Biomedical subjects

K Watabe

Publications and source records attributed to K Watabe.

At least 91 records · Page 5Linked to original sources

PubMed1994

The X-gene of human hepatitis B virus transactivates the c-jun and alpha-fetoprotein genes.

The X-gene product of human hepatitis B virus is a transacting transcriptional factor which activates a variety of heterologous viral and host promoters/enhancers. We have found that the X-gene product can significantly transactivate the regulatory sequences located at the 5'-upstream of the c-jun oncogene when a reporter plasmid containing the sequences was co-transfected to HepG2 cells with an X-gene expression plasmid. The results of mutational analysis indicate that the X-gene activation requires the AP-1 sequence of the c-jun gene. Furthermore, we also found that the X-gene is capable of activating the 5'-upstream sequence of the alpha-fetoprotein gene. There are at least two elements that respond to the X-gene transactivation. One is located in the sequences between -5,100 and -2,900, and the other is at the C/EBP site. Therefore, the X-gene activates the c-jun and alpha-fetoprotein genes through different host factors, namely AP-1 and C/EBP, respectively. The results of c-jun activation by the X-gene strongly support the previous hypothesis that the X-gene may play a critical role in the development of hepatocellular carcinoma.

Base Sequence↗

PubMed1994

Poisoning by the red alga 'ogonori' (Gracilaria verrucosa) on the Nojima Coast, Yokohama, Kanagawa Prefecture, Japan.

A food poisoning case due to the ingestion of 'ogonori', an edible red alga, occurred at Yokohama, Kanagawa Prefecture, Japan, in late October 1993, resulting in two victims, including one death (female). No causative agent present in the ogonori was found from a routine bioassay for marine toxins. From the production of increased amounts of prostaglandins (PGs), mainly PGE2, by the alga on stimulation by cutting or soaking in fresh water, and further increase of PGE2 by addition of arachidonic acid, it appeared that an enzyme, probably fatty acid cyclooxygenase, in the ogonori and the body of the victim, was acting on the highly unsaturated fatty acids in the oil of the ingested seafood and in the blood hemorrhaged from the stomach of the victim. This resulted in the production of over 30 mg of PGE2 and small amounts of other PGs in a comparatively short time. With this dosage the victim suffered from nausea, vomiting, and hypotension, and died of hypotensive shock. PGE2 seems to work more selectively on females. This type of poisoning is very unusual, and differs from the more familiar forms of poisoning occurring after ingestion of marine organisms.

Animals↗

PubMed1994

A thermostable hydantoinase of Bacillus stearothermophilus NS1122A: cloning, sequencing, and high expression of the enzyme gene, and some properties of the expressed enzyme.

A DNA fragment containing the gene for a thermostable hydantoinase was cloned from a thermophile, Bacillus stearothermophilus NS1122A in Escherichia coli. Nucleotide sequencing showed that the DNA fragment contains one open reading frame, which is predicted to encode a peptide of 471 amino acids, with a calculated molecular weight of 51,724. When the hydantoinase gene was under the control of both the lpp promoter and the lac promoter-operator, and its expression was induced by isopropyl-1-thio-beta-D-galactopyranoside, it was overexpressed in E. coli leading to the formation of an insoluble aggregate. The enzyme was purified to homogeneity from the insoluble aggregate. The molecular mass of the purified active enzyme was approximately 200 kDa by gel filtration. Although the monomer had no activity, the activity was restored by incubation with Mn2+ or Co2+ at pH 8.1. These findings suggested that the hydantoinase is a metalloenzyme and the oligomeric structure is required for activity. The oligomeric structure is suggested to contribute to thermostability.

Amidohydrolases↗

PubMed1993

Characterization of the 5'-flanking region of the hepatocyte growth factor gene.

The Hepatocyte growth factor is the most potent mitogen for hepatocytes in primary culture and is involved in liver regeneration. The expression of the gene appears to be tightly controlled by various humoral factors. To understand the molecular mechanism of the gene expression, we cloned and determined the nucleotide sequence of the 5'-flanking region of the gene. In this region, there are sequences homologous to responding elements of P53, Rb, IL-1, IL-6, glucocorticoids, TPA and TGF-beta. We also identified three major transcriptional initiation sites by primer extension analysis of this region. Functional analyses of this region by constructing CAT reporter plasmids indicate that the sequence functions in a tissue specific manner and there is a negative regulatory region which suppresses the gene expression in rat transformed kidney cells.

Animals↗

PubMed1993

Purification of Bacillus subtilis spore coat protein by electrophoretic elution procedure and determination of NH2-terminal amino acid sequences.

Spore coat protein of Bacillus subtilis was purified by electrophoretic elution procedure. Solubilized coat protein components were separated on SDS-PAGE and the desired protein was recovered from the gel pieces under the optimal condition examined. Two purified polypeptides with molecular weights of about 40 kDa were obtained; each of them was in very closed size on SDS-PAGE, both retaining antigenic activity against anti-spore coat protein serum on immunoblot analysis. The N-terminal 23 and 30 amino acid sequences of them were determined, and they were not identical to each other and also not homologous in the sequences of coat proteins previously reported.

Amino Acid Sequence↗

PubMed1993

Immunohistochemistry of neuronal inclusions in the cerebral cortex and brain-stem in Lewy body disease.

Three cases of Lewy body disease were investigated in order to compare the morphological and immunohistochemical characteristics of the neuronal inclusions in the cerebral cortex (CC) and brain-stem (BS). Ultrastructurally, the CC contained intermediate-sized filaments with variable amounts of granular material and other organelles, whereas the BS consisted of an electron-dense core and an outer area with radially oriented filaments. The cerebral cortex was immuno-reactive with antibodies against tyrosine hydroxylase (TH) and tau protein, and differed from BS. In addition, although the CC were antigenically similar to BS in their neurofilament (70, 160 and 200 kDa) and ubiquitin contents, the localization of neurofilament immunoreactivity differed between them, being confined positively to the core of CC, but to the periphery of the BS. Although Lewy bodies (LB) in idiopathic Parkinson's disease are morphologically similar to BS, they have been reported to differ in their immunoreactivity with antibodies against tau. It has been reported that CC differ from LB with regard to immunoreactivity with antibodies against TH and tropomyosin. It is inferred that these inclusions (CC, BS and LB) differ in morphogenesis.

Aged↗

PubMed1993

Ileal duplication cyst associated with heterotopic pancreas: report of a case and literature review.

A rare case of solitary ileal duplication cyst accompanied by heterotopic pancreas in the terminal ileum is presented. An 8 year old boy with an ovoid shaped, elastic soft and pendant-growing ileal duplication cyst associated with aberrant pancreatic tissue but not communicating with the lumen of the ileum, underwent an adhesiotomy for a small intestinal adhesion that caused bowel obstruction. The obstructive state of the intestine seemed to be caused by an inflammation of the duplication cyst which was 4.5 x 2.7 x 2.5 cm and oval in shape. Excision of the cyst and the heterotopic pancreas was curative. The histological findings were identical to heterotopic pancreas accompanied by ileal duplication without any clinical features of heterotopic pancreas. A review of the literature is presented along with the author's experience in a case of ileal duplication cyst associated with heterotopic pancreas located in the ileum.

Child↗

PubMed1993

Molecular cloning and sequencing of the gene for a thermostable N-carbamyl-L-amino acid amidohydrolase from Bacillus stearothermophilus strain NS1122A.

The gene for N-carbamyl-L-amino acid amidohydrolase was cloned from Bacillus stearothermophilus strain NS1122A into E. coli. This gene started with a TTG triplet and was predicted to encode a peptide of 409 amino acids, with a calculated molecular weight of 44,248. The deduced amino acid sequence shared moderate homology with that of the corresponding enzyme of Pseudomonas sp. strain NS671.

Amidohydrolases↗

PubMed1993

Clinical, biological and histological considerations on primary intracranial germinomas in the prevalent sites.

Twenty-four germinomas from which tumor sites were 11 pineal, 7 suprasellar, and 6 basal ganglial and thalamic (BG-T) were subjected. Humoral tumor markers of HCG, CEA and AFP were measured and compared with immunohistochemistries for these tumor markers along with PLAP, HPL and PKK1. Humoral tumor markers and immunohistochemistries did not always comprehend to each other. Difference of the survival rates among tumor sites was thought to be attributed to the variabilities in their histological, immunochemical and flow cytometric results.

Adolescent↗

PubMed1992

Organ distribution and stability of phosphorothioated oligodeoxyribonucleotides in mice.

Results of recent studies in our laboratory have suggested a potential role for antisense oligodeoxyribonucleotides (oligo(dN)s) as therapeutic agents in the treatment of human hepatitis B virus infection. As a first step towards assessing the potential utility of oligo(dN) in therapy, we have examined the organ distribution, stability and toxicity of a phosphorothioated oligo(dN) (S-oligo) of 20 nucleotides in length which was administered to mice via different routes. Among the various organs analysed, the liver retained the highest amount of S-oligo (1.3-2 per cent of the total injection) at the peak time (10-30 min) regardless of the route of injection. However, the S-oligo appeared to be degraded in the liver to about 40 per cent of its original length within 30 min of injection, presumably by the action of 3' exonucleases. Injection of doses of up to 5 mg kg-1 of S-oligo had no apparent toxic effects on the mice.

Animals↗

PubMed1992

Identification and sequencing of a gene encoding a hydantoin racemase from the native plasmid of Pseudomonas sp. strain NS671.

DNA fragments containing the genes involved in the conversion of 5-substituted hydantoins to their corresponding L-amino acids have been cloned from the 172-kb native plasmid (pHN671) of Pseudomonas sp. strain NS671. The largest recombinant plasmid, designated pHPB14, encoded the ability to convert D-5-substituted hydantoins to the corresponding L-amino acids, whereas the smallest one, designated pHPB12, encoded the ability to convert them to their corresponding N-carbamyl-D-amino acids. Restriction analysis suggested that the inserts of both recombinant plasmids are derived from the identical portion in pHN671 and that the insert of pHPB14, compared with that of pHPB12, has an extra 5.3 kb in length. DNA sequencing revealed that pHPB14 contains two additional complete open reading frames, designated ORF5 and hyuE. Analysis of deletion derivatives of pHPB14 indicated that hyuE is required for the ability to produce L-amino acids from the corresponding D-5-substituted hydantoins, but ORF5 is not. Cells of Escherichia coli transformed with a plasmid containing hyuE were capable of racemizing different 5-substituted hydantoins, indicating that hyuE is a gene encoding a hydantoin racemase.

Amino Acid Sequence↗

PubMed1992

Purification and characterization of the hydantoin racemase of Pseudomonas sp. strain NS671 expressed in Escherichia coli.

The hydantoin racemase gene of Pseudomonas sp. strain NS671 had been cloned and expressed in Escherichia coli. Hydantoin racemase was purified from the cell extract of the E. coli strain by phenyl-Sepharose, DEAE-Sephacel, and Sephadex G-200 chromatographies. The purified enzyme had an apparent molecular mass of 32 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By gel filtration, a molecular mass of about 190 kDa was found, suggesting that the native enzyme is a hexamer. The optimal conditions for hydantoin racemase activity were pH 9.5 and a temperature of 45 degrees C. The enzyme activity was slightly stimulated by the addition of not only Mn2+ or Co2+ but also metal-chelating agents, indicating that the enzyme is not a metalloenzyme. On the other hand, Cu2+ and Zn2+ strongly inhibited the enzyme activity. Kinetic studies showed substrate inhibition, and the Vmax values for D- and L-5-(2-methylthioethyl)hydantoin were 35.2 and 79.0 mumol/min/mg of protein, respectively. The purified enzyme did not racemize 5-isopropylhydantoin, whereas the cells of E. coli expressing the enzyme are capable of racemizing it. After incubation of the purified enzyme with 5-isopropylhydantoin, the enzyme no longer showed 5-(2-methylthioethyl)hydantoin-racemizing activity. However, in the presence of 5-(2-methylthioethyl)hydantoin, the purified enzyme racemized 5-isopropylhydantoin completely, suggesting that 5-(2-methylthioethyl)hydantoin protects the enzyme from inactivation by 5-isopropylhydratoin. Thus, we examined the protective effect of various compounds and found that divalent-sulfur-containing compounds (R-S-R' and R-SH) have this protective effect.

Cloning, Molecular↗

PubMed1992

Cloning and sequencing of the genes involved in the conversion of 5-substituted hydantoins to the corresponding L-amino acids from the native plasmid of Pseudomonas sp. strain NS671.

Pseudomonas sp. strain NS671, which produces L-amino acids asymmetrically from the corresponding racemic 5-substituted hydantoins, harbored a plasmid of 172 kb. Curing experiments suggest that this plasmid, designated pHN671, is responsible for the conversion of 5-substituted hydantoins to their corresponding L-amino acids by strain NS671. DNA fragments containing the genes involved in this conversion were cloned from pHN671 in Escherichia coli by using pUC18 as a cloning vector. The smallest recombinant plasmid, designated pHPB12, contained a 7.5-kb insert DNA. The nucleotide sequence of the insert DNA was determined, and three closely spaced open reading frames predicted to encode peptides with molecular masses of 75.6, 64.9, and 45.7 kDa were found. These open reading frames were designated hyuA, hyuB, and hyuC, respectively. Cell extracts from E. coli carrying deletion derivatives of pHPB12 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gene products of hyuA, hyuB, and hyuC were identified. The functions of these gene products were also examined with the deletion derivatives. The results indicate that both hyuA and hyuB are involved in the conversions of D- and L-5-substituted hydantoins to corresponding N-carbamyl-D- and N-carbamyl-L-amino acids, respectively, and that hyuC is involved in the conversion of N-carbamyl-L-amino acids to L-amino acids.

Amino Acid Sequence↗

PubMed1991

Binding of oligonucleotides to cell membranes at acidic pH.

Antisense oligodeoxynucleotides [oligo(dN)] have the ability to enter living cells and block the expression of specific genes. However, little is known about the mechanism of cellular uptake of oligo(dN). We have found that oligo(dN) can bind to the cell membranes of eukaryotic cells with much greater efficiency under acidic conditions (pH 4.0-4.5) than at neutral pH. The binding appears to be specific to poly nucleic acids since various sizes of oligo(dN), DNA and RNA, but not mononucleotides, compete for the binding. We have identified a 34 kDa membrane protein from T-cells, which binds to oligo(dT) cellulose at pH 4.5 and can be eluted at pH 7.5. This protein fraction blocked the binding of oligo(dN) to living T-cells in a competitive fashion. Our results suggest that eukaryotic cells have a receptor for oligo(dN) at acidic pH and that the 34 kDa dalton protein on the cell membrane may mediate such binding.

Binding Sites↗

PubMed1991

Increased susceptibility of human fetal astrocytes to human T-lymphotropic virus type I in culture.

Human T-lymphotropic virus type I (HTLV-I) has been considered as an agent responsible for tropical spastic paraparesis and HTLV-I associated myelopathy. However, the pathogenesis of the diseases remains unclear. In a previous study we demonstrated that HTLV-I could infect adult human astrocytes and oligodendrocytes in vitro, although the rates of infected cells were low, at a rate of 0.1% and 0.01-0.05% respectively. Since mother-to-child transmission has been proposed as one of the major pathways for the prevalence of HTLV-I endemic, in the present study we investigated the susceptibility of human fetal astrocytes to HTLV-I in culture. After two days of co-culturing fetal brain cells with irradiated MT-2 cells (an HTLV-I-producing T-cell line), immunofluorescence staining revealed many positive astrocytes for HTLV-I p19 antigen. Multinucleated giant cells doubly immunoreactive to glial fibrillary acidic protein and HTLV-I antigen were frequently observed and showed a characteristic feature of hairy or fluffy external appearance. The percentage of infected astrocytes became as high as 19.4% at Day 21 of co-culture and then decreased. Electron microscopic examination revealed type C virus-like particles in astrocytes. These results indicate that human fetal astrocytes are more susceptible to HTLV-I infection than adult human astrocytes in tissue culture.

Antigens, Viral↗

PubMed1991

Comparison of immunochemical specificities of Vibrio parahaemolyticus O10 and O12 antigens using monoclonal antibodies.

The monoclonal antibodies against Vibrio parahaemolyticus O10 and O12 antigens (lipopolysaccharide, LPS) were prepared and specificities of the antibodies were examined. Five of six anti-O10 antibodies reacted with O10 antigen, but none of them reacted with O12 antigen. On the contrary all of the five anti-O12 antibodies reacted with O10 antigen as well as homologous O12 antigen. O10 and O12 antigens were subjected to alkali treatment or periodate oxidation, and reactivities of these chemically modified preparations with the monoclonal antibodies were examined. Reactivities of O10 with anti-O10 and anti-O12 antibodies were reduced by the above two chemical treatments, but that of O12 with anti-O12 was not. O-Deacetylation of O10 LPS by the alkaline treatment was confirmed by NMR spectroscopy. These results suggest contributions to O10-specificity of O-acetyl group and periodate sensitive sugar residue. Inhibition experiments of O10 and O12 homologous precipitations were also carried out with various sugars. From the results we concluded that O10 and O12 antigenic determinants were distinct entities, although O10 and O12 antigens have been reported to be similar and cross-reactive.

Antibodies, Monoclonal↗

PubMed1990

Mutational analysis of enhancer domains responsive to trans-activation by the X gene of human hepatitis B virus.

The X gene product of human hepatitis B virus (HBV) trans-activates the HBV enhancer. In order to identify domains responsive to trans-activation by the X gene, we introduced a series of mutations into the HBV enhancer and assayed the enhancer activities in the presence of the X gene product. Our results suggest that the EP domain of the enhancer is essential for trans-activation by the X gene.

Base Sequence↗

PubMed1990

Organ-specific distribution of isozymes of 5'-nucleotide phosphodiesterase in mouse.

1. The distribution of isozymes of 5'-nucleotide phosphodiesterase (E.C.3.1.4.1) was examined in various organs of mouse, including liver, spleen, pancreas, heart, lung, kidney, brain and blood. 2. Five isozymes were identified and designated as isozymes I through V. 3. These isozymes are distributed unevenly with respect to the various organs and clear differences were observed in the patterns of distribution among the organs examined. 4. The level of these isozymes was compared in serum of neonate and adult mice, and a higher level of isozyme I and a lower level of isozyme IV were found in neonates compared to adults. 5. These results suggest that each isozyme has different functional roles in individual organs and that these isozymes may be involved in proliferation and development of cells.

Animals↗