Biomedical subjects

S Taguchi

Publications and source records attributed to S Taguchi.

At least 55 records · Page 3Linked to original sources

PubMed1998

Nucleotide sequencing analysis of the 146-kilobase segment around the IkBL and MICA genes at the centromeric end of the HLA class I region.

To elucidate the complete gene structure and to identify new genes involved in the development of HLA class I antigen-associated diseases in the class I region of the human major histocompatibility complex on chromosome 6, a YAC clone (745D12) covering the 146-kb segment around the IkBL and MICA loci was isolated from a YAC library constructed from the B-cell line, BOLETH. A physical map of this region was constructed by isolation of overlapping cosmid clones derived from 745D12. Of these, five contiguous cosmids were chosen for DNA sequencing by the shotgun strategy to give a single contig of 146,601 bp from 2.8 kb telomeric of the IkBL gene to exon 6 of MICA. This region was confirmed to contain five known genes, IkBL, BAT1, MICB, P5-1, and HLA-X (class I fragment), from centromere to telomere, and their exon-intron organizations were determined. The 3.8-1 homologue gene (3.8-1-hom) showing 99.7% identity with the 3.8-1 cDNA clone, which was originally isolated using the 3.8-kb EcoRI fragment between the HLA-54/H and the HLA-G genes, was detected between MICA and MICB and was suggested to represent the cognate 3.8-1 genomic sequence from which the cDNA clone was derived. No evidence for the presence of expressed new genes could be obtained in this region by homology and EST searches or coding and exon prediction analyses. One TA microsatellite repeat spanning 2545 bases with as many as 913 repetitions was found on the centromeric side of the MICA gene and was indicated to be a potential hot spot for genetic recombination. The two segments of approximately 35 kb upstream of the MICA and MICB genes showed high sequence homology (about 85%) to each other, suggesting that segmental genome duplication including the MICA and MICB genes must have occurred during the evolution of the human MHC.

Carrier Proteins↗

PubMed1998

Reduced reactivity of pancreatic exocrine secretion in response to gastrointestinal hormone in WBN/Kob rats.

We compared pancreatic exocrine secretion in 5-month-old WBN/Kob rats, a model of chronic pancreatitis, with that in Wistar rats of the same age in a conscious state. Basal pancreatic secretion and pancreatic wet weight in WBN/Kob rats were lower than the values for Wistar rats. There was no difference in plasma cholecystokinin (CCK) concentration between the two types of rats. When CCK-8 was intravenously administered, the stimulation of pancreatic protein secretion in WBN/Kob rats was weaker than that in Wistar rats. When bile and pancreatic juice were diverted from the duodenum, the resulting increase in the plasma CCK concentration was similar in both types of rats, but stimulation of the volume and protein output of pancreatic juice in WBN/Kob rats was weaker than that in Wistar rats. In addition, WBN/Kob rats exhibited little increase in pancreatic wet weight because of this diversion. When secretin was intravenously administered, the stimulation of fluid secretion in WBN/ Kob rats was also weaker than that in Wistar rats. The binding of CCK-8 to pancreatic membrane fractions in WBN/Kob rats was much weaker than that in Wistar rats. Histological findings in WBN/Kob rat pancreas showed proliferation of fibrous tissue and atrophy of acinar cells. In conclusion, pancreatic exocrine secretion in response to the gastrointestinal hormones, CCK and secretin, was lower in WBN/Kob rats than in Wistar rats. These findings suggest that the hyposecretion of pancreas in WBN/Kob rats is hyporeaction of pancreatic membrane to gastrointestinal hormones.

Animals↗

PubMed1998

Clinical significance of serum levels of CD44 variant exons 8-10 protein in colorectal cancer.

We examined serum levels of a CD44 splice variant that contained variant exons 8-10 (CD44v8-10) as a tumor marker in colorectal cancer patients. We performed enzyme-linked immunosorbent assays in 81 sera obtained from 71 colorectal cancer patients and 10 healthy controls. Serum CD44v8-10 levels were significantly higher in the colorectal cancer patients than in the healthy controls (0.209 +/- 0.098 versus 0.114 +/- 0.019 OD; P < 0.01). There was a close correlation between immunohistochemical expression and serum CD44v8-10 levels. Surgical resection of the tumors resulted in a reduction of serum CD44v8-10 levels. There was no significant correlation between serum CD44v8-10 level and serosal invasion or histologic type. However, a significant correlation was observed between serum CD44v8-10 level and lymphatic or venous invasion. In addition, serum CD44v8-10 levels were significantly higher in carcinomas associated with lymph node or liver metastasis than in those without metastasis. These findings suggest the usefulness of serum CD44v8-10 level in the prediction of colorectal cancer metastasis.

Biomarkers, Tumor↗

PubMed1998

A novel insect defensin from the ant Formica rufa.

By combination of size exclusion and reversed-phase chromatography, we have isolated a novel member of insect defensin-type antimicrobial peptides from the entire bodies of bacteria-challenged Formica rufa (hymenoptera, formicidae). The molecular mass of the purified peptide was estimated to be 4120.42 by matrix-assisted laser desorption/ionization-time of flight/mass spectrometry. Sequence analysis revealed that this peptide consisted of 40 amino acid residues with six cysteines engaged in the formation of three intramolecular disulfide bridges. This peptide is unique among the arthropod defensins in terms of the presence of asparatic acid and alanine at position 33 and as C-terminal residue, respectively. In addition, this novel defensin from Formica rufa has the particularity to have no C-terminal extension in contrast to those reported for other hymenoptera defensins.

Amino Acid Sequence↗

PubMed1998

An endogenous target protease, SAM-P26, of Streptomyces protease inhibitor (SSI): primary structure, enzymatic characterization, and its interaction with SSI.

We have been focusing on the potent involvement of the molecular interaction between a protease and a protease inhibitor in the physiological or morphological regulation of Streptomyces cells producing them [Taguchi et al. (1995) J. Bacteriol. 177, 6638-6643; Suzuki et al. (1997) J. Bacteriol. 179, 430-438]. In this study, an extracellular protease, termed SAM-P26, was isolated as a target of endogenous protease inhibitor (SSI) from the culture medium of an SSI non-producing mutant strain derived from Streptomyces albogriseolus S-3253. Complete amino acid sequence determination revealed that SAM-P26 is identical to a protein encoded by the SAM-P20D gene, which was previously found to be located downstream of the gene for SAM-P20, another target protease of SSI. Based on the sequence homology, SAM-P26 was categorized as a member of the chymotrypsin family like SAM-P20. Sequence similarity between SAM-P26 and SAM-P20 was immunologically demonstrated by Western blot analysis using anti-SAM-P20 antiserum. The molecular mass (26 kDa) of SAM-P26 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was much higher than that calculated from the amino acid sequence of SAM-P26 (18,376.8 Da) and that of the S-pyridylethylated form (18,808.4 Da) of SAM-P26 determined by Matrix-assisted Laser Desorption/Ionization-Time of Flight/Mass Spectrometry. Analytical gel-filtration analysis revealed that SAM-P26 exists as a monomer (18.8 kDa) in the native state. The results as to substrate specificity and inhibitor sensitivity indicated SAM-P26 exhibits chymotrypsin-like activity. For the proteolytic activity, the optimal pH was 10.5 and the optimal temperature was 60 degreesC. The complex formation of SAM-P26 with SSI was confirmed by native-PAGE analysis.

Amino Acid Sequence↗

PubMed1998

Engineering of a cold-adapted protease by sequential random mutagenesis and a screening system.

A cold-adapted protease subtilisin was successfully isolated by evolutionary engineering based on sequential in vitro random mutagenesis and an improved method of screening (H. Kano, S. Taguchi, and H. Momose, Appl. Microbiol. Biotechnol. 47:46-51, 1997). The mutant subtilisin, termed m-63, exhibited a catalytic efficiency (expressed as the kcat/Km value) 100% higher than that of the wild type at 10 degrees C when N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide was used as a synthetic substrate. This cold adaptation was achieved with three mutations, Val to Ile at position 72 (V72I), Ala to Thr at position 92 (A92T), and Gly to Asp at position 131 (G131D), and it was found that an increase in substrate affinity (i.e., a decreased Km value) was mostly responsible for the increased activity. Analysis of kinetic parameters revealed that the V72I mutation contributed negatively to the activity but that the other two mutations, A92T and G131D, overcame the negative contribution to confer the 100% increase in activity. Besides suppression of the activity-negative mutation (V72I) by A92T and G131D, suppression of structural stability was observed in measurements of activity retention at 60 degrees C and circular dichroism spectra at 10 degrees C.

Cold Temperature↗

PubMed1998

Mechanism of rethrombosis after thrombolytic therapy: angioscopic findings and investigation of the coagulation system in dogs.

The purpose of this study was to angioscopically observe the process of thrombolysis after intracoronary administration of thrombolytic agents and to investigate the effects of these agents on coagulation/fibrinolysis systems in dogs. The coronary endothelium was removed and thrombus formation was confirmed by angioscopy. In the tissue plasminogen activator (tPA) group (n=8), complete thrombolysis occurred in all animals, but thrombolysis was incomplete in the urokinase (UK) group (n=6). The plasma level of plasmin alpha2-plasmin inhibitor complex peaked at 15 minutes after treatment in both the tPA and UK groups. Plasma thrombin-antithrombin III (TAT) complex decreased transiently at 15 minutes after tPA administration but increased at 30 and 60 minutes (P<0.05). In the UK group, plasma TAT also showed a transient decrease followed by an increase, which was minimal compared with that in the tPA group. Plasma TAT decreased transiently after infusion of tPA and subsequently increased to above the pretreatment level, suggesting a risk of rethrombosis after successful recanalization.

Angioscopy↗

PubMed1998

Identification of a structural gene encoding a metallothionein-like domain that includes a putative regulator protein for Streptomyces protease gene expression.

An open reading frame (termed ORF-PR) encoding a metallothionein-like domain-including protein was found upstream of a previously identified Streptomyces chymotrypsin-type protease gene (sam-P20). Promoter and terminator activities of ORF-PR were detected using the promoterless Streptomyces tyrosinase gene as a reporter gene and expression of ORF-PR was supposed to occur before that of sam-P20 gene. Frameshift mutation analysis showed that the ORF-PR product might act as a repressive regulator of the sam-P20 gene.

Amino Acid Sequence↗

PubMed1997

Nucleotide sequence analysis of the HLA class I region spanning the 237-kb segment around the HLA-B and -C genes.

To elucidate the detailed gene organization of the human leukocyte antigen (HLA) class I region on chromosome 6, seven contiguous cosmid genomic clones covering the 237-kb segment around the HLA-B and -C loci were subjected to DNA sequencing by the shotgun strategy to give a single contig of 236,822 bp from the MICA gene (58.2 kb centromeric of HLA-B) to 90.8 kb telomeric of HLA-C. This region was confirmed to contain four known genes, MICA, HLA-17, HLA-B, and HLA-C, from centromere to telomere. Further, a new member of the P5 multicopy genes was found to be about 1.3 kb upstream of the HLA-17 gene and designated P5.8. Five novel genes designated NOB1-5 were identified by RT-PCR and Northern blot hybridization. In addition, two pseudogenes, dihydrofolate reductase pseudogene (DHFRP) and ribosomal protein L3 homologous gene (RPL3-Hom), were also found in the vicinity of the HLA-B and -C genes, respectively. The two segments (about 40 kb) downstream of the HLA-B and HLA-C genes showed high sequence homology to each other, suggesting that segmental genome duplication including the major histocompatibility complex (MHC) class I gene must have occurred during the evolution of the MHC.

Blotting, Northern↗

PubMed1997

17beta-estradiol, diethylstilbestrol, tamoxifen, toremifene and ICI 164,384 induce morphological transformation and aneuploidy in cultured Syrian hamster embryo cells.

To examine the ability of estrogens and anti-estrogens to induce cellular transformation and genetic effects, Syrian hamster embryo (SHE) cells were treated with estrogens, 17beta-estradiol (E2) or diethylstilbestrol (DES), or with anti-estrogens, tamoxifen (TAM), toremifene (TOR) or ICI 164,384. Treatment with each substance for 1-3 days suppressed cellular growth in a dose-dependent manner. Colony-forming efficiency (CFE) increased following treatment of cells with E2 or DES for 48 hr at 3 or 10 microM but decreased at 20 or 30 microM. In contrast, CFE was increased by treatment with TAM, TOR or ICI 164,348 over the concentration range examined (1-30 microM). Treatment with each chemical at 1-30 microM for 48 hr caused morphological transformation of SHE cells in a dose-related fashion. The highest frequency was exhibited in SHE cells treated with DES at 20 microM and was 2 times higher than that induced by treatment with benzo[alpha]pyrene (B[alpha]P) at 4 microM. Transformation frequencies induced by other substances (E2, TAM, TOR and ICI 164,348) did not exceed that induced by the B[alpha]P treatment. TOR showed a higher transforming ability over all concentrations examined when compared to the other anti-estrogens (TAM and ICI 164,348). No significant increases in the frequencies of chromosomal aberrations were observed in SHE cells that were treated with any of the chemicals. However, treatment of SHE cells with each chemical induced a dose-dependent increase of aneuploid cells in the near diploid range. Our results indicate that the ability of the estrogens and anti-estrogens to induce numerical chromosomal abnormality may be involved in their cell transformation activity and potential carcinogenicity.

Aneuploidy↗

PubMed1997

Molecular phylogenetic characterization of Streptomyces protease inhibitor family.

We previously found that proteinaceous protease inhibitors homologous to Streptomyces subtilisin inhibitor (SSI) are widely produced by various Streptomyces species, and we designated them "SSI-like proteins" (Taguchi S, Kikuchi H, Suzuki M, Kojima S, Terabe M, Miura K, Nakase T, Momose H [1993] Appl Environ Microbiol 59:4338-4341). In this study, SSI-like proteins from five strains of the genus Streptoverticillium were purified and sequenced, and molecular phylogenetic trees were constructed on the basis of the determined amino acid sequences together with those determined previously for Streptomyces species. The phylogenetic trees showed that SSI-like proteins from Streptoverticillium species are phylogenetically included in Streptomyces SSI-like proteins but form a monophyletic group as a distinct lineage within the Streptomyces proteins. This provides an alternative phylogenetic framework to the previous one based on partial small ribosomal RNA sequences, and it may indicate that the phylogenetic affiliation of the genus Streptoverticillium should be revised. The phylogenetic trees also suggested that SSI-like proteins possessing arginine or methionine at the P1 site, the major reactive center site toward target proteases, arose multiple times on independent lineages from ancestral proteins possessing lysine at the P1 site. Most of the codon changes at the P1 site inferred to have occurred during the evolution of SSI-like proteins are consistent with those inferred from the extremely high G + C content of Streptomyces genomes. The inferred minimum number of amino acid replacements at the P1 site was nearly equal to the average number for all the variable sites. It thus appears that positive Darwinian selection, which has been postulated to account for accelerated rates of amino acid replacement at the major reaction center site of mammalian protease inhibitors, may not have dictated the evolution of the bacterial SSI-like proteins.

Amino Acid Sequence↗

PubMed1997

Cold adaptation of a mesophilic serine protease, subtilisin, by in vitro random mutagenesis.

Artificial cold adaptation of a mesophilic protease, subtilisin BPN' was attempted by means of random mutagenesis of its entire gene coupled with screening of cleared-zone-forming colonies on skim-milk plates at a low temperature. Out of sixty clones screened at 10 degrees C, one mutant enzyme (termed M-15) was found to acquire higher proteolytic activities, specifically dependent on low temperatures ranging from 10 degrees C to 1 degree C, in comparison with those of the wild-type. DNA sequencing analysis revealed that, by this mutation, the 84th amino acid residue, valine, was substituted by isoleucine, which is located 1.5 nm from the center of the catalytic triad in the tertiary structure of subtilisin. By kinetic analysis of the purified enzyme samples, the higher proteolytic activities of M-15 at low temperatures were found to be due to the decrease in the Km value. There was no difference in thermostability between the wild-type and mutant enzymes, when tested by heat treatment. Circular dichroism spectra also showed no difference between them at 10 degrees C, indicating that the mutation of V841 had no effect on the secondary structure of subtilisin.

Adaptation, Biological↗

PubMed1997

Stimulative effects of saponin from kikyo-to, a Japanese herbal medicine, on pancreatic exocrine secretion of conscious rats.

Our previous report stated that kikyo-to, a Japanese herbal medicine, consisting of the roots of Platycodon grandiflorum and Glycyrrhiza sp., stimulates the pancreatic exocrine secretion of conscious rats. The present study focused on the effective components of kikyo-to and the mechanism of stimuli to pancreatic secretion of rats. When 10 to 100 mg of platycodin D, a saponin from the root of Platycodon grandiflorum, was intragastrically administered, the pancreatic secretion of rats was stimulated. At the same time, the plasma CCK concentration increased. On the other hand, the stimulative effects of glycyrrhizin, a saponin from the root of Glycyrrhiza sp. were weak compared to platycodin D. The effects of 10 mg/kg of platycodin D on pancreatic secretion were inhibited by loxiglumide (50 mg/kg, i.g.), a CCK receptor antagonist. In contrast, the suppressive effect of atropine (300 micrograms/kg/h, i.v.) on pancreatic secretion was reduced by administering 10 mg/kg of platycodin D. In addition, up to 1 mM of platycodin D did not inhibit the trypsin activities in vitro. In conclusion, kikyo-to serves to stimulate pancreatic exocrine secretion mainly because platycodin D causes gastrointestinal hormones, particularly, CCK to be released from the duodenum.

Animals↗

PubMed1997

Changes in cerebral blood flow accompanied with reduction of blood pressure treatment in patients with hypertensive intracerebral hemorrhages.

Blood pressure usually is reduced in patients with hypertensive intracerebral hemorrhage for the prevention of the expansion of the hematoma and recurrent hemorrhage in acute stage. However, disturbed autoregulation of cerebral circulation is expected, and decreased cerebral blood flow (CBF) caused by excessive hypotension has been pointed out. There are different mechanisms of action in hypotensives, thereby the influence of hypotension on CBF in patients with the thalamic hemorrhage was investigated using nitroglycerin (TNG), diltiazem hydrochloride (DH) and trimethaphan camsilate (TC). Average CBF in a hemisphere on the hematoma side, the hemisphere without hematoma, and around the hematoma showed a slight decline after administration of TNG or DH. However, CBF declined more, after TC than DH. DH and TNG are preferable in descending order to control blood pressure of patients with intracerebral hemorrhage in the acute stages in view of a smaller decline in CBF.

Adult↗

PubMed1997

Identification of a novel HLA-B allele (B*4202) in a Saudi Arabian family with Behçet's disease.

A new HLA-B antigen, tentatively called HLA-B42AND, was identified as a B42 serologic variant in a Saudi Arabian family. DNA sequencing analysis of the second and third exon of this new B allele revealed that B42AND was identical to B*4201 except for a single T to C substitution at position 97 of exon 2. This substitution results in histidine (CAC) at codon 9 in B42AND instead of tyrosine (TAC) in B*4201. The antigen frequency of B42AND in a Saudi Arabian population was around 10%. This novel B42AND has officially been named HLA-B*4202.

Alleles↗

PubMed1997

A novel member of the subtilisin-like protease family from Streptomyces albogriseolus.

We previously isolated three extracellular endogenous enzymes from a Streptomyces albogriseolus mutant strain which were targets of Streptomyces subtilisin inhibitor (SSI) (S. Taguchi, A. Odaka, Y. Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In the present study, of the three enzymes the largest one, with a molecular mass of 45 kDa (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), termed SAM-P45, has been characterized in detail. The entire gene encoding SAM-P45 was cloned as an approximately 10-kb fragment from S. albogriseolus S-3253 genomic DNA into an Escherichia coli host by using a shuttle plasmid vector. The amino acid sequence corresponding to the internal region of SAM-P45, deduced from the nucleotide sequence of the gene, revealed high homology, particularly in three regions around the active-site residues (Asp, His, and Ser), with the amino acid sequences of the mature domain of subtilisin-like serine proteases. In order to investigate the enzymatic properties of this protease, recombinant SAM-P45 was overproduced in Streptomyces coelicolor by using a strong SSI gene promoter. Sequence analysis of the SAM-P45 gene and peptide mapping of the purified SAM-P45 suggested that it is synthesized as a large precursor protein containing a large C-terminal prodomain (494 residues) in addition to an N-terminal preprodomain (23 and 172 residues). A high proportion of basic amino acids in the C-terminal prodomain was considered to serve an element interactive with the phospholipid bilayer existing in the C-terminal prodomain, as found in other membrane-anchoring proteases of gram-positive bacteria. It is noteworthy that SAM-P45 was found to prefer basic amino acids to aromatic or aliphatic amino acids in contrast to subtilisin BPN', which has a broad substrate specificity. The hydrolysis by SAM-P45 of the synthetic substrate (N-succinyl-L-Gly-L-Pro-L-Lys-p-nitroanilide) most preferred by this enzyme was inhibited by SSI, chymostatin, and EDTA. The proteolytic activity of SAM-P45 was stimulated by the divalent cations Ca2+ and Mg2+. From these findings, we conclude that SAM-P45 interacts with SSI and can be categorized as a novel member of the subtilisin-like serine protease family.

Amino Acid Sequence↗

PubMed1997

A gene homologous to the Streptomyces chymotrypsin-like protease (SAM-p20) gene is tandemly located.

A gene encoding a homolog of the Streptomyces chymotrypsin-like serine protease, SAM-P20, was identified downstream of the sam-p20 gene and designated SAM-P20D. This gene has two tandem Shine Dalgarno sequences and two initiation codons. We have established vector systems with the function of tyrosinase gene-bone melanin pigmentation as a reporter for sam-p20D gene expression in Streptomyces coelicolor in order to identify the promoter and terminator activities. Using this system, the sam-p20D gene was suggested to be transcribed monocistronically.

Amino Acid Sequence↗

PubMed1997

[Differential diagnosis between peripheral lung cancer invading the chest wall and chest-wall tumors].

Preoperative histological diagnosis of lung cancer is usually based on findings from a transbronchial biopsy specimen of the tumor obtained through a fiberoptic bronchoscope. However, peripheral pulmonary tumors can be difficult to diagnose in this way. We report two patients with lung cancer invading the chest wall for whom histological diagnoses were not possible by transbronchial biopsy. Treatment for lung cancer is different from that for chest-wall tumors, so preoperative differential diagnosis of these conditions is important. Other organs should be searched to exclude metastatic chest-wall tumors. Percutaneous needle biopsy done under ultrasonic guidance made the histological diagnosis of lung cancer possible in both cases.

Adenocarcinoma↗