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T Hirabayashi

Publications and source records attributed to T Hirabayashi.

At least 55 records · Page 3Linked to original sources
PubMed1996

Purification and characterization of bleomycin hydrolase, which represents a new family of cysteine proteases, from rat skin.

Bleomycin (BLM) hydrolase, which hydrolyzes the carboxyamide bond in the beta-amino-alanine moiety, was purified from newborn rat skin. The enzyme was purified 2,500-fold over the crude extract to apparent homogeneity in five steps in the presence of 2-mercaptoethanol: 45-55% ammonium sulfate fractionation, followed by chromatographies on Sephacryl S-200, DEAE-cellulofine, Phe-Superose, and Mono Q ion-exchange. The native enzyme had a molecular mass of 280 kDa according to gel filtration. The subunit molecular mass was estimated as 48 kDa by SDS-PAGE, indicating that the enzyme was comprised of six identical subunits. The amino acid sequence of its NH2-terminus was determined to be acetyl-Met-Asn-Asn-Ala-Gly-Leu-Asn-Ser-Glu-Lys-, which was not found in the amino acid sequence database. The optimum pH of the enzyme was 7.5 with pepleomycin (PLM). The Km and Vmax values were 2.1 mM and 6.8 mu mol center dot mg-1 center dot h-1 for PLM, and 1.8 mM and 7.2 mu mol center dot mg-1 center dot h-1 for BLM-A2, respectively. The enzyme activity was inhibited by iodoacetic acid, N-ethylmaleinimide (NEM), and p-chloromercuribenzoic acid (pCMB) as well as divalent cations such as Cu2+, Cd2+, Hg2+, and Zn2+. It was effectively inhibited by a cysteine protease inhibitor E-64. However, cystatins A and C did not inhibit the activity. BLM hydrolase exhibited broad aminopeptidase substrate specificity towards aminoacyl-beta-naphthylamides such as basic, neutral, and hydrophobic amino acid residues, as well as acidic residues. These results indicated that BLM hydrolase represents a new family of cysteine proteases. Western blotting and immunohistochemical analyses showed that BLM hydrolase is ubiquitous in various rat tissues but at low levels in lung and adult skin tissues, suggesting that this enzyme plays an important role in the metabolism of antibiotics.

Amino Acid Sequence↗
PubMed1996

Cloning and analysis of cDNA encoding rat bleomycin hydrolase, a DNA-binding cysteine protease.

We isolated and characterized almost the entire cDNA encoding BLM hydrolase from rat spleen cDNA libraries. The cDNA encoded a polypeptide composed of 454 amino acids, that had a slightly larger molecular mass than that was previously estimated by SDS-PAGE for purified BLM hydrolase subunit. Amino acid sequence alignments showed that rat BLM hydrolase is homologous to that of rabbit (94% identity of partial amino acid sequence), yeast cysteine protease (39% identity), and the pepC gene products of three bacteria (34-40% identity). In addition, it contained the three regions that are conserved in other cysteine proteases and thought to constitute the catalytic center. These results indicated that rat BLM hydrolase is a member of the papain superfamily of cysteine proteases. Sequencing revealed several putative sites phosphorylated by different types of protein kinases, but no signal sequence, transmembrane domain, N-linked glycosylation site or DNA-binding motif. The yeast homolog is a DNA-binding cysteine protease [Xu et al. (1994) J. Biol. Chem, 269, 21177-21183]. We demonstrated that rat BLM hydrolase also binds the single-stranded form of the Ga14 DNA-binding site oligonucleotide with high affinity compared with that of the double-stranded form. Northern blots revealed that the level of BLM hydrolase mRNA expression was very low in the rat skin, lung, and skeletal muscle. Furthermore, BLM hydrolase mRNA was ubiquitously expressed in the human cell lines, HeLa, SKG-IIIa, FL, KB, HEp-2, U373 GM, P3HR-1, Raji, THP-1, Jurkat, and Molt-4. These results suggested that BLM hydrolase plays important physiological roles, including the metabolism of antibiotics.

Amino Acid Sequence↗
PubMed1996

Expression of chicken troponin T isoforms in cultured muscle cells.

Cells prepared from chicken skeletal muscles of different developmental stages were cultured to study their troponin T isoform expression, using antisera specific to the fast- and slow-muscle-type isoforms. We found that the cultured myogenic cells from chickens and chick embryos were classified into two types, fast type and fast/slow type in which fast- and slow-muscle-type isoforms were coexpressed. Cells expressing only slow-muscle-type troponin T isoforms could not be found. Most cells prepared from pectoralis major (fast muscle) and gastrocnemius (mixed muscle) of 11-day old embryos belonged to the latter, with only a small fraction belonging to the former. The percentage of fast type cells in those cells prepared from pectoralis major increased along development to over 90% by the 17th day of incubation, while, in the cells prepared from gastrocnemius, it reached a plateau of 30-40% by the 13th day of incubation. All the cells from anterior latissimus dorsi (slow muscle) belonged to the fast/slow type. Ratios of these two types of muscle cells varied depending on their origins and stages. The in vitro expression of troponin T isoforms was different from the in vivo expression, and each muscle seems to be determined differently in the composition of cell types during the developmental course.

Animals↗
PubMed1996

Analysis of dystrophin in muscular diseases by two-dimensional gel electrophoresis using agarose gels in the first dimension.

We analyzed dystrophin in case of normal control, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD) and infectious muscular disease using two-dimensional gel electrophoresis and immunoblotting with 3 monoclonal dystrophin antibodies: Dys 1, a mid-rod-domain antibody; Dys 2, a C-terminal-domain antibody; and Dys 3, an N-terminal-domain antibody. In cases of normal control, a clearly separated doublet of bands was observed for Dys 1 and 3 at molecular weights 400 and 420 kDa. The isoelectric point was between pH approximately 5.7-approximately 5.9, similar to that for the myosin heavy chain. In one DMD case, a single faint band was observed for Dys 2. BMD presented a single-band pattern for each antibody. Infectious diseases cases showed 3- to 5-band patterns for Dys 1 and single or no bands for Dys 2 and 3. The pI of the Dys 1 band was almost identical. These results suggest coexistence of normal dystrophin and its proteolytic products, both containing triple helical segment, and show that two-dimensional gel electrophoresis may be applicable in the analysis of dystrophin in muscular disease.

Adolescent↗
PubMed1996

Use of enzyme-linked-immunosorbent assay for detection of IgG and IgM antibodies to Fusobacterium necrophorum in cattle.

An enzyme-linked-immunosorbent assay (ELISA) with HCl heat-extracted antigen of Fusobacterium necrophorum was conducted to detect specific immunoglobulins G and M in infected cattle. The ELISA revealed an increase (> 0.40) in specific IgG in most of the animals with hepatic abscesses but not that in specific IgM. All the lesions were positive for F. necrophorum. These findings indicated that the ELISA for immunoglobulin G detection may prove to be a useful tool for predictive serodiagnosis of F. necrophorum infection in cattle.

Animals↗
PubMed1995

Outcomes of ileocolic conduit for biliary drainage in infants with biliary atresia; comparison with Roux-en-Y type reconstruction.

This study was undertaken to define the role of an ileocolic conduit that was devised as anti-reflux procedure in comparative study with Roux-en-Y type reconstruction in infants with biliary atresia treated at a single institution. The ileocolic conduit (IC) consisted of 30 cm of distal ileum anastomosed to the portahepatis and a 10-cm segment of ascending colon vented through the abdominal wall, which was later anastomosed to the second portion of the duodenum. In the Roux-en-Y type reconstruction (RY), 40 to 60 cm of jejunum was used for the hepatic limb. Twenty-three infants in group IC and 16 infants in group RY were entered in this study. Mean ages at definitive surgery were 65.3 +/- 23.1 days for group IC and 65.0 +/- 19.5 days for group RY. The two groups were compared for the postoperative outcomes in terms of bile excretion, incidence of cholangitis, esophageal varices, survival rate, and the effects on physical development. The follow-up ranged from 4 to 15 years. Bile excretion was obtained in all infants in both groups. Twenty infants (87.0%) in group IC and 10 infants (62.5%) in group RY became anicteric. Cholangitis occurred in 60.9% of group IC, compared with 83.3% for group RY, in which 6 infants required revision to complete diversion of Roux-en-Y limb (Suruga II). There was the same occurrence rate of esophageal varices for both groups. However, the varices tended to develop later in group IC (53.0 +/- 18.4 months) than in group RY (12.7 +/- 6.0 months) (P < .02).(ABSTRACT TRUNCATED AT 250 WORDS)

Biliary Atresia↗
PubMed1995

Efficacy of traditional herbal medicines in combination with acyclovir against herpes simplex virus type 1 infection in vitro and in vivo.

Traditional herbal medicines have been safely used for the treatment of various human diseases since ancient China. We selected 10 herbal extracts with therapeutic antiherpes simplex virus type 1 (HSV-1) activity. Among these, Geum japonicum Thunb., Rhus javanica L., Syzygium aromaticum (L.) Merr. et Perry, or Terminalia chebula Retzus showed a stronger anti-HSV-1 activity in combination with acyclovir than the other herbal extracts in vitro. When acyclovir and/or a herbal extract were orally administered at doses corresponding to human use, each of the 4 combinations significantly limited the development of skin lesions and/or prolonged the mean survival times of infected mice compared with both acyclovir and the herbal extract alone (P < 0.01 or 0.05). These combinations were not toxic to mice. They reduced virus yields in the brain and skin more strongly than acyclovir alone and exhibited stronger anti-HSV-1 activity in the brain than in the skin, in contrast to acyclovir treatment by itself. Combinations of acyclovir with historically used herbal medicines showed strong combined therapeutic anti-HSV-1 activity in mice, especially reduction of virus yield in the brain.

Acyclovir↗
PubMed1995

Inhibitory effect of bafilomycin A1, a specific inhibitor of vacuolar-type proton pump, on the growth of influenza A and B viruses in MDCK cells.

We studied the effect of bafilomycin A1 (Baf-A1), a novel and highly specific inhibitor for vacuolar-type proton (V-H+) pump, on the growth of influenza A and B viruses in Madin-Darby canine kidney cells. Vital fluorescence microscopic study showed that Baf-A1 induced the complete disappearance of acidified compartments such as endosomes and lysosomes both in infected and uninfected cells by the treatment with 0.1 microM inhibitor for 1 h at 37 degrees C. In addition, virus growth was inhibited when Baf-A1 was present from 1 h before infection to the end of incubation, or added within as early as 5-10 min after infection. Conversely, the virus growth was recovered in correlation with the reappearance of acidified compartments after removal of Baf-A1. These data suggest that Baf-A1-sensitive V-H+ pumps are solely responsible for the acidification of endosomes and lysosomes, and thus Baf-A1 inhibits the growth of influenza A and B viruses by affecting the acidified compartments in which low pH is essential for the uncoating process of influenza virus growth at an early stage of infection.

Animals↗
PubMed1995

Inhibitory effect of ferulic acid and isoferulic acid on murine interleukin-8 production in response to influenza virus infections in vitro and in vivo.

We investigated the effect of ferulic acid (FA) and isoferulic acid (IFA), which are active components of the rhizoma of Cimicifuga species used frequently as anti-inflammatory drugs in Japanese Oriental medicines, on murine interleukin-8 (IL-8) production in response to influenza virus infections in vitro and in vivo by antibody-sandwich enzyme-linked immunosorbent assay. In the in vitro study, the murine macrophage cell line RAW 264.7 was infected with influenza virus at a dose of 10 plaque forming units (PFU)/cell and cultured in the presence or absence of drugs. Both FA and IFA reduced the IL-8 levels in the 20-h conditioned medium in comparison with control in a dose-dependent manner. The effect of IFA was greater than that of FA: IL-8 levels were reduced to 43% and 56% of the control in the presence of 100 micrograms/ml of IFA and FA, respectively. In the in vivo study, mice were infected with 1,000 PFU of virus and received daily oral administrations of Cimicifuga heracleifolia extract (5 mg/mouse/day), FA (0.5 mg/mouse/day), IFA (0.125 mg/mouse/day), or phosphate buffered saline. The three drugs showed a tendency to reduce IL-8 levels in bronchoalveolar lavage (BAL) obtained 2 days after infection. Moreover, both FA and IFA also significantly reduced the number of exuded neutrophils into BAL. However, the drug administrations did not affect the virus yields in BAL. These data suggest that FA and IFA are novel and potent inhibitors of murine IL-8 production and might act as one of the main components of anti-inflammatory rhizoma of Cimicifuga species.

Animals↗
PubMed1995

[Evaluation of 5-FU, leucovorin, etoposide, and cisplatin (FLEP) chemotherapy by hepatic artery injection in the treatment of multiple liver metastases from gastric cancer].

We performed FLEP chemotherapy (consisting of 5-FU, leucovorin, etoposide, and cisplatin) by hepatic artery injections for three patients with multiple liver metastases from gastric cancer, and two of three resulted in partial response (PR). We presented two PR cases. Case 1 is a 57-year-old male with multiple liver metastases from gastric cancer. Distal partial gastrectomy with regional lymphadenectomies were carried out, and an injection port was implanted in the hepatic artery. We performed FLEP chemotherapy from 16 days after the operation. Liver metastases subsided and resulted in PR after 3 months by CT scan. He is now healthy and working for 15 months after the operation. Case 2 was a 49-year-old female with multiple liver metastases from gastric cancer. Total gastrectomy with regional lymphadenectomies was carried out. We performed FLEP chemotherapy by hepatic artery injections from 21 days after the operation. The response of chemotherapy resulted in PR by CT scan, and she is now healthy and has been working for 11 months after the operation. Thus, this form of chemotherapy may be useful for patients with multiple liver metastases from gastric cancer.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1994

Coexistence of fast-muscle-type and slow-muscle-type troponin T isoforms in single chimeric muscle fibers induced by muscle transplantation.

Regenerated muscle fibers which appeared after transplantation of chicken slow muscle (anterior latissimus dorsi) into breast fast muscle (pectoralis major) of the same animal were studied by two-dimensional SDS-polyacrylamide gel electrophoresis, immunoblotting, and immunostaining with antisera against fast-muscle-type troponin T and slow-muscle-type troponin T. In the transplanted muscle, degeneration of muscle fibers was followed by regeneration of slow muscle, which was revealed by detecting slow-muscle-type troponin T with the antiserum. Furthermore, coexistence of fast-muscle-type and slow-muscle-type troponin T isoforms in single chimeric muscle fibers composed of partly fast and partly slow fibers was observed in the regenerated muscle. We suggested that the chimeric fibers were originated from the fusion of fast and slow myoblasts during regeneration after muscle transplantation and that two nuclei differently determined in troponin T expression were working independently in a single cell.

Animals↗
PubMed1994

Persistent expression of tissue-specific troponin T isoforms in transplanted chicken skeletal muscle.

This study attempted to investigate the expression of skeletal muscle troponin T isoforms in chicken reared for six months after muscle transplantations of breast muscle into leg muscle, leg muscle into breast muscle, and slow muscle into breast and leg muscles of the same animal. The regenerated muscle after transplantation was studied by histological observation, two-dimensional SDS-polyacrylamide gel electrophoresis, and immunoblotting with anti-troponin T antibodies. Persistent expression of troponin T isoforms specific to donor tissue was observed in the regenerated muscle, and compared with their expression in the normal developing muscles. During the regeneration, the cells grew up and expressed troponin T isoforms in a manner similar to that in normal developing muscles, and on around the 178th day after the transplantation, the regenerated muscle expressed the adult type troponin T isoforms. Based on the troponin T isoforms expressed in the transplants, we consider that one type of skeletal muscle has some inherent potential to grow in and coexist with other types for a long term.

Animals↗
PubMed1994

Ubiquitin C-terminal hydrolase as a putative factor involved in sex differentiation of fish (temperate wrasse, Halichoeres poecilopterus).

Temperate wrasse (Halichoeres poecilopterus) is known to undergo sex transition from female to male (protogyny). In order to detect factors related to male sex differentiation which appear during sex transition, we compared protein constituents between transitional and mature gonads by two-dimensional gel electrophoresis. Eight proteins were found to increase in amount considerably in the transitional gonads in comparison with the counterparts in ovaries. Five of the eight proteins were also found in mature testes. One of the proteins shared by testes and transitional gonads had an isoelectric point at pH 5.3 and an apparent molecular weight of 26 kDa. Thus we termed the protein p 26. It was abundant in degenerating ovary, transitional gonad, and mature testis and less abundant in mature ovary and brain. Therefore, the expression of p 26 seemed to start during sex transition and continue during maturation of the testis. Amino acid sequence analysis revealed high homology of p 26 to ubiquitin C-terminal hydrolase, which is supposed to cleave ubiquitin associated with nuclear proteins and a transcriptional repressor. Therefore, p 26 was supposed to regulate gene expression through the mediation of ubiquitin. Proteins which seemed to be counterparts of p 26 were detected in testes of two other fish species by an antiserum against p 26. These results suggest that p 26 is a possible candidate for the factor which is related to sex differentiation.

Amino Acid Sequence↗
PubMed1994

Detection and characterization of muscle-specific nuclear proteins.

To identify nuclear proteins related to muscle tissue specificity, we tried to prepare antibodies recognizing muscle-specific nuclear proteins. Taking advantage of the autoimmunity of some nuclear proteins, we prepared an antiserum against chick muscle nuclear proteins by injecting protein components of the nuclei isolated from chick breast muscles into breast muscle. Three proteins, named p30, p32, and p37, were detected with the antiserum in a two-dimensional SDS-PAGE pattern of the isolated nuclei. P30 and p32 were not detected in the nuclei of liver, brain, cardiac muscle, or slow type skeletal muscle (anterior latissimus dorsi). They were detected in those of fast type skeletal muscle (pectoralis major and semitendinosus) and smooth muscle (gizzard) at all developmental stages examined. On serial fractionation of muscle cell nuclei, they were detected in a fraction obtained after DNase I treatment of the sample, suggesting that the proteins weakly bind to chromatin. A homology search of amino acid sequences showed that there is no known protein similar to p32.

Amino Acid Sequence↗
PubMed1993

Synthesis and conformational study of peptides possessing helically arranged delta ZPhe side chains.

Z-Dehydrophenylalanine (delta ZPhe) possessing four oligopeptides, Boc-(L-Ala-delta ZPhe-Aib)n-OCH3 (n = 1-4: Boc, t-butoxycarbonyl; Aib, alpha-aminoisobutyric acid), were synthesized, and their solution conformations were investigated by 1H-nmr, ir, uv, and CD spectroscopy and theoretical CD calculation. 1H-nmr (the solvent accessibility of NH groups) and ir studies indicated that all the NH groups except for those belonging to the N-terminal L-Ala-delta ZPhe moiety participate in intramolecular hydrogen bonding in chloroform. This suggests that the peptides n = 2-4 have a 4-->1 hydrogen-bonding pattern characteristic of 3(10)-helical structures. The uv spectra of all these peptides recorded in chloroform and in trimethyl phosphate showed an intense maximum around 276 nm assigned to the delta ZPhe chromophores. The corresponding CD spectra of the peptides n = 2-4 showed exciton couplets with a negative peak at longer wavelengths, whereas that of the peptide n = 1 showed only weak signals. Theoretical CD spectra were calculated for the peptides n = 2-4 of several helical conformations, on the basis of exciton chirality method. This calculation indicated that the three peptides form a helical conformation deviating from the perfect 3(10)-helix that contains three residues per turn, and that their side chains of delta ZPhe residues are arranged regularly along the helix. The center-to-center distance between the nearest phenyl pair(s) was estimated to be approximately 5.5 A. The chemical shifts of the delta ZPhe side-chain protons (H beta and aromatic H) for the peptides n = 2-4 indicated anisotropic shielding effect of neighboring phenyl group (s); the effect also supports a regular arrangement of the delta ZPhe side chains along the helical axis.

Amino Acid Sequence↗
PubMed1993

Possible association of p53 overexpression and mutation with high-grade chondrosarcoma.

Overexpression and point mutation of the p53 protein/gene was investigated in a series of chondrosarcoma by an immunohistochemical approach, and direct sequencing of the genomic DNA, respectively. In 2 of the 16 cases studied, both of which were high grade chondrosarcomas (grade III), immunodetectable p53 was identified. Histologically, one was ordinary type and the other a clear cell variant. However, no positivity was observed in the other cases including nine of low grade, ordinary type, three of low grade, clear cell type, and two of extraskeletal myxoid chondrosarcoma. Direct sequencing, following polymerase chain reaction amplification of exons 5-9 of the p53 gene in 14 cases, in which fresh materials were available, successfully demonstrated base substitution mutations in only two cases with detectable p53 overexpression on immunohistochemistry. Their details were GTC (valine) to TTC (phenylalanine) at codon 157 in exon 5, and CGT (arginine) to CAT (histidine) at codon 273 in exon 8. No mutation was detected in the other 12 cases which were negative for p53 immunostaining. These findings strongly suggest that p53 mutation plays a crucial role in the biologically aggressive subtype, and possibly in the process of tumor progression in human chondrosarcoma.

Adult↗
PubMed1992

Histological distribution and developmental changes of tropomyosin isoforms in three chicken digestive organs.

Histological localization of tropomyosin isoforms in three digestive organs from embryonic and adult chickens was performed by using rabbit antisera against chicken skeletal muscle tropomyosin and against low-Mr-type tropomyosin from chicken small intestine mucosa. The former antiserum (named TM-SH) reacted with alpha, beta, and high-Mr-type isoforms, and the latter (named TM-HL) reacted with alpha, beta, high-Mr-type and low-Mr-type isoforms, alpha and beta Isoforms were detected in muscle cells of the muscular layer and the muscularis mucosa. Low-Mr-type isoforms, however, were detected along the cell membrane and cytoplasm of almost all nonmuscle cells, especially in terminal webs of epithelial cells. Developmental changes of tropomyosin isoforms in digestive organs were studied by two-dimensional gel electrophoresis and image analysis. The relative amounts of alpha and beta isoforms increased in the course of development, but those of low-Mr-type and high-Mr-type isoforms decreased.

Animals↗