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S Hata

Publications and source records attributed to S Hata.

At least 109 records · Page 6Linked to original sources
PubMed1995

cDNA cloning of a putative G protein-coupled receptor from brain.

Using degenerate oligonucleotide primers corresponding to conserved regions of the G-protein coupled receptor superfamily, we carried out a low-stringency polymerase chain reaction and obtained two novel partial-length clones from a rat brain cDNA library. We used one of these clones for conventional library screening and isolated a longer cDNA clone, designated as RBU-15, from another rat brain library. Although RBU-15 was truncated at its 5' end, Northern blot analysis revealed that the gene was expressed in the brain and spleen. Next, we isolated a full-length cDNA clone, designated as HB-954, from a human fetal brain library, using RBU-15 as a probe. The deduced amino acid sequence of HB-954 contained four putative glycosylation sites in the N-terminal part, seven transmembrane domains, and a large cytosolic domain in the C-terminal part. The protein products of RBU-15 and HB-954 likely belong to a distinctive subfamily, because no receptors in the superfamily were found to be closely related to them.

Amino Acid Sequence↗
PubMed1995

Cloning, expression, and characterization of cDNAs encoding Arabidopsis thaliana squalene synthase.

We have isolated and characterized two overlapping cDNA clones for Arabidopsis thaliana squalene synthase. Their nucleotide sequences contained an open reading frame for a 410-amino acid polypeptide (calculated molecular mass, 47 kDa). The deduced amino acid sequence of the Arabidopsis polypeptide was significantly homologous (42-44% identical) to the sequences of known squalene synthases of several species, from yeast to man, but it was much less homologous to that of tomato phytoene synthase. To express the Arabidopsis enzyme in Escherichia coli, the entire coding region was subcloned into an expression vector. A cell-free extract of E. coli transformed with the recombinant plasmid, in the presence of NADPH and Mg2+, efficiently converted [14C]farnesyl diphosphate into squalene. On the other hand, in the absence of NADPH and the presence of Mn2+, the cell-free extract formed dehydrosqualene as a secondary product. Another E. coli extract expressing mouse squalene synthase showed the same activity as the Arabidopsis enzyme. Therefore, both the structure and reaction mechanism of squalene synthases are markedly conserved in taxonomically remote eukaryotes.

Alkyl and Aryl Transferases↗
PubMed1995

Nucleotide sequence of a cDNA for mouse squalene epoxidase.

A full-length cDNA encoding mouse squalene epoxidase was isolated by screening a mouse liver cDNA library with the rat squalene epoxidase gene as a probe. The cDNA had an open reading frame for a 572 amino acid polypeptide with a calculated molecular mass of 63.8 kDa. The predicted amino acid sequence of the mouse enzyme contained an FAD-binding motif, and was 93% identical to that of the rat enzyme. The former is one amino acid shorter than the latter. Blotting analyses showed that the mRNA is 2.8 kb in size and that a single copy of the gene is present in the mouse genome.

Amino Acid Sequence↗
PubMed1995

Identification of two splice isoforms of mRNA for mouse hepatocyte nuclear factor 4 (HNF-4).

Hepatocyte nuclear factor 4 (HNF-4) is a liver-enriched transcription factor involved in the expression of many liver-specific genes. In the preceding communication (Hata, S., Tsukamoto, T. and Osumi, T. (1992) Biochim. Biophys. Acta 1131, 211-213), we reported the presence of two isoforms of mRNA for HNF-4 in rat liver and kidney. The longer isoform contained a segment of 30 bases which was not present in the shorter one. As an initial step to determine whether or not other mammals have these mRNA isoforms, we isolated a cDNA for mouse HNF-4 using the rat HNF-4 gene as a probe. The cDNA had an open reading frame for a 465 amino acid polypeptide. The deduced amino acid sequence was remarkably conserved between mouse HNF-4 and rat HNF-4 (99.6% identical). Moreover, like the cDNA for the larger rat isoform, the mouse cDNA contained an extra segment of 30 bp in the coding region near the C-terminus. Blotting analyses showed that the mRNA is about 3.7 kb in size and that a single copy of the gene is present in the mouse genome. Next we carried out the polymerase chain reaction (PCR) using primers located just upstream and downstream of the extra segment. Two PCR products were amplified from a mouse liver cDNA library. Determination of their nucleotide sequences proved that they exactly corresponded to the two rat isoforms. Finally, we amplified a DNA fragment (1.1 kb in size) from mouse genomic DNA using the same PCR primers as above. Its nucleotide sequence unequivocally confirmed that different splice donor sites were used to generate the two isoforms.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and functional expression of a cDNA for mouse squalene synthase.

Using a probe obtained by PCR amplification, a full-length cDNA encoding squalene synthase was isolated from a mouse liver cDNA library. Its nucleotide sequence had an open reading frame fro a 416 amino acid polypeptide (calculated molecular mass, 48 kDa). In vitro transcription of the cDNA followed by in vitro translation produced a protein of the expected size. The deduced amino acid sequence was 93%, 88% and 46% identical to those of the rat, human and budding yeast squalene synthases, respectively. Blotting analyses showed that the mRNA is 1.6 kb in size and that less than two copies of the gene are present in the mouse genome. To establish the enzyme activity, the entire coding region was subcloned into an expression plasmid so that it was in frame with the N-terminal region of beta-galactosidase. Escherichia coli, which was transformed with the recombinant plasmid, expressed high activity of converting farnesyl diphosphate into squalene.

Amino Acid Sequence↗
PubMed1995

Distinct classes of mitotic cyclins are differentially expressed in the soybean shoot apex during the cell cycle.

Protein phosphorylation by the complexes of cyclin and cyclin-dependent kinase plays a key role in cell cycle progression in all eukaryotes. The amplification by polymerase chain reaction of a cyclin box from developing root nodules and root apices of soybean showed the expression of a number of different molecular species of mitotic cyclins in plant meristems, and they were classified into five distinct groups based on their sequence similarities. The complete soybean cyclin cDNAs, cyc1Gm to cyc5Gm, corresponding to each group were isolated, and their predicted amino acid sequences showed clear similarities to mitotic cyclins identified from various organisms. These genes are expressed predominantly in such meristematic tissues as root and shoot apices and young developing nodules. Double-target in situ hybridization involving histone H4 as an S-phase marker allowed us to estimate the phases during which these cyclin genes are abundantly expressed. The results indicated that cyc5Gm is expressed in G2-to-M phases and cyc3Gm is expressed from late S-to-G2 phases. These expression patterns, together with the sequence criteria, strongly suggest that cyc3Gm and cyc5Gm encode the plant cognates for A- and B-type cyclins, respectively. In addition, the expression of cyc1Gm was restricted during a short period in S phase, suggesting that it belongs to a novel class of plant cyclins. Sequence comparison of 18 plant mitotic cyclins cloned thus far showed that they can be divided into four distinct structural groups with different functions in cell cycle progression.

Amino Acid Sequence↗
PubMed1995

Simultaneous monitoring of intracellular pH and proton excretion during glycolysis by Streptococcus mutans and Streptococcus sanguis: effect of low pH and fluoride.

A system was developed by which 2',7'-bis(carboxyethyl)-4 or 5-carboxyfluorescein could be used to monitor intracellular pH at the same time that proton excretion was being measured. Streptococcal cells were loaded with the dye, and after the addition of glucose protons were excreted and the intracellular pH increased quickly and remained higher than the extracellular pH of 7.0. The excretion of protons stopped and the intracellular pH returned to the original level when glucose was depleted. The intracellular level of ATP remained high during glucose metabolism and decreased with the depletion of glucose. At extracellular pH of 5.5, and 5.0, the intracellular pH of fasting cells was higher than the extracellular pH value. After addition of glucose there were initial lags of proton excretion and of increases in intracellular pH at the acidic extracellular pH values. In the presence of fluoride, a lag in proton excretion and a simultaneous decrease in intracellular pH were observed, indicating a partial and transient inhibition of proton-ATPase activity.

Adenosine Triphosphate↗
PubMed1995

cGMP upregulates nitric oxide synthase expression in vascular smooth muscle cells.

8-Bromo-guanosine 3':5'-cyclic monophosphate (8-Br-cGMP), an analogue of cyclic guanosine monophosphate (cGMP), induced a time- and dose-dependent enhancement of interleukin-1-induced nitric oxide production in vascular smooth muscle cells. Human atrial natriuretic polypeptide, which stimulates cGMP accumulation in vascular smooth muscle cells, also enhanced interleukin-1-induced nitric oxide release at a concentration of 100 nmol/L. In contrast, coincubation with 10 mumol/L methylene blue, an inhibitor of soluble guanylate cyclase, inhibited interleukin-1-induced nitric oxide release from vascular smooth muscle cells. Furthermore, coincubation with 8-Br-cGMP also enhanced the interleukin-1-induced increase in inducible nitric oxide synthase messenger RNA in vascular smooth muscle cells. However, the enhancement of nitric oxide production induced by 8-Br-cGMP was significantly prevented by coincubation with neutralizing antibody against tumor necrosis factor-alpha. Furthermore, 8-Br-cGMP enhanced the interleukin-1-induced increase in tumor necrosis factor-alpha messenger RNA level in vascular smooth muscle cells. These findings indicate that cGMP may upregulate inducible nitric oxide synthase gene expression through the stimulation of tumor necrosis factor-alpha production in vascular smooth muscle cells. Thus, there may be a positive feedback mechanism between nitric oxide and the cGMP system in vascular smooth muscle cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Endothelin-1 enhances nitric oxide-induced cytotoxicity in vascular smooth muscle.

Prolonged incubation with 1 nmol/L interleukin-1 induced high levels of nitric oxide release and cytotoxicity in vascular smooth muscle cells. NG-Monomethyl-L-arginine, an inhibitor of nitric oxide synthesis, inhibited interleukin-1-induced cytotoxicity at a concentration of 3 mmol/L. Furthermore, prolonged incubation with 0.1 mmol/L sodium nitroprusside, a nitric oxide donor, also induced cytotoxicity. On the other hand, endothelin-1 at concentrations from 10(-10) to 10(-7) mol/L induced a concentration-dependent enhancement of cytotoxicity induced by interleukin-1. However, endothelin-1 did not affect interleukin-1-induced nitric oxide production. Coculture study of vascular smooth muscle cells and endothelial cells without direct cell contact revealed that incubation for 72 hours with interleukin-1 induced high levels of nitric oxide release from cocultured vascular smooth muscle cells to the same degree as release from vascular smooth muscle cells alone. However, interleukin-1-induced cytotoxicity was more enhanced in cocultured vascular smooth muscle cells than in vascular smooth muscle cells alone. Furthermore, coincubation with 20 nmol/L BQ-485, an antagonist of one type of endothelin receptor (ETA), prevented the enhancement of interleukin-1-induced cytotoxicity in cocultured vascular smooth muscle cells. These findings suggest that endothelin-1 secreted from endothelial cells may enhance nitric oxide-induced cytotoxicity by means of the ETA receptor in vascular smooth muscle cells.

Animals↗
PubMed1994

Expression of functional proliferating-cell nuclear antigen from rice (Oryza sativa) in Escherichia coli. Activity in association with human DNA polymerase delta.

Proliferating-cell nuclear antigen (PCNA), the auxiliary protein for DNA polymerase delta, is one of the key factors for both PCNA-dependent DNA synthesis and cell-cycle progression. Plant PCNA genes have previously been cloned from rice, carrot, tobacco, and soybean cells by screening the cDNA libraries using similarity to the human or rat PCNA genes. We subcloned the relevant gene from the rice PCNA cDNA into an Escherichia coli expression vector pMAL, and the PCNA protein was expressed in the bacteria in the form of a fusion protein (70 kDa) with maltose-binding protein (MBP). Monoclonal antibody against human PCNA reacted with both purified fusion protein and a 32-kDa fragment, resulting from restriction protease (factor Xa) digestion of the fusion protein. The N-terminal amino acid sequence of the 32-kDa fragment was identical to that of rice PCNA sequence. Rice PCNA fusion protein was found to stimulate DNA synthesis catalyzed by DNA polymerase delta from human cells (although much less effectively), while having no effect on DNA polymerase alpha activity. The results indicate that plant PCNA functions as one of the cofactors of DNA synthesis as is the case with other eukaryotes.

Amino Acid Sequence↗
PubMed1994

Column-switching high-performance liquid chromatographic analysis of BO-2727, a new carbapenem antibiotic, in human plasma and urine by direct injection.

A column-switching high-performance liquid chromatographic method has been developed for the simple and sensitive analysis of BO-2727 (I) in human plasma and urine. Plasma samples were diluted with an equal volume of a stabilizer, and the mixture was directly injected onto the HPLC system. The analyte was enriched in a pre-treatment column, while endogenous components were eluted to waste. The analyte was then backflushed onto an analytical column and quantified with ultraviolet detection. Urinary concentrations were determined in a similar way except that the enriched analyte was eluted in the foreflush mode to a cation-exchange column used for chromatographic separation. The standard curves for the drug were linear in the range of 0.05-50 micrograms/ml in plasma and 0.5-100 micrograms/ml in urine. The limits of quantification for plasma and urine were found to be 0.5 micrograms/ml and 0.5 micrograms/ml, respectively. This method was used to support Phase I clinical pharmacokinetic studies.

Anti-Infective Agents↗
PubMed1994

Characterization of the signal peptide at the amino terminus of the rat peroxisomal 3-ketoacyl-CoA thiolase precursor.

The amino-terminal presequences of rat peroxisomal 3-ketoacyl-CoA thiolase precursors (types A and B) were reported to be cleavable signal peptides for peroxisomal protein translocation. In the present study, this was proven by immunoelectron microscopy of the cultured Chinese hamster ovary cells stably expressing fusion proteins of the amino-terminal sequences of the thiolase precursor and Escherichia coli dihydrofolate reductase. The fusion proteins were processed into mature forms of the apparently correct sizes. Site-directed mutagenesis studies of the charged residues in the B-type presequence (26 amino acid residues) revealed that arginine at position -24 and histidine at position -17 were both indispensable. Even replacement of these residues with other basic amino acids abolished the import activity. Both Arg-24 and His-17 were also required in a longer presequence (36 amino acid residues) of the thiolase A, thereby suggesting that the signal can function in an internal position. When glutamic acid at position -11 was changed to amino acids other than aspartic acid, the signal peptide became apparently effective in both peroxisomal and mitochondrial targeting. All of these data indicate that the thiolase signal peptide is a newly defined type of peroxisomal targeting signal recognized by a mechanism presumably different from that for a known peroxisomal signal, the carboxy-terminal Ser-Lys-Leu-COOH motif.

Acetyl-CoA C-Acyltransferase↗
PubMed1994

Effect of amino acids in total parenteral nutrition on cholestasis in newborn rabbits.

The effect of amino acids on total parenteral nutrition (TPN)-associated cholestasis was examined using a newborn rabbit TPN model. Twenty-nine newborn Japanese white rabbits were divided into four groups. Group I received a TPN solution with composition and total energy similar to that of rabbit breast milk. Group II received more dextrose than group I. Group III received a larger amount of amino acids than group II. Group IV was nourished by lactating mothers. After 7 days of TPN, blood samples and liver specimens were obtained. In group II, the serum total bilirubin level (1.44 +/- 0.68 mg/dL) was significantly higher than normal (0.28 +/- 0.07 mg/dL) and that of group I (0.49 +/- 0.12 mg/dL). In group II, histological cholestasis was present, characterized by bile plugs in bile ducts, bile pigments in Kupffer cells and hepatocytes, and nonprotein calorie overload changes in clear cell transformation of the hepatocytes. In group III, the serum total bilirubin level (0.23 +/- 0.05 mg/dL) was normal, and there were minimal cholestatic and nonprotein calorie overload changes (histologically) in the liver. These results indicate that cholestasis in this study was induced by nonprotein calorie overload and was prevented by an appropriate volume of amino acids.

Amino Acids↗
PubMed1994

Quick stretch increases the production of inositol 1,4,5-trisphosphate (IP3) in porcine coronary artery.

The present study was undertaken to know whether the formation of inositol 1,4,5-trisphosphate (IP3) is increased by quick stretch, a dynamic mechanical stimulus in porcine coronary artery in order to inquiry the possibility that IP3 could mediate Ca2+ release in the stretch-induced contraction. Quick stretching of a helical strip of porcine coronary artery at a rate of 10 cm/sec, the amount of stretch equivalent to 140% of the initial muscle length (= 100%), and the stimulus period of 30 sec with 20-min intervals, produced delayed contraction. Quick stretching increased the content of IP3 about three-fold over the control basal level, which always preceded the contraction. A putative phospholipase C inhibitor, 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC), abolished the increase in the formation of IP3 and partially inhibited the stretch-induced contraction. The results suggest that quick stretching increases the formation of IP3 through a possible mechanism for activation of phospholipase C, which may lead to release of Ca2+ into myoplasm and to further activation of the contractile elements.

Acetylcholine↗
PubMed1994

Antenatal diagnosis of sacrococcygeal teratoma with hydrops fetalis; a case report.

Fetal sacrococcygeal teratoma (SCT) with hydrops fetalis is known to have a poor prognosis. We report a case of SCT with hydrops fetalis which was diagnosed antenatally at 30 weeks gestation. After delivery by cesarean section, the tumor was removed and the infant is alive one year after surgery with no sign of recurrence. However, he has profound brain damage, perhaps caused by hypoxia secondary to the perinatal hypovolemia. This case demonstrates that a 30 weeks' gestation, survival is possible in fetal cases of SCT with hydrops fetalis, and illustrates the importance of perinatal circulatory management.

Adult↗
PubMed1994

Pharmacokinetics and safety of BO-2727, a new injectable 1-beta-methyl carbapenem antibiotic, and its effect on the faecal microflora in healthy male volunteers.

The safety and pharmacokinetics of BO-2727, a new injectable 1-beta-methyl carbapenem antibiotic, and its effect on the faecal microflora were evaluated in single- and multiple-dose studies involving twenty-four healthy male adult volunteers following a preliminary safety evaluation of 25, 50 and 125 mg iv doses in six volunteers. BO-2727 was administered by iv infusion over 30 min. There was a good correlation between BO-2727 concentrations assayed by HPLC and a microbiological method; the HPLC results were used in the pharmacokinetic analysis. The single-dose study indicated that plasma concentration versus time curves at doses of 250, 500 and 1000 mg were well described by a two-compartment open model. The mean (+/- S.D.) elimination half-life ranged from 1.41 +/- 0.21 h to 1.54 +/- 0.10 h, and the peak plasma concentrations (Cmax) and the area under the plasma concentration versus time curves increased linearly with the dose. The mean urinary recoveries within the first 24 h were 70.63 +/- 5.24% to 77.38 +/- 4.55% of the dose. The highest concentration of BO-2727 in saliva was 0.72 mg/L which was reached 1 h after the start of the 1000 mg infusion and accounted for approximately 1% of the Cmax. No BO-2727 was found in faecal samples collected 24 and 48 h after a single 1000 mg dose. In the multiple-dose study, BO-2727 500 mg bd was administered for 4.5 days. There were no obvious differences in plasma concentrations and urinary recoveries between the single- and multiple-dose regimens. BO-2727 did not accumulate as determined by plasma concentrations and urinary recoveries. No marked changes in the aerobic and anaerobic faecal microflora were observed during multiple-dose administration. There were no significant adverse reactions, and likewise no abnormalities in physical and laboratory examinations that were definitely related to the drug.

Adolescent↗
PubMed1994

Anti-human IgE monoclonal antibodies recognizing epitopes related to the binding sites of high and low affinity IgE receptors.

Anti-human IgE monoclonal antibodies (mAbs) were produced and eight clones recognizing epitopes on native IgE were selected. Epitopes were mapped by a competitive inhibition enzyme-linked immunosorbent assay, Western blotting and a multi-pin peptide technology. Four sites (one each in the C epsilon 1, C epsilon 2, C epsilon 2/C epsilon 3 junction and C epsilon 3) were recognized by the mAbs. The relationship between the four epitopes and the binding sites of high and low affinity IgE receptors (Fc epsilon RI and Fc epsilon RII, respectively) was studied using a monovalent Fab fragment of each mAb as a binding inhibitor. The IgE-Fc epsilon RII binding was clearly inhibited by the mAb recognizing the C epsilon 2/C epsilon 3 junction, suggesting that Fc epsilon RII binds to a rather limited area around the C epsilon 2/C epsilon 3 junction. The IgE-Fc epsilon RI binding, on the other hand, was scarcely inhibited by any single mAb. However, the binding was inhibited when the epitope in C epsilon 2 was blocked simultaneously with that at the C epsilon 2/C epsilon 3 junction or with that in C epsilon 3, indicating that these three distinct epitopes are related to the Fc epsilon RI binding sites. When these three epitopes were shown in the stereograph of human IgE, the Fc epsilon RI binding area was spread largely on the groove side between C epsilon 2 and C epsilon 3 domains. These results suggest that Fc epsilon RI acquires the high affinity through multiple bindings.

Amino Acid Sequence↗