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Biomedical subjects

H Shirai

Publications and source records attributed to H Shirai.

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

Structural classification of CDR-H3 in antibodies.

Large varieties in the lengths and the amino acid sequences of the third complementarity determining region of the antibody heavy chain (CDR-H3) have made it difficult to establish a relationship between the sequences and the tertiary structures, in contrast to the other CDRs, which are classified by their canonical structures. A total of 55 CDR-H3 segments from well determined crystal structures were analyzed, and we have derived several remarkable rules, which could partly govern the CDR-H3 conformation dependence on the sequence. Since the rules are physically reasonable, they are expected to be applicable to structural modeling and design of antibodies.

Amino Acid Sequence↗
PubMed1996

Apoptotic cell death and its relationship to carcinogenesis in colorectal carcinoma.

BACKGROUND: Apoptotic cell death plays an important role in the proliferation and turnover of cells in various tumors. The relationship between apoptosis and cell proliferation was studied to determine each of their roles in colorectal carcinogenesis. METHODS: Apoptotic cells were identified by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. The occurrence of apoptosis was examined in colorectal cancer that had invaded the submucosa. Specimens were obtained from 38 cases of cancer with adenoma and 29 cases of cancer de novo. Apoptotic indices (AIs) as percentages of TUNEL-positive cells relative to the number of tumor cells and Ki-67 labeling indices (PI) were investigated. The relationship between the frequency of apoptosis and the expression of p53 and c-myc proteins was also investigated. RESULTS: In cancer with adenoma, the ratio of AI/PI in adenoma cells was significantly higher than that of cancer cells (P < 0.0001). Mean AIs of cancer with adenoma were significantly higher than those of cancer de novo particularly in the flat-type cancers (P < 0.05). Among p53 negative tumors, the ratio of AI/PI for cancer de novo was significantly lower than that for cancer with adenoma (P < 0.05). AI of cancer de novo was lower than that of cancer with adenoma in cases with overexpression of c-myc protein (P < 0.05), whereas there was no significant difference in the ratio of AI/PI between cancer de novo and cancer with adenoma. CONCLUSIONS: Colorectal carcinogenesis is related to the inhibition of apoptosis and to the augmentation of proliferative activity both in cancer with adenoma and in cancer do novo. A reduction of the rate of apoptosis as compared with that of cell proliferation might explain the rapid-growing nature of cancer de novo particularly in cases with the flat-type appearance.

Antigens, Neoplasm↗
PubMed1996

Flow cytometric examination of p53 protein in primary tumors and metastases to the liver and lymph nodes of colorectal cancer.

PURPOSE AND METHODS: To confirm prognostic significance of overexpression of p53 in cases of colorectal cancer, expression of p53 protein was examined by flow cytometry in 113 cases of colorectal cancer and its metastasis to the liver and lymph nodes. RESULTS: Overexpression of p53 was found in 44 (39 percent) of the 113 primary tumors. There were no significant correlations among the level of p53 protein in the primary tumor, clinicopathologic features, and prognosis of colorectal cancer. Overexpression of p53 protein was detected in 72 percent (18/25) of liver metastases and in 40 percent (10/25) of lymph node metastases. Frequency of samples that were positive for p53 was significantly higher for liver metastases than for primary tumors and lymph node metastases (P < 0.01). By comparing overexpression of p53 in primary tumors with that in corresponding secondary tumors, a decrease of more than 5 percent in the fluorescence index, compared with primary tumor, was not found in liver metastasis but was found in 20 percent of lymph node metastases. Incidence of cases with lower level expression of p53, compared with primary tumor, was significantly higher in lymph node metastases (32 percent) than in liver metastases (8 percent; P < 0.05). CONCLUSIONS: From these results, it seems possible that overexpression of p53 may not be a good prognostic indicator of colorectal cancer and may be influenced by environments of the tumor.

Blotting, Western↗
PubMed1996

Neuroprotective effect of 4'-(4-methylphenyl)-2,2':6',2-terpyridine trihydrochloride, a novel inducer of nerve growth factor.

We have identified 4'-(4-methylphenyl)-2,2':6',2-terpyridine: trihydrochloride (SS701), which belongs to a family of a small unique neuroprotective agents. SS701 accelerated the production of nerve growth factor (NGF) in cultured astroglial cells, dose- and time-dependently. In in vivo studies, SS701, when administered 30 min after induced cerebral ischemia, neuroprotective effects on delayed neuronal death in Mongolian gerbils were evident. The neuroprotective effects of SS701 against ischemia-induced delayed neuronal death are attributed to stimulation of the production of NGF.

2,2'-Dipyridyl↗
PubMed1996

Lengthening of the forearm by callus distraction.

Ten patients aged 3 to 13 years (mean, 9 years and 7 months) underwent forearm lengthening by callotasis. The indications for lengthening were shortening and/or deformity of the forearm due to exostosis of the distal ulna in five cases, enchondroma of the distal ulna in one, growth disturbance after fracture of the distal radius in one, radial club hand in one, congenital amputation of the forearm in one and congenital dislocation of the radial head in one. Four had lengthening of the ulna, one of the radius and five of both the radius and the ulna. The average lengthening achieved was 30 mm. Complications encountered were pin track discharge in three cases, callus fracture in five, delayed consolidation of the callus in one and no callus formation in one. Review after 1 to 7 years follow-up (with a mean of 4 years and 9 months) showed satisfactory improvement in appearance and function especially in patients who had tumorous conditions or traumatic epiphyseal arrest.

Adolescent↗
PubMed1996

Typing of verotoxins by DNA colony hybridization with poly- and oligonucleotide probes, a bead-enzyme-linked immunosorbent assay, and polymerase chain reaction.

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.

Animals↗
PubMed1995

Tardy ulnar nerve palsy caused by cubitus varus deformity.

Fifteen patients with tardy ulnar nerve palsy caused by cubitus varus deformity were studied. All patients had a history of previous fracture of the humerus during childhood. The mean interval between fracture and onset of symptoms was 15 years. The severity of the palsy was classified as McGowan's grade I in 12 patients, grade II in 2 patients, and grade III in 1 patient. The mean carrying angle was -2 degrees before surgery. X-ray films showed a shallow ulnar nerve groove, a dysplastic humeral trochlea, medial shift of the ulna, and deformity of the medial epicondyle. The ulnar nerve was explored in all but one patient. Operative findings suggested that the main cause of the palsy was compression by a fibrous band running between the two heads of flexor carpi ulnaris. Surgical steps included release of the fibrous band in 14 patients with anterior subcutaneous transposition of the ulnar nerve in 5 of those patients. A corrective osteotomy was done in 11 patients who requested correction of the varus deformity. Traumatic cubitus varus deformity should be recognized as another cause of cubital tunnel syndrome.

Adolescent↗
PubMed1995

Mapping of G protein coupling sites of the angiotensin II type 1 receptor.

Angiotensin II type 1 (AT1) receptors have been identified in a wide variety of tissues, including the kidney, liver, adrenal gland, cardiovascular system, and brain. AT1 receptors also mediate complex signaling mechanisms that elicit a diversity of specific physiological effects. The rat AT1A receptor has seven transmembrane domains and couples with three distinct G proteins: Gq, Gi, and Go. But it is unknown which domains of AT1A couple with and activate each type of G protein. To identify the domains responsible for the activation of various types of G protein, we studied the effect of five different synthetic peptides representing different domains of cytosolic segments of the rat AT1A receptor on the binding of the 35S-labeled stable analogue of GTP, GTP gamma S. Peptides P-3, which is located in the N-terminal region of the putative third intracellular loop of AT1A (residues 216 through 230), and P-5 (residues 306 through 320), corresponding to the N-terminal region of the C-terminal tail, were found to activate purified Gi1, Gi2, and Go proteins. These results indicate that not only the third cytosolic loop but also the C-terminal cytosolic domain of AT1A is important for Gi1, Gi2, and Go protein coupling and activation.

Amino Acid Sequence↗
PubMed1995

Inhibition of protein kinase C prevents rapid desensitization of type 1B angiotensin II receptor.

The type 1B angiotensin II (AT1B) receptor cloned from rat kidney was stably expressed in Chinese hamster ovary cells. The stably expressed receptor was characterized by radioligand binding studies and functional coupling to inositol 1,4,5-triphosphate (IP3) formation. Exposure of cells expressing the AT1B receptor to angiotensin II (Ang II) resulted in a rapid and dose-dependent homologous desensitization of receptor-mediated production of IP3, with an essentially complete desensitization at an agonist concentration > 10 nmol/L. Binding studies revealed no significant change in the number of AT1B receptors in transfected cells exposed to 1 nmol/L Ang II, whereas exposure to 100 nmol/L Ang II caused a rapid decrease of cell surface receptors, with a 75% loss of receptor number seen at 1 hour. Rapid desensitization occurred in the absence of receptor internalization. Blockade of receptor internalization with concanavalin A had at most only a slight effect on the agonist-induced desensitization. This indicates that factors other than internalization are chiefly responsible for the rapid agonist-induced desensitization. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, caused rapid desensitization of the receptor-mediated IP3 response. Neither tyrosine kinase inhibitors nor a protein kinase A activator affected the receptor-mediated IP3 response. The specific PKC inhibitor GF109203X or PKC depletion by prolonged treatment with 1 mumol/L PMA completely blocked the PMA-dependent desensitization. Desensitization evoked by a low Ang II agonist concentration (1 nmol/L) was reversed by the PKC-specific inhibitor GF109203X or PKC depletion, whereas the desensitizing effect at a high agonist concentration (100 nmol/L) is only partially prevented by PKC inhibitory treatment. These results demonstrate that PKC plays a crucial role in the desensitization of the AT1B receptor. They also suggest that receptor internalization and an additional PKC-independent pathway also contribute to desensitization of the AT1B receptor in transfected cells.

Analysis of Variance↗
PubMed1994

Molecular cloning and expression of the gene encoding human angiotensin II type 2 receptor.

The gene of human angiotensin II type 2 (AT2) receptor was isolated from a genomic DNA library prepared from human placenta. The coding region of the human AT2 receptor gene was contained in a single exon coding segment of the gene indicating an intronless structure of the coding region. The amino acid sequence of human AT2 receptor deduced from its nucleotide sequence has 363 amino acids and shows a high degree of sequence identity to rat and mouse receptor sequences. Specific binding of [125I]Sar1Ile8-angiotensin II was demonstrated in COS-7 cells transfected with a plasmid containing the human AT2 sequence. Scatchard analysis and ligand displacement profile were typical of the AT2 receptor. Reverse transcription-polymerase chain reaction analysis showed that AT2 receptor mRNA was expressed in adult uterus and pheochromocytoma.

Amino Acid Sequence↗
PubMed1994

Protein tyrosine phosphatase inhibition by angiotensin II in rat pheochromocytoma cells through type 2 receptor, AT2.

Two major isoforms of angiotensin II receptors, AT1 and AT2, have been defined on the basis of their ligand selectivity. While AT1 is known to mediate typical biological actions of angiotensin II as a cardiovascular regulator, the biological function of AT2 has not yet been established. In the present study using a rat pheochromocytoma cell line, which expresses AT2 exclusively, we found that angiotensin II inhibits phosphotyrosine phosphatase activity in vivo as measured by the inhibition of hydrolysis of [32P]-phosphate from the 32P-labeled synthetic peptide substrate, Raytide. This phosphotyrosine phosphatase inhibition was completely reversed by pertussis toxin, which indicates a G-protein coupled mechanism. In SDS-polyacrylamide gel electrophoresis we found that the phosphotyrosine group of an 85 kDa protein was a substrate mainly preserved, presumably as a consequence of the plausible intracellular phosphotyrosine phosphatase inhibition by angiotensin II.

Adrenal Gland Neoplasms↗
PubMed1994

c-fos induction in the subnucleus caudalis following noxious mechanical stimulation of the oral mucous membrane.

c-fos expression in response to noxious mechanical stimulation of the peripheral receptive field was used to study the somatotopic representation of the oral mucous membrane and trigeminal cutaneous receptive fields in the trigeminal subnucleus caudalis and the cervical spinal dorsal horn. Fos-neurons (neurons with c-fos protein-like immunoreactivity) following stimulation of the cutaneous receptive field were found in laminae I and II of the first and second cervical segments of the spinal cord ipsilateral to the stimulation and exhibited a clear somatotopic segregation; i.e., the fos-neurons responding to receptive fields in the mandibular, maxillary, and ophthalmic divisions were arranged in a mediolateral sequence. On the other hand, fos-neurons responding to the oral mucous membrane were found in the similar laminae of the rostral pole of subnucleus caudalis bilaterally. They were all found in the dorsalmost part of the subnucleus without segregation; e.g., topographic distributions of fos-neurons resulting from the mandibular and maxillary gingival stimulations were indistinguishable. The central mechanism of trigeminal nociception is discussed with respect to the difference between intra- and extraoral receptive fields.

Animals↗
PubMed1994

Role of carboxyl tail of the rat angiotensin II type 1A receptor in agonist-induced internalization of the receptor.

Binding of angiotensin II (Ang II) to its receptor type 1A (AT1A) is known to trigger its internalization. We studied the role of cytosolic segments of AT1A in the internalization, and obtained results indicating a functional role of the cytosolic carboxyl terminal tail of AT1A in the internalization. Deletion of 50 amino acids from the carboxyl terminus abolished the receptor internalization. Deletion mutants lacking 13 and 32 amino acid residues in the carboxyl terminal cytosolic region were internalized to the same extent as wild type AT1A; however, internalization of a mutant lacking the last 42 residues was partially suppressed. Thus, residues 310 through 327 were shown to be essential for the internalization. We propose that a short domain in the cytoplasmic tail (residues 310 to 327) may play a dominant role in the agonist-induced receptor internalization of AT1A. Our results also suggest that the molecular determinants of the AT1A receptor involved in receptor internalization are distinct from those participating in the desensitization process.

Amino Acid Sequence↗
PubMed1994

Apoptotic cell death in human gastric carcinoma: analysis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling.

We have examined the occurrence of apoptotic cell death in formalin-fixed, paraffin-embedded human gastric carcinoma specimens by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) method. The specificity of the TUNEL signals was confirmed by the omission of either TdT or biotinylated dUTP as negative controls, and by pretreatment with DNase I as a positive control. Careful observation of routine hematoxylin and eosin-stained sections showed a few tumor cells with apoptosis, especially in well-differentiated carcinomas. Intense TUNEL signals were frequently observed even in ordinary, non-pyknotic nuclei of tumor cells, and occasionally also in nuclear fragments corresponding to apoptotic bodies. Apoptotic indices (number of apoptotic cells/total number of tumor cells) ranged between 7.7 and 14.5% (mean, 10.9%) in nine well-differentiated carcinomas and between 2.7 and 7.5% (mean, 4.0%) in five which were poorly differentiated, the mean number being significantly higher in the former (P < 0.01). No apparent correlation was found between apoptosis and the expression of proliferating cell nuclear antigen, P53 or Le(y) in the present study. This high frequency of apoptosis, implying cell loss, may be related to the slow-growing nature of well-differentiated carcinomas. Poorly differentiated carcinomas, including scirrhous gastric carcinomas, showed a lower incidence of apoptosis, indicating the existence of an escape mechanism from the process.

Adenocarcinoma↗
PubMed1994

Characterization of phenotypic, serological, and toxigenic traits of Vibrio cholerae O139 bengal.

Biochemical and physiological traits of a collection of strains of Vibrio cholerae O139 Bengal isolated from India, Bangladesh, and Thailand showed that these strains formed a phenotypically homogeneous group with identical characteristics that were essentially similar to those of the O1 serogroup. Resistance to 150 micrograms of the vibriostatic agent O/129 (2,4-diamino-6,7-diisopropylpteridine) and Mukherjee's El Tor phage 5 and classical phage IV and the nonagglutinability of the strains with O1 antiserum were the only discernible differences between the O139 and O1 serogroups. Extensive serological characterization further revealed the O139 serogroup to be distinct from the existing 138 serogroups of V. cholerae. Antiserum raised against the O139 serogroup required absorption with the R reference strain CA385 and with the reference strain representing serogroup O22 to remove cross-reacting agglutinins. All of the 223 representative strains of V. cholerae O139 examined hybridized with DNA probes specific for the cholera toxin (CT) gene, zonula occludens toxin gene, and El Tor hemolysin gene but not with the probe specific for the heat-stable enterotoxin gene. The amount of CT present in stool samples of patients infected with the O139 serogroup was higher than that found in stools of patients infected with O1 El Tor, and this echoed findings that the amount of CT produced by O139 strains in vitro was higher than that produced by the O1 El Tor strains. The nucleotide sequences of the genes encoding the A and B subunits of CT of the O139 serogroup were identical to the sequences reported for the CT gene of O1 El Tor. The CT gene of O139 strains could be amplified by using primers developed for detection of the CT gene of the O1 serogroup by a PCR assay, which could also be used to detect the CT gene in stool samples of patients infected with strains of the O139 serogroup.

Animals↗
PubMed1993

Sandwich enzyme immunoassay for rat retinol-binding protein using antibody against recombinant antigen and its application.

The development of a sandwich enzyme immunoassay for rat retinol-binding protein using molecular biological techniques was described. Rat retinol-binding protein gene cloned by the PCR method was expressed by a fusion vector pEZZ18 in Escherichia coli strain HB101. A recombinant retinol-binding protein fused with IgG-binding domain ZZ of protein A was purified with IgG-Sepharose. Antibody against the recombinant protein was found to be specific to rat retinol-binding protein in plasma by immunoblot analysis. Affinity-purified anti-recombinant protein IgG was biotinylated and used for the sandwich enzyme immunoassay. In this assay, the measurable range is 1.9-60 ng/ml and the coefficients of variation within and between the assay series (assay range: 4-30 ng/ml) are 4.30 +/- 4.33 and 5.32 +/- 1.45%, respectively. Cross-reactivity of the immunoassay was examined using bovine, human, and mouse serum. There was a cross-reaction only with mouse serum. In an in vitro experiment, retinol-binding protein produced by rat hepatocytes could be measured by the sandwich enzyme immunoassay.

Animals↗