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Biomedical subjects

H Nojima

Publications and source records attributed to H Nojima.

At least 127 records · Page 7Linked to original sources
PubMed1991

Phospholipase C-delta gene of the spontaneously hypertensive rat harbors point mutations causing amino acid substitutions in a catalytic domain.

This study was undertaken in order to investigate the newly discovered spontaneously hypertensive rat (SHR)-specific restriction fragment length polymorphism (RFLP) at the genomic locus of (poly)phosphoinositide-specific phospholipase C (PLC)-delta at a DNA sequence level. Our aim was to clone the PLC-delta complimentary DNA (cDNA) from SHR and analyse the genomic DNA obtained from two hypertensive rat strains such as SHR and its stroke-prone substrain (SHR-SP) and three normotensive rat strains such as Sprague-Dawley, Donryu and Wistar-Kyoto (WKY) by preparing an aortic cDNA library of SHR, hybridization cloning of PLC-delta cDNA and an analysis of the genomic DNA by polymerase chain reaction. By digesting with restriction enzyme XhoI, we discovered an RFLP band displaying only in SHR and SHR-SP, not in Sprague-Dawley, Donryu and WKY rats. DNA sequencing of PLC-delta cDNA cloned from an aortic cDNA library of SHR revealed a total of three SHR-specific point mutations, two of which resulted in amino acid substitutions. The first point mutation (A to T) was detected at the XhoI site, changing a threonine(ACG) to a serine(TCG), and the second point mutation (A to G) was discovered in the vicinity of the first one, changing an isoleucine(ATA) to a methionine(ATG). This is the first demonstration of the mutations in the SHR genome changing amino acid sequences. These amino acid substitutions, situated in the putative catalytic X domain of PLC-delta, may be the major cause of the augmented PLC activity observed in the SHR, possibly leading to hypertension-related phenonemoma such as abnormal calcium homeostasis and increased intracellular calcium ion concentrations.

Amino Acid Sequence↗
PubMed1991

The external Ca(2+)-dependence of acetylcholine-induced contracture in single innervated and denervated skeletal muscle cells in mice.

We examined morphological differences and compared the Ca(2+)-dependence between acetylcholine (ACh)-contractures in normal and denervated skeletal muscle cells. ACh (1 microM - 1 mM) contracted the normal cells into a jackknife-shape. The higher the concentration of external Ca2+, the greater the ratio of responding cells/total cells observed and the sharper the angles of the "jackknives". ACh contracted the denervated cells into a compression-form, and the contraction was dependent on the external Ca2+. These results indicate that ACh-contractures in both normal and denervated cells are external Ca(2+)-dependent.

Acetylcholine↗
PubMed1991

Possible role of the hormonal form of vitamin D3 in the granuloma-associated angiotensin-converting enzyme activity.

We studied effects of the hormonal form of vitamin D3 on the angiotensin-converting enzyme (ACE) activity of hepatic granulomas in mice infected with Schistosoma mansoni. During 7 to 11 weeks after infection, mice were given orally 0.1 or 1.0 microgram/kg of 1 alpha (OH)D3 or only medium solution every other day. Granulomatous livers were removed at 7, 9 and 11 weeks after infection, and ACE activity was measured in the granulomas isolated from each liver tissue using a fluorometric method. Oral administration of 0.1 or 1.0 microgram/kg/2 days of 1 alpha (OH)D3 for 4 weeks significantly enhanced ACE activity in the granuloma tissue. Since the DNA content relative to a unit weight of protein in the granulomas did not change with the 1 alpha (OH)D3 treatment, it is suggested that the elevated tissue ACE activity is due to an actual increase of the enzyme activity in each granuloma cell. The present observation may have relevance to sarcoid granulomas characterized by an increased tissue ACE activity, since macrophages from patients with sarcoidosis synthesize a biologically active hormonal form of vitamin D3. Namely hormonal form of vitamin D3 locally produced by macrophages is involved not only in systemic Ca++ metabolism but also in the stimulation of macrophages themselves to produce ACE in the granulomas.

Animals↗
PubMed1990

High efficiency transformation of Escherichia coli with plasmids.

We have re-evaluated the conditions for preparing competent Escherichia coli cells and established a simple and efficient method (SEM) for plasmid transfection. Cells (DH5, JM109 and HB101) prepared by SEM are extremely competent for transformation (1-3 x 10(9) cfu/microgram of pBR322 DNA), and can be stored in liquid nitrogen for at least 40 days without loss of competence. Unlike electroporation, transformation using these competent cells is affected minimally by salts in DNA preparation. These competent cells are particularly useful for construction of high-complexity cDNA libraries with a minimum expenditure of mRNA.

Buffers↗
PubMed1990

Cloning of a DNA fragment displaying restriction fragment length polymorphisms between the genomes of spontaneously hypertensive and Wistar-Kyoto rats.

A 3 kilobase (kb) EcoRI fragment cloned from the genome of the spontaneously hypertensive rat (SHR) displayed restriction fragment length polymorphism (RFLP) compared with the genome of the Wistar-Kyoto rat (WKY) when total genomic Southern blot analysis was performed for two restriction enzymes, PstI and PvuII. Sequencing of the DNA fragment cloned from genomic SHR and WKY libraries revealed that this 3 kb EcoRI fragment harbours three point mutations. Two of them (C to T and A to T) are situated in the middle of the restriction sites for PstI and PvuII, thus disrupting the recognition sites for these enzymes in the SHR genome. Southern blot analysis using total complementary (c) DNA obtained from cDNA libraries of aortic smooth muscle cells from SHR and a whole WKY kidney, with this 3 kb EcoRI fragment as a probe, showed polymorphic bands suggesting that these point mutations are reflected in the sequences of messenger (m) RNA transcribed from the gene encoded in this 3 kb fragment. Detection of two bands by a Northern blot analysis for RNA from various SHR tissues indicates that this 3 kb fragment is actively transcribed in vivo.

Animals↗
PubMed1990

Role of molecular genetics in the understanding of the pathogenesis of hypertension.

Possible roles for molecular genetical approaches to understand the pathogenesis of hypertension are briefly reviewed. To elucidate observed alterations in hypertensive subjects at both molecular and systemic levels in a comprehensive way, a model (a working hypothesis) for the molecular pathogenesis of hypertension is proposed. Prospects for identifying restriction fragment length polymorphisms observed in hypertensive rats but not in normotensive rats are also discussed.

Animals↗
PubMed1989

Sulphotransferase-mediated activation of the carcinogen 5-hydroxymethyl-chrysene. Species and sex differences in tissue distribution of the enzyme activity and a possible participation of hydroxysteroid sulphotransferases.

Sulphation of the carcinogen 5-hydroxymethyl-chrysene (5-HCR) to the active metabolite 5-HCR sulphate occurred at significant rates in all of hepatic cytosols prepared from the male and female experimental animals, rats, mice, guinea-pigs and hamsters. The 5-HCR-sulphating activity was also found in kidney cytosols of all the experimental animals used, while their activities were much less than those of hepatic cytosols. In the male mice, the enzyme activity of testis was higher than any other examined tissue. Small intestine and adrenal of male and female guinea-pigs had relatively high enzyme activities. Small enzyme activities were also found in a variety of extrahepatic tissues of some of these animals. Marked species and sex differences (female much greater than male in the rat and mouse) were observed in the hepatic enzyme activity. In the female rat liver which showed the highest 5-HCR-sulphating activity among the examined tissues of all the animals, a typical hydroxysteroid sulphotransferase inhibitor, dehydroepiandrosterone (DHA) sulphate (1 mM), potently and competitively inhibited the sulphation of 5-HCR as well as that of DHA, a typical substrate for hydroxysteroid sulphotransferases. On the contrary, the phenol sulphotransferase inhibitors, pentachlorophenol and 2,6-dichloro-4-nitrophenol, had only a little effect on these enzyme activities even at a concentration of 50 microM that showed a potent inhibition of the phenol sulphotransferase activity. These results suggest that 5-HCR be sulphated in the female rat liver by hydroxysteroid sulphotransferases, but not by phenol sulphotransferases.

Animals↗
PubMed1989

Structural organization of multiple rat calmodulin genes.

Elsewhere, we have reported the structure of a rat calmodulin gene and two distinct rat calmodulin cDNAs, pRCM1 and pRCM3. Here, I report the cloning and sequencing of the third calmodulin cDNA (pRCM4) and two additional rat calmodulin genes. The original calmodulin gene is named CaM I (pRCM1) and the newly discovered calmodulin genes are named CaM II (pRCM3) and CaM III (pRCM4). CaM II spans about 10 x 10(3) base-pairs and consisted of five exons, while CaM III spans about 7.2 x 10(3) base-pairs and consisted of six exons. One of the introns (intron 3) observed in CaM I and CaM III is lost in CaM II. Otherwise, the intron/exon organization of these genes is exactly the same. In all calmodulin genes, the first intron separates the initiation codon (ATG) from the coding region of the protein. Northern blotting showed that CaM I is transcribed primarily into 1.7 x 10(3) base-pair mRNA in various tissues examined and 4.0 x 10(3) base-pair mRNA mainly in skeletal muscle, CaM II is transcribed into 1.4 x 10(3) base-pair mRNA almost exclusively in brain and CaM III is transcribed predominantly into 2.3 x 10(3) base-pair mRNA and faintly into 1.0 x 10(3) base-pair mRNA mainly in skeletal muscle and brain. DNA sequences in the promoter-regulator regions of these genes are partly homologous but essentially distinct and possess a number of direct repeats, palindromes and feasible stem-loop structures. Together with these, I report here the structures of the third and fourth calmodulin retropseudogenes.

Animals↗
PubMed1989

Differential calmodulin gene expression in fetal, adult, and neoplastic tissues of rodents.

Differential expression during rat development of three genes for calmodulin (CaM I-III) was examined in amnion, decidua, embryo, liver, placenta, parietal and visceral yolk sacs and uterus. CaMI expression was constant except for increasing activity in VYS during gestation. CaMII expression increased in all tissues except for a decrease in embryo. CaMIII did not change dramatically. Differential expression was also found in chemically or virally induced rat tumors, and in metastatic lung nodules of mouse mammary carcinoma. CaMII was the major gene expressed in all these neoplastic tissues.

Amnion↗
PubMed1989

Structural organization of calmodulin genes in the rat genome.

In summary, we present a list of phage clones we have obtained from rat genomic libraries (from lamba SC1 to lambda SC31, lambda WC1 and lambda WC40) together with cDNA clones we have obtained from a rat brain cDNA library (PRCM1,5,3 and 4). pRCM5 corresponds to 4.0 kb mRNA species observed primarily in skeletal muscle. These clones can be classified into three groups. They belong to three bona fide calmodulin genes with five to six exons called CaM I, CaM II and CaM III and four intronless retropseudogenes, one derived from CaM I and three derived from CaM II. We have not obtained retropseudogenes for CaM III so far. These three bona fide genes are transcribed into multiple sized mRNA species in a tissue-specific manner, that is, CaM I is ubiquitous, CaM II is transcribed mainly in brain and CaM III is transcribed primarily in brain and skeletal muscle. Four retropseudogenes do not appear to be transcribed. They are probably relics of inactivated genes. The physiological meanings of multiple calmodulin mRNA species and mechanisms of transcriptional regulation of these three bona fide genes will be the main subjects of our future experiments.

Amino Acid Sequence↗
PubMed1989

The Na,K-ATPase alpha 2 subunit gene displays restriction fragment length polymorphisms between the genomes of normotensive and hypertensive rats.

The Na,K-adenosine triphosphatase (ATPase) alpha 2 subunit gene was found to display restriction fragment length polymorphisms (RFLPs) between the genomes of normotensive and hypertensive rats when digested with the restriction enzymes Bgl II and Hind III. In normotensive rats, we tested the spontaneously hypertensive rat (SHR) and its substrain, the stroke-prone spontaneously hypertensive rat (SHR-SP). Rat (SD) complementary (c) DNA encoding the alpha 2 subunit of Na,K-ATPase was used as a probe. When the probe was dissected these RFLPs were found to occur in the vicinity of the genomic locus encoding the middle part of the messenger (m) RNA for the alpha 2 subunit of Na,K-ATPase. A Northern blot analysis indicated that these RFLPs did not influence the alpha 2 subunit with regard to either size or amount of mRNA.

Animals↗
PubMed1989

The effects of single and repeated inoculations of various larval doses on Strongyloides ratti burden and distribution in rats.

The changes in worm burden, distribution, length, and fecundity after and during single and repeated inoculations of 10, 50, or 500 larvae of Strongyloides ratti were examined in rats. Worm burden after a single inoculation of a higher larval dose reduced rapidly. Repeated inoculations of lower larval doses at weekly intervals led to a delayed peak and slower reduction of worm burden; the repeated inoculations of 10 larvae did not induce worm expulsion for at least 7 wk. In repeated inoculations at 3-wk intervals, a primary inoculation of 500 larvae induced strong resistance to reinfection at week 3, whereas no resistance was induced until week 6 in rats receiving repeated inoculations of 10 or 50 larvae. Similar dose-dependent reductions in worm length and fecundity were observed in single and repeated inoculations, and the reductions began earlier than worm expulsion. Intestinal migration of worms from the upper small intestine to the large intestine was observed during the course of single and repeated inoculations. Earlier and clearer migration was observed in rats receiving higher doses. These findings indicate that in S. ratti infection, the changes of worm burden, distribution, length, and fecundity are dependent on the inoculated larval dose.

Animals↗
PubMed1988

Inhibition of post-decapitation convulsions in the rat by dibenzothiepin neuroleptics via alpha 1-adrenoceptor blockade.

The mechanisms involved in inhibitory effects of isofloxythepin, a newly synthesized dibenzothiepin neuroleptic, on post-decapitation convulsions were studied in rats. Isofloxythepin (0.05-2.0 mg/kg s.c.) inhibited post-decapitation convulsions in a dose-dependent manner as shown by the decrease in the incidence and the shortening of the duration of convulsions. The convulsions were also inhibited by oxyprothepin, zotepine or chlorpromazine but not by haloperidol. Prazosin and bunazosin, both alpha 1-adrenoceptor antagonists, suppressed the post-decapitation convulsions but a non-selective alpha 2-adrenoceptor agonist, tolazoline, was without effect. The convulsions were inhibited dose dependently by clonidine, an alpha 2-adrenoceptor agonist, but were prolonged in duration by yohimbine, an alpha 2-adrenoceptor antagonist. Yohimbine antagonized the inhibitory effects of isofloxythepin, prazosin and clonidine. The noradrenaline-induced contraction of rat vas deferens was inhibited by isofloxythepin, prazosin or chlorpromazine. Isofloxythepin bound to alpha 1-receptors as did chlorpromazine in the rat brain cortex. The results imply that post-decapitation convulsions seem to be inhibited by a block of postsynaptic alpha 1-adrenoceptors, enhanced by a block of presynaptic alpha 2-adrenoceptors and reduced by isofloxythepin via the blocking of postsynaptic alpha 1-adrenoceptors. The convulsions thus could serve as a good model for studying the actions of drugs on the central nervous system alpha-adrenoceptors.

Adrenergic alpha-Agonists↗
PubMed1988

Diabetic state-induced modification of resting membrane potential and conductance in diaphragm muscle of alloxan and diabetic KK-CAy mice.

The electrical properties of skeletal muscle membranes were investigated in genetically diabetic KK-CAy mice and alloxan-induced diabetic ddY mice. Using isolated phrenic nerve-diaphragm muscle or sciatic nerve-gastrocnemius muscle in situ preparations, nerve-stimulated twitch tensions (the maximal value) were obtained at lower voltage pulse in diabetic KK-CAy mice than in normal ddY mice. The diabetic state reduced resting membrane potentials (1.7-4.0 mV) and resting membrane conductance (0.37-0.44 mu siemen), decreased the amplitude (3.8-3.9 mV) and overshoot (4.5 mV) of directly induced-action potential, and prolonged action potential duration. In the diabetic state, resting membrane conductance was multiply-correlated with blood glucose level and resting membrane potential. In alloxan-induced diabetic mice, resting membrane potentials were significantly multiply-correlated with the weeks elapsed after alloxan injection and blood glucose level (p less than 0.01). Since the reduction of resting membrane potential correlated with the weeks, changes in resting membrane potential may be involved in the decrease in insulin-like growth factor action. The reduction of resting membrane conductance was correlated with the increase in blood glucose.

Action Potentials↗