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Biomedical subjects

K Shimizu

Publications and source records attributed to K Shimizu.

At least 541 records · Page 30Linked to original sources
PubMed1997

Novel germline RET proto-oncogene mutations associated with medullary thyroid carcinoma (MTC): mutation analysis in Japanese patients with MTC.

Germ-like and somatic mutations in the RET proto-oncogene are associated with inherited and sporadic medullary thyroid carcinoma (MTC). The majority of patients with multiple endocrine neoplasia type 2A (MEN2A) and familial medullary thyroid carcinoma (FMTC) carry germ-line point mutations that result in the substitution of one of five cysteine residues. We investigated exons 10, 11, 13, 14 and 16 of the RET proto-oncogene in 33 unrelated Japanese patients with MTC. Eleven of the 33 cases (33%) were found to have germ-line mutations. Three previously unreported mutations in exon 10 and 11 were identified: one in codon 620, (TGC-->GGC), resulting in a cysteine to glycine substitution, and two in codon 630, (TGC-->TCC) and (TGC-->TAC), resulting in cysteine to serine and cysteine to tyrosine changes, respectively. The new mutations were present in the germ-line DNA of four unrelated patients for whom a family history of MTC had not been documented. Because the new RET alleles described here involve cysteine residues in a region of protein previously associated with FMTC and MEN2A, it is very likely that they represent mutations that predispose to the development of MTC.

Adolescent↗
PubMed1997

Frequent mutation of the E2F-4 cell cycle gene in primary human gastrointestinal tumors.

The E2F group of transcription factors transactivates genes that promote progression through the G1-S transition of the cell cycle. Members of the retinoblastoma (Rb) family of proteins bind to E2Fs and inhibit this function. E2F-4, one example of the E2F group, functions as an oncogene when transfected into nontransformed cells in vitro. On the other hand, mice that are homozygously lacking a normal E2F-1 gene develop cancers, consistent with a tumor-suppressive role for this gene. The exact function of E2Fs has thus been unclear; moreover, direct involvement of this gene in primary human tumorigenesis has not been shown. We, therefore, investigated mutation within the E2F-4 coding region in 16 primary gastric adenocarcinomas, 12 ulcerative colitis-associated neoplasms, 46 sporadic colorectal carcinomas, 9 endometrial cancers, and 3 prostatic carcinomas. We limited our investigation to the serine repeat within E2F-4, reasoning that this tract might be altered in genetically unstable tumors (replication error-positive, or RER+). All tumors were RER+, with the exception of a control group of 15 RER- sporadic colorectal carcinomas. PCR with incorporation of [32P]dCTP was performed using primers flanking the serine trinucleotide (AGC) repeat. Twenty-two of 59 gastrointestinal tumors (37%) contained E2F-4 mutations; these comprised 5 of 16 gastric tumors (31%), 4 of 12 ulcerative colitis-associated neoplasms (33%, including 1 dysplastic lesion), and 13 of 31 sporadic colorectal cancers (42%). No mutation was present in any of the endometrial, prostate, or RER- colorectal tumors. Of note, homozygous mutations occurred in three cases, and two of seven informative patients showed loss of one E2F-4 allele in their tumors. Furthermore, the RER+ sporadic colorectal tumors were evaluated at trinucleotide repeats within the genes for N-cadherin and B-catenin; no tumors demonstrated mutation of these genes. These data suggest that E2F-4 is a target of defective DNA repair in these tumors.

Adenocarcinoma↗
PubMed1997

Metabolic flux distributions in recombinant Saccharomyces cerevisiae during foreign protein production.

A stoichiometric flux balancing analysis was applied to the recombinant yeast cultivation to examine the cellular physiology and relationship between the production of heterologous protein and metabolic fluxes. The fluxes in the metabolic pathway within a recombinant S. cerevisiae grown on galactose alone or mixtures of galactose and ethanol medium were calculated. It is found that an amplification of the PP (Pentose Phosphate) pathway activity resulted in an improvement of the foreign protein expression and cell yield on ATP. The carbon source used for biosynthesis from TCA cycle in the exponential growth phase was 2 and 5-fold higher, respectively, as compared with that in the late exponential growth phase and stationary phase in batch culture with galactose minimum medium. The metabolism of ethanol together with galactose in the recombinant cell looks like increasing the flux from Acetyl-CoA to TCA cycle, and amplifying the flux directing the synthesis of various kinds of precursors such as amino acids and nucleic acid which are necessary for production of a foreign protein. Metabolic flux distribution analysis also shows that the ATP synthesis rate under substrate-level phosphorylation in the mixed carbon source cultivation was lower than that in the sole carbon source (galactose) during the expression of foreign protein. However, the total ATP production rate was higher in the mixed carbon source cultivation.

Adenosine Triphosphate↗
PubMed1997

Scanning force microscopy of the interaction events between a single molecule of heavy meromyosin and actin.

How close an approach is necessary for two interactive protein molecules to recognize each other before association? How strong a force field is exerted between two proteins at the recognition distance? How extensive are the association interfaces? How strong a force is necessary to pull the associated proteins apart? By means of atomic force microscopy at a truly single molecule level, these fundamental and intriguing questions were answered with the muscle proteins actin and myosin.

Actins↗
PubMed1997

Expression of ubiquitin protein in each organ at death from hypothermia.

To examine the expression of heat shock protein (hsp) in each organ on autopsy in 11 deaths from hypothermia, we performed immunological staining of specimens from each organ using an antibody to hsp, ubiquitin (Ub). Staining of the liver, kidneys, lungs and pancreas revealed a high level of Ub expression in the cytoplasm and nuclei. However, staining of the same specimens of all controls revealed no Ub expression in the cytoplasm or nuclei. This finding suggests that cells are affected by stress even at a low temperature, and may be important in clarifying the cellular kinetics in death from hypothermia.

Adult↗
PubMed1997

Selective expression of HSP70-1 gene in the adrenal cortex but not in the medulla of thermally stressed rats.

The heat shock protein 70 (HSP70) gene expression in rat adrenal glands was investigated. In situ hybridization and Northern blotting revealed a marked increase of inducible HSP70 (HSP70-1) gene expression in the adrenal cortex, but not in the medulla, after whole body hyperthermia. In vivo footprinting assay of HSP70-1 promoter region suggested a possible involvement of other factor(s) in addition to the heat shock factor (HSF). We also detected significant increases of both ACTH and corticosterone in the plasma of the hyperthermic rats. These results suggested some physiological factors other than HSF may also participate in the tissue-specific induction of HSP70-1 gene in vivo.

Adrenal Cortex↗
PubMed1997

Overexpression of N-acetylglucosaminyltransferase III disrupts the tyrosine phosphorylation of Trk with resultant signaling dysfunction in PC12 cells treated with nerve growth factor.

beta-1,4-N-Acetylglucosaminyltransferase III (GnT-III: EC 2.4.1.144) is a pivotal glycosyltransferase which participates in branch formation by catalysis of the synthesis of a bisecting GlcNAc structure in N-glycans. These structures are thought to be one of the unique features of the N-glycans of neural tissues. To examine the intracellullar role of GnT-III expression and its product in neural cells, its gene was overexpressed in rat pheochromocytoma PC12 cells which normally express a low level of GnT-III. In the GnT-III gene-transfected cells, lectin blot analysis showed that some glycoproteins showed increased levels of bisecting GlcNAc structures. Following treatment with nerve growth factor (NGF) the control cells showed neurite outgrowth for differentiation whereas the transfectants showed no morphological response or change in the rate of cell growth. Transient tyrosine phosphorylation of the Trk/NGF receptor was detected at 5-15 min after NGF treatment in control cells, but not detected in the GnT-III gene-transfected cells despite the intact binding of NGF to the cells. Moreover the dimerization of Trk with NGF treatment was not induced in the GnT-III transfectant as compared with the dimerization seen in control cells. These results indicate that overexpression of GnT-III gene in PC12 cells affects some functions of glycoprotein receptors such as Trk by alteration of N-glycan structures, and results in changes in the intracellular signaling pathway of tyrosine phosphorylation modified by NGF.

Animals↗
PubMed1997

Studies on the contribution of c-fos/AP-1 to arthritic joint destruction.

Features characteristic to rheumatoid joint destruction, including synovial overgrowth and bone resorption, are experimentally produced by augmenting c-fos gene expression. We tested here if arthritic joint destruction was inhibited upon inactivation of the c-fos/AP-1 signal by administering short double-stranded AP-1 DNA oligonucleotides into mice with collagen-induced arthritis to compete for the binding of AP-1 in vivo at the promoter binding site. Arthritic joint destruction was inhibited in a sequence-specific and dose-dependent manner by oligonucleotides containing the AP-1 sequence. The oligonucleotides inhibited gene expression at the transcriptional level. Nucleotide sequences besides AP-1 also appeared to be important structurally for binding of AP-1 onto DNA and for the stability of oligonucleotides against nucleases. Immunohistochemical chase experiment administering biotinylated oligonucleotides into arthritic mice showed that AP-1 oligonucleotides reached the inflamed joint. Thus, activation of c-fos/AP-1 appears essentially important in arthritic joint destruction.

Animals↗
PubMed1997

Targeted killing of migrating glioma cells by injection of HTK-modified glioma cells.

The "bystander effect" describes the killing of nearby unmodified cells and herpes simplex thymidine kinase (HTK)-transduced cells by ganciclovir (GCV) treatment. This effect is thought to be produced by contact between these cells. In this study, we showed that injected glioma cells migrated rapidly to a place distant from the injection point whereas injected virus-producing fibroblast cells did not migrate in a murine brain model. Moreover, the initially injected glioma cells and glioma cells injected at a later time mix very well, even at a place distant from the injection point. This suggested that glioma cells migrating after injection could be targeted by HTK-modified glioma cells introduced in a second injection and be killed together by GCV treatment. Therefore, we injected HTK-modified glioma cells 3 days after injection of wild glioma cells to investigate whether wild-type glioma cells that migrated to a place distant from the injection point could also be killed by GCV treatment. Tumor growth was suppressed after the GCV treatment. Suppression of tumor growth of wild glioma cells is not solely mediated by the immune response, which may be triggered by the killing of HTK-modified glioma cells with GCV, because inoculation of HTK-modified glioma to the contralateral side followed by GCV treatment did not cure the initial wild glioma. Moreover, the migration of the second inoculum of glioma cells is necessary for effective killing, because early administration of GCV resulted in insufficient killing.

Animals↗
PubMed1997

Somatostatin and brain-derived neurotrophic factor mRNA expression in the primate brain: decreased levels of mRNAs during aging.

The expression of the genes for somatostatin (SRIF) and brain-derived neurotrophic factor (BDNF) was investigated in the central nervous system (CNS) of the macaque monkey (Macaca fuscata fuscata). Using Northern blot analysis, one SRIF mRNA transcript, 0.65 kb, and two BDNF mRNA transcripts, 1.6 and 4.0 kb in length, were detected in the monkey brain tissues. During the aging process (2 years, 10 years, and > 30 years), the ratio of SRIF mRNA/glyceraldehyde-3 phosphate dehydrogenase (G3PDH) mRNA significantly decreased (60-70%) in the hippocampus and in several cerebral subdivisions such as frontal cortex, temporal cortex, motor cortex, somatosensory cortex and visual cortex. BDNF mRNA was expressed in the various cerebral subdivisions and in the hippocampus. During the aging process, the gene expression of BDNF declined (20-50% for the 4.0 kb transcript, and 40-70% for the 1.6 kb transcript) in the various cerebral subdivisions. In the hippocampus, the level of the 1.6 kb mRNA at > 30 years old declined to 60% of the level at 2 years old, while the 4.0 kb mRNA did not change significantly during the aging process. Recent studies have shown that BDNF enhances the expression of SRIF mRNA in the rodent cerebral cortex (Nawa, H. et al., J. Neurochem., 60 (1993) 772-775; Nawa, H. et al., J. Neurosci., 14 (1994) 3751-3765). These studies and our present results suggest that the decrease in gene expression for a neurotrophic molecule, such as BDNF, might cause the levels of SRIF mRNA to decline in the primate brain during the aging process.

Aging↗
PubMed1997

Hypoxia, but not reoxygenation, induces interleukin 6 gene expression through NF-kappa B activation.

Interleukin (IL) 6 is one of major mediators of inflammation, and IL-6 gene activation during hypoxia/reoxygenation has been implicated in the pathogenesis of ischemia/reperfusion injury. However, molecular events involved in IL-6 gene expression during hypoxia/reoxygenation remain to be identified. We have previously shown that NF-kappa B plays an essential and indispensable role in the transcriptional activation of the IL-6 gene induced by various stimuli, including IL-1 and tumor necrosis factor-alpha. We show here that hypoxia, but not reoxygenation, induces the activation of NF-kappa B through the degradation of a major inhibitor of NF-kappa B, I kappa B alpha. This hypoxia-induced NF-kappa B activation resulted in the kappa B-dependent transcriptional activation of the IL-6 gene. Interestingly, the time course of hypoxia-induced NF-kappa B activation was rather slow as compared with those of NF-kappa B activation induced by other stimuli, such as IL-1: a significant NF-kappa B activation was not observed before 1 hr of hypoxia treatment and persisted for up to 7 hr of hypoxia treatment. However, hypoxia-induced NF-kappa B activation was not inhibited by cycloheximide, which indicates that hypoxia directly triggers NF-kappa B activation. Furthermore, while hypoxia is unlikely to generate reactive oxygen intermediates, pretreatment of cells with antioxidants such as N-acetyl cysteine and alpha-tocopherol inhibited NF-kappa B activation induced by hypoxia. Thus, we discuss possible implications of these results for a postulated role of reactive oxygen intermediates in NF-kappa B activation.

Animals↗
PubMed1997

Surface Monolayers of Poly(l-glutamic Acid)-Functionalized Amphiphiles: Effect of Attachment of Spiropyran to the Polymer Segment

A novel poly(l-glutamic acid)-functionalized amphiphile (2) containing spiropyran groups in the side chain was successfully prepared. Surface monolayers of 2 were affected by isomerization of the spiropyran group; i.e., 2sp, which is in the spiro form, gave an expanded monolayer comparable with that of the spiropyran-free 1, while 2mc in the cyanine form gave a well-condensed monolayer similar to the 1 monolayer. CD measurements for these deposited monolayers onto quartz plates showed that the helix content was higher for the 2sp monolayer than for the 2mc monolayer. Upon isomerization from the cyanine to the spiro form, the 2mc monolayer had a tendency to expand gradually. When d-tryptophan was added into the subphase, such a monolayer expansion was completely suppressed due to electrostatic interactions between zwitterions, whereas no effect of the addition of l-tryptophan on the monolayer was observed. This result suggests that the assembled structure of alpha-helical poly(l-glutamic acid) would provide a suitable site for capturing alpha-amino acid enantioselectively.

Journal Article↗
PubMed1997

Sexual selection in Japanese macaques I: female mate choice or male sexual coercion?

Mate choice is defined as a behavioural pattern in one sex which increases the probability of fertile matings with certain members of the opposite sex. The chosen sex may use force, however, to coerce matings from reluctant members of the choosing sex. In a confined group of Japanese macaques, Macaca fuscata fuscatahoused at the Primate Research Institute of Kyoto University, Japan, behavioural and endocrine data were combined to assess the validity of several mate-choice behaviours and the potential influence of sexual coercion. Preliminary evidence was found for both female mate choice of males and male sexual coercion of females. Both female proximity maintenance towards males and male aggression towards females were correlated with an increase in fertile matings. Most male aggression appeared to be the by-product of increased time spent in proximity due to female proximity maintenance, but some male aggression appeared to be sexual coercion. These results are interpreted in light of recent research on male sexual coercion, and a tentative model of mate choice for this species is proposed.1997The Association for the Study of Animal Behaviour

Journal Article↗
PubMed1997

Sexual selection in Japanese macaques II: female mate choice and male-male competition

Mating and reproductive outcomes are the product of the potentially conficting strategies of breeding males and females. In a captive group of Japanese macaques, Macaca fuscata fuscatabehavioural, endocrine and genetic data were combined to assess the independent effects of male-male competition and female mate choice on mating and reproductive success. Males were ranked by social dominance and by their attractivity to females. Computer simulation and logistic regression analyses showed that male attractivity had a stronger effect than male dominance on both mating and reproductive outcome. Although male dominance and attractivity both significantly predicted mating success during fertile periods, only male attractivity significantly predicted actual male reproductive success. These results provide evidence that female mate choice can be a stronger determinant of mating and reproductive outcome in this species when male and female strategies are in conflict. The lack of a consistent correlation between male dominance rank and reproductive success in this species may be accounted for in part by female mate choice of socially subordinate males. Sires who were observed to mate with the mother during her fertile period were more likely to be socially dominant than sires who were not observed to do so. This observation suggests the existence of alternative mating strategies by subordinate males, which may also contribute to the lack of a consistent correlation between male dominance rank and reproductive success in this species.1997The Association for the Study of Animal Behaviour

Journal Article↗
PubMed1997

Small intestinal cancer with extensive lymph node metastases showing complete remission by methotrexate/5-fluorouracil sequential therapy: report of a case.

A case of poorly differentiated adenocarcinoma of the small bowel with extensive lymph node metastases is herein presented, which responded to methotrexate/5-fluorouracil (MTX/5-FU) sequential therapy. The lymph node metastases disappeared completely after 10 months of treatment. After recurrence, combination therapy with radiation, hyperthermia, and cisplatinum were also effective in reducing the degree of nodal swelling while still allowing the patient to maintain her accustomed lifestyle for a prolonged period of time. Further multi-institutional studies are still needed, however, to fully assess this new therapeutic regimen for small bowel cancers.

Adenocarcinoma↗
PubMed1997

Feasibility of diagnosing chronic myocarditis by endomyocardial biopsy.

In studies of all the layers of autopsied hearts from patients with chronic myocarditis, local clusters of lymphocytes are frequently noted, in contrast with hearts obtained from patients with acute myocarditis. Myocardial biopsy specimens, however are no larger than about 2mm x 3mm. With this in mind, the present study was undertaken to determine whether chronic myocarditis can be diagnosed by endomyocardial biopsy. Specimens were obtained from seven patients in whom chronic myocarditis was confirmed by the clinical course and by autopsy findings. In H&E stained specimens, sites corresponding to the biopsy sites in both ventricles (right ventricular free wall, right ventricular side of the ventricular septum, left ventricular lateral wall) were selected at random (five sites each from the right and left ventricles in each patient) and examined under a light microscope. A mean of 5 or more lymphocytes per visual field (by light microscopy at 400-fold magnification), a proposed quantitative diagnostic criterion of myocarditis, was noted in the right ventricle in three patients (5 lymphocytes in two patients and 6 in one patient) and in the left ventricle in one patient (5 lymphocytes). Also, when the presence of lymphocyte clusters, considered to be a characteristic feature of chronic myocarditis, was determined, clusters of 20 or more lymphocytes per visual field were found in the same patients as those mentioned above, namely, in three patients (42.8%) in the right ventricle, as mentioned above, and in one patient (14.3%) in the left ventricle. At the sites of these lymphocyte clusters. findings such as degenerative changes of the myocardial cells and interstitial fibrosis were also associated, making possible a diagnosis of myocarditis. Therefore, in chronic myocarditis, even if five specimens are obtained by right ventricular biopsy, in approximately one half of patients the diagnosis of chronic myocarditis will be missed because of sampling errors.

Adolescent↗