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H Tojo

Publications and source records attributed to H Tojo.

At least 73 records · Page 4Linked to original sources
PubMed1997

Equine inhibin/activin beta A-subunit mRNA is expressed in the endometrial gland, but not in the trophoblast, during pregnancy.

The expression of both inhibin alpha- and inhibin/activin beta A-subunit mRNA was examined in equine uteroplacental tissues collected during pregnancy (days 90 to 300). Northern blot analysis revealed that 5 transcripts (7.0, 4.1, 3.4, 2.6, 1.5 kb) of beta A-subunit were present, and the most abundantly expressed transcript was the 1.5 kb one. Relatively high levels of the 1.5 kb transcript were seen in the second trimester of pregnancy compared to what was found in the third trimester. To identify the tissue localization of beta A-subunit mRNA, in situ hybridization was performed, and the positive signal was observed exclusively in the endometrial glands, but not in the fetal placental tissue (trophoblast) at days 150, 210, and 300 of pregnancy. On the other hand, inhibin alpha-subunit transcript could not be detected at any stage of pregnancy examined either by Northern blot analysis or in situ hybridization. Although the factor(s) regulating the gene expression of beta A-subunit in this equine tissue is currently unknown, these results suggest that activin, but not inhibin, is predominantly produced in the endometrial glands of the pregnant mare, and thus produced activin may play a paracrine or endocrine role during pregnancy in this species.

Animals↗
PubMed1997

Phospholipase A2 stimulates rat gastric epithelial cell line (RGM-1) migration.

OBJECTIVE AND METHODS: The effect of phospholipase A2 (PLA2) and its phospholipid metabolites on gastric epithelial migration was examined using an in vitro wounding model of confluent monolayers of rat gastric epithelial cell line RGM-1. RESULTS: Lysophosphatidylcholine (lysoPC) (0.01 100 ng/ml) as well as PLA2 (0.01-100 mU/ml) dose-dependently increased the cell migration. Lysophosphatidic acid (10 ng/ml) also increased the migration, but no significant increase in migration was observed when stimulated by lysophosphatidylethanolamine (10 ng/ml) or lysophosphatidylserine (10 ng/ml). Addition of 4-bromophenacyl bromide (BPB), a PLA2 inhibitor, completely blocked the effect of PLA2. However, addition of piroxicam (a cyclooxygenase inhibitor) or nordihydroguaiaretic acid (a lipoxygenase inhibitor) had no significant effect. Combination of PLA2 (10 mU/ml) with lysoPC (10 ng/ml) had no additive effect on migration. Moreover, lysoPC levels were increased in the cells after incubation with PLA2 (10 mU/ml). After pretreatment of RGM-1 cells with replication-inhibiting doses of mitomycin C. PLA2 and lysoPC still increased the cell migration. CONCLUSIONS: These data suggest that PLA2 may, independently of proliferation, increase gastric epithelial migration mainly via lysoPC production.

Acetophenones↗
PubMed1997

Growth hormone-releasing hormone (GHRH)-GH-somatic growth and luteinizing hormone (LH)RH-LH-ovarian axes in adult female transgenic mice expressing human GH gene.

We have examined alterations in the hypothalamo-pituitary GH-somatic growth axis and the hypothalamo-pituitary LH-ovarian axis in a line of transgenic ICR mice expressing human GH (hGH) under the influence of the whey acid protein promoter. Transgenic female mice weighed twice as much as control females and were infertile. The size of the anterior pituitary (AP) was 1/3 that of the controls. In transgenic mice, acinar cells in the mammary and mandibular glands displayed hGH-immunoreactivity, and plasma hGH was detected by radioimmunoassay. In the medial basal hypothalamus (MBH) of transgenic females, the immunoreactive-GHRH level was decreased (P<0.01). There was a corresponding reduction in the number of GHRH-immunoreactive neurons in the arcuate nucleus (ARC) and in the immunostaining of GHRH nerve terminals in the median eminence. The level of somatostatin (SRIH) in the MBH was increased (P<0.05), and SRIH-immunoreactive neurons in the periventricular nucleus (PeV) were increased in size and number in transgenic mice. The MBH level of LHRH in transgenic animals was greater (P<0.01) than in controls, although there was no apparent difference in the number of LHRH-immunoreactive neurons or in LHRH level in the preoptic area. There are fewer SRIH- and LHRH-immunoreactive neurons in the ARC in transgenic mice. Cells in the AP for GH, PRL, and LH were fewer in transgenic mice. The ovary suffered disturbance of follicular development and of corpora lutea formation. These results demonstrate that chronic overproduction of hGH may profoundly affect the organization of the GHRH/SRIH-GH-somatic growth axis and the LHRH-LH-ovarian axis due to reduction of GHRH-, SRIH- and LHRH-neurons in the ARC and increase of SRIH-neurons in the PeV.

Animals↗
PubMed1997

Efficient selection of preimplantation transgenic embryos by an improved procedure using Dpn I-Bal 31 digestion and the polymerase chain reaction.

Efficient selection of preimplantation transgenic embryos by an improved method after pronuclear injection of exogenous DNA is described. The method is based on subjecting DNA extracted from the embryos to restriction enzymes as well as the polymerase chain reaction (PCR). The incorporated procedure included recovery of the digested DNA with glassmilk before PCR, which markedly enhanced the rate of accurate detection of transgenic embryos. When exogenous DNA sequences in the mouse embryos were not integrated into the genome they were digested with both Dpn I and Bal 31, and subsequent PCR analysis generated DNA fragments of the injected DNA sequence in only 1.5% of cases examined. However, DNA extracted from mouse embryos containing the transgene sequences integrated into the genome evaded digestion by both enzymes and yielded transgene-specific PCR products in 68.6% of the embryos tested. When bovine embryos were used, sequences of the endogenous haemoglobin gene used as a control genomic DNA sequence were protected from enzyme digestion (PCR products in 70.5% of the embryos examined); by contrast, the non-integrated injected sequences were almost completely eliminated by the same treatment (PCR products in 1.4% of the embryos examined). It is suggested that this method might be useful for the selection of transgenic embryos before embryo transfer, thereby reducing the number of recipient females required.

Animals↗
PubMed1997

Different female reproductive phenotypes determined by human growth hormone (hGH) levels in hGH-transgenic rats.

The effect of continuous human GH (hGH) secretion on female reproduction was studied in adult female transgenic rats expressing the hGH gene with a mouse whey acidic protein (mWAP) promoter. Two lines of transgenic female rats carrying the mWAP/hGH gene were established and used in the study. One was characterized by relatively high levels of serum hGH (high line), and the other had relatively low levels (low line). High-line female rats had recurring, pseudopregnancy-like estrous cycles accompanied by increased serum progesterone levels for 2 wk after ovulation, and they were fertile. In these rats, luteinization occurred spontaneously without cervical stimulation, probably due to high levels of serum hGH, which has prolactin (PRL)-like activity in the rat. Although low-line female rats had recurring, regular 4-day estrous cycles, they were sterile. In these rats, pseudopregnancy could not be induced by mating or by mechanical cervical stimulation. PRL surges following cervical stimulation were not detected, and PRL secretion was not induced by a dopamine antagonist, metoclopramide. The ovarian tissue in this line had a much higher number of corpora lutea and grew much heavier than in normal littermates, suggesting impairment of PRL-induced structural luteolysis. Suppression of PRL secretion in low-line rats was, at least in part, due to a marked decrease in the number of lactotrophs in the pituitary. The present study shows that the serum hGH level plays a crucial role in regulating luteal function in female transgenic rats expressing the hGH gene.

Animals↗
PubMed1997

Expression and cellular localization of inhibin alpha-subunit mRNA in equine fetal gonads.

The expression of inhibin alpha-subunit mRNA in equine fetal gonads during pregnancy (Days 90 to 300) was examined by means of Northern blot analysis. In all samples examined, a single species of transcript was detected at the size of 1.5 kb. A digoxigenin-labeled antisense cRNA probe specific to equine inhibin alpha-subunit was synthesized and in situ hybridization analysis to locate the inhibin alpha-subunit mRNA positive cells was performed using frozen tissue sections of equine fetal ovary (day 150 of pregnancy) and equine fetal testis (day 180 of pregnancy). In the fetal ovary, positive cells were seen throughout the interstitial area but did not show any particular localization. In the fetal testis, on the other hand, the antisense cRNA hybridized almost exclusively to the interstitial cells surrounding developing seminiferous cords and Sertoli cells within the cords. Positive signals were also detected in a limited number of the interstitial cells located away from the cords. These results suggest that in equine fetal gonads, inhibin and/or inhibin alpha-subunit related molecules such as the monomeric form are produced and these molecules may have a paracrine/autocrine role within the gonads.

Animals↗
PubMed1997

Cell-cell contact down-regulates expression of membrane type metalloproteinase-1 (MT1-MMP) in a mouse mammary gland epithelial cell line.

Membrane type matrix metalloproteinase (MT-MMP), which possesses a C-terminal transmembrane domain, is expressed on the cell membrane (Sato et al., 1994, Nature 370: 61-65). It was suspected, therefore, that the expression of MT-MMPs might be regulated by cell-cell interactions. We examined the patterns of MT1-MMP expression in a mouse mammary gland epithelial cell line, HC11, which is capable of responding to prolactin in vitro. HC11 cells form well-differentiated monolayer of cuboidal epithelium at confluence. During the log growth phase, cells which are well dispersed and seemingly migrating actively, or located at the periphery of small colonies, reacted strongly with an anti-MT1-MMP antibody, whereas no MT1-MMP immunoreactivity was detected in the cells which established cell-cell contact with adjacent cells. At confluence, the HC11 cells lost MT1-MMP immunoreactivity completely. Northern blot analysis revealed that MT1-MMP mRNA is present at a high level in HC11 cells during the log phase of growth. Although MT1-MMP immunoreactivity disappeared by the 1st day confluence was reached, the decline of MT1-MMP mRNA levels started only a few days later. The discrepancy in the timing of decrease of MT1-MMP protein and that of the transcripts suggests the presence of translational control mechanisms for MT1-MMP expression during cell-cell interaction.

Animals↗
PubMed1997

Immunohistochemical localization of group II phospholipase A2 in colonic mucosa of patients with inflammatory bowel disease.

OBJECTIVES AND METHODS: We previously reported the increased contents of group II phospholipase A2 (PLA2) in inflamed colonic mucosa of patients with Crohn's disease and ulcerative colitis (UC). However, the cellular source of the increased group II PLA2 has remained to be clarified. In the present study, we examined immunohistochemical localization of group II PLA2 in inflamed colonic mucosal biopsy samples obtained from eight patients with Crohn's disease, seven with UC, and in normal colonic mucosa of six control subjects without inflammatory bowel disease using a monoclonal antibody raised against human group II PLA2. RESULTS: and CONCLUSIONS: On immunoblot analysis using the antibody, a single band was detected on each lane containing colonic mucosal homogenates from a control subject, a Crohn's disease patient, or a UC patient at the same position as human group II PLA2 purified from ileal mucosa. The results of immunohistochemistry showed that colonic epithelial cells in inflamed mucosa obtained from all active Crohn's disease patients examined (n = 4) contained immunoreactive group II PLA2. In four of seven patients with active UC, group II PLA2 immunoreactivity was similarly positive. No apparent group II PLA2 immunoreactivity was detected in all colonic mucosal biopsy samples obtained from inactive Crohn's disease patients (n = 4) and control subjects. The present study supports the presence of group II PLA2 in colonic epithelial cells in actively inflamed mucosa of patients with Crohn's disease and UC, which suggests that epithelial cells may serve as the cellular source of increased group II PLA2 content in inflamed colonic mucosa of these patients.

Adolescent↗
PubMed1996

Human group II phospholipase A2 in normal and diseased intervertebral discs.

We measured calcium-dependent phospholipase A2 (PLA2) activity and immunoreactive group II PLA2 levels of 54 normal discs obtained from cadavers and 73 disc samples surgically obtained from patients with spinal disorders, including intervertebral disc herniations, spondylosis, and spondylolisthesis. Both cadaveric and surgical disc specimens contained about two-fold greater PLA2 activity than the ileal mucosa, one of the richest sources of group II PLA2. Discs of middle-aged cases had significantly higher activity than those of younger and elder cases. In cadaveric normal discs, calcium-dependent PLA2 activity was significantly higher in females than in males. Annulus fibrosus and nucleus pulposus contained the same PLA2 levels. In diseased disc, herniated fragments that had extruded or protruded out of the discs possessed lower activity than other parts of discs in the intervertebral space. Immunoreactive group II PLA2 levels of intervertebral discs closely correlated with PLA2 enzymatic activity. We purified a PLA2 from human intervertebral disc to homogeneity to further identify the isozymic nature of discal PLA2. Its NH2-terminal amino acid sequences and molecular weight were identical to those of human group II PLA2. Immunohistochemical analysis using a monoclonal anti-group II PLA2 antibody showed that in both annulus fibrosus and nucleus pulposus chondrocytes contained intense group II PLA2 immunoreactivity in their cytoplasm, and that the matrix contained no substantial immunoreactivity. These results suggest that group II PLA2 in chondrocytes has important physiological roles in discal ordinary metabolism, maintaining discal homeostasis.

Adolescent↗
PubMed1996

Genetic analysis of the chromosomal region encoding lysophospholipase L2 of Vibrio cholerae O1.

From the Tn5-inserted mutant library of Vibrio cholerae O1, we found a mutant, NF404, which lost the production of both hemolysin and cholera toxin (CT) even though the Tn5-insertion site was out side from the structural genes for hemolysin and CT. Cloning and sequencing analysis of the homologous region from the wild-type strain, revealed that the sequence spanning the coding region of an ORF1 nominated as lypA, encoding a 39.5 kDa protein. Deduced amino acid sequence of the lypA gene had 37.6% identity to the lysophospholipase L2 (EC 3.1.1.5) of Escherichia coli. In the downstream of lypA, a second open reading frame (ORF2) encoding an unknown protein with molecular weight of 19.9 kDa was found. Assaying the lysophospholipase L2 activity in the cell extract of E.coli harboring lypA in an expression vector showed clear increase in the enzymatic activity.

Amino Acid Sequence↗
PubMed1996

Pancreaticobiliary maljunction-associated pancreatitis: an experimental study on the activation of pancreatic phospholipase A2.

Congenital dilatation of the bile duct (CDBD), or choledochalcyst, is often complicated by recurrent pancreatitis. Reflux of pancreatic juice into the bile duct through pancreaticobiliary maljunction (PBM), an anomaly commonly associated with CDBD, and ensuing activation of pancreatic enzymes could be involved in the pathophysiologic mechanism of recurrent pancreatitis. A study was undertaken to follow the time course of the activity of phospholipase A2 (PLA2) in animal models of PBM. The assay procedures for PLA2 were evaluated, as were the conditions for separating the active enzyme from its inactive proenzyme (pro-PLA2) by immunoblotting. A rat model was designed according to Block's method with some modifications. The kinetics of prophospholipase A2 (proPLA2) activation in bile was examined by measuring PLA2 activity and by immunoblotting using anti-rat pancreatic enzyme antibody after separating PLA2 from its zymogen under nonreducing conditions. Experimental animals were divided into three groups: group 1 (PBM group) in which bile and pancreatic juice were mixed with occlusion of the papilla; group 2, in which the papilla and hepatic hillus were occluded without mixing the two juices; and group 3, in which simple laparotomy was done. In group 1 animals, pro-PLA2 in bile was activated to its active form. In group 2 animals, where proPLA2 was predominant, there was only slight elevation of PLA2 activity in bile. In group 1 an immunohistologic study demonstrated localization of PLA2 around necrotic foci in the pancreatic parenchyma. These results suggest the involvement of activated PLA2 in the pathogenesis of choledochal cystassociated pancreatitis.

Animals↗
PubMed1996

Analysis of neurotrophin-3 expression using the lacZ reporter gene suggests its local mode of neurotrophic activity.

We replaced the mouse neurotrophin-3 gene with the Escherichia coli-derived lacZ gene by means of homologous recombination. The mice with this mutation were useful models for studying the distribution of neurotrophin-3 expression in vivo, because visualization by 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside (X-Gal) staining was simple and rapid compared with in situ hybridization or immunohistochemistry. Whole-mount staining of mutant embryos at embryonic day 10 revealed that lacZ, a reporter for the neurotrophin-3 gene, was expressed in the mesencephalon, mandibular arch and somites. In the embryos at days 13-17, lacZ was markedly expressed in the peripheral target tissues of sensory and sympathetic neurons. We also found that spinal motor neurons and sensory neurons in trigeminal and dorsal root ganglia express lacZ. Some of these X-Gal staining regions overlapped with the sites expressing trkC, a high-affinity receptor for neurotrophin-3. The distribution of X-Gal staining in heterozygotes and homozygotes was similar to that of neurotrophin-3 messenger RNA detected by in situ hybridization. However, there was less lacZ expression in the dorsal root ganglia of homozygotes than neurotrophin-3 expression in wild-type mice. These results suggest that the neurotrophin-3 produced in the dorsal root ganglia also plays a role in the survival of some of the neurotrophin-3-positive neurons and that the local mode of neurotrophic activity is widely distributed.

Amino Acid Sequence↗
PubMed1996

Quantitative analysis of the spreading of the mouse trophoblast in vitro: a model for early invasion.

The outgrowth of the mouse blastocyst in culture represents an in vitro model of trophoblastic invasion. In the present study we analysed trophoblast spreading by time lapse video microscopy. Trophoblast spreading consists of (1) the migration and (2) the giant cell transformation of trophoblast cells, (3) the proliferation of ectoplacental cone (EPC) cells and (4) the subsequent transformation of EPC cells into the secondary giant cells. During migration, ruffling of the trophoblast cell membrane is followed by the formation of lamellipodia. The mean surface areas of the spreading trophoblast, measured in more than 100 cultured blastocysts, increased linearly from 48 to 96 h of culture, while the linear migratory speed at the periphery of the outgrowth declined as the time of culture advanced. The EPC cells increased in size approximately eightfold during the giant cell transformation. The apparent nuclear:cytoplasmic ratios, i.e., ratios between the size of nucleus and that of the cytoplasm, measured as the surface areas on the photomicrographs, of EPC cells increased between 40-46 h of culture, but a sharp decline in the ratio occurred between 50 and 51 h of culture, reflecting either the sudden and tremendous increase in the cellular volume and/or spreading of the cytoplasm. The rates of trophoblast spreading varied considerably among the blastocysts of different genetic constitution examined (ICR, C57BL/6, C3H/He and (B6 x C3)F1. It was fastest in blastocysts obtained from matings of males and females of (B6 x C3)F1, and slowest in the C57BL/6 embryos. The differences in the rate of outgrowth observed may not simply be ascribed to difference in the developmental speed of the early embryos, because the rate of outgrowth reached a plateau at about 96-120 h and no "catch-up' was observed by leaving the blastocysts in culture longer. Our results strongly suggest the possible presence of genetic regulatory mechanisms underlying trophoblast outgrowth; further analysis of the phenomenon may provide clues to understand the molecular mechanisms of trophoblastic invasion during the early phase of implantation, hopefully leading to improved success rates of in vitro fertilization-embryo transfer.

Animals↗
PubMed1996

Lysophospholipase L2 of Vibrio cholerae O1 affects cholera toxin production.

The implication in cholera toxin (CT) production of the newly identified gene, lypA, that encodes the lysophospholipase L2 of Vibrio cholerae, was investigated. Introduction of lypA into the V. cholerae O1 mutant (NF404), which has a Tn5-insertion in lypA and has lost CT as well as haemolysin production, restored the lysophospholipase activity and CT production but not the haemolytic activity. Inactivation of the lypA gene of the wild-type strain by chromosomal integration of a plasmid containing a portion of the lypA gene decreased the lysophospholipase L2 activity and the production of CT but not the haemolytic activity. Furthermore, constructed mutants of E1 Tor-biotype and Classical-biotype strains which have a defective lypA failed to produce CT and exhibited decreased enterotoxicity in the ligated rabbit ileal loop test. These results suggest that lypA is possibly required for the expression of CT and may play a role in pathogenicity of V. cholerae.

Animals↗
PubMed1996

Quantitative analysis of striped coat-color patterns in Large White-->Duroc chimeric pigs with special reference to the genetic control mechanisms of the dominant black-eyed white phenotype.

Coat colors of four chimeric pigs produced by the microinjection of dissociated blastomeres of (Landrace x Large White) blastocysts to the blastocyst cavity of Duroc x Duroc) blastocysts (Kashiwazaki et al., 1992) exhibited characteristic horizontal stripe-patterns. We carried out quantitative analysis of those patterns in order to derive information concerning the genetic regulatory mechanisms of the dominant black-eyed white phenotypes in the pig. In the four chimeras, the theoretical mean widths of the single-clone stripe calculated from the estimated widths of minimal recognizable stripe (MRS) (Tachi, 1988) were 2.1 +/- 0.1, 2.23 +/- 0.15, 1.89 +/- 0.06, and 1.93 +/- 0.28 cm respectively. The estimated number of single-clone stripes in the thoracico-lumbar region of those animals were 42.3, 40.7, 46.3, 44.2, and about twice the mean number of vertebrae in the same region (Duroc, 20 or 21; Large White 21 or 22). Furthermore, the mean length of thoracico-lumbar vertebrae in two of the chimeric pigs, as measured on X-ray radiographs, was approximately twice the mean single-clone stripe width. It was concluded that the stripe-patterns of the chimeric pigs probably represented the dermatome patterns of epidermis; and in the pig, a single somite was likely to be derived from the clones of two primordial cells, as originally proposed by Gearhart & Mintz (1972) in the mouse. It was suggested, furthermore, that in the Large White-->Duroc chimeric pigs, melanocytes that migrated into the region of skin formed by a Large White dermatome could not survive, thus creating a clearly demarcated white stripe. Possible involvement of KL or c-kit in the dominant black-eyed white phenotype of the pig is discussed.

Animals↗
PubMed1995

Group II phospholipase A2 activates mitogen-activated protein kinase in cultured rat mesangial cells.

Group II phospholipase A2 (PLA2) is a mediator of inflammation in various disease including glomerulonephritis. We recently found that urinary excretion of PLA2 was increased in patients with mesangial proliferative glomerulonephritis and that interleukin-1 (IL-1) enhanced platelet derived growth factor-stimulated mesangial cell proliferation through the action of group II PLA2 secreted in response to IL-1 stimuli. Here we report signal transducing mechanism through group II PLA2 in mesangial cells. Group II PLA2 (1-15 U/ml) rapidly activated mitogen-activated protein (MAP) kinase. IL-1 beta activated MAP kinase in two phases and the slow activation in the late phase, proceeding in parallel with increased group II PLA2 secretion elicited by IL-1 treatment, was inhibited by the specific antibody raised against group II PLA2. This suggests that the late phase activation of IL-1-induced MAP kinase was mediated, at least in part, by secreted group II PLA2.

Animals↗
PubMed1995

Neurotrophin-3 is expressed in the posterior lobe of mouse cerebellum, but does not affect the cerebellar development.

We replaced the neurotrophin-3 (NT-3) gene with Escherichia coli-derived lacZ via homologous recombination in embryonic stem (ES) cells and generated the mutant mice. Here we show the in vivo expression of NT-3 in the cerebellum during the postnatal period. A high level of lacZ expression was found in the granular layer of posterior lobe (lobules VII to X) in the postnatal NT-3(+/-) cerebellum. The expression in these regions was reduced with age. Although the Purkinje cells are considered to be a target of NT-3 and the NT-3(-/-) mice displayed severe moving disorders like ataxia, no histological abnormality was observed in their cerebellum. These findings suggest that the NT-3 expressed in the cerebellum gives some trophic effects primarily to the posterior lobe, however, the deficiency does not affect its development.

Animals↗