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Biomedical subjects

H Nojima

Publications and source records attributed to H Nojima.

At least 91 records · Page 5Linked to original sources
PubMed1995

Accelerated desensitization of nicotinic receptor channels and its dependence on extracellular calcium in isolated skeletal muscles of streptozotocin-diabetic mice.

1. To elucidate the influence of the diabetic state on desensitization of nicotinic acetylcholine (ACh) receptor channels, we investigated the time course of the decrease in amplitude of ACh potentials elicited by iontophoretic application to isolated diaphragm muscle of streptozotocin-diabetic mice. We also investigated time- and extracellular Ca(2+)-dependent changes in the channel opening frequency of ACh-activated channel currents and the involvement of protein kinases by use of the cell-attached patch clamp technique in single skeletal muscle cells. 2. When ACh potentials were evoked at 10 Hz, the decline in trains of ACh potentials was accelerated in the diabetic state. 3. The time-dependent decrease in the channel opening frequency of diabetic muscle cells was greatly accelerated compared with normal cells in 2.5 mM Ca2+ medium. 4. This accelerated decrease in channel opening frequency was restored by pretreatment with a protein kinase C inhibitor, staurosporine (10 nM) but neither a protein kinase A inhibitor, H-89 (3 microM) nor a calmodulin kinase II inhibitor, KN-62 (5 microM) were able to restore the fall in opening frequency. 5. These results demonstrate that in the diabetic state the desensitization of nicotinic ACh receptor channels may be greatly accelerated by activating protein kinase C, which is caused by an increase in the amount of available intracellular Ca2+.

Acetylcholine↗
PubMed1995

The G1/S boundary-specific enhancer of the rat cdc2 promoter.

Multiple species of G1 cyclins and cyclin-dependent kinases are induced sequentially during G1 phase, and the expression of cyclin A and cdc2 genes is subsequently induced at the G1/S boundary. To analyze the mechanism of cdc2 promoter activation, the 5'-flanking region of the rat cdc2 gene was isolated and its structural features were characterized. The highly conserved sequence between human and rat cdc2 genes is present in the basal promoter region from positions -183 to -122, which contains the E box, SpI, and E2F motifs. The expression of 5' sequential deletion derivatives of the promoter fused to luciferase cDNA in rat 3Y1 cells revealed the presence of the enhancer element. The presumed enhancer region was further analyzed by the introduction of base substitutions and by the formation of DNA-protein complexes with cell extracts prepared at various times during the G1-to-S-phase progression. These analyses revealed that the enhancer sequence, AAGTTACAAATA, located from -276 to -265, confers strong inducibility on the basal promoter at the G1/S boundary. The base substitutions introduced into the motifs of transcription factors indicated that the E2F motif is essential for the enhancer-dependent activation of the cdc2 promoter at the G1/S boundary. Electrophoretic mobility shift assays and DNase I footprinting showed that a factor which interacts with the enhancer element is induced late in G1 phase.

Animals↗
PubMed1995

Potentiating effects of beta-eudesmol-related cyclohexylidene derivatives on succinylcholine-induced neuromuscular block in isolated phrenic nerve-diaphragm muscles of normal and alloxan-diabetic mice.

beta-Eudesmol, a sesquiterpenoid alcohol contained in Atractylodes lancea, potentiates succinylcholine (SuCh)-induced neuromuscular blockade. The potentiating effect is greater in diabetic muscles than in normal ones. As a ligand for affinity chromatography to study the potentiating mechanism, we designed and synthesized newly beta-eudesmol-related cyclohexylidene derivatives (2-(3-hydroxy-3-methylbutyl)cyclohexylidene; KTE-13, 2-(3-hydroxy-3-methylbutyl)-4-cyclohexylidene carboxylic acid; KTE-32 and 4-tert-butoxycarbonyl-2-(3-hydroxy-3-methylbutyl) cyclohexylidene; KTE-33). We examined the potentiating effects of those compounds in phrenic nerve-diaphragm muscle preparations of normal and alloxan-diabetic mice. KTE-33 (100 microM) potentiated more greatly SuCh-induced neuromuscular blockade in diabetic muscles than in normal ones (the potentiating ratios in normal and diabetic muscles were 6.7 and 10.6, respectively), while KTE-13 (100 microM) and -32 (200 microM) potentiated weakly. These results suggest that the ester group in KTE-33 rather than a carboxyl group in KTE-32 is important in inducing the potentiation of SuCh-induced neuromuscular blockade in diabetic state.

Animals↗
PubMed1995

Depolarizing neuromuscular blocking action of coryneine derived from aconite root in isolated mouse phrenic nerve-diaphragm muscles.

The mode of the neuromuscular blocking action of coryneine (a quaternary ammonium derivative of dopamine) derived from aconite root was investigated in isolated phrenic nerve-diaphragm muscles and denervated diaphragm muscles of mice. Coryneine (20-150 microM) blocked the nerve-evoked twitch response without affecting the contraction evoked by electrical stimulation of the muscle. The blocking effect was reversed by neostigmine, a cholinesterase inhibitor. The electrical charge-response curve on depolarization produced by iontophoretically applied acetylcholine (ACh) at the endplate regions in normal muscles was shifted to the right on decreasing the maximal response by 40 microM coryneine. The double-reciprocal plot revealed that coryneine reduced the apparent affinity of ACh for its receptor on decreasing the maximal response. Coryneine (20 microM-2mM) itself depolarized the endplate membrane and this effect was reversibly suppressed by 1 and 5 microM pancuronium. Coryneine 30 microM-10mM) produced contractions of denervated muscles in a concentration-dependent manner and the effects were reduced by 70nM pancuronium. These results indicate that coryneine is a depolarizing agent and a mixed-type competitive and noncompetitive neuromuscular blocker.

Animals↗
PubMed1994

Hac1: a novel yeast bZIP protein binding to the CRE motif is a multicopy suppressor for cdc10 mutant of Schizosaccharomyces pombe.

We cloned by phenotypic complementation a novel Saccharomyces cerevisiae's multicopy suppressor of the Schizosaccharomyces pombe cdc10-129 mutant which we call HAC1, an acronym of 'homologous to ATF/CREB 1'. It encodes a bZIP (basic-leucine zipper) protein of 230 amino acids with close homology to the mammalian ATF/CREB transcription factor and gel-retardation assays showed that it binds specifically to the CRE motif. HAC1 is not essential for viability. However, the hac1 disruptant becomes caffeine sensitive, which is suppressed by multicopy expression of the yeast PDE2 (Phosphodiesterase 2) gene. Although the mRNA level of HAC1 is almost constitutive throughout the cell cycle, it fluctuates during meiosis. The upstream region of the HAC1 gene contains a T4C site, a URS (upstream repression sequence) and a TR (T-rich) box-like sequence, which reside upstream of many meiotic genes. These results suggest that HAC1 may also be one of the meiotic genes.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Isolation and characterization of cDNA clones specifically expressed in testicular germ cells.

We have cloned cDNAs involved in germ cell-specific expression. For this, a subtracted cDNA library was generated by subtracting cDNAs derived from supporting cells of mutant testis from wild-type testis cDNAs. Detailed analyses of mRNA expression revealed that the genes corresponding to the cloned cDNAs were exclusively expressed in testes and were developmentally controlled.

Actin Depolymerizing Factors↗
PubMed1994

Monoclonal antibody to beta 2 subunit of neuronal nicotinic receptor depresses the postjunctional non-contractile Ca2+ mobilization in the mouse diaphragm muscle.

The involvement of subtypes of nicotinic acetylcholine receptor (nAChR) in the postjunctional non-contractile Ca2+ mobilization was investigated in mouse diaphragm muscles treated with an anticholinesterase, using monoclonal antibodies (mAbs) to nAChR subunits. mAb 210 (specific for alpha 1 subunit of muscle nAChR) depressed contractile Ca2+ transients without affecting non-contractile Ca2+ transients. mAb 270 (specific for beta 2 subunit of neuronal nAChR) depressed only non-contractile Ca2+ transients. mAb 210 did not completely block the ACh-activated channel currents in flexor digitorum brevis muscle cells. The present findings indicate that the anti-beta 2 mAb 270-related subtype of nAChR may postsynaptically operate the non-contractile Ca2+ mobilization at the neuromuscular junction, suggesting the involvement of a subtype different from the usual muscle-type nAChR.

Animals↗
PubMed1994

SCR: novel human suppressors of cdc2/cdc13 mutants of Schizosaccharomyces pombe harbour motifs for RNA binding proteins.

By phenotypic complementation of the cdc2 and the cdc13 mutants of the fission yeast Schizosaccharomyces pombe, we have cloned two novel multicopy suppressors from a cDNA library of the human fibroblast. They encode homologous proteins containing two regions that are highly conserved among RNA binding proteins. We named them scr2 and scr3, the acronyms of the suppressor of cdc2 (cdc13) with RNA binding motif. They encode proteins of 403 (Scr2) and 407 (Scr3) amino acids. Western blot analysis showed that the amount of Cdc2 increased when either rat kidney fibroblasscr2 or scr3 was introduced into the cdc2-L7 and cdc13-117 mutant cells of S.pombe. No conspicuous alteration in the transcript level was detected as judged by Northern analysis. Considering that the cdc2+ suppresses the cdc13 mutant and vice versa, one of the possible interpretations of these result is that these genes suppress the mutants through the induction of the translation of Cdc2.

Amino Acid Sequence↗
PubMed1994

A zinc finger protein controls the onset of premeiotic DNA synthesis of fission yeast in a Mei2-independent cascade.

In the fission yeast Schizosaccharomyces pombe, meiosis is initiated by the action of Mei2 in a complex cascade activated following conjugation. We have isolated a new gene named rep1+ that is required for the initiation of premeiotic DNA synthesis. rep1+ encodes a 53 kDa protein with one zinc finger motif that is essential for function, and effectively rescues a null mutant of the res1+ gene but only partially a temperature-sensitive mutant of the cdc10+ gene, both of which are required for the onset of mitotic, as well as premeiotic, S phase. Deletion of rep1+ has no apparent effects on the mitotic cell cycle or conjugation, but blocks the initiation of premeiotic DNA synthesis. However, this defect is partially suppressed when rapidly growing cells are induced to conjugate, indicating that the rep1+ function is at least partly substituted by those of the genes controlling the 'start' of the mitotic cell cycle. The rep1 null mutant fails to induce the res2+ gene, a newly identified res1+ homolog cooperating with Cdc10 and acting for the onset of mitotic and premeiotic DNA synthesis, as well as for meiotic division. The rep1+ gene itself is induced moderately during nitrogen starvation but highly during conjugation, and this induction is dependent on both ste11+ and mating pheromones but independent of mei2+. Thus, rep1+ controls the initiation of premeiotic DNA synthesis via induction and/or activation of Res2 and some other essential factors in a cascade independent of Mei2.

Amino Acid Sequence↗
PubMed1994

Cdc25A is a novel phosphatase functioning early in the cell cycle.

The cdc25+ tyrosine phosphatase is a key mitotic inducer of the fission yeast Schizosaccharomyces pombe, controlling the timing of the initiation of mitosis. Mammals contain at least three cdc25+ homologues called cdc25A, cdc25B and cdc25C. In this study we investigate the biological function of cdc25A. Although very potent in rescuing the S.pombe cdc25 mutant, cdc25A is less structurally related to the S.pombe enzyme. Northern and Western blotting detection reveals that unlike cdc25B, cdc25C and cdc2, cdc25A is predominantly expressed in late G1. Moreover, immunodepletion of cdc25A in rat cells by microinjection of a specific antibody effectively blocks their cell cycle progression from G1 into the S phase, as determined by laser scanning single cell cytometry. These results indicate that cdc25A is not a mitotic regulator but a novel phosphatase that plays a crucial role in the start of the cell cycle. In view of its strong ability to activate cdc2 kinase and its specific expression in late G1, cdc2-related kinases functioning early in the cell cycle may be targets for this phosphatase.

Amino Acid Sequence↗
PubMed1994

The evidence of accelerative interaction between cAMP-dependent protein kinase and external calcium for the desensitization of nicotinic acetylcholine receptor channel in mouse skeletal muscle cells.

We investigated the effect of an activator (AA373) and an inhibitor (H-89) of cAMP-dependent protein kinase (PK-A) on the time-dependent decline in the opening frequency of acetylcholine (ACh)-activated channel currents using the cell-attached patch-clamp technique in adult mouse skeletal muscle cells. Although the time-dependent decline was independent of the presence of external Ca2+, it was accelerated by AA373 (1 mM) in 2.5 mM Ca2+ but not in Ca-free solution. The acceleration induced by AA373 was prevented by pretreatment with H-89 (3 microM). The accelerative effect of AA373 was also prevented by pretreatment with staurosporine (10 nM), a protein kinase C inhibitor, but not affected by pretreatment with KN-62 (5 microM), a calmodulin kinase II inhibitor. These results demonstrate that the desensitization of nicotinic ACh receptor channels was accelerated by PK-A in the presence of extracellular Ca2+. The accelerative effect of PK-A may be induced indirectly via the phosphorylation process that is influenced through the intracellular Ca2+ mobilization.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Id-related genes encoding helix-loop-helix proteins are required for G1 progression and are repressed in senescent human fibroblasts.

Three complete cDNA clones encoding Id-related helix-loop-helix (HLH) proteins lacking a basic region were isolated from a pcD2 cDNA expression library prepared from TIG-3 human diploid fibroblasts (HDF). Of these cDNAs (Id-1H, Id-1H', and Id-2H), two (Id-1H and Id-1H') appeared to be derived by alternative RNA splicing. Id-1H and Id-2H seem to be human homologues of mouse Id-1 and Id-2, respectively, and have potential to encode 154 and 135 amino acid proteins. The Id-1H and Id-2H mRNAs were barely detectable in quiescent early passage HDF; serum coordinately induced both mRNAs, with two peaks of expression, in early and late in G1. Antisense oligomers complementary to Id-1H and Id-2H mRNA prevented early passage HDF from entering the S phase of the cell cycle. The treatment of serum-stimulated early passage cells with the antisense Id-1H oligomer completely abolished Id-1H. In senescent cells, serum barely induced the Id-1H and Id-2H mRNAs, although the levels of c-myc expression induced were similar in early passage and senescent cells. The expression levels of these Id genes vary among immortal human cell lines. Both genes were overexpressed in VA4 SV40-transformed lung fibroblasts and EJ-1 bladder carcinoma cells, while these genes were expressed at a very low level in SVts8 cells derived from SV40 tsA-transformed TIG-3 cells. SVts8 cells may acquire some function redundant to Id proteins. HT1080 fibrosarcoma cells expressed the Id-1H gene but not the Id-2H gene, suggesting these Id genes may subserve redundant functions.

Alternative Splicing↗
PubMed1994

The structure-activity relationship between phenylene-polymethylene bis-ammonium derivatives and their neuromuscular blocking action on mouse phrenic nerve-diaphragm muscle.

The structure-activity relationship of phenylene-polymethylene bis-ammonium (PMBA) derivatives, C6H4[X(CH2)nR]2, on isolated mouse phrenic nerve-diaphragm muscle was investigated to obtain more potent and stable compounds for use as pharmacological tools to clarify the mechanism of succinylcholine (SuCh)-induced neuromuscular blockade. The neuromuscular blocking effect of all the PMBA derivatives was not reversed by neostigmine, a cholinesterase inhibitor. The potency of the neuromuscular blockade was in the order p- > o- > m- with respect to the side-chain substituents. A PMBA composed of X = CH2, n = 5 and R = N+Et3 was 5.9- and 23-fold more potent than SuCh and decamethonium, respectively. The derivatives of R = N+Et3 were observed to be more potent than those of R = N+Me3, N-Me-piperidinio and pyridinio derivatives. Replacement of X = CH2 with O, CHOH and CHOAc decreased the neuromuscular activity while replacement with S, SO and SO2 increased it. Introduction of NO2 into the phenylene ring increased the activity, while the introduction of an alcohol, aldehyde and ketone group decreased it. Removal of a carbonyl or ether group from SuCh decreased its activity, whereas the introduction of these into PMBA failed to increase it. We managed to synthesize unhydrolyzable neuromuscular blocking agents which are more potent than SuCh.

Animals↗
PubMed1994

Structural components of beta-eudesmol essential for its potentiating effect on succinylcholine-induced neuromuscular blockade in mice.

beta-Eudesmol, a sesquiterpenoid alcohol isolated from Atractylodes lancea rhizoma, potentiates the neuromuscular blocking effect of succinylcholine (SuCh). The potentiating effect is greater in diabetic muscles than in normal ones. To identify the structural components of beta-eudesmol contributing to this action, we examined the potentiating effect of newly synthesized tertiary alcohols related to beta-eudesmol in phrenic nerve-diaphragm muscle preparations of normal and alloxan-diabetic mice. Potentiating effects were exhibited by cyclohexylidene derivatives but not by cyclohexanone or cyclohexanol derivatives. The compound 2-(3-hydroxy-3-methylbutyl)cyclohexylidene exhibited a potentiating effect, but 3-(3-hydroxy-3-methylbutyl)cyclohexylidene did not. These results indicate that both the presence of an exo-methylene attached to a cyclohexane ring and the distance between the exo-methylene and the hydroxy group in beta-eudesmol are involved in the potentiating effect on SuCh-induced neuromuscular blockade.

Animals↗
PubMed1993

The putative phosphoinositide-specific phospholipase C gene, PLC1, of the yeast Saccharomyces cerevisiae is important for cell growth.

Using the polymerase chain reaction technique, we have isolated a gene that encodes a putative phosphoinositide-specific phospholipase C (PLC) in the yeast Saccharomyces cerevisiae. The nucleotide sequence indicates that the gene encodes a polypeptide of 869 amino acid residues with a calculated molecular mass of 101 kDa. This polypeptide has both the X and Y regions conserved among mammalian PLC-beta, -gamma, and -delta, and the structure is most similar to that of mammalian PLC-delta. This putative yeast PLC gene has been designated PLC1. Disruption of PLC1 results in slow growth or lethality for cells, depending on their genetic background and the medium, indicating that PLC1 is important for cell growth. Expression of rat PLC-delta 1 cDNA suppressed the growth defect of plc1 disruptants, strongly suggesting that PLC1 encodes PLC.

Amino Acid Sequence↗
PubMed1993

Differential activation of cyclin and cyclin-dependent kinase genes by adenovirus E1A12S cDNA product.

The differential activation of cyclin and cyclin-dependent kinase genes by the adenovirus E1A gene product (E1A) or serum factors was studied with a rat 3Y1 derivative cell line, g12-21, in which the E1A12S cDNA can be expressed in response to dexamethasone (dex). The induction of DNA synthesis in quiescent g12-21 cells occurred within 12 h after serum stimulation, while it occurred within 8 h after treatment with dex. The expression of cyclin D1 and E genes in the serum-stimulated cells was induced in mid G1 and mid to late G1, respectively, while that of the cyclin D1 gene was not induced and the induction of the cyclin E gene was shifted to the G1/S boundary in the dex-treated cells. The cdk2 gene was induced in late G1 and cdc2 and cyclin A genes at the G1/S boundary in both serum-stimulated and dex-treated cells. These results suggest that E1A skips cell cycle events which normally occur in early to mid G1 and may directly activate late-response genes. Analysis of the transcription factor E2F complexes formed in the promoter regions of cdc2 and dihydrofolate reductase genes showed that the amount of complexes formed is maximal at the G1/S boundary, but decreases in S phase when these genes are transcribed extensively.

Adenovirus E1A Proteins↗