Biomedical subjects
Publications and source records attributed to T Mizuno.
Effects of brief treatment of growth factors on the urogenital sinus of embryonic rats were investigated and it was found that 8 hour-treatment in the beginning of 5-day cultivation with the epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) can provoke prostatic bud formation, in the medium deprived of androgens.
A survey was conducted in a highland community of Gifu prefecture on 815 residential elderly persons, over 65 years old, utilizing self-administered questionnaires to study the degree of association between life style factors and health conditions. A total of 718 valid participants (90.6% of the total) were analyzed and cross-examined by applying Breslow's seven health factors, daily behavior factors, physical health consciousness, social mobility factors (13 behavior factors which were determined by the Institute of the Elderly) to determine associations with respondents health conditions. Of the participants, 310 were male and 408 were female with mean ages of 73.8 +/- 6.9 years old and 74.0 +/- 6.9 respectively. The survey results indicated that there were no prominent gender differences over the mobility rate and physical health consciousness among participants. However, it was also found that in the male participants the per person prevalence of past history of illness was higher than in females. In the daily activity analysis, female participants indicated a higher prevalence of difficulty in walking and balance maintenance while ascending and descending stairs. In the general daily activity analysis, male participants had a higher requirement for an aide. In social mobility, male participants recorded a high degree of self-reliance by being physically, socially, and intellectually active and the female participants by fulfilling their social responsibilities. Breslow's seven health indicators were applied and their evaluation points were calculated as 3.9 +/- 1.7 for male participants and 3.5 +/- 1.8 for female participants which showed a significantly higher score for the male participants. The survey also indicated that factors which increase the Breslow score for males, were having a spouse, hobbies, a satisfactory life, a habit of exercising, and non-smoking which in females an appropriate sleeping habit (not less than 7 hours), and working, were the positively related factors.
A 68 year-old-man was first found to have CLL with IgG, kappa monoclonal gammopathy 6 years ago. Bestrabucil (total dose 35,150 mg) was taken orally from August 1989 to December 1989. Etoposide (total dose 23,100 mg) was then orally administered from January 1990 to December 1995. He was then referred to our hospital in January 1996 because of progressive anemia and thrombocytopenia. Peripheral blood showed a WBC of 21,200/microliter with 4% myeloblasts and 79% lymphocytes, Hb 7.9 g/dl and Plt 5 x 10(4)/microliter. The serum level of lysozyme was increased (75.6 micrograms/ml). Bone marrow aspiration disclosed hyper-cellularity with proliferation of the blasts and a monocytoid cell population, which cytochemical studies demonstrated to be of the myelo-monocytic series, thus indicating acute myelogenous leukemia (AML-M4) superimposed on CLL. Surface marker analysis of bone marrow mononuclear cells revealed reactivity for CD 11c, CD13, CD15, CD33, HLA-DR. The karyotype was normal. Southern blot analysis and reverse transcriptase-polymerase chain reaction did not reveal rearrangement of the MLL gene. Complete remission was achieved by chemotherapy consisted of BHAC, idarubicine, 6MP, vincristine and predonisolone. Long-term treatment with oral etoposide may contribute to secondary AML.
Csx/Nkx-2.5 is a murine homeobox gene expressed predominantly in cardiocytes and their progenitor cells. The highly lineage-restricted expression pattern of Csx/Nkx-2.5 gene suggests the existence of a positive autoregulatory loop in the transcriptional regulation of Csx/Nkx-2.5. The first intron of CSX1, a human homolog of Csx/Nkx-2.5 gene, had two potential CSX1-binding sequences. Activity of the CSX1 minimal promoter in cultured cardiac myocytes was significantly increased by placing the 3' half of the CSX1 first intron downstream of the reporter gene, suggesting that this region functions as a positive enhancer element. Transient transfection experiments in nonmuscle cells demonstrated that the reporter construct containing the CSX1 minimal promoter and the 3' half of the CSX1 first intron was strongly transactivated by overexpression of CSX1, whereas the CSX1 minimal promoter alone was not. Together these results suggest that the highly lineage-restricted expression of CSX1 is accomplished by autoactivation, which may be mediated by the enhancer element in the first intron.
Bacteria have devised sophisticated signaling systems for eliciting a variety of adaptive responses to their environment, which are generally referred to as the "two-component regulatory system." The widespread occurrence of the two-component systems in both prokaryotes and eukaryotes implies that it is a powerful device for a wide variety of adaptive responses of cells to their environment. The two-component signal transducers contain one or more of three conserved and characteristic phosphotransfer signaling domains, named the "transmitter, receiver, and alternative transmitter." The recently determined entire genomic sequence of Synechocystis sp. strain PCC 6803 allowed us to compile systematically a complete list of genes encoding such two-component signal transduction proteins. The results of such an effort, made in this study, revealed that at least 80 ORFs were identified as members of the two-component signal transducers in this single species of cyanobacteria.
Angiotensin II (AngII) induces cardiac hypertrophy through activating a variety of protein kinases. In this study, to understand how cardiac hypertrophy develops, we examined AngII-evoked signal transduction pathways leading to the activation of extracellular signal-regulated protein kinases (ERKs), which are reportedly critical for the development of cardiac hypertrophy, in cultured cardiac myocytes isolated from neonatal rats. Inhibition of protein kinase C (PKC) with calphostin C or down-regulation of PKC by pretreatment with a phorbol ester for 24 h abolished AngII-induced activation of Raf-1 and ERKs, and addition of a phorbol ester conversely induced a marked increase in the activities of Raf-1 and ERKs. Pretreatment with two chemically and mechanistically dissimilar tyrosine kinase inhibitors, genistein and tyrphostin, did not attenuate AngII-induced activation of ERKs. In contrast, genistein strongly blocked insulin-induced ERK activation in cardiac myocytes. Although pretreatment with manumycin, a Ras farnesyltransferase inhibitor, or overexpression of a dominant-negative mutant of Ras inhibited insulin-induced ERK activation, neither affected AngII-induced activation of ERKs. Overexpression of a dominant-negative mutant of Raf-1 completely suppressed ERK2 activation by AngII, endothelin-1, and insulin. These results suggest that PKC and Raf-1, but not tyrosine kinases or Ras, are critical for AngII-induced activation of ERKs in cardiac myocytes.
Escherichia coli cells that are deficient in the cAMP receptor protein (CRP) overproduce cAMP. We and others have previously found that transcription of the adenylate cyclase gene (cya) is negatively regulated by the CRP-cAMP complex. Here, we have investigated the contribution of this transcriptional regulation to the control of cAMP levels. Several variants of the cya gene have been constructed and characterized with respect to their expression and their ability to produce cAMP. Overproduction of cAMP in a crp- background was reduced from 200-fold to 50-fold when transcriptional regulation by CRP-cAMP was eliminated by replacing the cya promoter with the constitutive bla promoter. When the C-terminal 48 amino acids of adenylate cyclase were deleted without changing the promoter, the degree of overproduction of cAMP was reduced to 4-fold. Finally, the increase in cAMP level observed in crp- cells was almost completely abolished when the truncated cyclase was expressed from the bla promoter. We conclude that transcriptional regulation of cya does indeed play a role in the down-regulation of cAMP production by CRP, although the major regulation is exerted at the posttranscriptional level. The C-terminal region comprising the last 48 amino acids of cyclase is responsible for the posttranscriptional regulation. A simple new method for the determination of cAMP is also described.
RB mRNA increases during terminal differentiation of C2 myoblasts. We demonstrate that RB promoter activity increases about 4-fold during differentiation. The increase of RB promoter activity was reduced when a point mutation was designed in the ATF site. In a gel shift assay of the ATF site, two specific bands were observed. One of them, with the lower mobility, disappeared during differentiation. This band reacted with an antibody against ATF-1. We cotransfected an RB promoter-luciferase plasmid with the TREB36/ATF-1 plasmid. ATF-l suppressed the activity of the wild-type RB promoter but not of that with a point mutation at the ATF site. These results suggest that the ATF site of the RB promoter is a responsive element during myogenic differentiation of C2 cells. We hypothesize that RB promoter activity is stimulated partially due to the dissociation of ATF-1, which suppresses the promoter activity through the ATF site in C2 myoblasts.
Crystals of a novel histidine protein kinase domain of the anaerobic sensor protein ArcB from Escherichia coli have been obtained by a hanging-drop vapor-diffusion method with micro- and macroseeding techniques. Preliminary X-ray crystallographic analysis revealed that they belong to space group P2(1)2(1)2(1) with dimensions a = 30.56, b = 34.93 and c = 110.78 A, having one molecule in the crystallographic asymmetric unit. The crystals diffract to at least 2.0 A resolution.
The Escherichia coli H-NS protein is one of the major constituents of the nucleoid structure. This protein has been implicated not only in the compact organization of the nucleoid structure, but also in the global regulation of gene expression. H-NS negatively regulates the transcription of a number of apparently unlinked genes on the chromosome, suggesting that it functions as a global transcriptional repressor. In this study, on systematic mutational analysis of hns, three distinct functional domains were found in H-NS, which appear to be responsible for DNA-binding, transcriptional repression and protein-protein interaction (dimerization and/or oligomerization), respectively. We first isolated a number of hns mutations which resulted in derepression of the proVWX operon. These included 20 independent missence mutations each resulting in a single amino acid substitution, and six nonsense mutations each giving a C-terminally truncated form of H-NS. The substituted amino acids were revealed to be located non-randomly in the primary sequence of H-NS. This set of hns mutants was examined extensively in terms of phenotypes and biochemical properties. Based on the in vivo and in vitro results, together with the locations of the altered amino acids, three distinct functional domains were identified in H-NS. Mutations in the C-terminal domain resulted in a loss of its DNA-binding ability, suggesting that this domain is directly involved in its binding to DNA. The N-terminal domain was suggested to be involved in the ability to repress transcription. Mutations in this region abolished its ability to repress the transcription of proV, in vivo and in vitro, without loss of its DNA-binding activity. None of the mutants examined was impaired in the formation of a dimer and/or oligomers, suggesting that the central region of H-NS is involved in oligomerization. These results are discussed with special reference to the molecular mechanism underlying the function of H-NS as a transcriptional repressor. In addition, expression of the bgl operon was found to be affected by only a subset of hns mutations in a highly allele-specific manner. This finding is also addressed with regard to a unique regulatory mechanism (i.e. silencing) for the bgl operon, which is partly mediated by H-NS.
To clarify the involvement of nitric oxide (NO) derived from nitrosothiols (RSNO) in 5-hydroxytryptamine (5-HT)-induced Ca(2+)-independent cGMP formation (CIGF) in NG108-15 cells, we investigated the effects of 5-HT on intracellular contents of RSNO as well as of NO metabolites. 5-HT stimulation resulted in an increase in the intracellular contents of nitrate and cGMP. RSNO was detected in NG108-15 cells and was decreased by 5-HT stimulation. Furthermore, the time course of nitrate increase was coincident with that of RSNO decrease. CarboxyPTIO inhibited 5-HT-induced CIGF, whereas oxyhemoglobin failed to inhibit it. The data suggest that NO is stored in a stable form as RSNO and that 5-HT stimulates NO generation from endogenous RSNO, which is followed by elevation of cGMP via activation of cytosolic guanylyl cyclase by NO in NG108-15 cells. We suggest the existence of a novel 5-HT signal transduction pathway involved in NO generation in NG108-15 cells.
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Missense mutations are by the far the most common types of mutations found in p53 of human tumors, suggesting that mutant p53 proteins function either by abrogating wild-type function or by gaining new oncogenic functions. To distinguish between the dominant-negative effect and gain of new function of p53 missense mutants, we measured the ability of transfected missense mutant p53s in p53-null Jurkat cells to alter T-cell receptor (TCR) surface expression. The TCR is a key signal transduction moiety common to T lymphocytes and is one of the major sites for aberrations in T-cell leukemias/lymphomas. Three p53 mutants (248trp, 249ser, and 273his) enhanced the frequency of TCR mutants after graded doses of X-radiation compared to null p53 parent- and wild-type p53-possessing normal lymphocytes; the parent Jurkat and normal lymphocyte showed no difference. These enhancements were not the results of a change in radiosensitivity or in G1 checkpoint arrest characteristics. Therefore, the creation of this mutator phenotype by missense-type p53 mutations implies that a more direct mechanism, apart from changes of cell cycle kinetics or cell death, may be responsible for the selection of certain p53 point mutations, which eventually result in the tumorigenesis of the cell.
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The OmpR protein is a positive regulator specific for the Escherichia coli ompF and ompC genes. This protein functions in a phosphorylation-dependent manner through a presumed interaction with RNA polymerase. We previously isolated OmpR mutants which were suggested to be defective in transcription activation, but not in DNA binding (the so-called positive control (PC) mutant). In this study we isolated mutants of the alpha-subunit of RNA polymerase which can suppress one of the putative PC mutants of OmpR. A crucial amino acid substitution was identified as [V264G] in the alpha-subunit, which is located in the helix H1 of the C-terminal domain, which has been claimed, based on mutational and structural analyses, to be involved in the interaction with other positive regulators including the well-characterized cAMP receptor protein.
The CbpA protein is an analog of the DnaJ molecular chaperone of Escherichia coli. The dnaJ- cbpA- double-null mutant exhibits severe defects in cell growth, namely, a very narrow temperature range for growth. To gain insight into the functions of CbpA as well as DnaJ, we isolated a multicopy suppressor gene that permits this dnaJ- cbpA- mutant to grow normally at low temperatures. The suppressor gene was identified as rpoD, the gene that encodes the major sigma 70. The biological implications of this finding are examined and discussed.
The effects of injections of naloxone, a universal opioid receptor antagonist, into the medial septal nucleus on hippocampal acetylcholine (ACh) release and behavior were investigated in freely moving rats by means of the microdialysis method. The injection of naloxone (2, 10 and 20 micrograms) produced a marked increase in hippocampal ACh release in a dose-dependent manner. These effects of naloxone were reversed by the post-injection of [D-Ala2, N-Me-Phe4, Gly-ol]-enkephalin (DAGO; 10 micrograms), an opioid mu receptor agonist. Furthermore, basal release of hippocampal ACh was significantly reduced by the injection of DAGO alone. It was also found that rats given an injection of naloxone showed an increase in motor activity and occasionally exhibited behavioral seizures. These effects of naloxone were also reversed by the post-injection of DAGO. The present results suggest that endogenous opioids ionically inhibit the activity of septo-hippocampal cholinergic neurons via mediation of mu opioid receptors in the medial septal nucleus. They also suggest that endogenous opioids modulate the incidence of seizures, at least in part, through opioid mu receptors in the medial septal nucleus.
Rats from one month to 22 months of age were subjected to in vivo brain microdialysis under a free-moving condition. The 3,4-dihydroxyphenylacetic acid level in the striatum reached a maximum at 3 months old. However, both basal and potassium-induced dopamine release were maximal at 1.5 months, which represents sexual maturation (puberty). The inhibitory effect of monoamine oxidase (MAO) activity by pargyline was maximal at 1.5 months. The delayed maximum level of the metabolites may be due to different age-related changes in MAO-A and MAO-B. From these results, it is suggested that in the rat striatum, the release of dopamine is maximal at the age of sexual maturation, and that the whole synaptic function of the dopaminergic neurons is at its highest during late adolescence.