Biomedical subjects

M Katsuki

Publications and source records attributed to M Katsuki.

At least 73 records · Page 4Linked to original sources

PubMed1997

Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the Ah (dioxin) receptor.

BACKGROUND: The aryl hydrocarbon receptor (AhR or dioxin receptor) is a ligand-activated transcription factor that is considered to mediate pleiotropic biological responses such as teratogenesis, tumour promotion, epithelial hyperplasia and the induction of drug-metabolizing enzymes to environmental contaminants usually represented by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast to the role of AhR in the regulatory mechanism of xenobiotic-metabolizing enzymes, there is no direct proof that the AhR is involved in the teratogenic effects of TCDD. RESULTS: To gain insight into the physiological and teratogenic role of the AhR, we have used gene targeting in mice to disrupt the murine Ahr gene by homologous recombination. Ahr-null mice were viable and fertile and were apparently normal at birth, but displayed a slightly slower growth rate than wild-type mice for the first few weeks of life. When pregnant dams were administered with TCDD by gavage, at a dose of 40 microg/kg body weight at gestation day 12.5, none of the Ahr-null mutant foetuses were sensitive to the teratogenic effects of TCDD, although almost all wild-type foetuses suffered from cleft palate and hydronephrosis. In heterozygous Ahr+/- genotypes, nearly all foetuses suffered from hydronephrosis in response to TCDD treatment, while haplo-insufficiency was observed in the incidence of cleft palate. CONCLUSION: These results clearly show that the AhR is involved in the malformation of the palate and kidney in mouse embryos caused by TCDD and suggests that the mechanism of its involvement differs between the cleft palate and hydronephrosis.

Animals↗

PubMed1997

Dp260 disrupted mice revealed prolonged implicit time of the b-wave in ERG and loss of accumulation of beta-dystroglycan in the outer plexiform layer of the retina.

Dp260 is a C-terminal isoform of dystrophin and is expressed specifically in the retina. Abnormal electroretinograms (ERG) in some Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients are likely linked to a disruption of Dp260. To clarify the importance of Dp260 in the retina, we examined dystrophin exon 52 knock-out mice, whose expression of Dp260 is impaired. We also confirmed the localization of Dp260 in the outer plexiform layer (OPL) of the retina. Disruption of Dp260 causes a change in the localization of beta-dystroglycan, which is normally found in the OPL of the retina. This suggests a requirement for Dp260 for normal formation of the dystrophin-dystroglycan complex in the retina. Dp71, also expressed in the retina, was, however, not detected in the OPL. The difference in localization of Dp260 and Dp71 implies that the two isoforms have different functions. The dystrophin exon 52 knock-out mice had a prolonged implicit time of the b-wave in ERG, although no significant change was observed in amplitude. These ERG findings differed from those of DMD and BMD patients, especially with regard to amplitude of the b-wave, but make it clear that Dp260 is required for normal electrophysiology.

Animals↗

PubMed1997

Long term results using a modified King's method for cubital tunnel syndrome.

Forty-six elbows in 40 patients with cubital tunnel syndrome were evaluated after using a modified King's method. The average postoperative followup period was 79 months. The severity of cubital tunnel syndrome was graded before and after operation by the modified method of Stuffer et al which evaluated the amount of numbness, sensation, muscle strength, and muscle atrophy. The results were positively correlated with the preoperative severity of the disease and the ulnar nerve sulcus angle as measured in the axial view of the elbow. When the amount of the medial epicondylectomy was large, a good result was obtained, but the risk of medial collateral ligament insufficiency increased. Because the results of medial epicondylectomy are inconsistent with stability of the elbow, when doing a modified King's operation, careful repair of the medial collateral ligament is essential to minimize instability of the elbow.

Adult↗

PubMed1997

Simple and efficient vitrification procedure for cryopreservation of mouse embryos.

Mouse pronuclear oocytes and 2-cell embryos derived from in vitro fertilization were cryopreserved by a novel simple vitrification procedure. Most cryopreserved oocytes/embryos were morphologically normal after warming, and 89-92% of them developed to the blastocyst stage during the culture. Moreover, the rate of morphologically normal pronuclear oocytes after being repeatedly cooled and warmed three times was as high as that of oocytes cooled and warmed only once, and 85% of them developed to the blastocyst stage. In addition, 43-57% of the cryopreserved oocytes/embryos transferred to recipients had developed normally to live fetuses observed on day 18.5 of pregnancy.

Animals↗

PubMed1997

The effects of basic fibroblast growth factor, transforming growth factor-alpha, and transforming growth factor-beta on mesenchymal cells in the rabbit phallus: an in-vitro study.

Effects of bFGF, TGFalpha and TGFbeta on cultured mesenchymal cells of rabbit phalli were investigated. A remarkable cell proliferation reaching confluency was only obtained from the media containing bFGF by the first week of culture. By subsequent 4 weeks culture using such confluent cells in the media containing bFGF, TGFalpha or TGFbeta, respectively, cells cultured in the media containing bFGF showed an active transformation to endothelial precursor cells determined by positive immunoreactions for factor VIII-related antigen at 2 weeks. By electron microscopy, such vasoformative cells appeared in contact with each other, occasionally forming a solid cell cord. The use of Matrigel's basement membrane matrix (Matrigel) in each culture bed promoted the capillary ingrowth, implying that certain extracellular matrix (ECM) components included in Matrigel may accelerate the formation of neocapillaries. Enhancement in the production of ECM components and transformation to smooth muscle cells determined by positive immunoreactions for actin were also seen in cells cultured in the media containing TGFalpha and TGFbeta, respectively. The present results indicate that the above growth factors play crucial roles in the differentiation of the rabbit cavernous body.

Animals↗

PubMed1996

Motor discoordination results from combined gene disruption of the NMDA receptor NR2A and NR2C subunits, but not from single disruption of the NR2A or NR2C subunit.

NMDA receptors consist of two distinct classes of subunits. The NR1 subunit possesses all properties of the NMDA receptor-channel complex, whereas four NR2 subunits (NR2A-2D) potentiate and differentiate NMDA receptor responses by heteromeric assemblies with NR1. The mRNAs for the five NMDA receptor subunits are expressed in the cerebellum in a distinct temporospatial manner. To study functions of the NMDA receptors in the cerebellum, we generated knockout mice deficient in either NR2A or NR2C or both of these subunits. All three mutant mice developed normally and showed normal overall morphology of the cerebellum. The NMDA receptor-mediated components of EPSCs in granule cells, as assessed by whole-cell recordings of cerebellar slices, were reduced in NR2A- and NR2C-deficient mice and nearly abolished in mice lacking both NR2A and NR2C. The NR2A- and NR2C-deficient granule cells were different in the current-voltage relationship and time course of NMDA receptor responses. The NR2A and NR2C subunits thus contribute to distinct NMDA receptor-mediated excitatory transmission in mossy fiber-granule cell synapses in the mature cerebellum. Both NR2A- and NR2C-deficient mice showed no impaired movements in the motor coordination tasks tested. The mutant mice deficient in both NR2A and NR2C could also manage simple coordinated tasks, such as staying on a stationary or a slowly rotating rod, but failed more challenging tasks such as staying on a quickly rotating rod. These data demonstrate that the NMDA receptors play an active role in motor coordination.

Animals↗

PubMed1996

Perinatal expression and inducibility of human CYP3A7 in C57BL/6N transgenic mice.

The expression and inducibility of CYP3A7 transgene in the fetus and suckling neonates from one of the transgenic lines (M10) were investigated by Northern and Western blot analyses. The mRNA expression could be detected as early as the 15th embryonic day and increased gradually with advancing gestation but then remarkably so after birth. The protein expression was also detectable postnatally and increased. Inducibility was achieved in neonatal mice via maternal exposure to zinc sulfate. Midazolam hydroxylase activities could be detected in liver microsomes prepared from 14-day-old neonates. These activities were significantly higher in transgenic than nontransgenic lines of mice (p < 0.001).

Aging↗

PubMed1996

Impairment of hippocampal mossy fiber LTD in mice lacking mGluR2.

Subtype 2 of the metabotropic glutamate receptor (mGluR2) is expressed in the presynaptic elements of hippocampal mossy fiber-CA3 synapses. Knockout mice deficient in mGluR2 showed no histological changes and no alterations in basal synaptic transmission, paired-pulse facilitation, or tetanus-induced long-term potentiation (LTP) at the mossy fiber-CA3 synapses. Long-term depression (LTD) induced by low-frequency stimulation, however, was almost fully abolished. The mutant mice performed normally in water maze learning tasks. Thus, the presynaptic mGluR2 is essential for inducing LTD at the mossy fiber-CA3 synapses, but this hippocampal LTD does not seem to be required for spatial learning.

Animals↗

PubMed1996

Inactive allele-specific methylation and chromatin structure of the imprinted gene U2af1-rs1 on mouse chromosome 11.

The imprinted U2af1-rs1 gene that maps to mouse chromosome 11 is predominately expressed from the paternal allele. We examined the methylation of genomic sequences in and around the U2af1-rs1 locus to establish the extent of sequence modifications that accompanied the silencing of the maternal allele. The analysis of HapII or HhaI sites showed that the silent maternal allele was hypermethylated in a block of CpG sequences that covered more than 10 kb. By comparison, the expressed paternal allele was unmethylated from a CpG island upstream of the transcribed region through 2 kb. An analysis of DNaseI hypersensitivity of a putative promoter of U2af1-rs1 showed an open chromatin conformation only on the unmethylated, expressed paternal allele. These results suggest that allele-specific hypermethylation covering the gene and its upstream CpG island plays a role in maternal allele repression of U2af1-rs1, which is reflected in altered chromatin conformation of DNaseI hypersensitive sites.

Alleles↗

PubMed1996

Humanized prion protein knock-in by Cre-induced site-specific recombination in the mouse.

To establish humanized mice with a knock-in (gene replacement) technique, we constructed a targeting vector which consists of the human prion protein and the loxP sequences. The introduced human prion protein with the loxP system in the embryonic stem cells was transmitted through the mouse germ line. Transient expression of Cre recombinase in the fertilized eggs resulted in the prion protein humanized mice. The Cre-loxP-mediated gene replacement is a simple and efficient method which is generally applicable to make humanized animal models.

Animals↗

PubMed1996

A novel positive detection system of in vivo mutations in rpsL (strA) transgenic mice.

To positively detect the in vivo mutations accumulated in different mouse organs, we have developed a transgenic mouse system. This transgenic mouse carried an Escherichia coli (E. coli) plasmid pML4 as a shuttle vector that consisted of a replication origin (ori), the kanamycin-resistant gene (KanR) and the rpsL+ gene (strAS) derived from E. coli. These E. coli elements were expected to be inert in the transgenic mouse system; thus, neutral mutations would be accumulated on the shuttle plasmid in the transgenic mice. The shuttle plasmid vector was recovered from the mouse genomic DNA and introduced into kanamycin-sensitive (KmS) and streptomycin-resistant (SmR) E. coli cells by using electroporation. The original pML4 shuttle plasmid transformed the host E. coli to KmR and SmS, since both the KanR and rpsL genes exhibited dominant traits of KmR and SmS, respectively. On the other hand, when the retrieved pML4 shuttle plasmid carried a mutated rpsL gene, it could be positively detected as both KmR and SmR. Based on this principle, we were able to positively detect the in vivo mutations accumulated in the rpsL transgene of the shuttle vector pML4 integrated into the mouse genome. The total number of rescued shuttle plasmids were counted on the plates containing Km alone, while only mutants were detected on the plates containing both Km and Sm. We have so far established 22 independent transgenic mouse lines that carried up to approx. 750 copies of the shuttle plasmid pML4 in a haploid genome. By using high-copy-number transgenic mouse lines which carried 350 copies or more of the shuttle vector, we also developed a simple and proficient method for retrieving the shuttle plasmid from various tissues of the transgenic mice. The background mutant frequency was approx. 5 x 10(-5). In order to validate the applicability of the positive-detection transgenic system for the induced mutagenicity assay, methylnitrosourea (MNU) was administered to the transgenic mice, and an increase in the number of mutant frequencies was seen in all tested organs including spleen, liver and brain. The rpsL transgenic mouse system was therefore considered to provide a quick-and-easy risk assessment test for in vivo tissue-specific mutagenicity, using positive detection by streptomycin.

Animals↗

PubMed1996

Establishment of transgenic mice carrying human fetus-specific CYP3A7.

CYP3A7 is a cytochrome P450 isozyme expressed prenatally in humans. Six lines of mice transgenic for human CYP3A7 were established by microinjecting a CYP3A7 cDNA downstream of a mouse metallothionein-1 promoter gene into the male pronucleus of fertilized mouse oocytes. The inserted CYP3A7 transgene was expressed at a mRNA level in a variety of tissues including the liver, kidney, lung, spleen, testis, small intestine, thymus, brain, skin, and heart of adult mice. The protein expression of the transgene was also detected in the liver and testis of line M10 mice. A significantly higher level of total testosterone in the serum was found in line M10 male mice. In addition, this transgenic line exhibited weight increases in the liver, kidney, and uterus but a decrease in the testis (P < 0.01). The transcript of the integrated CYP3A7 gene possessed the ability to activate aflatoxin B1 in Ames test in which the his+ revertants of Salmonella typhimurium TA100 per plate were significantly higher (P < 0.01) when liver microsomes of line M10 transgenic mice were used. This result demonstrates that the CYP3A7 gene has been integrated into the mouse genome and translated into a catalytically active enzyme. These transgenic mice are expected to give useful information for studies on fetal toxicities in humans.

Aflatoxin B1↗

PubMed1996

Modulation of hematopoiesis in mice with a truncated mutant of the interleukin-2 receptor gamma chain.

The interleukin-2 (IL-2) receptor gamma chain is indispensable for IL-2-, IL-4-, IL-7-, IL-9-, and IL-15-mediated signaling. Mutations of the human gamma chain cause the X-linked severe combined immunodeficiency (XSCID), showing that T and natural killer cells absolutely require the gamma chain for their development in humans. To elucidate the roles of the gamma chain in hematopoiesis, we have generated mice, by gene targeting, that express a form of the gamma chain lacking the cytoplasmic region. Male mice carrying the truncated gamma-chain mutant, which mimics mutations in patients with XSCID, showed a decrease in the number of lymphocytes and an increase in monocytes; the number of T cells was profoundly reduced and no natural killer cells were detected, which is similar to the characteristic of human XSCID. Unlike human XSCID, the levels of B cells were also reduced. In spite of the severe decrease in CD45R+/sIgM+ B cells, the level of IgM in serum of the 8-week-old mutant mice was higher than that of control littermates. Interestingly, the stem cell population with surface phenotypes of CD34, c-kit, and Sca-1 was significantly increased. Furthermore, the colony-forming assay showed that the mutant mice had 15-fold higher numbers of hematopoietic progenitor cells in the spleen as compared with that of controls. These results indicate that functional loss of the gamma chain causes significant effects on the immunological system in mice.

Animals↗

PubMed1996

Inactivation of hepatitis C virus cDNA transgene by hypermethylation in transgenic mice.

Transgenic mice were produced by microinjection of a partial hepatitis C virus (HCV) genome sequence including the structural protein region, under the control of the albumin promoter and enhancer into fertilized eggs of C57BL/6 and BDF1 mice. Three founders carrying at least five copies of the transgene but not expressing HCV-specific RNA were generated. Methylation analysis indicated that the transgene was extensively methylated. Mapping of methylated cytosine residues of the transgenic mouse DNA showed that all C residues of a particular part of the HCV genome but not all the CpG island like sequences were methylated. Transiently expressed HCV cDNA in COS7 cells and the active endogenous albumin gene were not methylated. Furthermore, 5-azacytidine, a potent demethylating agent, induced HCV gene expression in a line of these transgenic mice. These results suggest that methylation of HCV cDNA is a cause of its inactive expression in transgenic mice, and that this phenomenon may occur in other stable systems for expression of the HCV genome.

Animals↗

PubMed1996

p53-dependent apoptosis suppresses radiation-induced teratogenesis.

About half of human conceptions are estimated not to be implanted in the uterus, resulting in unrecognizable spontaneous abortions, and about 5% of human births have a recognizable malformation. In order to find clues to the mechanisms of malformation and abortion, we compared the incidences of radiation-induced malformations and abortions in p53 null (p53-/-) and wild-type (p53+/+) mice. After X-irradiation with 2 Gy on day 9.5 of gestation, p53-/- mice showed a 70% incidence of anomalies and a 7% incidence of deaths, whereas p53+/+ mice had a 20% incidence of anomalies and a 60% incidence of deaths. Similar results were obtained after irradiation on day 3.5 of gestation. This reciprocal relationship of radiosensitivity to anomalies and to embryonic or fetal lethality supports the notion that embryonic or fetal tissues have a p53-dependent "guardian" of the tissue that aborts cells bearing radiation-induced teratogenic DNA damage. In fact, after X-irradiation, the number of cells with apoptotic DNA fragments was greatly increased in tissues of the p53+/+ fetuses but not in those of the p53-/- fetuses.

Animals↗

PubMed1996

Dopamine D2 receptor plays a critical role in cell proliferation and proopiomelanocortin expression in the pituitary.

BACKGROUND: It has been suggested that the dopaminergic system is involved in the inhibitory control of secretion of pituitary hormones and in the regulation of motor function in the striatum. To elucidate the specific role of the dopamine D2 receptor (D2R) in the pituitary and brain, we generated D2R deficient (D2R mutant) mice using the gene targeting method. RESULTS: Electrophysiological studies as well as ligand binding assays show no functional D2R in mutant mice. The D2R mutant mice display a hypoactivity and a slow, creeping movement. The expression of enkephalin mRNA in the striatum is increased in the mutant mice, but not that of dynorphin and substance P. D2R mutant mice have significantly darker coat colour than their wild-type littermates and show an elevation of plasma alpha-melanocyte stimulating hormone (alpha-MSH) levels. We found corresponding hyperplastic changes of intermediate lobe of the pituitary and the increased expression of pro opiomelanocortin (POMC) in the pituitary in D2R mutant mice. CONCLUSIONS: D2R plays a critical role in the inhibitory regulation of endocrine cell proliferation and the transcription of POMC mRNA, and consequently in the regulation of alpha-MSH in plasma. D2R might be involved in the regulation of enkephalin expression in the striatum, and hence might affect the movement and the behaviour.

Animals↗

PubMed1996

Rare activation of the human c-Ha-ras transgene of mice in hemangioendothelial sarcomas and liver tumors induced by Glu-P-1.

A transgenic mouse (Tg), having the human c-Ha-ras proto-oncogene, has been demonstrated to develop hemangioendothelial sarcomas (HESs) which are associated with the transgene mutation, but not to develop liver tumors. In this study, we examined the effects of 2-amino-6-methyldipyrido [1,2-a:3',2'-d] imidazole (Glu-P-1), a food-borne carcinogen, which has been demonstrated to induce HESs and liver tumors in CDF1 mice, on Tg mice. Chronic administration of 0.05% Glu-P-1 in the diet induced HESs in Tg (7/17), but not in 18 non-transgenic mice (N-Tg). With basal diet, two out of 17 Tg but none of 22 N-Tg, developed HESs. In contrast, Glu-P-1 administration induced liver tumors, both in Tg and in N-Tg; 16/17 in Tg and 13/18 in N-Tg. The incidence of hepatocellular carcinomas in Tg was higher than that in N-Tg (8/17 versus 3/18). With basal diet, only one out of 17 Tg and none of 22 N-Tg developed liver tumors. The Ha-ras mutation in tumors developed by the groups administered Glu-P-1, was examined. No mutations were detected in the transgene and mouse c-Ha-ras genes in all three HESs examined. In contrast, when 29 liver tumors taken from Tg were examined, two mutations of the transgene were detected in two HCCs. No mouse c-Ha-ras gene mutations were detected in any of the 47 liver tumors examined, which had developed in Tg and N-Tg mice. These results suggest that the transgene plays a role in the development of HESs induced by Glu-P-1, but not as a result of its mutation. Further, the transgene plays no significant role in the development of liver tumors induced by Glu-P-1, but does play a role in the malignant conversion of some liver tumors, as a result of its mutation.

Animals↗

PubMed1996

Characterization of excitatory amino acid neurotoxicity in N-methyl-D-aspartate receptor-deficient mouse cortical neuronal cells.

Roles and mechanisms of N-methyl-D-aspartate (NMDA) receptors in glutamate neurotoxicity were investigated in cultures of NMDA receptor-deficient cortical neuronal cells. Mutant mice lacking a functional NMDA receptor were generated by gene targeting of the NR1 NMDA receptor subunit. Cortical neuronal cells prepared from wild-type NR1+/+, heterozygous NR1+/- and homozygous mutant NR1-/- mice at 15-17 days of gestation grew indistinguishably from each other. Brief exposures (5 min) of both NR1+/+ and NR1+/- neuronal cells to glutamate or NMDA, but not kainate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), resulted in widespread neuronal degeneration by the following day. In contrast, neither glutamate nor NMDA treatment caused neuronal degeneration in NR1-/- cells, indicating that NMDA receptors are responsible for rapidly triggered glutamate neurotoxicity. The above four compounds were all effective in inducing the death of NR1+/+ and NR1+/- neuronal cells after prolonged exposure (20-24 h). However, NMDA had no neurotoxic effects on NR1-/- cells, although the other three compounds wer neurotoxic with potencies comparable to those for NR1+/+ and NR1+/- cells. The AMPA and kainate receptors are thus sufficient for inducing slowly triggered glutamate neurotoxicity. Brief exposure of a mixed population of NR1+/+ and NR1-/- neuronal cells to NMDA selectively killed the NMDA receptor-expressing cells without any appreciable effects on neighbouring NMDA receptor-deficient cells. This finding further supports a direct and indispensable role for NMDA receptors in NMDA-evoked neuronal cell death.

Animals↗