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Biomedical subjects

K Sugimoto

Publications and source records attributed to K Sugimoto.

At least 19 recordsLinked to original sources

Dosage suppressors of the dominant G1 cyclin mutant CLN3-2: identification of a yeast gene encoding a putative RNA/ssDNA binding protein.

Three G1 cyclins, CLN1, CLN2, and CLN3, have been identified in the budding yeast Saccharomyces cerevisiae. G1 cyclins are essential, albeit functionally redundant, rate-limiting activators of cell cycle initiation. We have isolated dosage-dependent suppressor genes (designated HMD genes) of the mating defect caused by CLN3-2, a dominant mutation in CLN3, HMD2 and HMD3 are identical to STE4 and STE5, respectively, HMD1 is an essential gene that encodes a protein containing a putative RNA binding domain. Overproduction of HMD1 results in a relatively specific reduction in the level of the CLN3 or CLN3-2 transcript. This reduction occurs subsequent to transcription initiation of CLN3 since overexpression of HMD1 did not affect expression of a heterologous transcript from the CLN3 promoter but did result in a reduction of CLN3 transcript expressed from a heterologous promoter. HMD1 has at least one essential role independent of its effect on CLN3 since HMD1 remains essential for viability in the absence of a functional CLN3 gene.

Amino Acid Sequence

Interleukin-6 inhibits the potent stimulatory action of androgens, glucocorticoids and interleukin-1 alpha on apolipoprotein D and GCDFP-15 expression in human breast cancer cells.

Our study was designed to investigate the potential interaction between steroid hormones and interleukin-6 (IL-6) in the regulation of apolipoprotein D (apo-D) and gross cystic disease fluid protein 15 (GCDFP-15) expression in ZR-75-1 human breast cancer cells. We first observed that exposure to IL-6 for 6-14 days decreased basal apo-D and GCDFP-15 secretion by 50% and 23%, respectively. In the same experiment, such treatment with IL-6 decreased cell proliferation by approximately 40% after 6 and 14 days of incubation. Exposure to IL-6 markedly decreased dihydrotestosterone (DHT)-induced apo-D and GCDFP-15 release, with a half-maximal effect measured at 13 U/ml. A similar inhibitory action of IL-6 was observed on the glucocorticoid dexamethasone (DEX)-induced apo-D and GC-DFP-15 secretion. The sensitivity of the apo-D and GCDFP-15 response to the stimulatory action of DHT or DEX was, however, not changed by concomitant exposure to IL-6. The inhibitory effect of IL-6 on the secretion of these two biochemical markers was additive to that of 17 beta-estradiol. In addition, IL-6 blocked the stimulatory effect of interleukin-1 alpha (IL-1 alpha) on apo-D and GCDFP-15 secretion. Our results show that IL-6 is a potent inhibitory of basal as well as androgen-, glucocorticoid- and IL-1 alpha-induced apo-D and GCDFP-15 secretion in ZR-75-1 human breast cancer cells, while cell proliferation is inhibited by this cytokine.

Androgens

HYS2, an essential gene required for DNA replication in Saccharomyces cerevisiae.

To investigate cell cycle regulation at the S or G2 phase in Saccharomyces cerevisiae, we have isolated mutants displaying supersensitivity to hydroxyurea (HU), a chemical that inhibits DNA replication. Such mutants, which we have named hydroxyurea sensitive (hys), defined four linkage groups and we characterized the hys2 mutation in this study. The hys2-1 mutant displays temperature sensitive growth and a constellation of phenotypes indicating defective DNA metabolism. At the restrictive temperature, hys2-1 cells arrest as large budded cells with a single nucleus at the neck of the bud and a short spindle. The hys2-1 mutant exhibits increased rates of chromosome loss and recombination. Additionally, hys2-1 appears to accumulate incompletely replicated DNA that can be detected by a pulse field electrophoresis assay. Finally, deletion of RAD9 in a hys2-1 strain decreases the percentage of arrested cells, suggesting that an intact RAD9-checkpoint is required for the cell cycle arrest in hys2-1 cells. HYS2 encodes a 55 kDa protein that is essential for viability at all temperatures. Taken together, these data suggest that Hys2 plays a role in DNA replication.

Base Sequence

Genome structure, mapping and expression of the tropomyosin gene tmy-1 of Caenorhabditis elegans.

The complete tropomyosin gene, designated tmy-1, of Caenorhabditis elegans was recovered by genome walking from a fragment that was obtained by exon-expression cloning using specific cloning using specific anti-tropomyosin antiserum as a probe. The genome structure of the tmy-1 gene has been determined by combining the DNA sequences of cDNA clones with those of the genomic fragments. The single-copy gene spans approximately 13 kb and include 14 exons. Comparison of cDNA and genomic sequences demonstrates that three isoforms are encoded by the gene tmy-1. Homology of the 27 C-terminal amino acid residues to those of Drosophila and vertebrates suggest that these may be the body wall, pharyngeal and non-muscle types. Tissue-specific expression of the tmy-1 gene was determined by microinjection of a promoter/lacZ fusion gene and with immunohistochemistry by using affinity-purified tissue-specific anti-tropomyosins. The 5' end promoter common to CeTMI and CeTMII is expressed in the body wall muscles, vulva, anus muscles and male tail muscles. Control sequences of the 5' end promoter are located 660 to 800 bp upstream of the initial methionine codon. The third isoform, CeTMIII, encoding 256 amino acids residues was expressed in the pharyngeal muscles by the promoter in the third intron. The mRNA of CeTMIII was trans-spliced with SL1 and SL2. These results allow is to solve the question of what is common from this worm to vertebrates, and also what are the cross-species complexities and the tissue-specific differences of tropomyosins. The tmy-1 gene is located on the C. elegans genomic YAC grid near the right end of chromosome I, in the region on the lev-11 gene.

Alternative Splicing

Effects of nicorandil on kinetics of oxygen uptake at the onset of exercise in patients with coronary artery disease.

The beneficial effects of coronary vasodilators on exercise capacity in patients with angina pectoris are well known. However, their effects on oxygen uptake (VO2) kinetics at the onset of exercise have not been elucidated. The present study was undertaken to determine the acute effects of nicorandil, a newer coronary vasodilator, on the kinetics of VO2 at the onset of exercise in patients with ischemic heart disease. Ten patients with significant coronary stenosis performed constant mild-intensity cycle exercise (32 +/- 3 W) for 6 minutes after oral administration of 10 mg of nicorandil or an identical placebo in a double-blind, crossover manner. Nicorandil had no effect on resting heart rate, blood pressure, or VO2. However, the time constant for the increase in VO2 during constant work rate exercise was significantly shorter (the kinetics of VO2 were faster) after administration of nicorandil than after placebo (46.5 +/- 13.3 vs 51.1 +/- 11.9 seconds; p = 0.039). The increase in VO2 at 6 minutes compared with 3 minutes of constant work, which reflects the VO2 kinetics, also was reduced with nicorandil (3.8 +/- 37.9 vs 27.5 +/- 27.1 ml/min; p = 0.022). Nicorandil was found to increase the rate of VO2, increase during the onset of constant work rate exercise, probably as a result of an improved response in cardiac output. Analysis of VO2 kinetics provides new and useful parameters for the evaluation of circulatory adjustments at the onset of exercise in patients with ischemic heart disease.

Adult

Molecular cloning of cDNA encoding mouse Cdc21 and CDC46 homologs and characterization of the products: physical interaction between P1(MCM3) and CDC46 proteins.

Two new mouse genes encoding proteins that belong to the yeast minichromosome maintenance (MCM) protein family, which is involved in the initiation of DNA replication, were isolated and their nucleotide sequence was determined. They were a putative CDC46/MCM5 homolog and a putative cdc21 homolog. About 30% amino acid identity was obtained between members in the family, and > 40% between the putative mouse and yeast homologs. The expression of these genes was cell-cycle specific at the late G1 to S phase. Immunochemical analyses showed the physical interaction between mouse P1MCM3 and CDC46 protein. These results suggest that MCM proteins function in co-ordination for DNA replication.

Animals

Cloning and sequencing of beta-mannanase gene from Bacillus subtilis NM-39.

A gene encoding beta-mannanase from Bacillus subtilis NM-39 was cloned into Escherichia coli DH5 alpha by using pUC 18 and its nucleotide sequence was determined. The beta-mannanase gene was 1080 base pairs long and encoded a mature protein of 336 amino acids and a signal peptide of 24 amino acids. The deduced amino acid sequence of the cloned mannanase showed sequence homology with mannanase from alkalophilic Bacillus sp. strain AM-001 (about 50%).

Amino Acid Sequence

Opioidergic inhibition of capsaicin-evoked release of glutamate from rat spinal dorsal horn slices.

We investigated the effects of opioid agonists on the capsaicin-evoked release of glutamate from nociceptive primary afferent fibers of the rat (6-8 weeks) using a fluorometric on-line continuous monitoring system for glutamate. In the presence of 0.3 microM tetrodotoxin, the application of 3 microM capsaicin to spinal dorsal horn slices produced an evoked glutamate release (55.9 +/- 4.02 pmol.mg-1 protein, n = 15). DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin; 0.3-10 microM) and morphine (1-30 microM), mu-opioid agonists, produced a concentration-dependent reduction (approximately 85 and approximately 77% reduction, respectively) in the capsaicin (3 microM)-evoked release of glutamate. These inhibitory effects were significantly antagonized by naloxone (1 microM). DPDPE ([D-Pen2,5]enkephalin; 1-10 microM), a delta-opioid agonist, also reduced the capsaicin-evoked release in a concentration-dependent manner (approximately 59% reduction). Naltrindole (1 microM), a selective delta-antagonist, significantly antagonized the inhibitory effect of DPDPE (10 microM). In contrast, neither U-50,488H (1-10 microM) nor U-69,593 (10 microM), kappa-opioid agonists, had any effects on the evoked release of glutamate. These results suggest that mu-, and delta-opioid agonists modulate pain transmission in the spinal dorsal horn, at least in part, by inhibiting the release of glutamate from capsaicin-sensitive primary afferents.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh

Effect of green tea rich in gamma-aminobutyric acid on blood pressure of Dahl salt-sensitive rats.

gamma-Aminobutyric acid (GABA) is known to be involved in the regulation of blood pressure by modulating the neurotransmitter release in the central and peripheral sympathetic nervous systems. This study investigated the antihypertensive effect of green tea rich in GABA (GABA-rich tea) in young and old Dahl salt-sensitive (S) rats. GABA-rich tea was made by fermenting fresh green tea leaves under nitrogen gas. In experiment 1, 21 11-month-old rats, fed a 4% NaCl diet for 3 weeks, were given water (group W), an ordinary tea solution (group T), or a GABA-rich tea solution (group G) for 4 weeks. The average GABA intake was 4.0 mg/rat per day. After 4 weeks of the treatment, blood pressure was significantly decreased in group G (176 +/- 4; P < .01) compared with group W (207 +/- 9) or group T (193 +/- 5 mm Hg). Plasma GABA levels were more elevated in group G (111 +/- 54) than in group W (not detectable) or group T (14 +/- 8 ng/mL; P < .01 v G). In experiment 2, 21 5-week-old rats, fed a 4% NaCl diet, were divided into groups W, T, and G. The average GABA intake was 1.8 mg/rat per day. Body weight or chow and beverage consumption did not differ significantly among the three groups. After 4 weeks of the treatment, although blood pressure was comparable in groups W and T (165 +/- 3 v 164 +/- 5 mm Hg, mean +/- SE), it was significantly lower in group G (142 +/- 3 mm Hg) than in the other groups (P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Brain histaminergic system in mast cell-deficient (Ws/Ws) rats: histamine content, histidine decarboxylase activity, and effects of (S) alpha-fluoromethylhistidine.

The mast cell-deficient [Ws/Ws (White spotting in the skin)] rat was investigated with regard to the origin of histamine in the brain. No mast cells were detected in the pia mater and the perivascular region of the thalamus of Ws/Ws rats by Alcian Blue staining. The histamine contents and histidine decarboxylase (HDC) activities of various brain regions of Ws/Ws rats were similar to those of +/+ rats except the histamine contents of the cerebral cortex and cerebellum. As the cerebral cortex and cerebellum have meninges that are difficult to remove completely, the histamine contents of these two regions may be different between Ws/Ws and +/+ rats. We assume that the histamine content of whole brain with meninges in Ws/Ws rats is < 60% of that in +/+ rats. So we conclude that approximately half of the histamine content of rat brain is derived from mast cells. Next, the effects of (S) alpha-fluoromethylhistidine (FMH), a specific inhibitor of HDC, on the histamine contents and HDC activities of various regions of the brain were examined in Ws/Ws rats. In the whole brain of Ws/Ws rats, 51 and 37% of the histamine content of the control group remained 2 and 6 h, respectively, after FMH administration (100 mg/kg of body weight). Therefore, we suggest that there might be other histamine pools including histaminergic neurons in rat brain.

Alcian Blue

Cloning and sequencing of sulfite reductase alpha subunit gene from Saccharomyces cerevisiae.

A DNA fragment of 2.1 kb was specifically amplified by PCR with primers based on the amino acid sequences obtained from the N-terminal region and the cyanogen bromide-derived peptide of the sulfite reductase alpha subunit in Saccharomyces cerevisiae. With this fragment as a probe, the gene coding for the sulfite reductase alpha subunit was isolated from a genomic library of S. cerevisiae. Sequencing analysis revealed that the gene contains a 3105-bp open reading frame, which is large enough to code for a protein of 1035 amino acid residues. The transcript of the sulfite reductase alpha subunit gene was detected by Northern analysis after methionine deprivation, but the amount of the transcript did not directly correlate with the enzyme activity. The DNA fragment containing the sulfite reductase alpha subunit gene rescued the met10 phenotype by complementation.

Amino Acid Sequence

Large-volume intraoperative peritoneal lavage with an assistant device for treatment of peritonitis caused by blunt traumatic rupture of the small bowel.

The benefits of large-volume intraoperative peritoneal lavage (IOPL), with an assistant lavage device, were evaluated retrospectively in 114 patients with peritonitis caused by blunt traumatic rupture of the small bowel. Postoperative complications caused by infection were a major problem after rupture of the small bowel (46 of 114, 39.4%). Both prolongation of the interval between injury and laparotomy and rupture of the lower part of the small bowel were risk factors for postoperative complications caused by infection. Large-volume IOPL (25.2 +/- 2.1 L) with an assistant lavage device reduced the rate of complications caused by infection from 30 of 58 (51.8%) to 15 of 56 (26.8%). The volume used for IOPL was closely related to the occurrence of postoperative complications resulting from infection. No complications from infection occurred in patients who received lavage with of 28.3 +/- 2.7 L of saline, whereas complications occurred in those patients treated with a smaller volume of lavage fluid (18.0 +/- 2.5 L). Large-volume IOPL should be considered in patients with blunt rupture of the small bowel who are at risk for infection, and the assistant device for IOPL may be useful for such treatment.

Adolescent

Purification and characterization of the Pseudomonas aeruginosa NfxB protein, the negative regulator of the nfxB gene.

The protein NfxB, involved in conferring resistance to quinolones in Pseudomonas aeruginosa, has a helix-turn-helix motif which is similar to that of other DNA-binding proteins. It appears to affect the membrane-associated energy-driven efflux of some antibiotics (H. Nikaido, Science 264:382-388, 1994). We constructed a plasmid that overproduced NfxB in Escherichia coli and purified the protein. Two species of NfxB (23 and 21 kDa), which are probably translated from different initiation codons, were isolated. Both proteins are also expressed in vivo in P. aeruginosa, with the 23-kDa NfxB being the major species. NfxB specifically binds upstream of the nfxB coding region as demonstrated by gel retardation and DNase I footprinting. Expression of the phi (nfxB'-lacZ+) (Hyb) gene was repressed in the presence of the nfxB gene product provided by a second compatible plasmid in E. coli. In the P. aeruginosa wild-type strain (PAO2142), NfxB was undetectable by immunoblotting; however, it was detected in the nfxB missense mutant (PK1013E). These results suggested that NfxB negatively autoregulates the expression of nfxB itself. Since the 54-kDa outer membrane protein (OprJ) (N. Masuda, E. Sakagawa, and S. Ohya, Antimicrob. Agents Chemother. 39:645-649, 1995) was overproduced in nfxB mutants, NfxB may also regulate the expression of membrane proteins that are involved in the drug efflux machinery of P. aeruginosa.

Amino Acid Sequence

The EGP1 gene may be a positive regulator of protein phosphatase type 1 in the growth control of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae GLC7 gene encodes the catalytic subunit of type 1 protein phosphatase (PP1) and is required for cell growth. A cold-sensitive glc7 mutant (glc7Y170) arrests in G2/M but remains viable at the restrictive temperature. In an effort to identify additional gene products that function in concert with PP1 to regulate growth, we isolated a mutation (gpp1) that exacerbated the growth phenotype of the glc7Y170 mutation, resulting in rapid death of the double mutant at the nonpermissive temperature. We identified an additional gene, EGP1, as an extra-copy suppressor of the glc7Y170 gpp1-1 double mutant. The nucleotide sequence of EGP1 predicts a leucine-rich repeat protein that is similar to Sds22, a protein from the fission yeast Schizosaccharomyces pombe that positively modulates PP1. EGP1 is essential for cell growth but becomes dispensable upon overexpression of the GLC7 gene. Egp1 and PP1 directly interact, as assayed by coimmunoprecipitation. These results suggest that Egp1 functions as a positive modulator of PP1 in the growth control of S. cerevisiae.

Amino Acid Sequence

Separate control of the survival, the self-renewal and the differentiation of hemopoietic stem cells.

Hemopoietic stem cells adhere to hemopoietic supportive (MS-5) cells, but not to non-supportive (MS-K) cells. Although a soluble stem cell factor (SCF) was produced by both of these cell lines, little activity was detectable in the supernatant from the cultures of either of these cells, indicating that SCF might be compartmentalized within the extracellular matrix (ECM), and transferred directly to the stem cells via the ECM (44). To probe this possibility, we studied the transfer of SCF from the ECM and the subsequent support of the survival of the hemopoietic stem cells. A stem cell-enriched bone marrow cell fraction was overlaid on SCF-containing ECM. The stem cells survived and proliferated for some days without differentiation under these conditions, whereas stem cells overlaid on ECM without SCF died within a few days. Addition of interleukin-3 (IL-3) to the ECM that contains SCF, induced differentiation of the stem cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced further differentiation of the stem cells, which was accompanied by a decrease in the number of colony-forming unit in spleen (CFU-S). These observations verified the above hypothesis, and indicated that the survival, the self-renewal, and the differentiation of hemopoietic stem cells can be separately controlled at least in vitro.

Animals

Purification and partial characterization of a spore cortex-lytic enzyme of Clostridium perfringens S40 spores.

A spore cortex-lytic enzyme was purified in an active form from the exudate of fully germinated spores of Clostridium perfringens S40. The enzyme caused attenuation of absorbance in coatless spore suspensions and phase-darkening of the spores, but had minimal activity on isolated peptidoglycan fragments. The enzyme was identified as a 31 kDa protein which is probably an N-acetylmuramyl-L-alanine amidase. The amino-terminal 15 residues of the enzyme were: VLPEPVVPEYIVVHN.

Amino Acid Sequence

Quinine-HCl-induced modification of receptor potentials for taste stimuli in frog taste cells.

After frog taste cells were adapted to 1 mM quinine-HCl (Q-HCl) for 10 sec, modification of receptor potentials in the taste cells induced by salt, acid, sugar and bitter stimuli was studied with microelectrodes. The phasic component of receptor potentials induced by 0.1 M NaCl, KCl, NH4Cl and MgCl2 was enhanced following adaptation to Q-HCl. The rate of rise of receptor potentials in response to the salts was increased after Q-HCl adaptation. The amplitude and the rate of rise of receptor potentials induced by 1 mM acetic acid were larger after Q-HCl adaptation than after water adaptation. The amplitude of phasic component and rate of rise of receptor potentials for 0.5 M sucrose after Q-HCl were the same as those after water. The amplitudes of tonic receptor potentials for 1 mM Q-H2SO4, brucine and picric acid after Q-HCl adaptation were the same as those after 1 mM NaCl adaptation. Correlation coefficient between taste cell responses induced by 1 mM Q-HCl and 1 mM Q-H2SO4 was very high, but those between 1 mM Q-HCl and 1 mM brucine responses and between 1 mM Q-HCl and 1 mM picric acid responses were low. This indicates that Q-HCl and Q-H2SO4 bind to the same receptor site, but brucine and picric acid bind to different receptor sites to which Q-HCl does not bind.

Animals