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Biomedical subjects

K Shiota

Publications and source records attributed to K Shiota.

At least 19 recordsLinked to original sources
PubMed1997

Beneficial effects of amlodipine in a murine model of congestive heart failure induced by viral myocarditis. A possible mechanism through inhibition of nitric oxide production.

BACKGROUND: Although calcium channel blockers have not been shown to be beneficial for the treatment of patients with heart failure, a recent clinical trial demonstrated a favorable effect of amlodipine on the survival of patients with heart failure resulting from nonischemic dilated cardiomyopathy. We investigated the effects of amlodipine on a murine model of congestive heart failure induced by the M variant of encephalomyocarditis virus (EMCV). METHODS AND RESULTS: Four-week-old male DBA/2 mice were inoculated with EMCV and administered amlodipine, diltiazem, or vehicle PO for 2 weeks. The heart weight-to-body weight ratio and the histopathological grades of myocardial lesions were significantly lower and survival was significantly increased in the amlodipine-treated group (P < .01, P < .05, and P < .05, respectively) than in the control group. In vitro, amlodipine added to murine J774A.1 macrophages concomitant with EMCV inhibited nitrite formation in a concentration-dependent manner, but diltiazem did not. Furthermore, NG-monomethyl-L-arginine, an inhibitor of NO synthesis, decreased myocardial lesions significantly in this murine model. Immunohistochemistry revealed that the number of cells stained with antibody against an inducible NO synthase decreased significantly in the amlodipine-treated group compared with that in the control group (P < .01). CONCLUSIONS: Amlodipine appears to have a protective effect against myocardial injury in this animal model of congestive heart failure. The therapeutic effect of amlodipine may be in part resulting from inhibition of overproduction of NO.

Amlodipine↗
PubMed1997

Morphological analysis of germ cell apoptosis during postnatal testis development in normal and Hsp 70-2 knockout mice.

The present study examined the occurrence of apoptotic cell death in the testis of wild-type mice from postnatal days 3 to 26 and in juvenile Hsp 70-2 knockout mice. Adult Hsp 70-2 knockout males are infertile and lack spermatids and spermatozoa (Dix et al. [1996a] Proc. Natl. Acad. Sci. U.S.A. 93:3264-3268). To identify the cell types undergoing apoptosis, we also examined the relationship between the occurrence of apoptotic cell death and the expression pattern of the Hsp 70-2 gene product (heat-shock protein 70-2 [HSP70-2]; marker for spermatocytes and spermatids), germ cell nuclear antigen 1 (GCNA1;marker for spermatogonia and spermatocytes), and vimentin (marker for Sertoli cells). This study shows that during postnatal development of the wildtype mouse testis (1) the percentage of apoptotic cell death detected by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method is higher in mice from days 8 to 22 than in younger or older mice, (2) the majority of apoptotic cells are spermatogonia and less frequently are spermatocytes, and (3) the degenerative cell death of spermatogonia and primary spermatocytes involves apoptosis with fragmentation of DNA. The analysis of apoptotic cell death in the testes of juvenile Hsp 70-2 knockout mice showed an additional increased level of apoptosis at day 17, during the first wave of spermatogenesis, in pachytene spermatocytes.

Animals↗
PubMed1997

Role of apoptosis in duct obstruction-induced pancreatic involution in rats.

It has been recognized in mammals that after pancreatic duct obstruction, acinar cells progressively disappear and pancreatic islets are preserved. Previous studies by electron microscopy have suggested the involvement of apoptosis in acinar cell deletion. In the present study, we employed molecular biological methods and investigated whether acinar cell deletion is due to apoptosis. In male Sprague-Dawley rats, pancreatic duct ligation was performed through a left paramedian incision. Pancreatic tissue was studied at each of the following intervals after ligation: 3, 6, 12, 18, and 24 h and 2, 3, 5, and 7 days. DNA fragmentation was determined by in situ labeling of DNA strand breaks on tissue sections [fluorescein-labeled terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method (TUNEL)] and by electrophoretic detection of the fragments of extracted DNA. Tissue sections were also examined by hematoxylin/eosin staining and immunohistochemical staining of insulin. Pancreatic duct ligation induced acinar cell deletion by day 5. Pancreatic tissue from control rats demonstrated no TUNEL-positive nuclei. In contrast, acinar cells from rats 12 h to 5 days after duct ligation showed TUNEL-positive nuclei. The number of TUNEL-positive nuclei was maximum 2 days after duct ligation. Electrophoresis showed DNA fragmentation after duct ligation. Control rats showed a genomic DNA pattern. Islets were preserved throughout the experimental period in duct-ligated rats and control rats. The results suggest that apoptosis may be the dominant form of acinar cell death in the rat pancreatic duct ligation model.

Amylases↗
PubMed1996

Targeted disruption of gp130, a common signal transducer for the interleukin 6 family of cytokines, leads to myocardial and hematological disorders.

gp130 is a ubiquitously expressed signal-transducing receptor component shared by interleukin 6, interleukin 11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin 1. To investigate physiological roles of gp130 and to examine pathological consequences of a lack of gp130, mice deficient for gp130 have been prepared. Embryos homozygous for the gp130 mutation progressively die between 12.5 days postcoitum and term. On 16.5 days postcoitum and later, they show hypoplastic ventricular myocardium without septal and trabecular defect. The subcellular ultrastructures in gp130-/- cardiomyocytes appear normal. The mutant embryos have greatly reduced numbers of pluripotential and committed hematopoietic progenitors in the liver and differentiated lineages such as T cells in the thymus. Some gp130-/- embryos show anemia due to impaired development of erythroid lineage cells. These results indicate that gp130 plays a crucial role in myocardial development and hematopoiesis during embryogenesis.

Animals↗
PubMed1996

Fetal and postnatal development of palmar, plantar, and digital pads, and flexion creases of the rat (Rattus norvegicus).

Fetal development of the hands and feet of rats was investigated to determine the feasibility of using rats as an experimental model for studying the factors influencing early development of the hands and feet, and especially the dermatoglyphics in humans. Eighty rats fetuses of 14-21 days gestational age and 80 newborn rats of 0-7 days of age were used to study the morphological features of the palmar, plantar, and digital areas and to determine the timing of appearance and the location of the volar pads and flexion creases. Comparisons between analoguous developmental stages of rat and human fetuses demonstrate striking similarities in overall fetal development. Marked differences, however, were found between rat and human fetuses in the timing of developmental milestones and in some morphological features. The results indicate that rats can serve as a useful experimental model in studies of the utility of the epidermal ridge configurations and flexion creases in medical disorders, provided that the differences in the timing of development are taken into consideration.

Animals↗
PubMed1996

Sequential changes of programmed cell death in developing fetal mouse limbs and its possible roles in limb morphogenesis.

Apoptotic cell death in the developing limb of mouse fetuses was examined sequentially on days 11-15 of gestation by means of Nile blue (NB) sulfate staining with special reference to its relation to limb morphogenesis. With some exceptions, programmed cell death (PCD) in the hand and foot was observed in the mesenchyme but not in the surface ectoderm. We found that during digital formation PCD begins at the proximal portion of the interdigital mesenchyme and subsequently expands distally. Therefore, the initial PCD that occurs in the interdigital zones may determine the proximal ends of digital separation and also contribute to the demarcation between the palm (sole) and digits (toes). During digital separation, the areas of PCD in the interdigital zones were found to become larger and expand distally on day 13, which may be necessary for the separation of digits and for determining the interdigital area to disappear. PCD in presumptive phalangeal joints was also found to proceed from proximal to more distal joints. The PCD in presumptive joints may be required for the separation of phalanges and metacarpal (metatarsal) bones and for the formation of joint cavities. In addition, intense PCD was observed in the radial (tibial) and ulnar (fibular) margins of the hand and foot plates for 4-5 days. Such PCD at marginal areas seems to prevent the formation of supernumerary digits (preaxial and postaxial polydactyly) and other digital malformations. Therefore, the timing when PCD commences and ends, the sites where PCD occurs, and the intensity, duration, and proximo-distal progress of PCD appear to be genetically determined, and the elimination of unnecessary cells by PCD may be essential for normal limb morphogenesis. The present findings also suggest that the normal progress of PCD in the hand and foot plates of rodent fetuses may prevent the formation of some limb malformations such as webbing fusion of digits, polydactyly, or cleft hand/foot.

Animals↗
PubMed1996

Testis-specific expression of mRNAs for a unique human type 1 hexokinase lacking the porin-binding domain.

Several enzymes in the glycolytic pathway are reported to have spermatogenic cell-specific isozymes. We reported recently the cloning of cDNAs representing three unique type 1 hexokinase mRNAs (mHk1-sa, mHk1-sb, and mHk1-sc) present only in mouse spermatogenic cells and the patterns of expression of these mRNAs (Mori et al., 1993: Biol Reprod 49:191-203). The mRNAs contain a spermatogenic cell-specific sequence, but lack the sequence for the porin-binding domain that somatic cell hexokinases use to bind to a pore-forming protein in the outer mitochondrial membrane. We now report the cloning of cDNAs representing three unique human type 1 hexokinase mRNAs (hHK1-ta, hHK1-tb, and hHK1-tc) expressed in testis, but not detected by Northern analysis in other human tissues. These mRNAs also contain a testis-specific sequence not present in somatic cell type 1 hexokinase, but lack the sequence for the porin-binding domain. The hHK1-tb and hHK1-tc mRNAs each contain an additional unique sequence. The testis-specific sequence of the human mRNAs is similar to the spermatogenic cell-specific sequence of the mouse mRNAs. Furthermore, Northern analysis of RNA from mouse, hamster, guinea pig, rabbit, ram, human, and rat demonstrated expression of type 1 hexokinase mRNAs lacking the porin-binding domain in the testes of these mammals. These results suggest that hexokinase may have unique structural or functional features in spermatogenic cells and support a model proposed by others for hexokinase gene evolution in mammals.

Amino Acid Sequence↗
PubMed1996

Ganglioside composition of the rat choriocarcinoma cell line, Rcho-1.

The Rcho-1 cell line, originally established from a rat choriocarcinoma, shows differentiation into placental trophoblastic giant cell-like cells and has been used to study the mechanism of placental function control. In the present study, we analysed the ganglioside composition of Rcho-1 cells by HPTLC orcinol/H2So4, TLC/ immunostaining and immunohistochemistry. Rcho-1 cells expressed GM3 and GD3 as the major gangliosides and CTH as major neutral glycolipid when they were cultured in growth medium (20% FCS) or transplanted beneath the kidney capsule. The expression of these gangliosides was strong in the undifferentiated small cells, whereas the completely differentiated giant cells showed poor staining with antibodies against the gangliosides. Under culture conditions to induce cell differentiation using horse serum (1-20% HS), the expression of GD3 was suppressed and re-expressed when the medium was changed to growth medium, suggesting that a change of ganglioside components may trigger and define the direction of terminal differentiation. Thus the composition of glycolipids is conserved in Rcho-1 cells and is similar to that of the rat placenta, where GM3 is dominant in mid-pregnancy and decreased in late pregnancy, whereas GD3 is low in mid-pregnancy and increased in late pregnancy.

Animals↗
PubMed1996

Molecular cloning and characterization of a new member of the rat placental prolactin (PRL) family, PRL-like protein D (PLP-D).

The rat placental PRL family consists of molecules structurally similar to PRL and GH, and to date, seven members have been identified. During investigation of pregnancy stage-specific placental factors by the differential display method, we obtained a complementary DNA (cDNA) fragment (199 bp) encoding a peptide homologous to PRL-like protein (PLP)-C. By using the 3' and 5' rapid amplification of cDNA ends method, a full-length cDNA was cloned and tentatively named PLP-D. The cDNA encoded a mature protein of 240 amino acids, including a 29-amino acid signal sequence. PLP-D contains one putative N-glycosylation site and six cysteine residues that are highly conserved in the placental PRL family. Sequence comparison between PLP-D and other members of the placental PRL family showed that PLP-D is highly homologous to PLP-C (80%) and decidual PRL-related protein (73%). Northern blot analysis revealed that PLP-D messenger RNA (mRNA) first appeared at day 14 of pregnancy, and that its expression increased until term. In situ hybridization analysis indicated that PLP-D mRNA was specifically expressed in spongiotrophoblast cells and trophoblast giant cells of the placental junctional zone. Differentiated Rcho-1 cells also expressed PLP-D mRNA, whereas undifferentiated Rcho-1 cells did not.

Amino Acid Sequence↗
PubMed1996

Effects of 20 alpha-hydroxysteroid dehydrogenase and its inhibitors on canine osteosarcoma cell growth in vitro.

Cytotoxic effect of progesterone on the neoplastic cells was investigated using POS cells, a cell line established from a spontaneous osteosarcoma in a dog. The 20 alpha-HSD activity of POS cells was 0.85 +/- 0.26 NADPH nmol/min mg protein, demonstrating that a considerable activity was present in this tumor cell. When progesterone was added to the medium and cultured for 48 hr, progesterone dose-dependently inhibited the cell growth, showing that progesterone was cytotoxic on this osteosarcoma cells in vitro. In order to assure the role of 20 alpha-HSD on the cell growth, various concentrations of four types of steroid derivatives, STZ 20, 23, 25, and 26, potent inhibitors of 20 alpha-HSD, were added to the medium with 0.1 microM progesterone. Then, the POS cell growth was more strongly inhibited, which may suggest that the cytotoxicity of progesterone was enhanced by inactivation of 20 alpha-HSD by STZs. From these results, POS osteosarcoma cells have 20 alpha-HSD, which might play an important role in tumor cell growth against the cell toxicity of progesterone.

20-Hydroxysteroid Dehydrogenases↗
PubMed1996

Site-directed mutagenesis of recombinant equine chorionic gonadotropin/luteinizing hormone: differential role of oligosaccharides in luteinizing hormone- and follicle-stimulating hormone-like activities.

Equine chorionic gonadotropin (eCG) consists of highly glycosylated alpha- and beta-subunits and belongs to the glycoprotein hormone family that includes LH and FSH. eCG is a unique member of the gonadotropin family because it elicits response characteristics of both FSH and LH in other species than the horse. To determine the biological role of the N-linked oligosaccharide at Asn 56 of the alpha-subunit and O-linked oligosaccharides at the carboxyl-terminal peptide (CTP) of the beta-subunit, two mutant eCGs, in which Asn 56 of the alpha-subunit was replaced with Gln (eCG alpha 56/beta) or CTP was deleted (eCG alpha/ beta-CTP), were produced by site-directed mutagenesis and transfecting chinese hamster ovary (CHO-K1) cells. LH- and FSH-like activities were assayed in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells and granulosa cells, respectively. The wild type eCG showed similar LH- and FSH-like activities to native eCG in the in vitro bioassays. The LH-like activity of eCG alpha 56/beta was greatly reduced, whereas that of eCG alpha/beta-CTP was unaffected, demonstrating that the oligosaccharide at Asn 56 of the alpha-subunit of eCG plays an indispensable role in LH-like activity. Interestingly, the FSH-like activity of eCG alpha 56/beta was increased markedly in comparison with the wild type, and that of eCG alpha/beta-CTP was also considerably increased. These data indicate that the dual activities of eCG, LH- and FSH-like activities, could be separated by removal of the N-linked oligosaccharide on the alpha-subunit Asn 56 or CTP-associated O-linked oligosaccharides.

Animals↗
PubMed1996

[Usefulness of Dumon-type endotracheal stent for left main bronchial obstruction caused by thoracic aortic aneurysm--a case report].

We used a Dumon-type endotracheal stent for left main bronchial stenosis occurred after the operation of combined true and Type III dissecant aneurysm. A 65-year-old man with acute Type III dissection with true thoracic aneurysm was operated on by means of arch replacement with thoracic aortic replacement. He complained of dyspnea and the respirator support was not able to discontinued. The cause of dyspnea was oppression of the left main bronchus with remnant thoracic dissecting aneurysm together with a hematoma. A Dumon-type stent was successfully inserted into the left main bronchus under bronchoscopic guide via tracheostomy. It is a choice of conservative therapy after major surgery such as operation of thoracic aneurysm to relieve the bronchial obstruction.

Aged↗
PubMed1995

Analysis of rat placental plasma membrane proteins by two-dimensional gel electrophoresis.

The placenta plays an essential role in fetal growth and the maintenance of pregnancy and its functions are strictly controlled in a stage-specific manner. To gain an insight into placental functions and their regulation, we analyzed the plasma membrane proteins of rat placenta by two-dimensional polyacrylamide gel electrophoresis (2D/E). Plasma membrane fractions of the placenta obtained on days 12, 14, 16, 18 and 20 of pregnancy were purified by Percoll gradient centrifugation, and subjected to 2D/E analysis. After the proteins on the 2D/E gels had been visualized by silver staining, the patterns on the gels at different stages of pregnancy were compared using image analysis software. Proteins within an isoelectric point (pI) range of 4.0 to 7.0 and a molecular weight (Mw) range of 20-100 kDa were analyzed in detail, and about 800 proteins on average were recognized on each gel. Of these, the expression of 150 proteins was found to change dramatically according to the stage of pregnancy. According to their expression patterns, these proteins were categorized into two groups, Group I and Group II. The proteins belonging to Group I showed a higher intensity of expression on day 12 and disappeared on day 20. They included 119 plasma membrane proteins and were divided into five subgroups. Group II, which consisted of three subgroups, included 31 proteins showing a low or negligible expression on day 12 and higher expression on day 20. Most of the other membrane proteins (about 600) were expressed constantly during pregnancy. On the basis of our data, we constructed a database for plasma membrane proteins of the rat placenta.

Animals↗
PubMed1995

Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to up-regulation of neural helix-loop-helix factors, premature neurogenesis, and severe neural tube defects.

Mammalian hairy and Enhancer of split homolog-1 (HES-1) encodes a helix-loop-helix (HLH) factor that is thought to act as a negative regulator of neurogenesis. To directly investigate the functions of HES-1 in mammalian embryogenesis, we performed a targeted disruption of the HES-1 locus. Mice homozygous for the mutation exhibited severe neurulation defects and died during gestation or just after birth. In the developing brain of HES-1-null embryos, expression of the neural differentiation factor Mash-1 and other neural HLH factors was up-regulated and postmitotic neurons appeared prematurely. These results suggest that HES-1 normally controls the proper timing of neurogenesis and regulates neural tube morphogenesis.

Animals↗
PubMed1995

Glycosphingolipid composition of rat placenta: changes associated with stage of pregnancy.

The composition of glycolipids and their changes in the placenta were investigated in the normal pregnant rat. Total lipid fractions extracted from the placenta between days 12 and 20 of pregnancy (day 0 = oestrus) were subjected to glycolipid analysis using DEAE-Sephadex chromatography, silica-gel HPLC, silica-gel TLC, TLC/immunostaining, matrix-assisted secondary-ion mass spectrometry in the negative-ion mode and 1H NMR. Glycolipids identified in the rat placenta were: gangliosides GM3 (NeuAcLacCer and NeuGcLacCer) and GD3 (NeuAcNeuAcLacCer, NeuAcNeuGcLacCer and NeuGcNeuAcLacCer), and neutral glycolipids ceramide monosaccharide (CMH) (GlcCer), ceramide disaccharide (CDH) (LacCer), ceramide trisaccharide (CTH) (Gb3Cer) and ceramide tetrasaccharide (CQH) (Gb4Cer). The content of neutral glycolipids was higher than that of gangliosides throughout pregnancy. Of the neutral glycolipids, CMH and CTH predominated and the level of CDH was low at mid-pregnancy. During late pregnancy, CMH and CTH decreased and CDH increased markedly. CQH remained at a low level throughout pregnancy. Of the gangliosides, GM3 was predominant on days 12-16 and then decreased, whereas GD3, which was low on day 12, increased slightly on day 16 and maintained the same level thereafter. Immunohistochemical studies indicated that these changes in the expression of major gangliosides from GM3 to GD3 occurred in labyrinthine trophoblasts. Thus expression of these glycolipids appears to change markedly during pregnancy.

Animals↗
PubMed1995

Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor.

Hepatocyte growth factor/scatter factor (HGF/SF) functions as a mitogen, motogen and morphogen for a variety of cultured cells. The genes for HGF/SF and its receptor (the c-met proto-oncogene product) are expressed in many tissues during the embryonic periods and in the adult. HGF/SF is thought to mediate a signal exchange between the mesenchyme and epithelia during mouse development. To examine the physiological role of HGF/SF, we generated mutant mice with a targeted disruption of the HGF/SF gene. Here we report that homozygous mutant embryos have severely impaired placentas with markedly reduced numbers of labyrinthine trophoblast cells, and die before birth. The growth of trophoblast cells was stimulated by HGF/SF in vitro, and the HGF/SF activity was released by allantois in primary culture of normal but not mutant embryos. These findings suggest that HGF/SF is an essential mediator of allantoic mesenchyme-trophoblastic epithelia interaction required for placental organogenesis.

Allantois↗
PubMed1995

Programmed cell death in the interdigital tissue of the fetal mouse limb is apoptosis with DNA fragmentation.

BACKGROUND: Programmed cell death is an essential event during mammalian morphogenesis which eliminates unnecessary cells to accomplish histogenesis and organogenesis. Cell death in interdigital spaces of the developing limb is a classical example of morphogenetic cell death. We investigated whether classical programmed cell death in the interdigital tissue of the developing limb in mice is apoptosis with fragmentation of nuclear DNA and also examined sequentially the occurrence of programmed cell death and cell proliferation in the developing limb of mouse fetuses to analyze their interrelation. METHODS: We examined the occurrence of apoptotic cell death in the developing limbs of mouse fetuses by using Nile blue sulphate staining, agarose gel electrophoresis for detecting DNA laddering, and a cytochemical labeling of DNA fragmentation. We also labeled proliferating cells using BrdU/anti-BrdU immunohistochemistry and examined the interrelation between apoptotic programmed cell death and cell proliferation. RESULTS: DNA ladders, a biochemical evidence of apoptosis, were detected in DNA extracts from the interdigital tissue of day 13 mouse fetuses by agarose gel electrophoresis. Programmed cell death and DNA fragmentation were detected by Nile blue staining and cytochemical labeling of DNA fragmentation, respectively, in the interdigital mesoderm and in the regions of presumptive joints of the digit. BrdU/anti-BrdU immunohistochemistry for identifying proliferating S-phase cells revealed that interdigital mesenchymal cells cease DNA synthesis before programmed cell death and DNA fragmentation begin. CONCLUSIONS: We confirmed that both cytological apoptotic alterations and fragmentation of nuclear DNA occur in the interdigital tissue and presumptive joint areas of fetal mouse limbs, and they appear to play a significant role in the separation of digits as well as the formation of joint cavities.

Animals↗