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H Tojo

Publications and source records attributed to H Tojo.

At least 55 records · Page 3Linked to original sources
PubMed1999

Efficient selection of transgenic mouse embryos using EGFP as a marker gene.

We have established a reliable method that uses the EGFP (Enhanced Green Fluorescent Protein) gene as a marker for selecting transgenic embryos from preimplantation embryos. Embryos that were subjected to the pronuclear microinjection of the CMV/beta-actin/EGFP fusion gene were cultured in vitro until they developed into the morulae- or blastocyst-stage. The expression of EGFP was easily observed by a fluorescent microscopy. There appeared to be no damage to the in vivo developmental ability of the embryos in response to the EGFP excitation light, which utilized an IB filter for a period of 30 min. Modified PCR analysis using Dpn I and Bal 31 digestion of the embryonic DNA showed that all of the embryos expressing EGFP in all their cells were transgenic, while more than half with mosaic expression of EGFP were not transgenic. Approximately 77% of pups born from the embryos that uniformly expressed the EGFP gene were transgenic, while 21.4% of pups from the embryos with mosaic expression were transgenics. The results showed that the use of EGFP as a marker is very useful and reliable for selecting transgenic embryos, and that it is important to transfer the embryos expressing EGFP in all their cells to obtain truly transgenic animals.

Actins↗
PubMed1999

Expression of milk protein genes is induced directly by exogenous STAT5 without prolactin-mediated signal transduction in transgenic mice.

STAT5 is a member of the family of STAT (signal transducer for activating transcription), which was isolated from nuclear extracts of the lactating gland of sheep. It was reported that, in vitro, STAT5 could be activated directly by prolactin (PRL) without PRL-mediated signal transduction. The present study was conducted to investigate above the possibility in vivo, using the transgenic mice overexpressing ovine STAT5 (oSTAT5) under the control of MMTV promoter. Three transgenic mouse lines that expressed the exogenously introduced oSTAT5 in their mammary glands were established. The expression levels of exogenous oSTAT5 were higher than those of endogenous STAT in the mammary glands of all of the three transgenic lines. Although the expression levels of endogenous milk protein genes, WAP, and beta-casein genes, were not correlated with oSTAT5 expression, the expression levels of WAP and beta-casein were induced by exogenous oSTAT5 in the transgenic mice. The present study demonstrated, at very least, that STAT5 expression can directly activate the expression of milk protein genes, and particularly the WAP gene without PRL-mediated signal transduction.

Animals↗
PubMed1999

Inactivation of p34cdc2 kinase by the accumulation of its phosphorylated forms in porcine oocytes matured and aged in vitro.

Culturing of matured porcine oocytes in vitro results in the enhancement of their cytoplasmic ability for oocyte activation (so-called ageing), although they are arrested at metaphase II. The enhanced ability for oocyte activation is related to decreased activity of the maturation promoting factor (MPF). In the present study we clarified the molecular mechanism of MPF inactivation during ageing, especially the changes in the phosphorylation status of p34cdc2, a catalytic subunit of MPF, compared with that in fertilised oocytes. The MPF activity decreased gradually when maturation culture was prolonged from 36 to 72 h, confirming the decreasing MPF activity in aged oocytes. The activity of 48 h matured oocytes also decreased after in vitro fertilisation. Immunoblotting of p34cdc2 with anti-PSTAIRE antibody revealed that the culturing of matured oocytes induces a gradual increase in pre-MPF, which is a p34cdc2 and cyclin B complex inactivated by phosphorylation at the inhibitory phosphorylation site of p34cdc2. In contrast, pre-MPF decreased after fertilisation, indicating the degradation of cyclin B. These results suggest that the molecular mechanisms of inactivation of MPF are different between oocyte activation and ageing, and that the mechanism during ageing might be based on the inhibitory phosphorylation of p34cdc2, whereas that of oocyte activation is based on the degradation of cyclin B.

Amino Acid Sequence↗
PubMed1999

High-linoleate and high-alpha-linolenate diets affect learning ability and natural behavior in SAMR1 mice.

Semipurified diets incorporating either perilla oil [high in alpha-linolenate, 18:3(n-3)] or safflower oil [high in linoleate, 18:2(n-6)] were fed to senescence-resistant SAMR1 mouse dams and their pups. Male offspring at 15 mo were examined using behavioral tests. In the open field test, locomotor activity during a 5-min period was significantly higher in the safflower oil group than in the perilla oil group. Observations of the circadian rhythm (48 h) of spontaneous motor activity indicated that the safflower oil group was more active than the perilla oil group during the first and second dark periods. The total number of responses to positive and negative stimuli was higher in the safflower oil group than in the perilla oil group in the light and dark discrimination learning test, but the correct response ratio was lower in the safflower oil group. The difference in the (n-6)/(n-3) ratios of the diets reflected the proportions of (n-6) polyunsaturated fatty acids, rather than those of (n-3) polyunsaturated fatty acids in the brain total fatty acids, and in the proportions of (n-6) and (n-3) polyunsaturated fatty acids in the total polyunsaturated fatty acids of the brain phospholipids. These results suggest that in SAMR1 mice, the dietary alpha-linolenate/linoleate balance affects the (n-6)/(n-3) ratio of brain phospholipids, and this may modify emotional reactivity and learning ability.

Aging↗
PubMed1999

Molecular cloning of cDNA for equine follistatin and its gene expression in the reproductive tissues of the mare.

A cDNA clone encoding equine follistatin was isolated from an equine ovarian cDNA library. Out of 1.2 x 10(5) independent clones screened, one positive clone was isolated and its cDNA sequence determined. The isolated clone, named EQ-FS-1, contained a complete open reading frame encoding 344 amino acid residues. The similarity of its deduced amino acid sequence to these of other mammalian species was greater than 95%. Although its expression level varied among the tissues examined, follistatin mRNA was detected in the equine uteroplacental tissues, follicles and corpora lutea by Northern blot analysis. In situ hybridization revealed that the expression of follistatin mRNA in the equine follicle was restricted exclusively to granulosa cells. When the expression pattern of follistatin mRNA in the equine uteroplacental tissues from mid- to late-pregnancy was examined, it was shown that its expression level tended to decrease after mid-pregnancy. These results suggest that follistatin acts in the reproductive tissues of the mare in maintaining pregnancy.

Amino Acid Sequence↗
PubMed1999

Reappraisal of potassium permanganate oxidation applied to Lowicryl K4M embedded tissues processed by high pressure freezing/freeze substitution, with special reference to differential staining of the zymogen granules of rat gastric chief cells.

The high pressure freezing/freeze substitution technique is known to yield a deep vitreous freezing of tissues. Combination of this technique with Lowicryl K4M embedding allows us histochemical studies of dynamic cellular processes with improved structural preservation. The disadvantage of Lowicryl K4M embedding is its poor electron density in electron microscopy. To address this problem, we examined the effects of KMnO4 oxidation applied to Lowicryl K4M embedded rat gastric glands processed by high pressure freezing. The KMnO4 oxidation-uranyl acetate-lead citrate sequence succeeded not only in contrast enhancement of cellular components, but also in differential staining of the zymogen granules of rat gastric chief cells. This technique could be applied to semi-thin sections of Lowicryl K4M embedded rat gastric glands. The KMnO4 oxidation-toluidine blue staining provided sufficient contrast with regard to the zymogen granules. Various experiments used in this study verified that the KMnO4 oxidation plays an essential role in the differential staining of the zymogen granules. Combined use of the KMnO4 oxidation with phospholipase A2-immunostaining demonstrated that gold labeling was localized to the zymogen granules without the loss of immunolabeling. Energy dispersive X-ray microanalysis revealed some manganese depositions on the zymogen granules. It is highly anticipated that the KMnO4 oxidation will become a useful tool for histochemical investigations combined with cryofixation/freeze substitution and low temperature embedding techniques.

Acrylic Resins↗
PubMed1999

[Assessment of oxygen transport in individual splanchnic organs using critical oxygen extraction].

Vulnerability in individual organs under ischemic stress can be assessed by critical oxygen extraction (critical O2ER). We measured critical O2ER in the whole-body, total splanchnic organs, liver and gut using a biphasic regression model of the oxygen delivery-consumption relationship in 7 dogs subjected to hemorrhagic stress under 1% isoflurane anesthesia. The difference in critical O2ER between the whole-body and total splanchnic organs was not significant, while hepatic critical O2ER was significantly larger than that of the gut (P < 0.05), 89.7 +/- 2.4% (mean +/- SD) and 66.1 +/- 5.1%, respectively. This finding indicated that the liver functions well under ischemic conditions due to its excellent ability to extract oxygen. The anatomical character of the sinusoid and the microcirculatory improving effect of isoflurane may affect the ability of oxygen extraction of the liver. The critical threshold of hepatic venous oxygen saturation as a marker of hepatic oxygen imbalance was considered to be below 10%.

Anesthesia, Inhalation↗
PubMed1998

Kainic acid-induced perirhinal cortical seizures in rats.

Seizures induced in rats by kainic acid microinjection into the perirhinal cortex were studied electrophysiologically and behaviorally and compared with known features of seizures following kainic acid injection into the amygdala. Unlike amygdalar seizures, perirhinal cortical seizures did not generalize to become limbic seizures but rather spread to sensorimotor cortex to become manifest as motor seizures. Perirhinal cortical seizures also required larger kainic acid doses for provocation and were briefer.

Amygdala↗
PubMed1998

Purification and characterization of a catalytic domain of rat intestinal phospholipase B/lipase associated with brush border membranes.

A brush border membrane-associated phospholipase B/lipase was solubilized from the distal two-thirds of rat small intestine by autolysis during storage at -35 degrees C over 1 month, and then the enzyme was purified to homogeneity and characterized enzymatically and structurally. The purified enzyme exhibited broad substrate specificity including esterase, phospholipase A2, lysophospholipase, and lipase activities. SDS-gel electrophoretic and reverse-phase high performance liquid chromatographic analyses demonstrated that a single enzyme catalyzes these activities. It preferred hydrolysis at the sn-2 position of diacylphospholipid and diacylglycerol without strict stereoselectivity, whereas it apparently exhibited no positional specificity toward triacylglycerol. Diisopropyl fluorophosphate, an irreversible inhibitor of serine esterases and lipases inhibited purified enzyme. When the position of enzyme on SDS-gel electrophoresis under the non-reducing conditions was determined by assaying the activity eluted from sliced gels, brush border membrane-associated enzyme corresponded to a approximately 150-kDa protein; autolysis gave a 35-kDa product, in agreement with the results of immunoblot analysis. The purified 35-kDa enzyme consisted of a 14-kDa peptide and a glycosylated 21-kDa peptide. Their NH2-terminal amino acid sequences were determined and found in the second repeat of 161-kDa phospholipase B/lipase with 4-fold tandem repeats of approximately 38 kDa each, which we cloned and sequenced in the accompanying paper (Takemori, H., Zolotaryov, F., Ting, L., Urbain, T., Komatsubara, T., Hatano, O., Okamoto, M., and Tojo, H. (1988) J. Biol. Chem. 273, 2222-2231). These results indicate that the purified enzyme is the catalytic domain derived from the second repeat of brush border membrane-associated phospholipase B/lipase.

Amino Acid Sequence↗
PubMed1998

Identification of functional domains of rat intestinal phospholipase B/lipase. Its cDNA cloning, expression, and tissue distribution.

A cDNA encoding a rat intestinal Ca(2+)-independent phospholipase B/lipase (PLB/LIP) was cloned from an ileac mucosa cDNA library using a probe amplified by polymerase chain reaction based on the purified enzyme's sequence. PLB/LIP consists of an NH2-terminal signal peptide, four tandem repeats of about 350 amino acids each, and a hydrophobic domain near the COOH terminus. The enzyme purified previously was found to be derived from the second repeat part. To examine the function of each domain, the full-length PLB/LIP, individual repeats, and a protein lacking the COOH-terminal hydrophobic stretch were expressed in COS-7 cells. The results showed that the second repeat, but not the other repeats, had all the activities (phospholipase A2, lysophospholipase, and lipase) found in the purified natural and expressed full-length enzymes, suggesting repeat 2 is a catalytic domain. The full-length enzyme was mainly present in membrane fractions and efficiently solubilized by treatment with 1% Triton X-100, but not with phosphatidylinositol-specific phospholipase C. Deletion of the COOH-terminal hydrophobic stretch caused the secretion of > 90% of synthesized PLB/LIP into culture media. These results suggest the hydrophobic domain is not replaced by a glycosylphosphatidylinositol anchor but serves as a membrane anchor directly. A message of the full-length PLB/LIP was abundantly expressed in the ileum and also, in a smaller, but significant amount, in the esophagus and testis. Immunohistochemistry showed that PLB/LIP is localized in brush border membranes of the absorptive cells, Paneth cells, and acrosomes of spermatid, suggesting its roles related and unrelated to intestinal digestion.

Amino Acid Sequence↗
PubMed1998

Expression and localization of membrane type matrix metalloproteinase-1 (MT1-MMP) in trophoblast cells of cultured mouse blastocysts and ectoplacental cones.

Membrane type matrix metalloproteinases (MT-MMPs) possess a C-terminal transmembrane domain and are expressed on the cell membrane. It was suspected, therefore, that MT1-MMP might play an important role in the trophoblastic invasion during implantation. The patterns of expression and localization of membrane type matrix metalloproteinase-1 (MT1-MMP) were examined immunocytochemically in cultured mouse blastocysts and excised extoplacental cones (EPCs). MT1-MMP immuno-reactivity was present in the giant trophoblast cells located at the periphery of the spreading trophoblast of cultured blastocysts and the outgrowths of cultured EPCs, but not in the densely packed trophoblast cells in both the blastocysts and the EPCs. It appears likely that MT1-MMP expressed on the edge of the invading trophoblast facilitates the trophoblastic invasion by cleaving proMMP-2, a known substrate of MT1-MMP, in the decidua. Immunohistochemical examination of early conceptuses confirmed that the trophoblast cells actively invading the endometrium in vivo express MT1-MMP strongly. It is suggested, furthermore, that the expression of MT1-MMP might be downregulated by cell-cell contact in mouse trophoblast cells, as in the mouse mammary epithelial cell line HC11.

Animals↗
PubMed1998

Functions of the N-glycans of rat leukemia inhibitory factor expressed in Chinese hamster ovary cells.

Leukemia inhibitory factor (LIF) is a pluripotent growth factor which acts in various cell systems. LIF is a glycoprotein containing six putative N-glycosylation sites. We established Chinese hamster ovary (CHO) cell lines to evaluate the biological roles of the N-glycosylation in rat LIF (rLIF). The bioactivity of rLIF was evaluated in two different bioassay systems using F9 and DA-1a cells. Employing site directed mutagenesis, six N-glycosylation-deficient LIF mutants were generated by replacing each asparagine residue (N) (at positions N9, N34, N63, N73, N96, and N116) by glutamine (Q). The resultant mutants showed similar activity in the bioassay using F9 cells. However, N34Q was about 3 times more potent than the wild-type rLIF in the assay using DA-1a cells. These findings suggest that the presence of N-glycan at N34 suppresses cell proliferation. In contrast, N63Q was about 2.5 times less potent than the wild-type rLIF indicating the pivotal role of N63 glycosylation for rLIF bioactivity. Taken together, our data suggest that the N-glycans of LIF play different roles depending on the cell line and that glycosylation of each specific residue contributes differently to its bioactivity.

Amidohydrolases↗
PubMed1998

Wilms' tumor suppressor gene (WT1) as a target gene of SRY function in a mouse ES cell line transfected with SRY.

With the aim of identifying the gene(s) located downstream from SRY, we transfected an ES cell line with XX karyotype, TMA-18, with a Sry DNA construct and established cell lines, TS18-1 and TS18-2, where the transfected Sry was expressed in the functional linear mRNA form. Among the five potential SRY-target genes examined, i.e., MIS, SF1, P450arom, Sox9 and WT1, only the expression of WT1 was induced de novo by the unscheduled expression of Sry in the transfected cell lines. No clear indication of Sry-induced enhancement of Sox9 expression was obtained in the present series of experiments. Function of a yet unidentified gene(s) located on the Y chromosome might be needed for the up-regulation of Sox 9 expression which takes place during the development of male gonads. Quantitative RT-PCR analysis of the patterns of WT1 expression in developing fetal gonads revealed that although both male and female fetal gonads express WT1, male gonads invariably expressed WT1 mRNA at higher levels than female ones after the Sry expression. Immunohistochemical analysis of the male fetal gonads between 10.5 and 13.5 dpc demonstrated the presence of strong WT1 immunoreactivity in Sertoli cells of the primordial testes. Suggestions were made in the past indicating that both SF1 and WT1 proteins might be active in a common pathway upstream from Sry. Our results showed that WT1 is located downstream, rather than upstream from Sry and behaves independently from SF1. Analysis using an appropriate in vitro system will be essential to understand the molecular mechanisms of SRY action within cells.

Animals↗
PubMed1997

Molecular cloning of cDNA encoding the c-kit receptor of Shiba goats and a novel alanine insertion specific to goats and sheep in the kinase insert region.

The complete open reading frame (ORF) of the c-kit cDNA was cloned from a cerebellar cDNA library of the Shiba goat (Capra hircus var Shiba) with the dominant black-eyed white phenotype. The analysis of the deduced amino acid sequence revealed the presence of a single amino acid insertion (alanine) in the kinase insert (KI) region. While the newly found alanine insertion is not correlated with the coat color phenotype of goats, it appears to be characteristic of the c-kit genes in goats and sheep. Although the biological significance of the insert remains to be investigated, its phylogenetically limited distribution will provide us with a useful and interesting tool to analyze the problems of evolution of sheep and goats in bovidae.

Alanine↗
PubMed1997

Group II phospholipase A2 as an autocrine growth factor mediating interleukin-1 action on mesangial cells.

The proliferation of mesangial cells plays a central role in the progression of glomerulonephritis. We studied the role of group II phospholipase A2 in interleukin-1-stimulated proliferation of mesangial cells. Cultured rat mesangial cells secreted 5.3 units group II phospholipase A2/24 h per 10(5) cells in response to stimulation of 200 U/ml of interleukin-1. Northern hybridization analysis showed that mRNA for group II phospholipase A2 was induced by exogenously added group II phospholipase A2 (15 U/ml) as well as interleukin-1. The pretreatment of quiescent mesangial cells with interleukin-1 augmented [3H]thymidine incorporation caused by platelet derived growth factor. Exogenous group II phospholipase A2 (5-36 U/ml) purified homogeneously from rat spleen also increased [3H]thymidine incorporation by platelet derived growth factor-stimulated mesangial cells in a dose dependent manner (36 U/ml phospholipase A2; 1.9-fold). The stimulatory effect of interleukin-1 on DNA synthesis of mesangial cells was specifically blunted by immunoglobulin raised against group II phospholipase A2. Group II phospholipase A2 (16 U/ml) amplified a platelet derived growth factor-stimulated increase in the mesangial cell number by 1.5-fold. Among the products of the phospholipase A2-catalyzed reaction, lysophospholipids including lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidic acid, but not fatty acids, mimicked the stimulatory effect of interleukin-1 and phospholipase A2. These results suggest that group II phospholipase A2 acts as a signaling molecule that mediates interleukin-1-induced growth of rat mesangial cells through yielding lysophospholipids.

Animals↗