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Biomedical subjects

K Murayama

Publications and source records attributed to K Murayama.

At least 55 records · Page 3Linked to original sources
PubMed2000

Possible involvement of cyclophilin B and caspase-activated deoxyribonuclease in the induction of chromosomal DNA degradation in TCR-stimulated thymocytes.

TCR engagement of immature CD4(+)CD8(+) thymocytes induces clonal maturation (positive selection) as well as clonal deletion (negative selection) in the thymus. However, the cell death execution events of thymocytes during the negative selection process remain obscure. Using a cell-free system, we identified two different DNase activities in the cytosol of in vivo anti-TCR-stimulated murine thymocytes: one that induced chromosomal DNA fragmentation, which was inhibited by an inhibitor of caspase-activated DNase, and another that induced plasmid DNA degradation, which was not inhibited by an inhibitor of caspase-activated DNase. We purified the protein to homogeneity that induced plasmid DNA degradation from the cytosol of anti-CD3-stimulated thymocytes and found that it is identical with cyclophilin B (Cyp B), which was reported to locate in endoplasmic reticulum. Ab against Cyp B specifically inhibited the DNA degradation activity in the cytosol of anti-CD3-stimulated thymocytes. Furthermore, recombinant Cyp B induced DNA degradation of naked nuclei, but did not induce internucleosomal DNA fragmentation. Finally, we demonstrated that TCR engagement of a murine T cell line (EL4) with anti-CD3/CD28 resulted in the release of Cyp B from the microsome fraction to the cytosol/nuclear fraction. Our data strongly suggest that both active caspase-activated DNase and Cyp B may participate in the induction of chromosomal DNA degradation during cell death execution of TCR-stimulated thymocytes.

Animals↗
PubMed2000

Solvent extraction coupled on-line to a reversed micellar mediated chemiluminescence detection system for trace-level determination of atropine.

A fast and sensitive method for the determination of atropine, an alkaloid closely related to cocaine, is proposed. The principles of on-line ion-pair formation of alkaloid-metal complexes and liquid-liquid extraction are applied to the chemiluminescence determination of atropine. On mixing with a reversed micellar medium of cetyltrimethylammonium chloride in dichloromethane-cyclohexane (1:1 v/v)-water (0.3 M Na2CO3) containing luminol, the ion-pair complex of tetrachloroaurate(III) with atropinium produced an analytical chemiluminescence signal when it entered the reversed micellar water pool. Using the reverse-flow injection and chemical conditions optimized for atropine in aqueous samples, a detection limit of 1 ng/mL was achieved and a linear calibration graph was obtained with a wide dynamic range from 10 ng/mL to 100 micrograms/mL. The proposed method is simple and provides a good precision with a relative standard deviation (n = 6) of ca. 3% at the atropine concentration of 100 ng/mL. After a preliminary study involving the potential interference from species of organic, inorganic, and metallic nature, the method was applied to the determination of atropine in artificial urine samples and of atropine and scopolamine in pharmaceutical formulations.

Atropine↗
PubMed2000

Stage-specific isoforms of Ascaris suum complex. II: The fumarate reductase of the parasitic adult and the succinate dehydrogenase of free-living larvae share a common iron-sulfur subunit.

Complex II of adult Ascaris suum muscle exhibits high fumarate reductase (FRD) activity and plays a key role in anaerobic electron-transport during adaptation to their microaerobic habitat. In contrast, larval (L2) complex II shows a much lower FRD activity than the adult enzyme, and functions as succinate dehydrogenase (SDH) in aerobic respiration. We have reported the stage-specific isoforms of complex II in A. suum mitochondria, and showed that at least the flavoprotein subunit (Fp) and the small subunit of cytochrome b (cybS) of the larval complex II differ from those of adult. In the present study, complete cDNAs for the iron-sulfur subunit (Ip) of complex II, which with Fp forms the catalytic portion of complex II, have been cloned and sequenced from anaerobic adult A. suum, and the free-living nematode, Caenorhabditis elegans. The amino acid sequences of the Ip subunits of these two nematodes are similar, particularly around the three cysteine-rich regions that are thought to comprise the iron-sulfur clusters of the enzyme. The Ip from A. suum larvae was also characterized because Northern hybridization showed that the adult Ip is also expressed in L2. The Ip of larval complex II was recognized by the antibody against adult Ip, and was indistinguishable from the adult Ip by peptide mapping. The N-terminal 42 amino acid sequence of Ip in the larval complex II purified by DEAE-cellulofine column chromatography was identical to that of the mature form of the adult Ip. Furthermore, the amino acid composition of larval Ip determined by micro-analysis on a PVDF membrane is almost the same as that of adult Ip. These results, together with the fact, that homology probing by RT-PCR, using degenerated primers, failed to find a larval-specific Ip, suggest that the two different stage-specific forms of the A. suum complex II share a common Ip subunit, even though the adult enzyme functions as a FRD, while larval enzyme acts as an SDH.

Amino Acid Sequence↗
PubMed2000

Characterization of native and recombinant peptidyl prolyl cis-trans isomerases derived from Methanococcus thermolithotrophicus based on cDNA sequence.

It is important to establish whether a recombinant protein is an authentic copy of the predicted cDNA sequence. In this study, recombinant protein for native peptidyl prolyl cis-trans isomerase (N-PPIase) and double-labeled (13C- and 15N-) protein (DL-PPIase) appeared on the sodium dodecyl sulfate (SDS) electropherograms as two bands for N-PPIase and four bands for DL-PPIase. Since the N-terminal amino acid residues of all bands were the same, we characterized these bands using the peptide mapping method and amino acid composition analysis. Peptide mapping of the proteins seemed to be almost identical but they could not reflect the whole amino acid sequences of the protein. The bands on the polyvinylidene difluoride (PVDF) membrane, electroblotted after SDS-polyacrylamide gel electrophoresis (SDS-PAGE), were hydrolyzed and their amino acid composition was analyzed using a highly sensitive 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) amino acid analysis and compared with the cDNA sequences for proteins. The matching score (sigma(T%-E%)2) for similarity of proteins was calculated by summation of the square difference between the theoretical (T%) and the experimental (E%) amino acid composition of the recombinant protein. The amino acid composition of all bands of both proteins showed more than 93% of the theoretical values. The major molecular weights of both proteins were 16812 and 17694 by electrospray ionization (ESI)-mass spectrometry. However, the purified proteins also contained minor compounds with Mr of 3721 for N-PPIase and 5285 for DL-PPIase. These compounds were considered to be nonpeptidyl products that comigrated with the protein. Similarities of the amino acid composition of the four bands were more than 98%. Our results indicate that AQC amino acid analysis is the most suitable method for characterization of a recombinant protein.

Amino Acids↗
PubMed2000

Automatic semi-microcolumn liquid chromatographic determination of catecholamines in rat plasma utilizing peroxyoxalate chemiluminescence reaction.

A fully automated and highly sensitive method with a semi-microcolumn liquid chromatography system for the determination of rat plasma catecholamines (CAs) was developed. Automated on-line extraction of CAs in diluted plasma using a precolumn packed with strong acidic cation exchange resin was coupled with separation of CAs on a semi-microcolumn (250 x 1.5 mm id). fluorogenic derivatization with ethylenediamine and finally postcolumn peroxyoxalate chemiluminescence detection utilizing bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl]oxalate (TDPO) and hydrogen peroxide. The detection limits were 0.91, 0.36 and 1.1 fmol for norepinephrine (noradrenaline), epinephrine (adrenaline) and dopamine, respectively, at a signal-to-noise ratio of 3. A good linearity of the calibration curve for each CA was observed in the range of 5.0 to 500 fmol for each CA using N-methyldopamine (N-MeDA) as an internal standard. The RSD for the proposed method (n = 5) were 3.7-9.5% for the intra-day assay and 6.6-10.0% for the inter-day assay. The volume of rat plasma required for the determination of CAs was 10 microliters.

Animals↗
PubMed2000

Rapid determination of parvalbumin amino acid sequence from Rana catesbeiana (pI 4.78) by combination of ESI mass spectrometry, protein sequencing, and amino acid analysis.

The complete amino acid sequence of beta-type parvalbumin (PA) from bullfrog Rana catesbeiana (pI 4.78) was determined by tandem mass spectrometry in combination with amino acid analysis and peptide sequencing following Arg-C and V(8) protease digestion. The primary structure of the protein was compared with that of beta-type PA from R. esculenta (pI 4.50), with which it is highly homologous. Compared with R. esculenta beta-type PA4.50, R. catesbeiana beta-type parvalbumin (PA 4.78) differed in 15 out of 108 amino acid residues (14% displacement), PA4.78 had Cys at residue 64 and was acetylated at the amino terminus, but 25 residues of the carboxyl terminus were completely conserved. Several amino acid displacements were found between residues 51 and 80 (30% displacement), although the functionally important sequence of PA was completely conserved. The amino acids residues of putative calcium-binding sites were Asp-51, Asp-53, Ser-55, Phe-57, Glu-59, Glu-62, Asp-90, Asp-92, Asp-94, Lys-96, and Glu-101, which were conserved in all a and b-types of R. catesbeiana as well as other parvalbumins. In addition, Arg-75 and Glu-81, which are thought to form a salt bridge located in the interior of the molecule [Coffee, C.J. et al. (1976) Biochim. Biophys. Acta 453, 67-80], were also conserved in PA4.78.

Amino Acid Sequence↗
PubMed2000

[Quantitative analysis of indocyanine green angiographic image in central serous chorioretinopathy].

PURPOSE: To disclose the possible involvement of choroidal vascular change in development of central serous chorioretinopathy (CSC). METHODS: Indocyanine green (ICG) angiography was performed in 31 eyes with acute CSC, and 21 eyes from normal subjects. The maximum diameter of the choroidal veins and intensity of background fluorescence in the posterior fundus with ICG video images were measured for further analysis using IMAGEnet (Topcon). Then the results from CSC affected eyes, their fellow eyes, and normal eyes were compared. Aging factors were taken into consideration when we analyzed the data. RESULT: The maximum diameters of the choroidal veins were larger in both affected and fellow eyes than in the normal eyes (p < 0.001), and had a positive correlation with aging particularly in fellow eyes (r = 0.36). Both in the affected and fellow eyes, the background fluorescein intensity in the posterior pole of the late phase images was lower than in the normal eyes (p < 0.001), and was correlated with aging (r = 0.28, r = 0.43). CONCLUSION: This quantitative study showed that choroidal venous dilatation and the residual background fluorescence in the posterior fundus might be positive findings reflecting the pathogenesis of CSC.

Adult↗
PubMed2000

[Transurethral microwave thermotherapy for benign prostatic hyperplasia].

The clinical efficacy of transurethral microwave thermotherapy using the PROSTCARE apparatus was evaluated in 60 patients with bladder outlet obstruction associated with benign prostatic hyperplasia. All of the 60 patients received a single thermal session for 60 minutes with an average intraprostatic temperature of 45 degrees C. They were evaluated by analyzing the international prostate symptom score (IPSS), quality of life (QOL) index, maximum urinary flow rate and prostate volume at 2 months after the treatment to estimate criteria for efficacy of treatment in BPH. Both IPSS and QOL index improved significantly. The maximum urinary flow rate improved but there was no significant change. There was no significant change in prostate volume. The rates of improvement in IPSS, QOL index and maximum flow rate were 73%, 78% and 47% of the patients respectively. The rate of overall improvement was 78% of the patients. As a complication after the treatment, 2 patients complained of ejaculation disturbance.

Aged↗
PubMed2000

[Clinical effects of cefoselis (CFSL) on infections in obstetric and gynecologic field and prevention of postoperative infections].

Clinical effects of cefoselis (CFSL) on various infections and prevention of postoperative infections in the field of obstetrics and gynecology were investigated with a total of 100 patients of 8 facilities in Yamagata and the following results were obtained: 1. For the patients (n = 70) who received the treatment with CFSL at 2 g/day for 5 days to prevent postoperative infections, the treatment was effective for such infections in 68 of 69 (98.6%) except one to whom the treatment was discontinued during the course. 2. For the patients with infections (n = 30) who were treated with CFSL at 2-4 g/day for 5-7 days, the treatment was markedly effective in 8/30 (26.7%), effective in 21/30 (70%) and not effective in 1/30 (3.3%). The overall rate of efficacy was 29/30 (96.7%). Based on the clinical effects for each isolate, the bacteriological efficacy was evaluated as 29/29 and the rate of bacterial eradication for each isolate was 23/29 (79.3%). 3. Laboratory test revealed liver functional abnormalities in one patient and eruption, a subjective/objective symptom caused by CFSL was noted in two patients. These results suggest that CFSL is effective for various infections in obstetric and gynecologic field and also the prevention of postoperative infections.

Adult↗
PubMed1999

Tandem repeat structure of rhamnose-binding lectin from catfish (Silurus asotus) eggs.

The primary structure of catfish (Silurus asotus) egg lectin (SAL) was determined. SAL cDNA contained 1448-bp nucleotides and 308 amino acid residues, deduced from open reading frame. The SAL mature protein composed of 285-amino acid residues was followed by a predicted signal sequence having 23 residues. The mRNA of SAL was found to be expressed in eggs, but not in liver. SAL is composed of three tandem repeat domain structures divided into exactly 95 amino acid residues each, and all cysteine positions of each domain were completely conserved. Sequence homologies between the three domains, termed D1 (1-95), D2 (96-190) and D3 (191-285), were as follows; D1-D2, 28%; D2-D3, 33%; D1-D3, 43%. Two conserved peptide motifs, -(AN)YGR(TD)S(T)XCS(TGR)P- and -DPCX(G)T(Y)KY(L)-, appear to exist at the N- and C-terminal regions of each domain, respectively. The kinetic parameters of SAL obtained by measuring surface plasmon resonance were as follows: K(a) (M(-1)) for neohesperidosyl-BSA, 7. 1 x 10(6); for melibiosyl-BSA, 4.9 x 10(6); and for lactosyl-BSA, 5. 2 x 10(5). These results show that RBLs including SAL comprise a family of alpha-galactosyl binding lectins having characteristic tandem repeat domain structures.

Amino Acid Sequence↗
PubMed1999

Free-living nematodes Caenorhabditis elegans possess in their mitochondria an additional rhodoquinone, an essential component of the eukaryotic fumarate reductase system.

The respiratory chain of Caenorhabditis elegans was characterized in mitochondria isolated from aerobically grown nematodes. Nematode mitochondria contain ubiquinone-9 as a major component and rhodoquinone-9 as a minor component. The ratio of ubiquinone-9/rhodoquinone-9 is higher in C. elegans mitochondria than in mitochondria from second-stage larvae of Ascaris suum, the free-living stage of porcine gut-dwelling nematode. The individual oxidoreductase activities comprising succinate oxidase and the amount of substrate-reducible cytochromes are comparable to those of mitochondria from second-stage larvae of A. suum. The specific activity of fumarate reductase is lower in C. elegans mitochondria than in mitochondria from second-stage larvae of A. suum, but still higher than in mammalian mitochondria. These results indicate that the free-living nematode C. elegans is capable of synthesizing rhodoquinone, as distinguished from aerobic mammalian species, although its mitochondria appear more aerobic than A. suum larval mitochondria.

Aerobiosis↗
PubMed1999

Molecular recognition of adenophostin, a very potent Ca2+ inducer, at the D-myo-inositol 1,4,5-trisphosphate receptor.

The recognition mode of adenophostin A at the D-myo-inositol 1,4, 5-trisphosphate [Ins(1,4,5)P(3)] receptor was investigated. Comparison of conformations of Ins(1,4,5)P(3) and adenophostin A by using the combination of NMR and molecular mechanics (MM) calculations demonstrated that adenophostin A adopted a moderately extended conformation regarding the distance between the 2'-phosphoryl group and the 3' ',4' '-bisphosphate motif, as suggested previously [Wilcox, R. A. et al. (1995) Mol. Pharmacol. 47, 1204-1211]. Based on the nuclear Overhauser effect (NOE) observed between 3'-H and 1' '-H and on MM calculations, the molecular shape of adenophostin A proved to be an extended form at least in solution, in contrast to Wilcox's compactly folded, preliminary hairpin model. GlcdR(2,3',4')P(3), an adenophostin analogue without adenine moiety, was found to be less potent than adenophostin A and almost equipotent to Ins(1,4,5)P(3). We propose the possibility that (i) the optimal spatial arrangement of the three phosphoryl groups and/or (ii) the interaction of the adenine moiety of adenophostin A with the putative binding site, if it exists in the vicinity of the Ins(1,4,5)P(3)-binding site, might account for the exceptional potency of adenophostin A.

Adenosine↗
PubMed1999

Autolysosomal membrane-associated betaine homocysteine methyltransferase. Limited degradation fragment of a sequestered cytosolic enzyme monitoring autophagy.

We compared the membrane proteins of autolysosomes isolated from leupeptin-administered rat liver with those of lysosomes. In addition to many polypeptides common to the two membranes, the autolysosomal membranes were found to be more enriched in endoplasmic reticulum lumenal proteins (protein-disulfide isomerase, calreticulin, ER60, BiP) and endosome/Golgi markers (cation-independent mannose 6-phosphate receptor, transferrin receptor, Golgi 58-kDa protein) than lysosomal membranes. The autolysosomal membrane proteins include three polypeptides (44, 35, and 32 kDa) whose amino-terminal sequences have not yet been reported. Combining immunoblotting and reverse transcriptase-polymerase chain reaction analyses, we identified the 44-kDa peptide as the intact subunit of betaine homocysteine methyltransferase and the 35- and 32-kDa peptides as two proteolytic fragments. Pronase digestion of autolysosomes revealed that the 44-kDa and 32-kDa peptides are present in the lumen, whereas the 35-kDa peptide is not. In primary hepatocyte cultures, the starvation-induced accumulation of the 32-kDa peptide occurs in the presence of E64d, showing that the 32-kDa peptide is formed from the sequestered 44-kDa peptide during autophagy. The accumulation is induced by rapamycin but completely inhibited by wortmannin, 3-methyladenine, and bafilomycin. Thus, detection of the 32-kDa peptide by immunoblotting can be used as a streamlined assay for monitoring autophagy.

Animals↗
PubMed1999

Differential involvement of mu-opioid receptor subtypes in endomorphin-1- and -2-induced antinociception.

We investigated the role of mu-opioid receptor subtypes in both endomorphin-1 and endomorphin-2 induced antinociception in mice using supraspinally mediated behavior. With tail pressure as a mechanical noxious stimulus, both intracerebroventricularly (i.c.v.) and intrathecally (i.t.) injected-endomorphins produced potent and significant antinociceptive activity. Antinociception induced by i.t. and i.c.v. injection of endomorphin-1 was not reversed by pretreatment with a selective mu1-opioid receptor antagonist, naloxonazine (35 mg/kg, s.c.). By contrast, antinociception induced by i.t. and i.c.v. endomorphin-2 was significantly decreased by mu1-opioid receptor antagonist. Antinociception of both i.t. and i.c.v. endomorphin-1 and -2 was completely reversed by pretreatment with beta-funaltrexamine (40 mg/kg, s.c.). The results indicate that endomorphins may produce antinociception through the distinct mu1 and mu2 subtypes of mu-opioid receptor.

Analgesics, Opioid↗
PubMed1999

Pelizaeus-Merzbacher disease: three novel mutations and implication for locus heterogeneity.

We report a mutational and polymorphic analysis of the proteolipid protein gene in members of 27 Japanese families with Pelizaeus-Merzbacher disease. We found causative mutations in 6 members of 27 families (22.2%); 5 of the 6 mutations, including two novel mutations, Leu45Arg and 231 + 2T --> G, resulted in the typically severe clinical symptoms. Paradoxically, the Cys219Tyr mutation, presumed to disrupt the tertiary structure of proteolipid protein by removing the disulfide bond between Cys200 and Cys219, was associated with a mild clinical presentation wherein the patient could walk with assistance and speak. It was inferred that the structural change prevented the toxicity associated with a gain of function mutation. Moreover, in one family 3 patients exhibited a intragenic polymorphism that did not segregate with the disease, suggesting a locus heterogeneity for Pelizaeus-Merzbacher disease.

Adult↗
PubMed1999

Plasminogen activator inhibitor 1 in central serous chorioretinopathy.

PURPOSE: To describe the plasma level of plasminogen activator inhibitor 1, the major antifibrinolytic agent, in eyes with central serous chorioretinopathy, in which choroidal thrombosis is suspected as the underlying condition based on the findings of choroidal hyperpermeability in indocyanine green angiograms. METHODS: Plasminogen activator inhibitor 1 concentrations in the plasma of 17 patients with central serous chorioretinopathy were measured by enzyme-linked immunosorbent assay and compared with those in 12 age-matched normal volunteers. RESULTS: The plasma levels of plasminogen activator inhibitor 1 in patients with central serous chorioretinopathy (range, 25 to 439 ng/ml; median, 87 ng/ml) were significantly increased compared with those in normal volunteers (range, 7 to 84 ng/ml; median, 36 ng/ml) (P = .0013, Mann-Whitney U test). CONCLUSION: Increased plasminogen activator inhibitor 1 concentrations in patients with central serous chorioretinopathy support the hypothesis that the choroidal hyperpermeability disclosed by indocyanine green angiography is caused from impaired fibrinolysis and the resulting thrombotic occlusion in the choroidal veins.

Adult↗
PubMed1999

Cholecystectomy is an effective treatment for biliary dyskinesia.

BACKGROUND: An increasing number of reports indicate symptomatic relief of biliary colic symptoms after cholecystectomy for biliary dyskinesia. Despite this, cholecystectomy as a treatment for biliary dyskinesia remains controversial. Our aim was to determine efficacy of cholecystectomy in alleviating biliary dyskinesia symptoms and the correlation with histologic findings. METHODS: Records of patients with gallbladder ejection fraction <35% between January 1994 and February 1999 were reviewed. Gallbladder pathology and degree of symptomatic improvement were determined on follow-up. RESULTS: Of the 27 cholecystectomy patients, 24 (89%) had significant improvement, 2 (7%) had partial improvement, and 1 (4%) had minimal improvement. Ten patients (43%) had normal gall-bladder, and 9 (90%) of them had significant improvement after cholecystectomy. Of the 6 nonsurgical patients, none had significant improvement, 4 (67%) had partial improvement, and 2 (33%) had minimal improvement. CONCLUSIONS: Biliary dyskinesia patients who underwent cholecystectomy had significantly greater symptom improvement compared with nonsurgical patients. Pathologic correlation suggests chronic inflammation may not be the only cause of gallbladder dysfunction. Cholecystectomy should be a first-line therapy for biliary dyskinesia patients.

Algorithms↗