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Biomedical subjects

K Takami

Publications and source records attributed to K Takami.

At least 73 records · Page 4Linked to original sources
PubMed1997

New ulcerative colitis model induced by sulfhydryl blockers in rats and the effects of antiinflammatory drugs on the colitis.

We tried to produce a new ulcerative colitis model in rats by topical administration of sulfhydryl blockers. After male SD rats were fasted for 24 hr, 100 microliters of 3% N-ethylmaleimide (NEM) or iodoacetamide (IA) was introduced into the colon via a Nelaton's catheter. Both NEM and IA caused severe diarrhea with rectal bleeding and decreased body weight for about 7 days. At autopsy, adhesions and dilatation of the colon and severe mucosal lesions were observed. Both the weight and myeloperoxidase activity of the colon increased markedly. Maximum changes were observed within 1-3 days followed by gradual recovery, but even on day 21, some abnormalities were still observed. The ulceration and inflammation of the colon were confirmed by histological studies. Antiinflammatory drugs such as indomethacin inhibited the inflammation of the colon by NEM, but aggravated the ulceration. These results revealed that sulfhydryl blockers instilled into the colon caused ulcerative colitis in the rat. This model may be useful in studies on the pathogenesis of ulcerative colitis and the evaluation of drugs for therapy. Furthermore, it was suggested that antiinflammatory drugs may delay the healing of colonic ulcers.

Animals↗
PubMed1997

Unmasking of an unusual myelin basic protein epitope during the process of myelin degeneration in humans: a potential mechanism for the generation of autoantigens.

A rabbit antiserum (anti-EP), induced against a synthetic peptide corresponding to residues 68 to 86 of guinea pig myelin basic protein, powerfully immunostained abnormal-appearing oligodendrocytic processes and cell bodies in demyelinating areas associated with multiple sclerosis plaques. However, it failed to recognize any structures in normal human, rat, or guinea pig brain. The antiserum recognized the synthetic peptide QDENPVV, which corresponds to human myelin basic protein residues 82 to 88. Immunoabsorption with this peptide eliminated immunohistochemical staining. By contrast, several commercial antibodies recognizing nearby sequences of human myelin basic protein intensely stained all myelinated structures in both normal and multiple sclerosis tissue. The unusual epitope recognized by anti-EP appears to be accessible only in areas of myelin degeneration. If insults occur that repeatedly expose a region of MBP normally sheltered from immunosurveillance, a self-sustaining immune reaction might result.

Adult↗
PubMed1996

Determination of the allele-specific antigen-binding site on I-Ak and I-Ab molecules.

Residues 46 and 54 on a pigeon cytochrome c 43-58 analog, 50E, function as major histocompatibility complex class II contact sites. A peptide, 46F50E54A, with phenylalanine (F) at position 46 and alanine (A) at 54 on 50E bound to Ab and a peptide, 46D50E54A, with aspartic acid (D) at 46 and alanine at 54, bound to Ak. To determine the allele-specific peptide contact sites on I-A molecules corresponding to the I-A contact sites of the peptides, we analyzed responses of Ak- and/or Ab-restricted T cell hybridomas to 46F50E54A or 46D50E54A using L cell transfectants expressing recombinant I-A molecules between Ak and Ab or point mutants of Ak as antigen presenting cells. It was shown that the N-terminal half of the alpha helix of the A alpha chain determined the allele-specific T cell responses. Furthermore, with arginine (k type amino acid) or alanine (b type amino acid) at position 56 of the Ak alpha chain, these T cell hybridomas were stimulated predominantly by 46D50E54A (Ak binding peptide) or 46F50E54A (Ab binding peptide), respectively. Thus, the amino acid at position 56 of the A alpha chain determines allele-specific antigen presentation. This postulate was confirmed by direct binding analysis of 50E analogs of various I-A molecules. A single amino acid change (arginine to alanine) at position 56 of the Ak alpha chain altered the peptide binding specificity (46D50E54A to 46F50E54A).

Alleles↗
PubMed1996

Analysis of neurotrophin-3 expression using the lacZ reporter gene suggests its local mode of neurotrophic activity.

We replaced the mouse neurotrophin-3 gene with the Escherichia coli-derived lacZ gene by means of homologous recombination. The mice with this mutation were useful models for studying the distribution of neurotrophin-3 expression in vivo, because visualization by 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside (X-Gal) staining was simple and rapid compared with in situ hybridization or immunohistochemistry. Whole-mount staining of mutant embryos at embryonic day 10 revealed that lacZ, a reporter for the neurotrophin-3 gene, was expressed in the mesencephalon, mandibular arch and somites. In the embryos at days 13-17, lacZ was markedly expressed in the peripheral target tissues of sensory and sympathetic neurons. We also found that spinal motor neurons and sensory neurons in trigeminal and dorsal root ganglia express lacZ. Some of these X-Gal staining regions overlapped with the sites expressing trkC, a high-affinity receptor for neurotrophin-3. The distribution of X-Gal staining in heterozygotes and homozygotes was similar to that of neurotrophin-3 messenger RNA detected by in situ hybridization. However, there was less lacZ expression in the dorsal root ganglia of homozygotes than neurotrophin-3 expression in wild-type mice. These results suggest that the neurotrophin-3 produced in the dorsal root ganglia also plays a role in the survival of some of the neurotrophin-3-positive neurons and that the local mode of neurotrophic activity is widely distributed.

Amino Acid Sequence↗
PubMed1996

Fgr expression restricted to subpopulation of monocyte/macrophage lineage in resting conditions is induced in various hematopoietic cells after activation or transformation.

The c-fgr gene product (Fgr) is a member of the src-family of protein tyrosine kinases. We have established a monoclonal antibody (2H2) which recognizes the unique N-terminal domain of the murine Fgr. In the present study, using immunohistochemical analysis and immune complex kinase assay with the 2H2, we investigated expression of Fgr in various cell populations and tissues in a murine system. In resting conditions, Fgr expression was confined to subsets of a monocyte/macrophage lineage. Thus, Fgr+ cells were detected in paracortical areas and medullas of lymph nodes, but seen only in marginal zones of the spleen and the medulla of the thymus. No Fgr+ macrophage was detected in other tissues, Peyer's patches, brain, heart, lung, liver, pancreas, kidney and peritoneal cavity. However, immune complex kinase assay revealed that, upon stimulation, T and B cells as well as peritoneal macrophages expressed significant levels of Fgr molecules. Transformed cell lines of lymphoid origin, EL-4 and LK35.2, which are T and B lineage lymphomas, respectively, also expressed Fgr molecules. Thus, various cells of hematopoietic origin appeared to possess a potentiality to express Fgr following activation or transformation. The present findings may help elucidate the functional significance of Fgr in immunologically committed cells in either activated or non-activated conditions.

Animals↗
PubMed1995

Biallelic inactivation of the APC gene in hepatoblastoma.

Familial adenomatous polyposis (FAP) is an inherited disorder caused by germline mutation of the adenomatous polyposis coli (APC) gene. Increased risk of hepatoblastoma (HBL) in FAP kindreds has been reported. To determine whether inactivation of the APC gene plays a role in development of HBL, 13 sporadic infantile hepatic tumors were analyzed for genetic alterations in the APC gene. A PCR-mediated RNase protection analysis was performed to detect subtle genetic alterations in the mutation cluster region and in exons 3 and 4 of the APC gene. The results showed that a G to T transversion at the splice acceptor site of the intron 3-exon 4 junction had occurred in one HBL. Sequence analysis of normal tissue of the patient proved the mutation to be germinal. Southern blot analysis at the APC locus revealed that the tumor had lost the opposite allele and was isodisomic at this locus. RNA analysis indicated that the tumor contained only the small APC transcript, from which exon 4 was entirely absent. Since abnormal splicing causes termination due to frameshift, it was hypothesized that only the truncated APC protein was expressed in this tumor. These findings suggest that inactivation of the APC gene is closely related to tumorigenesis of HBLs in FAP patients.

Alleles↗
PubMed1995

Neurotrophin-3 is expressed in the posterior lobe of mouse cerebellum, but does not affect the cerebellar development.

We replaced the neurotrophin-3 (NT-3) gene with Escherichia coli-derived lacZ via homologous recombination in embryonic stem (ES) cells and generated the mutant mice. Here we show the in vivo expression of NT-3 in the cerebellum during the postnatal period. A high level of lacZ expression was found in the granular layer of posterior lobe (lobules VII to X) in the postnatal NT-3(+/-) cerebellum. The expression in these regions was reduced with age. Although the Purkinje cells are considered to be a target of NT-3 and the NT-3(-/-) mice displayed severe moving disorders like ataxia, no histological abnormality was observed in their cerebellum. These findings suggest that the NT-3 expressed in the cerebellum gives some trophic effects primarily to the posterior lobe, however, the deficiency does not affect its development.

Animals↗
PubMed1995

Targeted disruption of the neurotrophin-3 gene with lacZ induces loss of trkC-positive neurons in sensory ganglia but not in spinal cords.

We have replaced the NT-3 gene with Escherichia coli-derived lacZ gene by means of homologous recombination in embryonic stem cells and thus produced null mutant mice. Mice homozygous for this mutation developed to birth, but most of them could not suck well and died within 2 days after birth. The surviving homozygous mutant mice displayed movement disorder similar to ataxia. The expression of lacZ was widely distributed in the target tissues of peripheral nerves, spinal motor neurons, lumbar dorsal root ganglia and trigeminal ganglia during the prenatal periods. A neuroanatomical examination revealed that there was marked cell reduction present in trigeminal and lumbar dorsal root ganglia in the developing homozygous mutant mice. In these tissues, the expression of trkC, a high-affinity receptor for NT-3, was markedly reduced. In contrast, we did not find any morphological abnormalities, significant cell loss or decreased levels of trkC expression in the motor neurons present in the ventral horn of the spinal cord. These results indicate that the absence of the NT-3 gene leads to a defect in the sensory nervous system, but it may be complemented by other neurotrophins in the motor nervous system during the development.

Animals↗
PubMed1995

An EcoRI RFLP in human APC gene.

A novel EcoRI-restriction fragment length polymorphism in the genomic sequence of the human adenomatous polyposis coli (APC) gene is described. This polymorphism in the APC gene would be very useful for examination of segregation of the mutated APC gene in the kindreds of familial adenomatous polyposis, and also for studying the loss of heterozygosity at the APC region in certain tumors.

Deoxyribonuclease EcoRI↗
PubMed1995

[Determination of a peptide deduced from the influenza virus haemagglutinin that induces human class II-restricted T cell responses in DQ6 transgenic mice].

HLA-DQ6 (DQ6) transgenic mice were immunized with a set of peptides deduced from either a fragment of the haemagglutinin (HA) of influenza A/Aichi/2/68 virus (HA123-138) or pigeon cytochrome c 43-58 (p43-58) analogues. HA123-138 induced a moderate level of T cell responses in DQ6 transgenic mice. The T cell responses were shown to be restricted to either the DQ6 or I-Ab molecule. We then established T cell hybridomas specific for HA123-138 and restricted to DQ6 from DQ6 transgenic mice immunized with the HA123-138. These T cell hybridomas were activated by the stimulation with HA123-138 in the presence of an HLA-DQ6 transfectant or a paraformaldehyde-fixed HLA-DQ6 positive EB cell line. Using one of these T cell hybridomas we searched for potent peptides which showed high antigenicity as compared with HA123-138. We found that a hybrid peptide antigen, 46F/HA126-134/54A, which was composed of HA126-134 and the I-Ab binding component (46F/54A) previously determined on p43-58 analogues evoked considerable responses in the HA123-138 specific T cell hybridoma. Competitive inhibition assay with this T cell hybridoma and direct binding assay using a DQ6 positive EB cell line or DQ6-transfected L cells showed consistently that the agretopic position 46 of the p43-58 analogues and hybrid peptide antigens was preserved among DQ6-restricted T cell responses and murine class II, I-A, restricted T cell responses. Phenylalanine (F) at the position 46 appeared to be a critical residue for binding to HLA-DQ6 as had been shown for binding to I-Ab.

Amino Acid Sequence↗
PubMed1995

Multistep carcinogenesis in colorectal cancers.

Recent advances in molecular genetics have revealed that multiple genetic alterations including activation of oncogenes and inactivation of tumor suppressor genes are required for tumor development and progression. Tumorigenesis of colorectal cancer, in which most cancers are considered to arise from preceding benign adenomas, has been well documented at the molecular level. Familial adenomatous polyposis (FAP), which is characterized by the development of hundreds to thousands of adenomatous polyps in the colon and rectum, one or more of which can progress to cancer if left without surgical treatment, is a good model for elucidation of genetic alterations involved in colorectal tumorigenesis. The adenomatous polyposis coli (APC) gene responsible for FAP was isolated in 1991, and germinal and somatic mutations of the APC gene have been identified. Moreover, activation of K-ras oncogene and inactivation of several tumor suppressor genes such as MCC, p53, and DCC are supposed to play important roles at specific stages of colorectal tumorigenesis. More recently, two genes, MSH2 and MLH1, responsible for hereditary non-polyposis colorectal cancer (HNPCC) have been identified. Thus the molecular mechanism of colorectal tumorigenesis now seems to be more complicated than has been supposed.

Adenomatous Polyposis Coli↗
PubMed1994

Low Grade Amplification of MDM2 Gene in a Subset of Human Breast Cancers without p53 Alterations.

MDM2 protein is thought to bind to p53 tumor suppressor protein leading to inhibition of p53-mediated transactivation. Amplification of the MDM2 gene has been frequently observed in human sarcoma, and relevant overexpression of the MDM2 protein is assumed to contribute to tumorigenesis through inactivation of the p53 function. In order to determine whether MDM2 amplification plays a role in the development of human breast cancer without genetic alteration of p53, we analyzed, MDM2 gene amplification by quantitative hybridization and genetic alteration of p53, in 32 primary tumors and 26 metastatic lymph nodes. Low grade amplification of the MDM2 gene (2-6 fold) was observed in four cases, none of which showed even subtle genetic alterations of p53 or loss of alleles on 17p. Moreover, in three of the four cases with MDM2 gene amplification, the level of gene amplification in the metastatic lymph nodes was slightly higher than that in the primary tumors. These results, taken together with previous findings, suggest that a subset of breast cancers without genetic alteration of p53 may also arise by inactivation of the p53 function through interaction with the overexpressed MDM2 protein induced by gene amplification.

Journal Article↗
PubMed1994

A frequent alteration of p53 gene in carcinoma in adenoma of colon.

In general, colorectal carcinoma is thought to originate mainly from adenoma, and this pathway is called the adenoma-carcinoma sequence. Carcinoma in adenoma is an appropriate model for analysis of this mechanism, because adenoma and carcinoma tissues coexist in the same polyp and the carcinoma is thought to have originated from the surrounding adenoma. Expression of the p53 protein was analyzed in 36 cases of carcinoma in adenoma in the colon by immunohistochemistry using an anti-human p53 monoclonal antibody (PAb1801). Alterations of the p53 gene were analyzed by the polymerase chain reaction for microanalysis of normal mucosa, adenoma, and carcinoma from histological slides. Mutations were assessed by the polymerase chain reaction-single strand conformation polymorphism analysis and identified by DNA sequencing in some cases. Loss of heterozygosity was studied by polymerase chain reaction-restriction fragment length polymorphism analysis. Positive staining for p53 was detected in three (8%) of 37 adenomas and 20 (53%) of 38 focal carcinomas. One (7%) of 15 adenomas with mild dysplasia, three (14%) of 22 adenomas with moderate dysplasia, and 16 (42%) of 38 focal carcinomas had a mutation in exon 5 through exon 8 of the p53 gene. As for allelic loss in the p53 gene locus, only one adenoma with moderate dysplasia had loss of heterozygosity, whereas six (40%) of 15 focal carcinomas had loss of heterozygosity. Of those tumors (3 of 37 adenomas and 20 of 38 focal carcinomas) that reacted with PAb1801, 78% (18 of 23) showed genetic alterations. Among 52 tumors which showed negative staining, five tumors had a p53 mutation and four of them were nonsense mutations. Putting all of these results together, 71% (24 of 34) of the cases underwent p53 gene and protein alterations during the conversion from adenoma to focal carcinoma. These data clearly indicate that genetic alterations of p53 are involved mainly in the malignant transformation from adenoma to focal carcinoma in colon carcinogenesis. In addition, some cases show heterogeneity of the p53 gene in carcinoma in adenoma of the colon. There may be other pathways than p53 responsible for malignant change in the colon.

Adenocarcinoma↗
PubMed1994

A meiotic DNA polymerase from a mushroom, Agaricus bisporus.

A meiotic DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7], which likely has a role in meiotic DNA repair, was isolated from a mushroom, Agaricus bisporus. The purified fraction displays three bands in SDS/PAGE, at molecular masses of 72 kDa, 65 kDa and 36 kDa. Optimal activity is at pH 7.0-8.0 in the presence of 5 mM Mg2+ and 50 mM KCl and at 28-30 degrees C, which is the temperature for meiosis. This enzyme is resistant to N-ethylmaleimide and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, suggesting that it is a beta-like DNA polymerase. These characteristics are similar to those of Coprinus DNA polymerase beta [Sakaguchi and Lu (1982) Mol. Cell. Biol. 2, 752-757]. In Western-blot analysis, the antiserum against the Coprinus polymerase reacts only with the 65 kDa band, which coincides with the molecular mass of the Coprinus polymerase. Western-blot analysis also showed that the antiserum could react with crude extracts not only from the Agaricales family, to which Agaricus and Coprinus belong, but also from different mushroom families and Saccharomyces. The Agaricus polymerase activity can be found only in the meiotic-cell-rich fraction, but the enzyme is also present in the somatic cells in an inactive state.

Agaricus↗