recombinant leptin on insulin secretion and synthesis in HIT-T 15 cells by using static culture system. The addition of recombinant leptin significantly increased insulin secretion for 20 min at the highest concentration (100 ng/ml). The addition of recombinant leptin dose-dependently increased insulin secretion for 24 h in the 7 mM glucose-containing F-12 K medium. The incubation with recombinant leptin for 24 h increased preproinsulin mRNA expression, assessed with reverse transcription-polymerase chain reaction (RT-PCR) method. It was furthermore demonstrated that HIT-T 15 cells possessed the specific binding site for [125I]-labeled leptin. The present study demonstrated the existence of the leptin-specific binding sites that mediate its stimulatory effect on insulin secretion and synthesis in HIT-T 15 cells.

Actins↗

PubMed1997

Selective depression of the spinal polysynaptic reflex by the NMDA receptor antagonists in an isolated spinal cord in vitro.

1. The effects of N-methyl-D-aspartate (NMDA) receptor glycine-binding site antagonists 7-chlorokynurenate (7-Clkyn) and (+/-)-3-amino-1-hydroxy-2-pyrrolidone (HA-966) on spinal reflexes in an isolated spinal cord that was maintained in Mg(2+)-free medium in vitro were examined. The actions of 7-Clkyn and HA-966 were compared with those of the channel-site antagonist (i.e., dizocilpine) and NMDA-binding site antagonists--that is, 3-[(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonate (CPP) and DL-2-amino-5-phosphonovalerate (APV). 2. 7-Clkyn and HA-966 produced a selective depression of the polysynaptic reflex (PSR) while negligibly affecting the activity of the monosynaptic reflex (MSR). The PSR was also differentially suppressed by dizocilpine, CPP and APV. The PSR inhibitory activity of the NMDA antagonists was in the following order: dizocilpine > CPP > APV = 7-Clkyn > HA-966. 3. The inhibitory effects of 7-Clkyn on PSR were markedly antagonized by the simultaneous application of D-serine, an agonist for the NMDA receptor glycine-binding sites. However, PSR inhibition by dizocilpine and CPP was unaffected. 4. Inhibition of the PSR by 7-Clkyn persisted in the presence of strychnine, which markedly increased the PSR activity by itself. 5. These findings suggest that the NMDA receptor glycine-binding sites play a role in generating the NMDA receptor-mediated PSR in the spinal cord in vitro.

Animals↗