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Y Mitsui

Publications and source records attributed to Y Mitsui.

At least 181 records · Page 10Linked to original sources
PubMed1994

Cellular mortality to immortalization: mortalin.

The roots of cellular mortality-limited capacity of normal cells to divide, and immortalization-unabated proliferation of cancerous cells remain undefined so far. Out of a variety of experimental strategies employed, the cell fusion approach has been proven to be significantly informative. The present article reviews some of the more important recent results and describes the use of natural and conditional aging systems obtained by the fusion of mortal and immortal mouse fibroblasts to identify putative senescence-determining and/or senescence-escaping genes. The strategy has led to the isolation of a novel 66-kDa protein, mortalin- a unique member of the mouse heat shock protein 70 (hsp 70) family. The intracellular distributions of mortalin, i.e., cytosolic and perinuclear, distinguish the mortal phenotype from the immortal one, respectively. Consistently, the cytosolic mortalin is seen to have a senescence-inducing function in contrast to the perinuclear mortalin which has no detectable effect on cellular phenotype. It is suggested that mortalin can be exploited to unravel some aspects of cellular mortality and immortality and also for the early detection of cancerous cells.

Animals↗
PubMed1994

Changes in erythrocyte deformability in normal pregnancy and pregnancy-induced hypertension, as revealed by electron spin resonance.

To study changes in hemorheologic properties during pregnancy, erythrocyte deformability was measured by an electron spin resonance (ESR) method. The results obtained by this method showed that erythrocyte deformability in normal pregnancy decreased significantly in the first trimester compared with nonpregnant controls, and continued to decrease slightly as pregnancy progressed. On the other hand, erythrocyte deformability in severe pregnancy-induced hypertension (PIH) was significantly lower than that in the third trimester of normal pregnancy. Additionally, we found that the hematocrit level needed for erythrocytes to exhibit high deformability is lower during pregnancy. These results suggest that hemodilution in normal pregnancy, so-called hydremia, compensates for the decrease in erythrocyte deformability. Conversely, since erythrocytes become less deformable in a hemoconcentration condition in severe PIH, microcirculatory disturbance of various organs, including the uteroplacental unit, may occur. The lowered erythrocyte deformability may be one of the important pathologic features in PIH.

Blood Viscosity↗
PubMed1994

[A familial Charcot-Marie-Tooth disease type 1B (CMTD1B) manifesting a new mutation of myelin P0 gene].

A 15-year-old girl (case 1) was admitted to our hospital because of progressive muscle weakness of the lower limbs and numbness of the upper limbs. She noted these symptoms beginning at 13 years of age. Neurological examination revealed that deep tendon reflexes were absent and hypesthesia of touch and pain sensation were distributed in a glove-and-stocking pattern. Muscle weakness and atrophy were predominantly present in the distal portions of the extremities. There were obvious pes cavus and champagne-bottle shape deformities. The motor conduction velocity of the median nerve was markedly delayed and sensory potentials were not evoked in any nerves examined. Lumbar MRI showed thickening of the nerve radices. Cranial MRI showed thickening of the acoustic nerves, as well. Histological studies of a biopsied sural nerve revealed a marked decrease in the number of myelinated and unmyelinated fibers and remarkable onion bulb formation. The patient's clinical manifestations and histological findings were more severe than that seen in the usual case of CMT1A. Her mother (case 2, 39 years old) had similar neurological and electrophysiological findings. DNA duplication encoding peripheral myelin protein 22, was not detected in either case 1 or 2. Sequencing of DNA from these patients revealed the presence of a mutant allele containing an A- to G-substitution of nucleotide 245, which replaced tyrosine with cysteine in the extracellular Ig-domain of the P0 protein.

Adolescent↗
PubMed1994

Maturation of human megakaryocytic cell line (CMK-7) on the human umbilical vein endothelial cell (EC) monolayer.

Human umbilical vein endothelial cells (EC) were studied to determine whether they were capable of stimulating the maturation of a human megakaryocytic cell line CMK-7. CMK-7 overlaid on EC monolayers in direct contact conditions (CMK-7EC1) and in non-contact conditions (CMK-7EC2), were compared with unstimulated CMK-7 (CMK-7US) and CMK-7 treated with 10(-8) M phorbol ester (CMK-7TPA). The increases in cell-size, CD41b and CD42 positivity, adenosine triphosphate (ATP) and serotonin (5-HT), platelet factor 4 (PF4), beta-thromboglobulin (beta-TG), and DNA contents, indicated both stimulated endomitosis and cytoplasmic maturation in the CMK-7TPA. In contrast, CMK-7EC1 increased in size, in ATP and 5-HT contents and in PF4 and beta-TG contents, without an increase in DNA content, indicating that only cytoplasmic maturation occurred. These findings showed that EC was able to stimulate the cytoplasmic maturation of CMK-7 only under direct contact conditions. Furthermore, the CMK-7EC1 on the EC monolayer developed long cytoplasmic processes that resembled those of megakaryocytes in the terminal stage.

Cell Line↗
PubMed1994

[Painful muscle cramps in liver cirrhosis and effects of oral taurine administration].

We administered 3 g of taurine orally for four weeks to 35 patients suffering from liver cirrhosis with repeated muscle cramp (MC). Improvement of MC was noted in 22 cases (62.9%). We also determined the plasma taurine concentration in eight cases of liver cirrhosis with MC. The plasma taurine concentration before ingestion was 54.1 +/- 20.7 nmol/ml, whereas that of four weeks after ingestion was 125.1 +/- 59.1 nmol/ml, which was significantly elevated by 2.3 fold. As the concentration increased, the frequency of MC decreased, suggesting the good correlation between ingestion and the decrease in frequency of MC. In liver cirrhosis without MC the plasma taurine concentration was 81.0 +/- 16.7 nmol/ml, which was significantly higher than in liver cirrhosis with MC. In a few cases with taurine ingestion, serial plasma taurine concentrations were detected. Plasma taurine reached the peak value during the first week of ingestion and plasma taurine levels were maintained 2-5 fold higher during ingestion.

Administration, Oral↗
PubMed1993

Circadian regulation of per repeat mRNA in the suprachiasmatic nucleus of rat brain.

We have recently reported fluctuations in the expression of the period repeat sequence (pp2.5) during light-dark cycles in the suprachiasmatic nucleus (SCN) of rat [Neurosci. Lett., 122 (1991) 113-116]. Presently, we performed in situ hybridization which shows that the fluctuation of pp2.5 expression continues during constant darkness conditions in the SCN of rat. The light exposure during subjective night but not subjective day triggered its elevated expression in a time-dependent manner which is parallel to that of c-fos expression. The results suggest the involvement of pp2.5 in the entrainment pathway of the circadian rhythm in rodent SCN.

Animals↗
PubMed1993

Spontaneous immortalization of mouse fibroblasts involves structural changes in senescence inducing protein, mortalin.

Mortalin, a novel member of mouse heat shock protein 70 (hsp70) family, is seen to distinguish the cellular mortal and immortal phenotypes by virtue of its cytosolic and perinuclear distribution, respectively. We report here that the cytosolic and perinuclear forms of mortalin from CD1-ICR mouse embryonic fibroblasts and NIH 3T3 cells, respectively, differ by two amino acids, can be distinguished on two-dimensional SDS-polyacrylamide gel. The perinuclear mortalins from RS-4 and Balb/c 3T3 cells harbor the same two amino acids as that of NIH 3T3 cells. However, these when analyzed with C-MEF mortalin did not exhibit the mobility shift equivalent to C-MEF and NIH 3T3 mortalins. The data indicate that the perinuclear mortalin from different immortal fibroblasts are not identical and implicate the possibility of additional structural changes in mortalin during immortalization. Such differences may also contribute to the differential in vitro growth characteristics of these immortal cells.

3T3 Cells↗
PubMed1993

Crystal structure of ribonuclease Ms (as a ribonuclease T1 homologue) complexed with a guanylyl-3',5'-cytidine analogue.

A ribonuclease T1 homologue, ribonuclease Ms (RNase Ms) from Aspergillus saitoi, has been crystallized as a complex with a substrate analogue GfpC where the 2'-hydroxyl (2'-OH) group of guanosine in guanylyl-3',5'-cytidine (GpC) is replaced by the 2'-fluorine (2'-F) atom to prevent transesterification. The crystal structure of the complex was solved at 1.8-A resolution to a final R-factor of 0.204. The role of His92 (RNase T1 numbering) as the general acid catalyst was confirmed. Of the two alternative candidates for a general base to abstract a proton from the 2'-OH group, His40 and Glu58 were found close to the 2'-F atom, making the decision between the two groups difficult. We then superposed the active site of the RNase Ms/GfpC complex with that of pancreatic ribonuclease S (RNase S) complexed with a substrate analogue UpcA, a phosphonate analogue of uridylyl-3',5'-adenosine (UpA), and found that His12 and His119 of RNase A almost exactly coincided with Glu58 and His92, respectively, of RNase Ms. Similar superposition with a prokaryotic microbial ribonuclease, RNase St [Nakamura, K. T., Iwahashi, K., Yamamoto, Y., Iitaka, Y., Yoshida, N., & Mitsui, Y. (1982) Nature 299, 564-566], also indicated Glu58 as a general base. Thus the present comparative geometrical studies consistently favor, albeit indirectly, the traditional as well as the most recent notion [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Biochemistry 29, 9064-9072] that Glu58, rather than His40, must be the general base catalyst in the intact enzymes of the RNase T1 family.

Amino Acid Sequence↗
PubMed1993

Induction of cellular senescence by transfection of cytosolic mortalin cDNA in NIH 3T3 cells.

We have recently identified a novel member of hsp70 family (mortalin) as a mortality marker (Wadhwa, R., Kaul, S. C., Ikawa, Y., and Sugimoto, Y. (1993) J. Biol. Chem. 268, 6615-6621). It has distinct intracellular distribution in mortal and immortal fibroblasts. Here, we report that the cytosolic (mot-1) and the perinuclear (mot-2) forms of mortalin cDNA cloned from mortal and immortal cells, respectively, differ by only two bases in the open reading frame, resulting in two amino acid changes. The induced expression of the cytosolic form by transfection of mot-1 cDNA (isolate from CD1-ICR mouse embryonic fibroblasts) to NIH 3T3 cells induced cellular senescence. However, the perinuclear form expressed by mot-2 cDNA (isolate from NIH 3T3 cells) did not yield an equivalent effect. The data suggest the senescence-inductive function of cytosolic mortalin and implicitly point to a genetic event involved in immortalization.

3T3 Cells↗
PubMed1993

Spleen-derived growth factor, SDGF-3, is identified as keratinocyte growth factor (KGF).

A heparin-binding mitogen rat for rat hepatocytes was partially purified from bovine spleen by a combination of heparin-affinity, cation-exchange and gel-filtration chromatography. Besides stimulating rat hepatocytes, this factor, which was designated spleen-derived growth factor-3 (SDGF-3), exhibited mitogenic activity for mouse epidermal keratinocytes but not mouse fibroblasts. Its apparent epithelial specificity and heparin-binding properties corresponded to those of keratinocyte growth factor (KGF). These findings, together with the fact that the mitogenic activity of SDGF-3 was abolished by a neutralizing monoclonal antibody specific for KGF, identify this bovine spleen-derived hepatocyte mitogen as KGF.

3T3 Cells↗
PubMed1993

Transendothelial migration activity of lymphokine-activated killer (LAK) cells.

With an in vitro static system using HUVEC (human umbilical vein-derived endothelial cells) cultured on type I collagen gel, we investigated the transendothelial migration activities of I1-2 activated killer (LAK) cells. Our results indicate that in comparison with unstimulated T cells, LAK cells exhibit strong transendothelial migration activity, as well as increased adhesiveness to HUVEC. Pretreatment of HUVEC for 24 h with rINF-gamma, rTNF-alpha and rIL-1 beta enhanced the LAK cell migration. The increase in the percentage of migration of LAK cells was greater than that of the percentage of adhesion but significantly less than the increase in the percentage of migration of resting T cells. The results of blocking studies using mAb strongly suggest that the enhanced migration of LAK cell was probably attributed to nonspecifically increased binding to HUVEC and markedly enhanced chemokinetic activity that was dependent primarily on the LFA-1 molecule. Among LAK cells, there were considerable differences in the migration activities of the various phenotypes. CD8+T-LAK migrated preferentially to CD4+T-LAK. CD16+ NK-LAK showed increased adhesion but somewhat decreased migration activities. However, rINF-gamma treatment of HUVEC for 24 h promoted vigorous migration of CD16+ NK-LAK, which suggests that endothelium regulate the migration of LAK cells. Based on these observations, we proposed that LAK cells, if transferred into tumor feeding vessels, can migrate into tumor tissue in considerable numbers and efficiently make contact with individual tumor cells to produce preferable clinical effects.

Cell Adhesion↗
PubMed1993

On the cytosolic and perinuclear mortalin: an insight by heat shock.

We have identified, cloned and characterized a 66-kD protein from cytosolic fractions of mouse embryonic fibroblasts and named it mortalin (Wadhwa et al., J. Biol. Chem., 268, in press, 1993). Immortal fibroblasts were seen to harbor the same or very similar protein, however, localized in the perinuclear locale. The present report is on the differentially localized forms of p66 protein which are biochemically and structurally found to be widely the same. In fact, heat shock treatment could translocate the cytosolic form to the perinuclear position without any detectable biochemical modification. The observed phenomenon adds to the unique identity of mortalin in hsp70 family. Besides, it confers that only the minor differences in the protein probably enroute its differential cellular distribution and the associated function.

3T3 Cells↗
PubMed1993

Keratinocyte growth factor as a mitogen for primary culture of rat hepatocytes.

In a series of our studies on identification of novel mitogens for hepatocytes, keratinocyte growth factor (KGF, FGF-7) is found to have a stimulatory effect on DNA synthesis of adult rat hepatocytes in primary culture. Stimulation of DNA synthesis with KGF was seen to be dose dependent with its maximum effect at 15-50ng/ml. Studies on nuclear labeling with BrdU confirmed the mitogenic effect of KGF on rat hepatocytes. The mitogenic effect of KGF was greater than those of EGF, FGF-1 and comparable to that of HGF. Further, it exhibited additive mitogenic effect when present along with the optimum concentration of HGF, EGF or FGF-1. Thus, it is concluded that KGF is a potent mitogen for rat hepatocytes in vitro.

Animals↗
PubMed1993

Endothelin-1 is produced and secreted by neonatal rat cardiac myocytes in vitro.

Production of endothelin-1 in the cultured neonatal rat cardiac myocytes was investigated. Studies on the cultured cardiac myocytes with reverse transcription-polymerase chain reaction followed by Southern hybridization indicated that mRNA of ET-1(preproET-1 mRNA) was expressed by cardiac myocytes. ET-1 polypeptide was also detected in the cultured cardiac myocytes. Moreover, ET-1 was secreted into the culture medium by them. These results suggest that the cardiac myocytes, which have been regarded as target cells of ET-1, are able to synthesize and secrete ET-1. Furthermore, there may be a possible autocrine mechanism of ET-1 in the myocytes of a heart.

Animals↗
PubMed1993

Enhanced expression of fibronectin during in vivo cellular aging of human vascular endothelial cells and skin fibroblasts.

Vascular endothelial cells are thought to play an important role in human aging as their senescence and/or detachment from vascular wall contribute to arteriosclerosis and high blood pressure in elderly persons. Since fibronectin is necessary for cell attachment and spreading and its increased expression has been reported in aging fibroblasts, we checked its expression in aortic endothelial cells aged in vivo. We found that the steady-state level of fibronectin expression increases with increasing donor age, while the labeling index of cultured cells decreases with age. The increased level of fibronectin expression correlated well with an increase in cell area. To explore whether these changes were a reflection of exhaustion of proliferation potential in vivo, we examined fibronectin expression in human umbilical vein endothelial cells aging in vitro. Very similar results were obtained, supporting the idea that vascular endothelial cells age in vivo by using up division potential. When we examined the expression of fibronectin in human skin fibroblasts aged in vivo and fetal lung fibroblasts aged in vitro, we obtained similar results. In conclusion, the level of expression of fibronectin and cell size increase during in vivo and in vitro aging of both endothelial cells and fibroblasts in a coordinate manner.

Aged↗
PubMed1993

Differential subcellular distribution of mortalin in mortal and immortal mouse and human fibroblasts.

Mortalin, p66mot-1, from the cytosolic fractions of mouse embryonic fibroblasts (MEF) has been identified. We have cloned and characterized it as a novel member of mouse hsp70 family [R. Wadhwa, S. C. Kaul, Y. Ikawa, and Y. Sugimoto (1993) J. Biol. Chem. 268, 6615-6621]. We report that immortal clones from MEF such as NIH 3T3, RS4, and Balb/c 3T3 harbor the same or a very similar protein but in the perinuclear locale. Biochemical analysis revealed that the proteins from mortal (MEF) and immortal (NIH 3T3) fibroblasts do not show gross differences. However, the phenomenon of differential distribution of p66 protein in mortal and immortal fibroblasts is conserved in three different strains of mouse and, interestingly, in human fibroblasts, too. The data suggest that at least one common step is involved in immortalization of mouse and human fibroblasts and also point to the practical implication(s) of such easily detectable differences.

Animals↗