Biomedical subjects

H Araki

Publications and source records attributed to H Araki.

At least 19 recordsLinked to original sources

PubMed1992

Functional analysis of Box II mutations in yeast site-specific recombinases Flp and R. Significance of amino acid conservation within the Int family and the yeast sub-family.

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that share approximately 30% amino acid matches. They exhibit a common reaction mechanism that appears to be conserved within the larger Integrase family of site-specific recombinases. Two regions of the proteins, designated as Box I and Box II, harbor, in addition to amino acid conservation, a significantly high degree of nucleotide sequence homology within their coding segments. Box II also contains two amino acids, a histidine and an arginine, that are invariant throughout the Int family. We have performed functional analysis of Flp and R variants carrying point mutations within the Box II segment. Several positions within Box II can tolerate substitutions with no effect, or only modest effects on recombination. Alterations of the Int family residues, His305 and Arg308, in the R protein lead to the arrest of recombination at the strand cleavage or the strand exchange step. This is very similar to previously observed "step-arrest" phenotypes in Flp variants altered at these positions and has strong implications for the catalytic mechanism of recombination. Flp and R variants at His305 and His309 can be complemented in half-site strand transfer by a corresponding Tyr343 to phenylalanine variant. In contrast to Arg308 Flp variants, which are efficiently complemented in half-site strand transfer by Flp(Y343F), no strong complementation has been observed between Arg308 variants of R and R (Y343F).

DNA Mutational Analysis↗

PubMed1992

Automatic elimination of unnecessary bacterial sequences from yeast vectors.

Most vectors for Saccharomyces cerevisiae are shuttle vectors which can be both propagated and selected in Escherichia coli. The DNA segments, however, which are required for propagation in E. coli are unnecessary and moreover toxic in S. cerevisiae. To delete these harmful DNA fragments from the vector after it is introduced into S. cerevisiae cells, we propose a specific gene conversion mechanism of a yeast plasmid, pSR1. Plasmid pSR1 has a pair of inverted repeats (IRs) that divides the plasmid molecule into two unique regions. Intramolecular recombination frequently occurs at a pair of specific recombination sites in IRs catalyzed by recombinase R, encoded by a pSR1 plasmid gene. This R-mediated recombination is often accompanied by gene conversion in IRs. Thus, a 2.1-kb pBR322 sequence for the E. coli host ligated into one of the IRs of a composite plasmid was automatically and effectively eliminated when the plasmid was introduced into S. cerevisiae cells.

DNA, Bacterial↗

PubMed1992

Half-site recombinations mediated by yeast site-specific recombinases Flp and R.

The Flp recombinase of Saccharomyces cerevisae and the related R recombinase of Zygosaccharomyces rouxii can efficiently catalyze strand cleavage and strand exchange reactions in half recombination sites. A half-site consists of one recombinase binding element, a recombinase cleavage site on one strand and a 5' spacer hydroxyl group on the other that can initiate the strand exchange reaction. We have studied the various types of strand exchanges that half-sites can participate in. Reaction between a left half-site and a right half-site generates a full recombination site. Strand transfer between two left half-sites or between two right half-sites produces pseudo-full-sites. Strand transfer within a half-site results in a stem-loop or hairpin product. The half-site strand transfer reaction is fairly indifferent to the spacer sequence of the substrate per se and is less sensitive to variations in spacer lengths than a full-site recombination reaction. The optimal spacer length of eight to ten nucleotides observed for the Flp half-site reaction likely permits the most productive catalytic interactions between two Flp monomers bound to each of two partner half-sites. When reacted with a full-site, the half-site can give rise to a normal or reverse recombinant, corresponding to homologous or non-homologous alignments of the spacer sequences during substrate synapsis. The contrary recombination (resulting from non-homologous spacer alignment), whose level is low relative to normal recombination, is partly suppressed when the half-site spacer ends in a 5'-phosphate rather than a 5'-hydroxyl group. Thus, the early steps of recombination, namely synapsis and initial stand transfer, are not dependent on complete spacer homology between the two recombining substrates. The selection of properly aligned substrate partners must occur at the homology dependent branch migration step. In reactions containing a mixture of Flp and R half-sites, Flp and R catalyze strand transfer, almost exclusively, within or between their respective cognate substrates. However, under conditions where self-crosses are inhibited, strand exchange between a Flp half-site and an R half-site appears to be stimulated by a combination of R and Flp.

Base Sequence↗

PubMed1992

Site-specific recombinase, R, encoded by yeast plasmid pSR1.

The R gene product (R protein) of Zygosaccharomyces rouxii plasmid pSR1 catalyzes site-specific recombination within a 58 base-pair (bp) sequence present in the 959 bp inverted repeats of this plasmid. The R protein was produced in Escherichia coli and partially purified. The partially purified protein catalyzed site-specific recombination in vitro without the supply of an energy source. Recombination resulted in intramolecular inversion or deletion, depending on whether the orientations of the two recombination sites on the substrate plasmid were the same or opposite. Presumably, R protein is the only protein required for the recombination reaction. A circular DNA molecule appears to be a better substrate than a linear molecule in R-mediated in vitro intramolecular recombination. The R protein binds to a set of six 12 bp elements within the inverted repeats of pSR1. Two of these 12 bp elements are arranged in an inverted configuration with a 7 bp spacer in the 58 bp sequence. The R protein mediates strand cleavage in vitro at the junction between the 12 bp elements and the 7 bp spacer. The cleavage sites on the top and bottom strands are staggered and flanked by polypurine tracts that form part of the 12 bp elements.

Base Sequence↗

PubMed1992

DNA polymerase II, the probable homolog of mammalian DNA polymerase epsilon, replicates chromosomal DNA in the yeast Saccharomyces cerevisiae.

Two temperature-sensitive DNA polymerase II mutants (pol2-9 and pol2-18) of the yeast Saccharomyces cerevisiae were isolated by the plasmid shuffling method. DNA polymerase II activity partially purified from both mutants was thermolabile, while DNA polymerase I and III activities remained thermotolerant. At the restrictive temperature, the pol2 mutants were defective in chromosomal DNA replication and exhibited the dumbbell terminal morphology typical of DNA replication mutants. The POL2 transcript accumulated periodically during the cell cycle, peaking at the G1/S boundary in the same manner as the transcripts of more than 10 other DNA replication genes. These results indicate that DNA polymerase II participates in nuclear DNA replication. The similarities in structure and activities between the DNA polymerases of yeast and mammals make it likely that mammalian DNA polymerase epsilon too is required for chromosomal DNA replication.

Amino Acid Sequence↗

PubMed1992

The CDC26 gene of Saccharomyces cerevisiae is required for cell growth only at high temperature.

We have cloned and sequenced the wild-type CDC26 gene and a mutant allele, cdc26-1, of Saccharomyces cerevisiae. Nucleotide sequence analysis revealed that the gene we cloned was the same as SCD26, a dosage-dependent suppressor of cdc26. However, the cloned gene is in fact the CDC26 gene, because a nucleotide substitution in cdc26-1 was found to be a nonsense mutation in this sequence. Disruption of this gene conferred thermosensitive cell growth and the disrupted cdc26 gene could not complement the cdc26-1 mutant allele. Thus, the CDC26 gene is required for cell growth only at high temperature.

Amino Acid Sequence↗

PubMed1992

Effects of ketanserin and mianserin on delayed neuronal death induced by cerebral ischemia in Mongolian gerbils.

We examined whether ketanserin and mianserin, drugs with 5-HT2 receptor antagonistic effects, would have protective effects in Mongolian gerbils on delayed neuronal death induced by cerebral ischemia. When training and test sessions in the passive avoidance task were carried out 6 and 7 days after 3 min of ischemia, passive avoidance impairment was apparent. Destruction and disappearance was apparent in the hippocampal CA1 neurons at 7 days after the induced ischemia. Administration of ketanserin (5-20 mg/kg) and mianserin (5-20 mg/kg) led to better passive avoidance and prevented the delayed neuronal death induced by the ischemia. These effects of ketanserin and mianserin were not inhibited by treatment with the cholinergic blocker scopolamine (5 mg/kg). Thus, ketanserin and mianserin have a protective effect on delayed neuronal death in gerbils and the effects of these drugs are not mediated by serotonergic and cholinergic systems.

Analysis of Variance↗

PubMed1992

Cholinomimetic activity of minaprine is related to the amelioration of delayed neuronal death in gerbils.

We attempted to determine if the cholinomimetic activity of the psychotropic drug minaprine was related to the amelioration of the delayed neuronal death induced by cerebral ischemia in Mongolian gerbils. Minaprine improved the passive avoidance deficit induced by cerebral ischemia, and the histopathological ischemic neuronal changes in the hippocampal CA1 neurons were diminished. These effects were completely inhibited by treatment with the cholinergic blocker scopolamine. Rectal temperature fell about 1.5 degrees C immediately after cerebral ischemia and hyperthermia occurred 30 and 60 min after recirculation. Minaprine had no effect on body temperature before or after ischemia. Physostigmine and tetrahydroaminoacridine (THA), drugs which stimulate the cholinergic system, improved passive avoidance deficits and prevented the delayed neuronal death. These effects of physostigmine and THA were completely inhibited by scopolamine. Pentobarbital and diazepam also improved the passive avoidance deficit and prevented the destruction of CA1 neurons. In contrast with minaprine, these effects of pentobarbital and diazepam were not inhibited by scopolamine. As the protective effect of minaprine against ischemia-induced delayed neuronal death is related to cholinomimetic activities, these events differ from those seen with pentobarbital and diazepam.

Animals↗

PubMed1992

Identification of candidate sequences that determine virulence in Coxsackievirus B4.

We have previously shown that a major determinant of virulence for coxsackievirus B4 mapped to the 5' end of the viral genome. Comparison of the corresponding cDNA sequences of a virulent and a non-virulent virus has allowed the identification of candidate determinants of virulence in the 5' untranslated region and the capsid proteins VP1, VP2 and VP4. Thirteen nucleotide substitutions were observed in a region spanning 3298 nucleotides. Four mutations were detected in the non-coding region. Of the remaining nine mutations, four were silent while five resulted in amino acid substitutions in VP1, VP2 and VP4. The amino acid substitutions in the virulent virus were analyzed in relation to the three-dimensional structures of the capsid proteins of poliovirus. Two substitutions mapped to the amino termini of VP1 and VP4. Of the two substitutions observed in VP2, one mapped to the large loop that connects beta strand E with the radial helix on the back surface of the eight-stranded antiparallel beta barrel while the other mapped to beta strand G. One amino acid substitution in VP1 mapped to the loop connecting beta strands D and E at a site close to a major determinant of attenuation in poliovirus type 2.

Amino Acid Sequence↗

PubMed1992

Sigma receptor modulation of the muscle relaxant action of eperisone.

We examined the mechanism of the muscle relaxant action of eperisone, using Straub tail and binding studies. In vivo, eperisone (50 and 100 mg/kg i.p.) produced a dose-dependent inhibition of the Straub tail in mice and this inhibitory effect was significantly reversed by haloperidol (0.5 mg/kg i.p.). This drug in itself had no effect on the Straub tail. Sulpiride (50 mg/kg i.p.) failed to reverse the inhibitory effect of eperisone. In vitro, (+)-3-(3-[3H]hydroxyphenyl)-N-(1-propylpiperidine) ((+)-[3H]3-PPP) specific binding, in rat brain membrane, was prevented by eperisone and the IC50 value was 0.43 nM. The muscle relaxant action of eperisone may be modulated by sigma receptors.

Animals↗

PubMed1992

Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine.

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.

Binding Sites↗

PubMed1992

Immunocytochemical localization of alpha-fetoprotein in the developing and carbon tetrachloride-treated rat liver.

The immunocytochemical localization of alpha-fetoprotein (AFP)-producing cells was observed in pre- and postnatal and carbon tetrachloride (CCl4)-treated rat livers in comparison with that of albumin (ALB)-producing cells. According to immunoblotting data, considerable numbers of AFP-positive hepatocytes were observed in the differentiating liver between prenatal day 19 and postnatal day 0 (6 h after birth). Analyses by serial section profiles of these cells revealed that certain AFP-positive hepatocytes are also stained with ALB antiserum. Immunoelectron microscopy of the AFP-producing cells revealed that immunoreactive gold particles are preferentially localized in rough endoplasmic cisternae, Golgi apparatus and Golgi-derived vesicles near the cell surface. In addition, the release of the content of the Golgi-derived vesicles into the differentiating bile canaliculi as well as into the space of Disse by exocytosis is apparent. In CCl4-treated rat liver, immunoreactions to AFP are localized exclusively in newly formed hepatocytes of the regenerative tissue. These AFP-positive cells have not established the hepatic cell cords, and the adjacent ones are conjugated to each other mainly by simple attachment devices as in the case of those in pre- and postnatal rat liver.

Albumins↗

PubMed1992

Heparin adheres to the damaged arterial wall and inhibits its thrombogenicity.

Heparin binds to thrombogenic extracellular matrices as well as to smooth muscle cells of the vascular wall in vitro. The inhibitory effects of heparin on thrombogenicity of the damaged arterial wall were examined in vivo using small mesenteric arteries of rats and a video recording system attached to a microscope. To induce thrombosis, we damaged the vessel wall over a short segment by compression and exposed the media to the blood stream. A platelet-rich thrombus enlarged gradually at the damaged site, occluded the vascular lumen for a short period, and then flowed away. Compression damage induced such thrombus formation several times. Heparin (500 units/ml) was given in three different ways: intravenous and intra-arterial administration (both 300 units/kg) and intraluminal application under stopped-flow conditions (less than 0.01 ml) for 1-2 minutes with subsequent draining out. Intravenous heparin significantly decreased both the total duration and the number of thrombotic occlusions, whereas intra-arterial heparin abolished thrombotic occlusion. Both routes of heparin administration similarly prolonged the blood coagulation time. Intraluminal application of heparin significantly inhibited subsequent thrombus formation after restoring the flow without changes in the blood coagulation time. After an intra-arterial administration or intraluminal application of fluorescein isothiocyanate-bound heparin, strong fluorescence was observed only at the damaged vascular segment. A heparin fraction with low affinity to antithrombin III or chondroitin sulfate A did not inhibit thrombosis. To clarify anticoagulant activity of vascular wall-bound heparin, damaged carotid arterial segments of rats were incubated (inside out) in platelet-poor plasma with thrombin, and fibrin clot formation around the segments with or without heparin binding was measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Beneficial effect of intravenous taurine infusion on electroretinographic disorder in taurine deficient rats.

We investigated the effect of intravenous taurine infusion on the electroretinogram (ERG) of taurine-deficient rats produced by treatment with guanidinoethyl sulfonate (GES), a taurine transport inhibitor. Mother rats were fed a taurine-free diet and given drinking water containing 1% GES from 2 weeks of gestation to weaning. The same feeding conditions were applied to male offspring after weaning. Both ERG measurement and continuous infusion of taurine at a dose of 10, 30 or 100 mg/animal/day were performed for 3 weeks from 7 to 10 weeks of age. GES-treatment reduced a- and b-wave amplitudes to 50% of the control levels and also increased b-wave latencies. Intravenous infusion of taurine improved these ERG abnormalities in a dose-dependent manner. Taurine concentrations in plasma, eyes and brain were also decreased by treatment with GES, and dose-dependent recovery was observed after infusion with taurine, although the concentrations of other amino acids were not affected by GES-treatment and infusion of taurine. Observations of morphological changes revealed that the retinal damage in GES-treated animals was decreased by taurine infusion. These results indicate that the changes in ERG and retinal structure observed in taurine deficiency are improved by intravenous infusion of taurine.

Amino Acids↗

PubMed1992

Pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) on the endothelium-dependent relaxation in rat mesenteric arterioles.

Effects of exchange-transfusion with a modified hemoglobin (pyridoxalated hemoglobin polyoxyethylene conjugate; PHP) on diameter of rat mesenteric arterioles responding to various vasoactive substances were examined using the image-splitter television microscopy method. The mesentery of the anesthetized rat was spread over the stage of a microscope and the microvasculature was visualized by transillumination. Exchange-transfusion with a 6%PHP solution (30 ml/kg) was performed and effects of topical application of norepinephrine (NE) and acetylcholine(ACh) on diameter of mesenteric arterioles were examined. By exchange-transfusion with PHP, NE induced a dose-dependent decrease in diameter, while ACh induced a dose-dependent increase in diameter of the arterioles preconstricted by NE. There was no difference in vascular responsiveness to ACh and NE before and after exchange-transfusion with PHP solution. Results suggest that circulating PHP moiety does not exert any action on function of endothelial cell to release endothelium-derived relaxing factor (EDRF) in rat mesenteric microvasculature in situ.

Acetylcholine↗

PubMed1992

[A case of multiple pulmonary metastases from duodenal cancer showing partial response using 5'-DFUR and a small dose of MMC].

A 77-year-old female with primary duodenal cancer had undergone pancreatoduodenectomy in May, 1989. Postoperative chemotherapy was done in combination with MMC (mitomycin C), lentinan and UFT (combined medicine of tegafur and uracil). In August, 1991, the patient complained of a cough and then was examined for multiple pulmonary metastases from duodenal cancer by chest X-ray and CT-scan. Then, she received 5'-DFUR (400-800 mg) and MMC (total 6 mg). Two months from the start of this therapy, the cough almost vanished and pulmonary lesions were diminished markedly. For about five months, this case corresponded to partial response (PR) according to the response criteria proposed by Koyama-Saitoh. The side effects of 5'-DFUR were diarrhea and anorexia. Therefore, we think that 5'-DFUR and a small dose of MMC yielded a partial response to multiple pulmonary metastases from duodenal cancer.

Adenocarcinoma↗

PubMed1992

[Innovative elastomeric bio-adhesive (pup-201) synthesized by urethane-prepolymer].

Properties of PUP-201 for the application to vital organs were estimated. Basic properties of PUP-201 were compared with two kinds of commercial fibrin and synthetic adhesives. Its initial and wet adhesive strength was 40 and 150 times stronger than those of fibrin adhesives respectively. Its curing time was 50 seconds, and elongating rate was 150% nearly as same as that of the intestine. This adhesive had not antimicrobial activities. The strength of adhesive bonded pig skin in shear by tension loading by way of JIS K6850 recorded 2.0-2.7kg/cm2 five minutes after, and 2.0-2.8kg/cm2 after 24 hours. The results of the same test in small intestine defined 0.4-1.0kg/cm2 in sero-sero attachment. There was no toxicity on the pig skin. Practical examinations for application to hemostasis on the section of dog liver and sealing of the lung section were performed successfully. PUP-201 was demonstrated excellently useful material for the adhesive, hemostasis and sealant of vital tissue.

Animals↗

PubMed1992

[Pharmacokinetic and clinical evaluation of levofloxacin in obstetrics and gynecology].

Levofloxacin (LVFX, DR-3355), a new synthetic quinolone derivative antibacterial agent, was evaluated for its pharmacokinetics and clinical efficacy in obstetric and gynecological infections, and the following results were obtained. Concentrations of LVFX in serum and intrapelvic genital organs such as uterine and adnexal tissues were determined following oral administration of 100 mg. Tissue penetration of LVFX was found to be good, with its tissue levels (Cmax) of 1.17-2.16 micrograms/ml or g after inhalation anaesthesia and 1.15-2.17 micrograms/ml or g after lumbar spinal anaesthesia. LVFX was given to 22 cases of obstetric and gynecological infections with a daily dose of 200-600 mg for 3-14 days and its clinical efficacy was 95% and bacteriological response was 100%. No side effect was observed. From these findings, we consider that LVFX will be a useful antibacterial agent against obstetric and gynecological infections.

Administration, Oral↗