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Biomedical subjects

T Mizuno

Publications and source records attributed to T Mizuno.

At least 253 records · Page 14Linked to original sources
PubMed1996

Endothelin-1 is involved in mechanical stress-induced cardiomyocyte hypertrophy.

We have recently shown that mechanical stress induces cardiomyocyte hypertrophy partly through the enhanced secretion of angiotensin II (ATII). Endothelin-1 (ET-1) has been reported to be a potent growth factor for a variety of cells, including cardiomyocytes. In this study, we examined the role of ET-1 in mechanical stress-induced cardiac hypertrophy by using cultured cardiomyocytes of neonatal rats. ET-1 (10(-8) approximately 10(-7) M) maximally induced the activation of both Raf-1 kinase and mitogen-activated protein (MAP) kinases at 4 and 8 min, respectively, followed by an increase in protein synthesis at 24 h. All of these hypertrophic responses were completely blocked by pretreatment with BQ123, an antagonist selective for the ET-1 type A receptor subtype, but not by BQ788, an ET-1 type B receptor-specific antagonist. BQ123 also suppressed stretch-induced activation of MAP kinases and an increase in phenylalanine uptake by approximately 60 and 50%, respectively, but BQ788 did not. ET-1 was constitutively secreted from cultured cardiomyocytes, and a significant increase in ET-1 concentration was observed in the culture medium of cardiomyocytes after stretching for 10 min. After 24 h, an approximately 3-fold increase in ET-1 concentration was observed in the conditioned medium of stretched cardiomyocytes compared with that of unstretched cardiomyocytes. ET-1 mRNA levels were also increased at 30 min after stretching. Moreover, ET-1 and ATII synergistically activated Raf-1 kinase and MAP kinases in cultured cardiomyocytes. In conclusion, mechanical stretching stimulates secretion and production of ET-1 in cultured cardiomyocytes, and vasoconstrictive peptides such as ATII and ET-1 may play an important role in mechanical stress-induced cardiac hypertrophy.

Animals↗
PubMed1996

Locomotion of neutrophil fragments occurs by graded radial extension.

The only kinematic description of cell locomotion that relates the dynamics of actin filaments to whole cell movement is the graded radial extension (GRE) model for fish keratocytes, which glide without changing their shapes or sizes. To test whether the GRE model is applicable to other cell types, we analyzed the detailed shape changes during locomotion of heat-induced motile fragments of human polymorphonuclear leukocytes (PMNs). These fragments, called cytokineplasts, were loaded uniformly with a fluorescent cytoplasm-staining dye and their motility and shape changes were analyzed by fluorescence-video microscopy and digital image processing. Two-dimensional (2-D) analysis showed that cytokineplasts only changed their shapes and sizes slightly and apparently maintained their roughly circular shapes, whereas fluorescence-intensity analysis revealed distinct changes in their cytoplasmic thickness profiles. Furthermore, small structures on the cytoplasmic margins behaved as predicted by the GRE model, which therefore is probably also applicable to the parental PMNs, which show complex shape changes. This is the first indication that the GRE model operates in non-fish-keratocyte cells and may, therefore, be a universal model of cell locomotion.

Cell Movement↗
PubMed1996

alpha-Fetoprotein-producing (hepatoid) carcinoma of the fallopian tube.

This report describes a case of alpha-fetoprotein (AFP)-producing carcinoma of the fallopian tube. The patient was a 52-year-old woman who presented with an abnormally high serum level of AFP. A left adnexal mass was discovered in the subsequent close examination. Hysterectomy and adnexectomy revealed a 3 x 3 x 4-cm tumor in the fimbriae of the left tube. Histologically and immunohistochemically, the tumor was diagnosed as an AFP-producing (hepatoid) carcinoma arising in the fallopian tube. To our knowledge, this is the first report of such a tumor appearing in the tube. This case supports the view that hepatoid cells may also arise from the müllerian epithelium.

Adenocarcinoma↗
PubMed1996

Trehalase in the spermatophore from the bean-shaped accessory gland of the male mealworm beetle, Tenebrio molitor: purification, kinetic properties and localization of the enzyme.

Trehalase from the bean-shaped accessory glands of the male mealworm beetle, Tenebrio molitor, was purified by acid treatment, with subsequent chromatography on columns of DEAE-cellulofine and Sephacryl S-300. The molecular masses of the native and the denatured forms were estimated to be 43 and 62 kDa by gel filtration and SDS-PAGE, respectively, an indication that the trehalase may be composed of a single polypeptide. The optimum pH of the reaction catalyzed by trehalase was 5.6-5.8. The Km for trehalose was 4.4 mmol.1(-1). Immunohistochemical experiments with trehalase-specific antiserum showed that the enzyme was localized in one specific type of secretory cell in the bean-shaped accessory gland epithelium and within the semisolid secretory mass that was a precursor to the wall of spermatophore. SDS-PAGE and immunoblotting analysis revealed the presence of a polypeptide of about 62 kDa in the spermatophore. Immunohistochemical observations showed that the trehalase was located at the outgrowth in the anterior portion of the spermatophore. When a fresh spermatophore was immersed in phosphate-buffered saline it discharged sperm in the same manner as in the bursa copulatrix of the female. Before the rupture of the expanded bulb of the spermatophore, almost all of the trehalase had dissolved in the phosphate-buffered saline. The addition of validoxylamine A to the saline, a specific inhibitor of trehalase, did not affect the expansion and evacuation of the spermatophore. These results demonstrate that trehalase, synthesized by a specific type of secretory cell in the bean-shaped accessory gland epithelium, is actively passed into the lumen of the bean-shaped accessory gland and then incorporated into the spermatophore. Trehalase appears to be one of the structural proteins of the spermatophore, although the possibility can not yet be completely ruled out that the trehalase-trehalose system functions for the nourishment and/or activation of the sperm in the bursa copulatrix of the female.

Acetates↗
PubMed1996

Molecular characterization of feline immunodeficiency virus genome obtained directly from organs of a naturally infected cat with marked neurological symptoms and encephalitis.

Feline immunodeficiency virus (FIV) was first isolated from cats with immunodeficiency syndrome. Recently, neurological abnormalities and brain lesions were shown in cats infected with FIV. To investigate the FIV genome associated with central nervous system (CNS) lesions, proviral DNA sequences from the V3-V6 region of the FIV env gene were directly amplified from uncultured necropsy tissues of a 2-year-old naturally FIV-infected cat with marked neurological symptoms and encephalitis. By in situ hybridization, FIV RNA was detected mainly in the astrocytes. Fifteen clones isolated from cerebrum, bone marrow and lymph node samples showed only a small number of mutations or deletions in this region. A representative clone, JN-BR1, was distantly related to the previous Japanese strain (TM2) belonging to the subtype B. However, it was relatively close to the Petaluma strain which is known to infect feline brain-derived culture cells and induce brain lesions in inoculated cats. By phylogenetic analysis, the JN-BR1 strain was placed in subtype A that included Petaluma strain and several other American and European strains. The JN-BR1 strain derived from brain with encephalitis in this study and the Petaluma strain may share a common genetic structure that is related to their neuropathogenicity.

Amino Acid Sequence↗
PubMed1996

Detection of apoptosis induced in peripheral blood lymphocytes from cats infected with feline immunodeficiency virus.

Peripheral blood lymphocytes (PBL) from cats infected with feline immunodeficiency virus (FIV) were examined for the occurrence of apoptosis after short-term culture. In the PBL from FIV-infected cats, changes in flow-cytometry scattergram, morphological characteristics of apoptosis and nucleosomal DNA fragmentation were observed. Percentages of apoptotic cells by flowcytometry analysis in PBL from FIV-infected cats (22.4% +/- 9.4%) were significantly higher than those in PBL from uninfected control cats (9.2% +/- 3.5%). The lymphocytes which underwent apoptosis included CD5+, CD4+ and sIgM+ cells, indicating that induction of apoptosis was not restricted to a special subset of lymphocytes. These findings provide evidence of the apoptotic state of PBL in cats with FIV infection.

Animals↗
PubMed1996

Age-related changes in diurnal acetylcholine release in the prefrontal cortex of male rats as measured by microdialysis.

Extracellular levels of acetylcholine in the prefrontal cortex were measured using the micro-dialysis method in freely moving young (three to four months old) and old (23 to 24 months old) male rats over a period of 24 h to examine the effect of aging on prefrontal acetylcholine release. Prefrontal acetylcholine release during a 24 h period exhibited a diurnal variation with higher levels during the dark cycle than during the light cycle in young rats but not in old rats. In addition, prefrontal acetylcholine release was closely associated with spontaneous activity in young rats but not in old rats. The present study suggests that aging reduces diurnal changes in the prefrontal acetylcholine release and that there is a cross-correlation between the prefrontal acetylcholine release and spontaneous locomotor activity in male rats.

Acetylcholine↗
PubMed1996

Effects of nutritional status and aging on leptin gene expression in mice: importance of glucose.

The factors regulating leptin mRNA under physiological conditions have not been fully elucidated, although both insulin and glucose have been implicated. Since, in male mice, plasma glucose decreases with age without a change in body weight or insulin, aging mice constitute a model to examine effects of glucose independent of effects of insulin or body weight. Therefore, we measured leptin mRNA in adipose tissue of 6-, 15- and 24-month-old C57BL/6J male mice, sacrificed either after a 48 h fast (nutritional deprivation) or 15 min after an intraperitoneal injection of glucose (2 mg/g body weight) (nutritional stimulation). There was a significant effect of both age and nutritional status on leptin mRNA, correlated with effects of age and nutritional status on plasma glucose. Leptin mRNA correlated with body weight, plasma glucose and plasma insulin. After statistically removing effects of plasma glucose, the remaining effects of age, nutritional status, and plasma insulin on leptin mRNA were no longer significant. However, after statistically removing effects of plasma insulin, the remaining effects of age, nutritional status, and plasma glucose continued to be significant. When nutrition-deprived and nutrition-stimulated mice were analyzed separately, plasma glucose significantly correlated with leptin mRNA in both groups, but body weight and plasma insulin correlated with leptin mRNA only in nutrition-deprived mice. When mice at each age were analyzed separately, glucose correlated with leptin mRNA at every age, and after statistical removal of the effects of glucose, the remaining effects of insulin on leptin mRNA were no longer significant at any age. These results support the hypothesis that plasma glucose is important in the regulation of leptin gene expression.

Aging↗
PubMed1996

SomA, a novel gene that encodes a major outer-membrane protein of Synechococcus sp. PCC 7942.

The outer membrane of a cyanobacterium (Synechococcus sp. strain PCC 7942) contains only a few major proteins. A gene encoding one of them, somA, was cloned and characterized. Based on the nucleotide sequence, SomA was predicted to comprise 531 amino acids with a calculated molecular mass of 57,136 Da. The deduced amino acid sequence of SomA shares similarities with two bacterial cell-surface proteins, the S-layer protein of Thermus thermophilus and the flagellin of Campylobacter coli. The predicted amino acid sequence of SomA revealed also that it contains a signal peptide-like sequence at its N terminus. This signal peptide-like sequence was capable of mediating protein translocation across the cytoplasmic membrane into the outer membrane of Escherichia coli, provided that this sequence was fused to the E. coli outer-membrane protein, OmpF. The signal peptide-like sequence was cleaved upon the translocation of the SomA::OmpF protein. We suggest that SomA is synthesized as a precursor and that its N-terminal 24 amino acid sequence is a cleavable signal peptide involved in protein targeting into the outer membrane. To our knowledge, this is the first example of cleavable signal peptides for proteins transported into the outer membrane of cyanobacteria.

Amino Acid Sequence↗
PubMed1996

A novel member of the cspA family of genes that is induced by cold shock in Escherichia coli.

Escherichia coli contains a major cold shock protein, CspA (or CS7.4), whose production is predominantly induced at low temperatures. This bacterium is known to possess five additional genes, each encoding a protein highly similar to CspA (referred to as the CspA family). Here we identified a gene that encodes a cold-shock-inducible analog of CspA and CspB. This newly cloned cspG gene is located at 22 min on the E. coli genetic map, apart from the other cspA family genes. Its gene product (70 amino acids) is 73 and 77% identical to CspA (70 amino acids) and CspB (71 amino acids), respectively. Analyses of a cspG-lacZ transcriptional fusion and Northern (RNA) hybridization revealed that cspG is a low-temperature-responsive gene. Its low-temperature-inducible promoters were determined, and the results indicated that the cspG sequence is highly similar to both the cspA and cspB sequences not only in the coding regions but also in the 5'-upstream noncoding regions surrounding their own promoters.

Amino Acid Sequence↗
PubMed1996

H-NS regulates OmpF expression through micF antisense RNA in Escherichia coli.

H-NS is a major constituent of the Escherichia coli nucleoid. Expression of the major outer membrane proteins, OmpC and OmpF, is influenced by hns mutations such that OmpC expression increases whereas OmpF expression decreases irrespective of the osmolarity of the medium (K. A. Graeme-Cook, G. May, E. Bremer, and C. F. Higgins, Mol. Microbiol. 3:1287-1294, 1989). In this study we show that the effect of an hns::neo mutation on OmpF expression is largely diminished in a deletion mutant carrying the micF gene that encodes the ompF mRNA-specific antisense RNA. In addition, the micF transcript levels in the hns::neo mutation are high compared with transcript levels in wild-type cells. On the basis of these results, we provide evidence for a link between OmpC/OmpF expression and the regulatory function of H-NS. We suggest that H-NS most likely affects OmpC expression directly at the level of transcription, but OmpF expression is indirectly regulated by micF antisense RNA.

Bacterial Outer Membrane Proteins↗
PubMed1996

Effects of H-NS and potassium glutamate on sigmaS- and sigma70-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli.

We have used supercoiled DNA templates in this study to demonstrate that transcription in vitro from the P1 and P2 promoters of the osmoresponsive proU operon of Escherichia coli is preferentially mediated by the sigma(s) and sigma70-bearing RNA polymerase holoenzymes, respectively. Addition of potassium glutamate resulted in the activation of transcription from both P1 and P2 and also led to a pronounced enhancement of sigma(s) selectivity at the P1 promoter. Transcription from P2, and to a lesser extent from P1, was inhibited by the nucleoid protein H-NS but only in the absence of potassium glutamate. This study validates the existence of dual promoters with dual specificities for proU transcription. Our results also support the proposals that potassium, which is known to accumulate in cells grown at high osmolarity, is at least partially responsible for effecting the in vivo induction of proU transcription and that it does so through two mechanisms, directly by the activation of RNA polymerase and indirectly by the relief of repression imposed by H-NS.

Amino Acid Transport Systems↗
PubMed1996

Molecular cloning and characterization of human cardiac homeobox gene CSX1.

Accumulating evidence has suggested that homeo-domain-containing proteins play critical roles in regulating the tissue-specific gene expression essential for tissue differentiation and in determining the temporal and spatial patterns of development. In order to elucidate the mechanisms of human heart development, we have isolated a human homologue of the murine cardiac homeobox gene Csx (also called Nkx-2.5) and denoted it as CSX1. The amino acid sequence of the CSX1 homeodomain is 100% and 67% identical to that of murine Csx/Nkx-2.5 and Drosophila tinman, respectively. CSX1 has at least three isoforms generated by an alternative splicing mechanism. One of these isoforms (CSX1a) encodes a protein of approximately 35 kD that possesses the homeodomain, whereas the other two (CSX1b and CSX1c) encode a truncated protein of approximately 12 kD that is identical to the CSX1a protein at the amino-terminal 112 amino acids but lacks the homeodomain. Northern blot analysis showed that CSX1 transcripts are abundantly expressed in both fetal and adult hearts, but no signal was detected in other human tissues examined. Amplification of each isoform by reverse transcriptase-polymerase chain reaction revealed that all of the three isoforms are expressed in fetal and adult hearts and that the homeobox-containing isoform CSX1a is most abundant. The homeodomain-containing protein encoded by CSX1a binds to Csx/Nkx-2.5 binding sequences and transactivates the sequence-containing luciferase reporter gene. Unexpectedly, the homeodomain-lacking protein encoded by CSX1b also transactivates the reporter gene, although CSX1b does not bind to the Csx/Nkx-2.5 binding sequences. The highly conserved homeodomain sequence in evolution and the restricted expression in the heart suggest that CSX1 plays an important role in the development and differentiation of the human heart and that there may be two different mechanisms in transcriptional regulation by the CSX1 protein, homeodomain-dependent and -independent mechanisms.

Base Sequence↗
PubMed1996

Identification of the nuclear localization signal of mouse DNA primase: nuclear transport of p46 subunit is facilitated by interaction with p54 subunit.

DNA polymerase alpha-primase is a replication enzyme necessary for DNA replication in all eukaryotes. Mouse DNA primase is composed of two subunits: a 46 kDa protein (p46), which is the catalytic subunit capable of RNA primer synthesis, and a 54 kDa protein (p54), whose physiological role is not clear. To understand the structure-function relationship of DNA primase, we set out to characterize these two subunits individually or in combination using a cDNA expression system in mammalian cultured cells, and determined the subcellular distribution of ectopically expressed DNA primase. The p54 expressed in COS-1 cells after transfection was predominantly localized in the nucleus, whereas p46 was retained in the cytoplasm as shown by indirect immunofluorescence analysis. Using several mutant proteins with deletions or substitutions as well as chimeric constructs, we identified the nuclear localization signal of p54 as RIRKKLR, encoded near the amino terminus (residues 6-12). Furthermore, co-expression of both p46 and p54 subunits markedly altered the subcellular distribution of p46; co-expressed p46 was transported into the nucleus as efficiently as p54. These results demonstrate that p54 has a nuclear localization signal and is able to be translocated into the nucleus independently of DNA polymerase alpha subunits. In contrast, p46 lacks a nuclear localization signal, and its nuclear translocation is facilitated by interaction with p54. We present here first evidence for a novel role of p54 in the nuclear translocation process, and a piggy-back binding transport mechanism of mouse DNA primase.

3T3 Cells↗
PubMed1996

A novel gene that interferes with the phosphotransfer signal transduction mediated by the EnvZ osmosensor in Escherichia coli.

In Escherichia coli, expression of the major outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity and other environmental stimuli. A two-component signal transduction system, mediated by EnvZ and OmpR, is crucially responsible for this osmotic regulation of the ompC and ompF genes. In this study, an E. coli gene was cloned, which interferes with expression of both the ompC and ompF genes at the level of transcription, provided that the cloned gene was introduced in E. coli cells by a multicopy plasmid. The gene product was identified as F107, which was previously characterized as a hypothetical protein in E. coli genome databases. F107 containing 107 amino acids appears to be highly hydrophobic, and has a sequence similarity to the eukaryotic type of cytochrome-c oxidase subunit III. The mechanism by which F107 inhibits transcription of ompC and ompF was examined extensively, mainly by using a set of envZ and ompR mutants. These results suggested that F107 interferes specifically with a function of the EnvZ osmosensory kinase. Possible mechanisms by which F107 affects the EnvZ function are discussed.

Amino Acid Sequence↗
PubMed1996

A cyanobacterial gene that interferes with the phosphotransfer signal transduction involved in the osmoregulatory expression of ompC and ompF in Escherichia coli.

Synechococcus sp. PCC7942 is a phototrophic cyanobacterium. In this study we cloned a Synechococcus gene that has a striking effect on the production of the Escherichia coli outer membrane proteins, OmpC and OmpF, provided that this gene was introduced by a multicopy plasmid into the heterologous cells. This multicopy gene in E. coli cells was able to specifically shut off the production of both OmpC and OmpF at the level of transcription. The nucleotides were sequenced for this gene, named sis, and its gene product was purified from E. coli to near homogeneity. A computer-aided search found that the deduced amino acid sequence consisting of 138 residues is novel, with no significant similarity to any other protein in the databases. Since the transcription of ompC and ompF is regulated by the regulatory factors EnvZ and OmpR, through phosphotransfer signal transduction, we explored the inhibitory effect of Sis in various genetic backgrounds as to envZ and ompR. In particular, the inhibitory effect of Sis was observed even in an DeltaenvZ background, but was not observed in a certain background in which the ompC and ompF transcription was supported by a mutant OmpR that can function in a phosphorylation-independent manner. These results suggested that the EnvZ kinase may not be the direct target of Sis, but rather that the process(es) concerning the phosphorylation and/or dephosphorylation of the OmpR protein may be affected by Sis. However, no direct effect of Sis was seen in an in vitro OmpR-phosphorylation assay with the purified OmpR and Sis proteins. Based on these results, possible functions of Sis are discussed with special reference to the phosphotransfer signal transduction in E. coli.

Amino Acid Sequence↗
PubMed1996

Antitumor activity and chemical modification of polysaccharides from niohshimeji mushroom, Tricholma giganteum.

Two chemical modification procedures, Smith degradation and formolysis processes, were used in an attempt to improve the antitumor activity of water-soluble and water-insoluble polysaccharides prepared from the fruiting body of niohshimeji, Tricholoma giganteum. The chemically modified products were examined for their antitumor effects on Sarcoma 180 solid tumor implanted in mice. The following results were as follows: Some Smith degradation products, i.e., O-R-Flo-c-beta and O-R-FA-2 prepared from water-soluble polysaccharides, and O-R-FII-1 and O-R-FIII-2-c from water-insoluble polysaccharides, had higher antitumor activities than the original polysaccharides. None of the formolysis products, F-Flo-a, F-W-Flo-a, F-FA-3, F-W-FA-3 from water-soluble polysaccharides, and F-FII-2, F-W-FII-2, F-FIII-2-b, and F-W-FIII-2-b prepared from water-insoluble polysaccharides had improved antitumor activities.

Animals↗