Biomedical subjects

Y Tsutsui

Publications and source records attributed to Y Tsutsui.

At least 73 records · Page 4Linked to original sources

PubMed1993

Immunohistochemical localization of calmodulin in the testes of patients with idiopathic male infertility.

The localization of calmodulin in testes of patients with idiopathic male infertility was studied using the indirect immunoperoxidase method. Specimens were obtained by testicular biopsy from 55 patients. They were divided into 26 cases of hypospermatogenesis, 11 cases of maturation arrest (8 of primary spermatocyte arrest and 3 of spermatid arrest) and 18 cases of Sertoli cell-only syndrome. Regardless of the type of testicular pathology, the types of immunoreactive cell and the intensities of staining were the same as those in the normal testis. That is, staining for calmodulin was first found to be positive in early pachytene primary spermatocytes. It became intense in late pachytene primary spermatocytes and round spermatids. By contrast, elongated spermatids and spermatozoa were not stained. Sertoli cells were stained slightly or not at all. A calmodulin-staining index (CaM-S index) was defined as the proportion of primary spermatocytes that were stained intensely for calmodulin relative to the total number of primary spermatocytes. The indices for the testes of men with complete spermatocyte maturation arrest were significantly lower than those for the testes of normal controls and of men with hypospermatogenesis. Degenerating late pachytene spermatocytes observed in the testes of men with spermatocyte arrest showed low calmodulin-specific immunoreactivity. Such a decrease in numbers of normal late pachytene spermatocytes might be responsible for the low CaM-S index in cases of complete spermatocyte arrest.

Adult↗

PubMed1993

Microphthalmia and cerebral atrophy induced in mouse embryos by infection with murine cytomegalovirus in midgestation.

Microphthalmia and cerebral atrophies were induced in mouse embryos after injection of murine cytomegalovirus (MCMV) into the conceptus at midgestation. The concepti of ICR mice on day 8.5 of gestation were injected with MCMV through the uterine wall, then pregnancies were allowed to continue. On day 15.5 of gestation, microphthalmia was observed in 19.2% of the MCMV-injected embryos (1 x 10(4) plaque-forming units). As the survival rate decreased when pregnancies were allowed to continue further, incidence of microphthalmia decreased, whereas cerebral atrophies, determined by examining the histological sections, were observed in 17.6% of the surviving mouse fetuses on day 18.5 of gestation. Microphthalmia was confirmed by microscopically measuring the eyes on the serial coronal sections. There were two types of microphthalmia: one with marked hypoplastic eye with periglobular mesenchymal proliferation, the other with small eye and lens without the mesenchymal proliferation. Immunohistochemical analysis was performed using antibodies specific to the nuclear antigen of MCMV. Viral antigen-positive cells were widely distributed in the mesenchymes around the oral and nasal cavities and in the mesenchymes around the brain, especially in the endothelial cells of the vessels and the perivascular mesodermal cells. In the eyes, viral antigen-positive cells were observed in mononuclear blood cells in the cavities of the vitreous bodies. These results suggest that the primary target of congenital cytomegalovirus infection may be the mesenchymal cells; then the infection extends to the eyes and brain. In addition, the mesenchymal infection may also disrupt their organogenesis, resulting in microphthalmia and cerebral atrophy. This experimental system may provide a model similar to congenital cytomegalovirus infection in humans.

Animals↗

PubMed1992

Pathogenesis of cytomegalovirus-associated pneumonitis in ICR mice: possible involvement of superoxide radicals.

We have studied the pathogenesis of murine cytomegalovirus (MCMV) pneumonitis in immunocompetent ICR mice and in mice treated with cyclophosphamide (CP). Intranasal infection of immunocompetent mice with MCMV resulted in transient and self-limited pulmonary lesions. When mice were given 200 mg/kg of CP one day before virus infection, transient splenic atrophy and subsequent splenic hypertrophy were induced, and the lesions in the lung were markedly augmented in their number and size although there was no significant enhancement of the virus growth. The augmentation coincided with the period of splenic hypertrophy. A marked increase in the number of pulmonary lesions was also induced in mice given 100 mg/kg of CP every 4 days following the initial dose of 200 mg/kg. In these mice, however, continuous splenic atrophy and augmented replication of MCMV in the lung were observed. When the activity of xanthine oxidase (XO) in lung tissue homogenates was measured, the activity was found to significantly increase after intranasal infection with MCMV irrespective of CP administration and there was a good correlation between the elevation of XO activity and the degree of pathological changes in the lung. In addition, we found that the administration of allopurinol, a specific inhibitor of XO and superoxide dismutase, a superoxide radical scavenger, reduced the number of the pulmonary lesions. These results suggest that superoxide radicals are involved in the pathogenesis of MCMV-associated pneumonitis in ICR mice.

Allopurinol↗

PubMed1992

Susceptibility of mouse embryo to murine cytomegalovirus infection in early and mid-gestation stages.

The susceptibility of mouse embryonic cells to murine cytomegalovirus (MCMV) infection was studied by injecting the virus in the early and mid-gestation stages. For the early stage, blastocysts from BDF1 mice were injected with MCMV or minimal essential medium (MEM) by micromanipulator and returned to the uteri of pseudopregnant ICR mice. On day 11 of gestation, the embryos were examined immunohistochemically, using antibody specific to the early antigen of MCMV, and the placentae were examined by plaque assay. No infection was detected by either method. Furthermore, no infection was detected in MCMV-infected blastocysts that were cultured and examined for infection by immunofluorescence. For mid-gestation embryos, the conceptus was injected with MCMV on day 8.5 of gestation and was subjected to immunohistochemical analysis from day 10.5 to 12.5 of gestation. Viral antigen-positive cells were first observed in the placentae, then antigen-positive cells appeared among the blood cells, endothelial and mesodermal cells of the embryos. On day 12.5 of gestation, clusters of viral antigen-positive cells were sometimes observed in the hearts and livers. Although the incidence was lower, viral antigen-positive cells were also observed in the neuroectoderm and the eyes. These results suggest that MCMV does not infect early embryos and that infection first occurs in the placenta of postimplantation embryos, whence it extends through the blood cells to the endothelial and mesodermal cells of different embryonic regions, eventually extending to the neuroectoderm.

Animals↗

PubMed1992

Relationship between HOX2 homeobox gene expression and the human cytomegalovirus immediate early genes.

The human embryonal carcinoma cell line NT2/D1 is known to be non-permissive for human cytomegalovirus (HCMV) but becomes permissive after being induced to differentiate by retinoic acid (RA). Because homeobox genes have been reported to be specifically activated in the RA-differentiated NT2/D1 cells, we investigated the possible correlation between expression of homeobox (HOX) 2 genes and expression of the immediate early (IE) genes of HCMV both in NT2/D1 cells and in HCMV permissive human embryonic lung (HEL) cells. HCMV infection did not induce activation of the HOX2A, HOX2E and HOX2I genes in undifferentiated NT2/D1 cells nor affect their activation in the RA-differentiated NT2/D1 cells. By in situ hybridization using a HOX2A RNA probe, HOX2A transcript-positive cells appeared as clusters in RA-differentiated NT2/D1 cells. Viral antigen-positive cells detected by immunofluorescence using an antibody specific for the IE-1 antigen of HCMV appeared as clusters among the population of cells in which the HOX2A transcript was detected. The HOX2A gene only was expressed in HEL cells, however none of the HOX2 genes was expressed in non-permissive HeLa, Raji or mouse embryonic cells. These results suggest that activation of the HOX2A may be necessary for the expression of IE genes. HCMV infection markedly increased the expression of the HOX2E gene in HEL cells in the presence, but not in the absence, of cycloheximide. Ultraviolet-inactivated HCMV also displayed this effect. On the other hand, HCMV infection suppressed expression of the HOX2A gene to some degree at the early and late phases of infection in HEL cells. Activation of the HOX2E gene by HCMV might possibly have a role in virus-induced abnormal embryogenesis.

Animals↗

PubMed1992

Immunohistochemical localization of calmodulin in the human testis [corrected].

The localization of calmodulin in the normal human testis was studied by the indirect immunoperoxidase method. Three different types of fixative were used: phosphate-buffered formalin, Bouin's solution or Carnoy's solution. Immunoreactivity specific for calmodulin was not detectable in the testis fixed in Carnoy's solution. The specimens fixed in phosphate-buffered formalin and in Bouin's solution were stained. The immunostaining for calmodulin was observed in pachytene spermatocytes, secondary spermatocytes and round spermatids but not in spermatogonia, or in pre-leptotene, leptotene and zygotene spermatocytes, elongated spermatids, spermatozoa or Leydig cells. Sertoli cells were not stained or were stained slightly. Among pachytene spermatocytes, the cells at the early stage were barely stained but those at the middle and late stages were slightly and intensely stained, respectively. The present report provides the first confirmation of the localization of calmodulin in the human testis.

Adult↗

PubMed1991

Postnatal porencephaly induced in mouse by murine cytomegalovirus.

Mouse embryos were infected with murine cytomegalovirus (MCMV) by injecting the virus into the cerebral ventricles at the late gestation. After deliveries, offspring were fed by the mothers until 4 weeks. Cystic brain lesions, regarded as porencephaly or paraventricular cysts, were observed in about 20% of the MCMV-injected offspring 3 to 4 weeks after birth. The porencephaly involved the cerebral cortex and the white matter, and sometimes opened to the ventricles, while the paraventricular cysts involved the white matter. The inner surfaces of the cysts were covered with thin monolayer cells. Around the cystic lesions, perivascular cuffings were sometimes observed in the meninges and the basal regions. Viral antigen-positive cells were observed in the cortex and the hippocampus but were hardly observed along the cystic walls. Immunohistochemical double staining using antibodies specific for the viral antigen and specific for factor VIII-related antigen showed that the brain capillary endothelial cells had susceptibility to MCMV infection, and in addition that some neurons in the cortex and the hippocampus had the same susceptibility. These findings suggest that there are at least two ways by which MCMV induce abnormalities in the developing mouse brains; migration of MCMV-infected neurons and affinity to the endothelial cells of the brain vessels to this virus.

Animals↗

PubMed1991

Subcellular distribution of the major immediate early proteins of human cytomegalovirus changes during infection.

The 72K immediate early (IE) 1 protein of human cytomegalovirus and the 68K protein, also encoded by the IE1 gene, were detected by immunoelectron microscopy using monoclonal antibodies specific for the 68K or 72K proteins. Early after infection, both proteins were localized to the nucleus, but with different localization patterns. Late after infection, both of the proteins decreased markedly in the nucleus, in which nucleocapsids appeared. Simultaneously, the 68K protein became diffusely distributed in the cytoplasm and the plasma membrane, whereas the 72K protein was distributed in the nuclear envelope and the plasma membrane. Both proteins were also observed in the coating membrane of the extracellular dense bodies.

Antigens, Surface↗

PubMed1991

Viral DNA detected by in situ hybridization in the developing mouse brain infected with murine cytomegalovirus.

To investigate the pathogenesis of brain abnormalities caused by congenital cytomegalovirus (CMV) infection, we previously reported experimental murine models of brain damage induced by intraventricular injection of murine CMV (MCMV) at the late stage of gestation. In the present study, viral DNA-positive cells in the damaged brain at different postnatal stages detected by in situ hybridization were compared with viral antigen-positive cells detected by an immunoperoxidase method using a monoclonal antibody against the immediate early antigen. At birth, the number of viral DNA-positive cells almost equalled that of viral antigen-positive cells. Seven to ten days after birth, the number of viral DNA-positive cells in the brain of MCMV-injected mice was one-fifth that of viral antigen-positive cells. Viral DNA-positive cells were more numerous in the hippocampus than in the cortex, and their density was dependent on the presence of viral antigen-positive cells. Dotted reaction products were observed in the nuclei of viral DNA-positive cells. These cells were rarely detected in lesions of later stages such as atrophy of the cortex and hippocampus, or the wall of the cystic lesions. These results suggest that viral DNA-positive cells detected by in situ hybridization are infected cells in which viral DNA replication is occurring actively.

Animals↗

PubMed1991

Angiotensinogen excretion in rat urine: effects of lipopolysaccharide treatment and sodium balance.

Rat urine was found to contain a component showing cross-reactivity with antibody against rat plasma angiotensinogen. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of rat urine revealed antigenic bands corresponding to the molecular weights of plasma angiotensinogen. The urinary angiotensinogen excretion in 8 rats, determined by direct radioimmunoassay, was 2.70 +/- 0.21 micrograms/day. Induction of acute inflammation in rats by injection of lipopolysaccharide caused about a 7-fold increase of urinary angiotensinogen excretion in the 24 hr after injection, with a concomitant elevation of plasma angiotensinogen. Neither sodium depletion nor loading by a low- or high-sodium diet altered the urinary excretion of angiotensinogen. These results suggest that the angiotensinogen present in rat urine is derived from that in plasma, although the level of excretion is too low to have any influence on the plasma level of angiotensinogen.

Angiotensinogen↗

PubMed1990

Secretion and clinical significance of atrial natriuretic peptide in patients with muscular dystrophy.

The plasma concentration of atrial natriuretic peptide was measured in patients with muscular dystrophies to study its relationship with congestive heart failure. In patients with Duchenne muscular dystrophy, the plasma atrial natriuretic peptide concentration was 35.5 +/- 3.3 pg/mL (mean +/- SE), which was higher than that in age-matched normal subjects (9.8 +/- 0.6 pg/mL). It increased with progression of disability and showed significant correlations with the cardiothoracic ratio and the ratio of the preejection period to the left ventricular ejection time. In patients with other types of muscular dystrophy, the plasma atrial natriuretic peptide concentration showed no significant change. Immunohistochemical examination demonstrated many atrial natriuretic peptide-positive cells in atrial muscle of an autopsied patient, indicating preservation of the peptide until the end stage. These findings suggest that measurement of the plasma atrial natriuretic peptide concentration is useful for evaluating heart failure in patients with Duchenne muscular dystrophy.

Adolescent↗

PubMed1990

Characterization of cell fusion in XC cells induced by Suncus murinus mammary tumor virus.

Syncytium formation in a rat tumor cell line (XC) induced by Suncus murinus mammary tumor virus (Sm-MTV) was studied. Multinucleate giant cells containing 20-30 nuclei were formed in a monolayer of XC cells by cocultivation with X-ray-irradiated Sm-MTV producing cells (Sm-MT-1). By fluorescent antibody staining. Sm-MTV antigens were demonstrated in the cytoplasm of syncytia, and budding particles and intracytoplasmic A particles were found in syncytial giant cells by electron microscopy. Cell-free supernatant of Sm-MT-1 was capable of inducing syncytia at a much lower incidence than cocultivation of Sm-MT-1 cells. Syncytium formation was completely inhibited when anti-Sm-MTV bovine serum was added to the coculture medium. Pretreatment of XC cells with actinomycin-D caused a partial reduction of Sm-MTV-induced cell fusion, but syncytium formation did occur at a reduced rate even when cellular RNA synthesis was completely inhibited. Dexamethasone increased virus production in Sm-MT-1 cells, resulting in the enhancement of Sm-MTV mediated syncytium formation. Sm-MTV was found to have a unique characteristic of cell fusion activity on XC cells with striking enhancement by dexamethasone.

Animals↗

PubMed1990

Differential expression of the major immediate early gene of human cytomegalovirus.

We prepared a murine monoclonal antibody reactive to a human cytomegalovirus (HCMV)-induced nuclear protein with an Mr of 68,000. Expression of the 68K protein was compared with the major immediate early (IE) 72K protein in various cell types after infection with HCMV or microinjection of plasmid DNA containing the major IE gene. The 68K nuclear protein was detected 2 to 3 h after appearance of the 72K protein in human embryonal lung (HEL) cells infected with HCMV. The 68K protein was distributed throughout the cytoplasm in the late phase of infection, while the 72K protein remained chiefly in the nucleus. The 68K protein was barely detected in the cells under IE conditions by immunoprecipitation, but, together with the 72K protein, it was expressed after microinjection of cloned DNA, containing only the major IE region (region 1), into the nuclei of HEL cells. The 72K protein was expressed in nuclei 2 h after microinjection, whereas the 68K protein was detected 4 to 5 h after the injection. The 68K protein was expressed after microinjection in non-permissive rodent fibroblasts or non-permissive transformed human cells in which these proteins were not expressed after viral infection. Immunoprecipitations after chase-labelling from IE conditions or after partial digestions suggested that the 68K protein is neither a degradation nor a modification product of the major IE 72K protein.

Antibodies, Monoclonal↗

PubMed1990

Antiviral effect of oxetanocin G against guinea pig cytomegalovirus infection in vitro and in vivo.

The antiviral activity of a new guanosine analog, 9-(2-deoxy-2-hydroxymethyl-beta-D-erythrooxetanosyl)guanine (OXT-G), against guinea pig cytomegalovirus (GPCMV) was evaluated both in vitro and in vivo. In the plaque reduction assay, the median effective concentration (EC50) of OXT-G, ganciclovir (DHPG) and acyclovir (ACV) against GPCMV was 0.03, 6.4 and 52 micrograms/ml, respectively. The selectivity index, based on the ratio of the median inhibitory concentration for cell growth of guinea pig embryo fibroblasts to the median effective concentration for GPCMV plaque formation, was about 100-fold higher than that of DHPG. In an in vivo study, Hartley guinea pigs infected with GPCMV were treated with OXT-G or DHPG (20 mg/kg/day) for 2 weeks, and it was found that virus titers in the salivary gland were 70-fold lower in OXT-G-treated guinea pigs than in DHPG-treated animals. The results indicate that OXT-G was more potent and selective against GPCMV infection than DHPG.

Acyclovir↗

PubMed1989

Brain abnormalities induced by murine cytomegalovirus injected into the cerebral ventricles of mouse embryos exo utero.

Murine cytomegalovirus (MCMV) was injected into the cerebral ventricle of mouse embryos on day 13 of gestation after exposing the embryos out of the uterus in the abdominal cavity of the mother. The embryos were allowed to develop to day 18 of gestation, then taken out from the abdominal cavity. Macroscopically, there were four expanded and three distorted brains out of 19 surviving embryos, whereas no brain abnormality was noticed in 13 embryos injected with culture medium instead of MCMV in the same way. Histopathological examination showed hydrocephalic lesions with strong dilatation of the ventricles and atrophy of the cerebral cortex, and inflammatory lesions with granulomatous proliferation of the ventricular walls with disappearance of the cortical zonation. Immunohistochemically, MCMV-induced nuclear antigen-positive cells were frequently observed in the wall of the ventricles and occasionally scattered in the cerebral cortex, white matter, and the nucleus basalis. Some fetuses injected with MCMV in the same way were recovered from the abdominal cavities on day 18 of gestation and transferred to foster nurse mothers. They showed massive cerebral necrosis after feeding for 9 days after birth. Brain abnormalities of mouse embryos after intraventricular injection with MCMV may provide an experimental model of brain damage induced by congenital cytomegalovirus infection.

Animals↗

PubMed1989

Susceptibility of brain cells to murine cytomegalovirus infection in the developing mouse brain.

Mouse embryos were infected with murine cytomegalovirus (MCMV) by injecting the virus into the cerebral ventricles in the late stage of gestation; the brains of the offspring were than analyzed using the histological and immunohistochemical methods. Brains of the offspring, which were injected with relatively high titers of MCMV [1 X 10(4) plaque-forming units (pfu)] on day 13 of gestation exo utero or on day 15 of gestation in utero, showed massiv necrosis of the cerebral cortex with gliomesodermal proliferation around 9 to 10 days after birth. In these brains, viral antigen-positive cells were observed in zonal arrangement in the lesion-free cortex and in the hippocampus. Immunohistochemical double staining showed that some of the viral antigen-positive cells had also reacted with antibody to neuron-specific enolase at the same time, but had hardly reacted with antibodies to brain-type creatine kinase or glial fibrillary acidic protein. Brains of the offspring, which were injected with relatively low titers of virus (1 x 10(3) pfu) on day 15 of gestation, showed zonal arrangement of viral antigen-positive cells mainly in the cerebral cortex and in the hippocampus 7 days after birth, although the numbers of the positive cells were low. Fourteen days after birth, some of these offspring showed atrophy of the cerebral cortex and the hippocampus. These results suggest that some of the neuronal cells in the cerebral cortex and the hippocampus have special susceptibility to MCMV infection.

Animals↗

PubMed1988

Major structural polypeptides of retrovirus produced by a Suncus murinus mammary tumor cell line and type D retrovirus share interspecies antigenic determinants.

Constitutive polypeptides of retrovirus produced by a Suncus murinus mammary tumor cell line (Sm-MTV) were characterized by immunofluorescence and immunoblotting. The major structural polypeptide of Sm-MTV, molecular weight 44/43K, immunologically cross reacted with the structural polypeptides of Mason-Pfizer monkey virus (MPMV) (molecular weights 27K and 12/10K). Sm-MTV constitutive polypeptides did not show antigenic relatedness with those of the following mammalian retroviruses: murine mammary tumor virus, Rausher murine leukemia virus, Molony murine leukemia virus, Osborn-Mendel rat leukemia virus, feline leukemia virus, RD114 virus, simian sarcoma virus 1, and human T-cell leukemia virus type I. Among the Sm-MTV structural polypeptides, those with molecular weights of 72/69K and 47K were glycosylated.

Animals↗

PubMed1988

Identification and characterization of a 50K DNA-binding protein of guinea-pig cytomegalovirus.

In a previous study we showed that cells infected with guinea-pig cytomegalovirus (GPCMV) contain large amounts of a non-structural 50K nuclear protein that is detectable by immunoelectron microscopy using monoclonal antibodies. The present study shows that this 50K protein is a DNA-binding protein, as determined by single-stranded DNA affinity chromatography, and a phosphorylated protein as demonstrated by immunoprecipitation using [32P]orthophosphate-labelled cells. This protein binds both viral and host cellular double-stranded and single-stranded DNA, assayed by a simple method using DNA linked to a nylon membrane. Induction of the 50K protein in GPCMV-infected cells was highly dependent on viral DNA synthesis, which was detected by dot hybridization using a cloned GPCMV DNA probe. Synthesis of the 50K protein was significantly impaired when phosphonoacetic acid was added to the culture medium. Induction of the 50K protein was detected about 6 h before the appearance of the 76K viral matrix protein.

Animals↗