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Biomedical subjects

M Miyagi

Publications and source records attributed to M Miyagi.

At least 73 records · Page 4Linked to original sources
PubMed1996

Effects of the new class I antiarrhythmic agent Ro 22-9194, (2R)-2-amino-N-(2,6-dimethylphenyl)-N-[3-(3-pyridyl)propyl]propionamide D-tartrate, on ischemia- and reperfusion-induced arrhythmias in dogs: involvement of thromboxane A2 synthase inhibitory activity.

We investigated the effects of (2R)-2-amino-N-(2,6-dimethylphenyl)-N-[3-(3-pyridyl)propyl]propionamide D-tartrate (Ro 22-9194), a novel class I antiarrhythmic agent, on myocardial ischemia- and reperfusion-induced arrhythmias in dogs. The incidence of ventricular fibrillation induced by reperfusion after a 30-min coronary ligation was significantly reduced by an i.v. infusion of Ro 22-9194 (10 mg/kg for 5 min before and an additional 20 mg/kg for 30 min during coronary ligation: total, 30 mg/kg) from 73% in the vehicle-treated group to 13%. Ro 22-9194 (20 and 30 mg/kg) also dose-dependently reduced the incidence of ventricular arrhythmias, including ventricular tachycardia and ventricular fibrillation, after coronary reperfusion. Other class I antiarrhythmic agents, mexiletine (15 mg/kg) and disopyramide (7.5 mg/kg), did not inhibit the development of ventricular fibrillation. In in vitro studies, Ro 22-9194, but neither mexiletine nor disopyramide (approximately 10(-3) M), inhibited thromboxane A2 synthase and arachidonic acid-induced aggregation of human platelets (IC50: 1.2 x 10(-5) M and 3.4 x 10(-5) M, respectively). Furthermore, Ro 22-9194 (30 mg/kg) attenuated the increase in venous thromboxane B2 concentrations in the local coronary vein during coronary ligation in dogs. A thromboxane A2 synthase inhibitor, OKY-046 (2.5 mg/kg administered for 5 min before coronary ligation) also showed no evident increases in thromboxane B2 concentrations as well as an antifibrillatory effect. Venous 6-keto-prostaglandin F1 alpha concentrations were not affected by either Ro 22-9194 or OKY-046. These results demonstrate that, unlike mexiletine and disopyramide, Ro 22-9194 protects against reperfusion-induced fatal ventricular arrhythmias in dogs. They also suggest that, in addition to the class I antiarrhythmic effect, the thromboxane A2 synthase inhibitory activity may contribute to the antiarrhythmic properties of Ro 22-9194.

6-Ketoprostaglandin F1 alpha↗
PubMed1995

Reaction mechanism of L-2-haloacid dehalogenase of Pseudomonas sp. YL. Identification of Asp10 as the active site nucleophile by 18O incorporation experiments.

L-2-Haloacid dehalogenase (EC 3.8.1.2) catalyzes the hydrolytic dehalogenation of L-2-haloacids to produce the corresponding D-2-hydroxy acids. We have analyzed the reaction mechanism of the enzyme from Pseudomonas sp. YL and found that Asp10 is the active site nucleophile. When the multiple turnover enzyme reaction was carried out in H2(18)O with L-2-chloropropionate as a substrate, lactate produced was labeled with 18O. However, when the single turnover enzyme reaction was carried out by use of a large excess of the enzyme, the product was not labeled. This suggests that an oxygen atom of the solvent water is first incorporated into the enzyme and then transferred to the product. After the multiple turnover reaction in H2(18)O, the enzyme was digested with lysyl endopeptidase, and the molecular masses of the peptide fragments formed were measured by an ionspray mass spectrometer. Two 18O atoms were shown to be incorporated into a hexapeptide, Gly6-Lys11. Tandem mass spectrometric analysis of this peptide revealed that Asp10 was labeled with two 18O atoms. Our previous site-directed mutagenesis experiment showed that the replacement of Asp10 led to a significant loss in the enzyme activity. These results indicate that Asp10 acts as a nucleophile on the alpha-carbon of the substrate leading to the formation of an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon atom.

Amino Acid Sequence↗
PubMed1995

Identification of histidine 31 and cysteine 95 in the active site of self-incompatibility associated S6-RNase in Nicotiana alata.

S-RNase is associated with the gametophytic self-incompatibility of flowering plants in Solanaceae and, on the basis of sequence homology, belongs to the RNase T2 family. To identify the active site residues in S-RNase, Nicotiana alata S6-RNase was studied by chemical modification. S6-RNase was inactivated with iodoacetic acid under conditions similar to those used for the inactivation of RNase T2. No inactivation took place in the presence of 2'GMP. Analysis of carboxymethylated S6-RNase revealed that the S-carboxymethylation of Cys95 caused inactivation of the enzyme and that the two histidine residues corresponding to two essential histidine residues of RNase T2 remained intact. Treatment of S6-RNase with diethyl pyrocarbonate (DEPC) resulted in loss of enzyme activity, and the enzyme was protected from inactivation in the presence of 2'GMP. The ethoxycarbonylated residues in DEPC-inactivated S6-RNase were analyzed by mass spectrometry, which also provided structural information on sugar moieties attached to Asn27 and Asn37. His31 was modified with DEPC in the absence of 2'GMP and was not modified in its presence. His31 and His91 are conserved in all members of the RNase T2 family sequenced so far, but Cys95 is not conserved in all known Solanaceae S-RNases. These results suggest that His31, possibly together with His91, corresponding to His115 at the active site of RNase T2, is essential to the function of S6-RNase, but Cys95 is not essential though its S-carboxymethylation causes inactivation.

Amino Acid Sequence↗
PubMed1995

Lymphocyte isoforms of mouse p50 LSP1, which are phosphorylated in mitogen-activated T cells, are formed through alternative splicing and phosphorylation.

p50 is phosphorylated in mitogen-stimulated T cells, and translocated from the membrane to the cytosol after activation of protein kinase C. Sequence analysis of p50 revealed that it is identical with LSP1, a putative calcium-binding and actin-binding protein. lymphocyte form of p50 exhibits heterogeneity in the apparent molecular mass on SDS-PAGE, 50 and 52 kDa (pp50 and pp52), and each isoform exhibits heterogeneity in the isoelectric point, when examined by two-dimensional PAGE. When the two molecular mass variants of p50 were dephosphorylated with alkaline phosphatase, both isoforms showed the same apparent molecular mass of 50 kDa on SDS-PAGE, but could be distinguished by their distinct isoelectric points. Dephosphorylated pp50 (p50a) has an acidic pI compared with dephosphorylated pp52 (p50b). Comparison of the peptide maps of purified p50a and p50b on HPLC revealed that the difference was limited to one peptide peak. NH2-terminal sequence and mass spectrometric analyses of these peptides showed that the peptides derived from p50a and p50b had the same NH2-terminal amino acid sequence up to eight residues, but had distinct molecular masses, 5,533.4 and 6,318.6 Da, respectively. These data suggested that pp52 (p50b) is the product of the previously cloned cDNA and the reduction in the molecular mass of the p50a-derived peptide could be explained by deletion of six amino acid residues, EHLIRH or HLIRHQ.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1995

Complete covalent structure of porcine liver acylamino acid-releasing enzyme and identification of its active site serine residue.

The complete covalent structure of porcine liver acylamino acid-releasing enzyme (AARE) [EC3.4.19.1], which catalyzes the hydrolysis of an N-terminally acylated peptide to release an N-acylamino acid, has been established. On basis of the amino acid sequence deduced from the cDNA sequence of porcine liver AARE [Mitta, M. et al. (1989) J. Biochem. 106, 548-555], sequence determination has been achieved by automated Edman degradation of peptides generated by chemical or enzymatic cleavages of the reduced and S-carboxymethylated protein. Ion-spray mass spectrometry was also successfully used to confirm the amino acid sequences of the peptides determined above and to elucidate both the N-terminal blocking group and the status of half-cystine residues of this protein. The protein consists of 732 amino acid residues, and the N-terminal methionine residue is blocked by an acetyl group. All of 18 half-cystine residues of this protein were proved to exist as cysteine residues. A serine residue reactive with diisopropyl fluorophosphate (DFP) was also identified as Ser587 by preparation of the AARE labeled with tritiated DFP followed by isolation and sequence analysis of a radioactive peptide obtained from its endoproteinase Asp-N digest.

Amino Acid Sequence↗
PubMed1994

A peptide that stimulates phosphorylation of the plant insulin-binding protein. Isolation, primary structure and cDNA cloning.

The soybean seed basic 7S globulin (Bg) is capable of binding bovine insulin and insulin-like growth factors, and has protein kinase activity which corresponds to about two thirds of the tyrosine kinase activity of the rat insulin receptor. A 4-kDa peptide named leginsulin, which can bind to Bg and compete with insulin for binding to Bg, was isolated from radicles of germinated soybean seeds. The leginsulin had a stimulatory effect on the phosphorylation activity of Bg, suggesting that it is involved in cellular signal transduction. The leginsulin was sequenced by automated Edman degradation and electrospray ionization mass spectrometry. It consisted of 37 amino acid residues with six half-cystines in three disulfide bridges. The mass spectrometric analysis revealed that a portion of the peptide is processed to delete the C-terminal glycine like a number of animal peptide hormones, but not C-terminally amidated. The cDNA encoding the leginsulin was cloned, sequenced and considered to code for a precursor polypeptide consisting of a putative signal peptide, the leginsulin, a linker peptide, a 6-kDa peptide and a C-terminal peptide. Although there is no sequence similarity between the leginsulin and insulin or insulin-like growth factors, the leginsulin is a possible candidate for plant peptide hormones.

Albumins↗
PubMed1994

Effects of fluoride intake on the mineral content, acid solubility and resorption caused by experimental periodontitis of rat alveolar bone.

Adult rats were given either distilled water or drinking water containing 100 parts/10(6) of fluoride. The alveolar bone of rats given fluoride for 90 days showed an increased mineral content and decreased acid solubility compared to the bone of rats given distilled water. Experimental periodontitis was initiated in both groups after 110 days of treatment to cause alveolar bone resorption. Fourteen days later, the rats were killed and it was found that the alveolar bone resorption caused by experimental periodontitis was significantly smaller in the rats given fluoride in their drinking water than in those given distilled water. The findings suggest that fluoride intake might have a protective effect on rapidly progressing alveolar bone resorption.

Alveolar Bone Loss↗
PubMed1994

Overproduction of Thermus aquaticus DNA polymerase and its structural analysis by ion-spray mass spectrometry.

We cloned the pol gene from the Thermus aquaticus YT-1 strain into a plasmid vector and constructed a high-level expression system of the gene in Escherichia coli. Six codons in the translational start region were changed to simple AT-type codons or codons which are most frequently used in E. coli by the genetic engineering techniques with retention of the amino acid sequence of the native enzyme. The modified pol genes were expressed under the lac promoter of pUC-type plasmid and 266,418 units of activity was obtained in a sonicated and heated crude extract from 2 g of E. coli cells bearing one of the recombinant plasmids, pTAQ9. Highly purified protein was subjected to structural analysis using a protein sequencer and an ion-spray mass spectrometer combined with reversed-phase HPLC (LC-MS). The primary structure of the DNA polymerase was identical with the amino acid sequence deduced from the nucleotide sequence of the pol gene as far as examined (about 95% of the sequence); though, several regions where small peptides of less than 5 residues were produced by lysyl endopeptidase digestion could not be sequenced.

Amino Acid Sequence↗
PubMed1994

[Flow cytometric DNA analysis and chemosensitivity in squamous cell carcinoma of the head and neck].

Cellular DNA content and succinate dehydrogenase activity of 92 human head and neck (34 laryngeal, 24 pharyngeal, 21 oral cavity, 13 maxillary) squamous cell carcinomas were examined, and DNA ploidy status and chemosensitivity were analyzed and compared. DNA aneuploidy was observed in 54 tumors (58.7%). The aneuploid pattern was most common in tumors of the maxillary sinus (84%), and least common in tumors of the larynx (41.3%). Histologically, aneuploidy was detected in 71.4% of poorly-differentiated, 63.8% of moderately-differentiated and 37.5% of well-differentiated squamous cell carcinomas. There was a statistically significant difference between the survival rates of patients with diploid and aneuploid patterns. Chemosensitivity was determined by exposing fresh tumor material to five antitumor drugs: adriamycin (ADM), cisplatin (CDDP), carboquone (CQ), 5-fluorouracil (5-FU) and mitomycin C (MMC). The average decrease in succinate dehydrogenase (SD) activity was 49.8% with ADM, 33.6% with CDDP, 39.9% with CQ, 68.4% with 5-FU and 45.5% with MMC. Histologically, poorly-differentiated squamous cell carcinomas were most sensitive to these five antitumor drugs. We also compared average SD activity in tumors from different organs and found that pharyngeal tumors tend to be most sensitive to these drugs, except for MMC. The chemosensitivity of a tumor with DNA diploidy tended to be higher among well- and moderately-differentiated squamous cell carcinomas. In contrast, tumors with DNA aneuploidy tended to have higher chemosensitivity in the poorly-differentiated type. The results of this study indicate that simultaneous analysis of DNA ploidy and chemosensitivity will be helpful in understanding the characteristics of tumors as well as in predicting the most effective chemotherapy agents for head and neck cancer patients.

Adult↗
PubMed1994

[Evaluation of myocardial viability and efficacy of reperfusion therapy by dual-SPECT using 99mTc-PYP and 201Tl in acute myocardial infarction].

UNLABELLED: Dual SPECT using 99mTc-PYP and 201Tl was performed in 70 patients with acute myocardial infarction, and the ratio of overlap zone between 201Tl and 99mTc-PYP uptake in the infarcted area was determined (the Y-ratio). The Y-ratio was significantly higher in the group with redistribution in the infarcted area on exercise thallium myocardial scintigraphy in the chronic stage of infarction, as well as in the group with normal left ventricular wall motion. Thus, the Y-ratio value was apparently useful in the evaluation of myocardial viability. Patients from 60% of the Y-ratio upward are suggested that myocardial viability had been survived. When the Y-ratio was determined in 30 patients who underwent early reperfusion therapy (ICT or direct-PTCA), no significant difference was found between the ICT group and the direct-PTCA group. However, the Y-ratio was significantly higher when reperfusion was performed within 6 hours than when it was performed after 6 hours, and a significant positive correlation (r = 0.63, p < 0.01), was found between reperfusion time from cardiac event within 9 hours and the value of (100--the Y-ratio). CONCLUSION: The Y-ratio was grateful appeared to be useful for the quantitative evaluation of myocardial viability in the acute stage of myocardial infarction. In addition, it appears to be important to perform reperfusion as soon as possible to improve myocardial salvage.

Adult↗
PubMed1993

Effects of sex hormones on production of prostaglandin E2 by human peripheral monocytes.

The effects of sex hormones on the vitro production of prostaglandin (PG) E2 by monocytes were investigated. Monocytes were obtained from heparinized peripheral blood of healthy adults and incubated for 24 hours with lipopolysaccharide (LPS) and sex hormones. After incubation, the medium was assayed for PGE2 by means of radioimmunoassay. PGE2 production by monocytes was enhanced by progesterone. Estradiol reduced PGE2 production at 0.4 ng/ml, but enhanced it at 20 ng/ml. Testosterone reduced PGE2 production. The reduced PGE2 production by monocytes treated with 0.4 ng/ml of estradiol was restored to the control level by addition of progesterone at 20 ng/ml. These results suggest that sex hormones may modulate gingival inflammation mediated by PGE2.

Adult↗
PubMed1993

[Clinical studies of ofloxacin 300 mg once a day administration in chronic respiratory tract infections].

Forty patients with chronic respiratory infections were randomly assigned to 2 groups to compare the effect of once daily administration of 300 mg each and 3 times daily administration of 600 mg each of ofloxacin (OFLX). Twenty patients were administered with 300 mg OFLX a day and 18 cases received 600 mg. The number of underlying diseases in the 300 mg group was greater than that in the 600 mg group. The ratios of general amelioration of clinical symptoms were 80.0% in the 300 mg group and 88.9% in the 600 mg group. For bacteriological effects, the eradication rate was 80.0% in the 300 mg group and it was 84.6% in the 600 mg. The incidence of side effects in the 300 mg group was 0% and that of the 600 mg group was 5.6% (1 patient) but the symptom was mild. The incidence of abnormal laboratory test results was 15.0% in the 300 mg group and it was 11.2% in the 600 mg group, but all of these abnormalities were slight and transient. The safety rates in the 300 mg and the 600 mg groups were 95.0% and 94.4%, respectively. Efficacy rates in the 300 mg and the 600 mg groups were 80.0% and 88.9%, respectively. There was no statistically significant difference in all the results between the 2 treatment groups, and the both treatments were highly effective. From the above results, we consider that once daily administration of 300 mg is a useful therapy in respiratory tract infections.

Adult↗
PubMed1992

Correlation of loss of alleles on the short arms of chromosomes 11 and 17 with metastasis of primary breast cancer to lymph nodes.

To examine the role of loss of heterozygosity (LOH) during tumor development and/or progression, we looked for correlations between metastasis of breast cancer to a regional lymph node(s) and LOH of chromosomal arms 11p, 13q, 16q, 17p, and 17q, where frequent losses in primary tumors have been detected. No correlation between lymph node metastasis and LOH of chromosomes 13q, 16q, or 17q was observed. However, tumors showing LOH of chromosomes 11p (chi 2 = 10.82, P less than 0.01) and 17p (chi 2 = 6.78, P less than 0.01) revealed a significantly higher incidence of metastasis to a regional lymph node(s) than tumors without LOH on these chromosomal arms. Furthermore, only four of 30 (13%) patients with tumors that retained both 11p and 17p had metastasis to a regional lymph node(s), compared with 24 of 32 (75%) patients with tumors that had lost both 11p and 17p. Analysis of LOH with markers on chromosomes 11p and 17p in a large number of tumors indicated that the peritelomeric region of each of these chromosomal arms contains a tumor suppressor gene that may be associated with tumor progression, particularly metastasis to a regional lymph node(s).

Alleles↗
PubMed1992

NH2-terminal acetylation of ribosomal proteins of Saccharomyces cerevisiae.

Using a mutant of Saccharomyces cerevisiae defective in the NAT1 gene, that encodes one of the NH2-terminal acetyltransferases, we have identified 14 ribosomal proteins whose electrophoretic mobility at pH 5.0 suggests they carry an additional charge, presumably due to the lack of NH2-terminal acetylation. At least 30 other ribosomal proteins from the mutant are electrophoretically normal. Attempted NH2-terminal analysis of most of the presumed acetylated proteins from wild type cells indicated that all were blocked. NH2-terminal analysis of the same proteins from the nat1 mutant strain yielded unique sequences. Each one carries an NH2-terminal serine. We conclude that these are normally acetylated due to the presence of the NAT1 gene product. It seems surprising that cells whose ribosomes have been altered to this degree grow rather well and synthesize the same spectrum of proteins as do wild type cells (Mullen, J. R., Kayne, P. S., Moerschell, R. P., Tsunasawa, S. Gribskov, M., Sherman, F., and Sternglanz, R. (1989) EMBO J. 8, 2067-2075). Finally, this analysis has provided the first sequence information available for several of the acetylated ribosomal proteins and for one non-acetylated ribosomal protein, which is clearly the product of the MFT1 gene (Garrett, J. M., Singh, K. K., Vonder Haar, R. A., and Emr. S. D. (1991) Mol. Gen. Gen. 225, 483-491).

Acetylation↗
PubMed1992

Antibodies against 70-kD heat shock cognate protein inhibit mediated nuclear import of karyophilic proteins.

Previously, we found that anti-DDDED antibodies strongly inhibited in vivo nuclear transport of nuclear proteins and that these antibodies recognized a protein of 69 kD (p69) from rat liver nuclear envelopes that showed specific binding activities to the nuclear location sequences (NLSs) of nucleoplasmin and SV-40 large T-antigen. Here we identified this protein as the 70-kD heat shock cognate protein (hsc70) based on its mass, isoelectric point, cellular localization, and partial amino acid sequences. Competition studies indicated that the recombinant hsc70 expressed in Escherichia coli binds to transport competent SV-40 T-antigen NLS more strongly than to the point mutated transport incompetent mutant NLS. To investigate the possible involvement of hsc70 in nuclear transport, we examined the effect of anti-hsc70 rabbit antibodies on the nuclear accumulation of karyophilic proteins. When injected into the cytoplasm of tissue culture cells, anti-hsc70 strongly inhibited the nuclear import of nucleoplasmin, SV-40 T-antigen NLS bearing BSA and histone H1. In contrast, anti-hsc70 IgG did not prevent the diffusion of lysozyme or 17.4-kD FITC-dextran into the nuclei. After injection of these antibodies, cells continued RNA synthesis and were viable. These results indicate that hsc70 interacts with NLS-containing proteins in the cytoplasm before their nuclear import.

Amino Acid Sequence↗
PubMed1992

Cloning and characterization of an interstitial deletion at chromosome 11p15 in a sporadic breast cancer.

In a number of types of cancer including breast, hepatocellular, and bladder carcinoma, frequent losses of heterozygosity (LOH) on chromosome 11p15 have indicated the presence of one or more tumor suppressor genes in this region. In the present study, we report the detection and characterization of a rearrangement at 11p15 in a sporadic breast carcinoma. Genomic DNA clones encompassing the rearranged region were isolated; localization of both flanking clones to 11p15 by two-color fluorescent in situ hybridization (FISH) indicated that the rearrangement was caused by an interstitial deletion in the affected allele. Although it is uncertain whether the region between the flanking two loci was missing from tumor cells, our result implied that a putative tumor suppressor gene on chromosome 11p15 is located between the loci on either side of the interstitial deletion or may be interrupted by one of the breakpoints.

Breast Neoplasms↗
PubMed1992

Effects of sex hormones on chemotaxis of human peripheral polymorphonuclear leukocytes and monocytes.

The effects of sex hormones on the in vitro chemotaxis of polymorphonuclear leukocytes (PMNs) and monocytes were investigated using fMLP as the chemoattractant. PMNs, monocytes, and plasma were obtained from heparinized peripheral blood of healthy adults. Chemotaxis of PMNs or monocytes treated with sex hormones were tested using 48-well chemotaxis microchambers. The correlation between sex hormone levels in plasma and the chemotactic ability of PMNs from the same donor was also investigated. The chemotaxis of PMNs was enhanced by progesterone, while it was reduced by estradiol. Random migration of PMNs was also enhanced by progesterone and reduced by estradiol. The effect of estradiol on PMN chemotaxis was inhibited by addition of antiestrogens or progesterone. Testosterone did not have a measurable effect on PMN chemotaxis. A significant positive correlation was found between the concentration of progesterone in plasma of females and PMN chemotactic ability in vitro. For males, there was no significant relationship between plasma levels of sex hormones and PMN chemotactic ability. Estradiol and testosterone levels in plasma did not correlate with PMN chemotactic ability. Sex hormones had no effect on the chemotaxis of monocytes. These results suggest that the altered PMN chemotaxis associated with gingival inflammation may be due to the effects of sex hormones.

Adult↗