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H Nojima

Publications and source records attributed to H Nojima.

At least 109 records · Page 6Linked to original sources
PubMed1993

Expression of the rat calmodulin gene II in the central nervous system: a 294-base promoter and 68-base leader segment mediates neuron-specific gene expression in transgenic mice.

Deletion analysis of the rat CaMII promoter demonstrated that the segment from -294 to +68 bases of CaMII was efficient as a promoter in NIH3T3 by transient assay. We developed transgenic mice carrying a fusion gene of this promoter segment and a beta-galactosidase reporter gene. This short CaMII promoter mediated the transgene expression in pyramidal cells of the cerebral neocortex, the pyriformcortex and the hippocampal regions CA1 to CA3, in granule cells of the dentate gyrus, in Purkinje cells of the cerebellum, and in neurons of the lateral vestibular nucleus of pons and the spinal cord of adult transgenic mice. The expression of endogenous CaMII was precisely analyzed by in situ hybridization in the nervous tissues. The localization of transgene expression was consistent with those of the endogenous CaMII in the adult transgenic mice. In the embryos at 13.5-15.5 days of gestation, the transgene was expressed in various neurons similarly to the endogenous CaMII but certain subtle differences were observed in the localization of expression. This short promoter of rat CaMII carried two sequence stretches highly conserved in the mouse, dog, chicken and Xenopus CaMII promoters. These conserved stretches may be involved in the observed neuron-specific expression of rat CaMII gene.

Animals↗
PubMed1993

Different modes of blockade by p-phenylene-polymethylene bis-ammonium compounds of the nicotinic acetylcholine receptor channel in skeletal muscle cells of mice.

The structure-activity relationships of five newly synthesized p-phenylene-polymethylene bis-ammonium (PMBA: C6H4[(CH2)nN+R3]2) compounds were investigated on the blockade of the nicotinic acetylcholine receptor (nAChR) channel. The cell-attached patch clamp configuration was used to measure single-channel currents in the endplate region of single flexor digitorum brevis muscle cells of adult mice. The bis-trimethylammonium compounds PMBA-1 (n = 4, R = CH3) and PMBA-23 (n = 6, R = CH3) produced channel opening above 0.3 microM and open channel blockade above 10 and 3 microM, respectively. The bis-triethylammonium compounds PMBA-43 (n = 1, R = CH2CH3) and PMBA-24 (n = 6, R = CH2CH3) showed no channel opening action, but PMBA-21 (n = 4, R = CH2CH3) opened channels weakly at 3 and 10 microM. These bis-triethylammonium compounds exerted different blocking actions on acetylcholine-activated channel currents. Above 10 microM PMBA-43, like tetraethylammonium, blocked open channels by decreasing the mean open time by rapid partial closing of the channel during the open-phase. At 10 microM, PMBA-21 blocked open and closed channels by decreasing the opening frequency by means of an irregular sequence of short pulses. At 0.3 microM, PMBA-24 blocked closed or nonconducting channels by decreasing the opening frequency without producing changes in mean open time. These results indicate that by lengthening the distance between two nitrogen atoms in the bis-triethylammonium group of PMBA, open channel blockade changes to closed channel blockade.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Isolation of flat revertants from human papillomavirus type 18 E6E7 transformed 3Y1 cells by transfection with a rat embryo fibroblast cDNA expression library.

A rat embryo fibroblast (REF) cDNA expression library was transfected into 3Y1 cells transformed by human papillomavirus type 18 E6 and E7 genes and 10 flat revertants were isolated. These revertants expressed the same levels of E6 and E7 mRNA as the parent cells, but had greatly reduced ability to form colonies in soft agar. Suppression of transformation was dominant in cell hybrids generated by fusing each revertant with the parental transformed cells. Furthermore, loss of transfected cDNA was observed in re-transformed cell hybrids derived from one flat revertant. Overexpression of the cDNA suppresses the colony-forming efficiency of the cells transformed by E6 and E7 genes.

Animals↗
PubMed1993

Cyclin G: a new mammalian cyclin with homology to fission yeast Cig1.

A new gene encoding a cyclin-like protein has been isolated from a rat fibroblast cDNA library by cross-hybridization with a mixture of c-src family proto-oncogene kinase domains as a probe. This putative cyclin, called cyclin G, contains a typical cyclin box at the N-terminus but no apparent 'destruction box' or 'PEST' sequence. Interestingly, in its C-terminus region, it has a sequence homologous with a tyrosine phosphorylation site of the epidermal growth factor receptor. Although this cyclin is phylogenetically related to HCS26 of Saccharomyces cerevisiae, it most resembles Cig1, a B-type cyclin, of Schizosaccharomyces pombe, which has been suggested to act at the G1/S phase of the cell cycle. Cyclin G mRNA is induced within 3 h after growth stimulation and remains elevated with no apparent cell cycle dependency, indicating its close association with growth stimuli but not with the cell cycle.

Amino Acid Sequence↗
PubMed1992

Hypotensive effect associated with a phospholipase C-delta 1 gene mutation in the spontaneously hypertensive rat.

To identify the genes responsible for blood pressure in the spontaneously hypertensive rat strain, we performed a cosegregation analysis between the genotype and blood pressure in a set of male F2 rats obtained by crossmating SHR with Wistar-Kyoto rats, a parental normotensive strain. Our investigation revealed that the phospholipase C-delta 1 polymorphism, which resulted in missense mutation, cosegregates with the lower blood pressure in SHR, and that PLC-delta 1 gene is located on chromosome 8. On the other hand, we found the lack of cosegregation between blood pressure and the nerve growth factor receptor gene, which is linked to a hypertensinogenic gene locus (denoted as BP/SP-1) on chromosome 10. We propose that PLC-delta 1 gene itself of closely linked gene on chromosome 8 is a new candidate with the hypotensive effect, and that BP-SP1 locus does not directly contribute to blood pressure elevation in original SHR.

Animals↗
PubMed1992

Elongation factor-1 alpha gene determines susceptibility to transformation.

Elongation factor-1 alpha (EF-1 alpha), an essential component of the eukaryotic translational apparatus, is a GTP-binding protein that catalyses the binding of aminoacyl-transfer RNAs to the ribosome. Expression of the EF-1 alpha gene decreases towards the end of the lifespans of mouse and human fibroblasts, but forced expression of EF-1 alpha prolongs the lifespan of Drosophila melanogaster. Eukaryotic initiation factor-4E, another component of the translational machinery, is mitogenic or oncogenic when constitutively expressed in some mammalian cells. Thus, components of the protein synthesis apparatus seem to be involved in the control of cell proliferation. Using expression cloning, we have isolated a complementary DNA clone from a BALB/c 3T3 mouse fibroblast variant, A31-I-13 (ref. 10), which specifies a factor determining the susceptibility of BALB/c3T3 to chemically and physically induced transformation. Here we report that the factor is EF-1 alpha and that its constitutive expression causes BALB/c 3T3 A31-I-1 (ref. 10), C3H10T1/2 (ref. 11) and Syrian hamster SHOK fibroblasts to become highly susceptible to transformation induced by 3-methylcholanthrene and ultraviolet light. EF-1 alpha messenger RNA is also constitutively expressed in a quiescent culture of the highly susceptible variant A31-I-13. We conclude that the removal of regulation of the expression of these components of the translational machinery may predispose cells to become more susceptible to malignant transformation.

3T3 Cells↗
PubMed1992

Blocking action of succinylcholine with beta-eudesmol on acetylcholine-activated channel activity at endplates of single muscle cells of adult mice.

The neuromuscular blockade produced by succinylcholine (SuCh) is potentiated by beta-eudesmol, a sesquiterpenoid alcohol isolated from Atractylodes lancea. beta-Eudesmol blocks the nicotinic acetylcholine receptor (nAChR) channel in both open and closed conformations. To clarify the mechanism of potentiation, we investigated the blocking effect of SuCh (0.1-10 microM) with beta-eudesmol on nAChR channel activity using the cell-attached patch clamp technique. Pretreatment with beta-eudesmol (20 microM) affected neither resting membrane potential nor ACh-activated channel activities. beta-Eudesmol produced the following changes in ACh-activated channel currents modulated by SuCh: reduction of SuCh (above 0.3 microM)-induced prolongation of channel open time and decrease in the frequency of channel opening in the presence of SuCh (above 3 microM). These results suggest that the potentiating effect of beta-eudesmol is postsynaptically due to accelerated desensitization of the nAChR, presumably resulting from preferential blocking action during the closed state of the receptor channel.

Acetylcholine↗
PubMed1992

A new cdc gene required for S phase entry of Schizosaccharomyces pombe encodes a protein similar to the cdc 10+ and SWI4 gene products.

We have isolated a new cell division cycle gene (res1+) required for entry into S phase, as a multicopy dual suppressor of the pat1 and cdc10 mutants of the fission yeast Schizosaccharomyces pombe. The res1+ gene specifies a 72 kDa protein with two copies of the cdc10/SWI6 motif. A disruptant of res1+ grows poorly at 30 degrees C with severe heat- and cold-sensitivities, and completely arrests in G1 at 36 degrees C and 23 degrees C. The arrested disruptant retains a full conjugation ability. In addition to the cdc10/SWI6 motif, Res1 and SWI4 proteins share a remarkable homology in their amino-terminal region, whereas Cdc10 and SWI6 do so in their carboxy-terminal region. Moreover, the amino-terminal region is essential for the function of Res1 as it is for the function of SWI4. Furthermore, analogous to the relationship of SWI4 to SWI6, the res1+ gene effectively rescues cdc10 mutants, but the cdc10+ gene cannot rescue the res1- phenotype. Thus, striking similarities exist in both structural and functional relationships between Res1 and SWI4, and between Cdc10 and SWI6. In view of the fact that SWI4 and SWI6 form a transcription factor complex and activate promoters containing the SWI4/SWI6 dependent cell-cycle box, Res1 might be a putative association partner of Cdc10 which appears to be involved at least in the activation of promoters containing a MluI cell-cycle box.

Amino Acid Sequence↗
PubMed1992

Indirect inhibitory effect of succinylcholine on acetylcholine-activated channel activities and its modulation by external Ca2+ in mouse skeletal muscles.

1. The effect of extracellular calcium on single acetylcholine (ACh)-activated channel activities when desensitizing concentrations of succinylcholine (SuCh) were applied to the surrounding endplate membrane was investigated by the cell-attached patch-clamp technique at endplates of single skeletal muscle (flexor digitorum brevis) fibres of adult mice. 2. Bath-applied SuCh (0.1-3 microM, in 2.5 mM Ca2+) increased in a concentration-dependent manner the mean open time of ACh-activated channel currents recorded at membrane potentials which cancelled the SuCh-induced depolarizations. 3. In the presence of 0.5 and 2.5 mM external Ca2+, SuCh (3 microM) applied outside the patch pipette prolonged the mean open time of ACh-activated channel currents in a time-dependent manner (by 45% and 52%, respectively), and simultaneously significantly decreased the single channel conductance (by 14% and 10%, respectively). These SuCh-induced effects did not occur in a nominally Ca(2+)-free extracellular medium. 4. Under the same conditions, SuCh (3 microM) augmented the time-dependent decline in the opening frequency of ACh-activated channel currents obtained in nominally Ca(2+)-free medium. 5. These results suggest that external calcium ions act to modulate nicotinic ACh receptor channel activity, and accelerate desensitization of the receptor.

Acetylcholine↗
PubMed1992

Raf-1 protein kinase is an integral component of the oncogenic signal cascade shared by epidermal growth factor and platelet-derived growth factor.

Our recent studies with cell mutants indicate that a cascade shared by the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) signals exists in NRK cells and mediates oncogenic signals induced by many oncogenes (A. Masuda, S. Kizaka-Kondoh, H. Miwatani, Y. Terada, H. Nojima, and H. Okayama, New Biol. 4:489-503, 1992). We have employed the antisense RNA technique to investigate possible involvement of Raf-1 kinase in this signal transduction cascade. NRK cell clones highly reduced in the Raf-1 production are generated by the expression of a c-raf-1 antisense RNA. They have no apparent growth defects and retain proper mitotic responses to growth factors but are refractory to transformation by EGF or PDGF plus transforming growth factor beta, v-erbB, v-fms, v-K-ras, v-mos, v-fos, v-src, simian virus 40 large T, and polyomavirus middle T but not by v-raf or adenovirus E1A. These results not only support our model for the oncogenic signal cascade but also lead to the conclusion that Raf-1 protein kinase is a downstream component of this oncogenic signal cascade shared by EGF and PDGF.

Blotting, Western↗
PubMed1992

Different modes of potentiation by beta-eudesmol, a main compound from Atractylodes lancea, depending on neuromuscular blocking actions of p-phenylene-polymethylene bis-ammonium derivatives in isolated phrenic nerve-diaphragm muscles of normal and alloxan-diabetic mice.

The essential moieties in p-phenylene-polymethylene bis-ammonium (PMBA) derivatives, C6H4[X(CH2)nN+R3]2, on the potentiating effects by beta-eudesmol, a main component of Atractylodes lancea, of their neuromuscular blockades were investigated in isolated phrenic nerve-diaphragm muscle preparations of normal and alloxan-diabetic mice. PMBA derivatives were separated into the following three groups based on the patterns of the potentiating effects: group I: PMBA-23 (n = 6, R = Me) and PMBA-24 (n = 6, R = Et); group II: PMBA-1 (n = 4, R = Me), PMBA-21 (n = 4, R = Et) and PMBA-2 (X = O, n = 3, R = Me); and group III: PMBA-31 (X = S, n = 3, R = Me), PMBA-3 (X = CO, n = 3, R = Me) and PMBA-4 (X = CHOH, n = 3, R = Me). The pretreatment with 80 microM beta-eudesmol for 60 min did not affect group I-induced neuromuscular blocking action, and it potentiated group II- and group III-induced ones. The potentiating effect of beta-eudesmol on group III was greater in diabetic muscles than in normal one and that on group II was to the same extent in both muscles. These results suggest that the four-methylene length of the side chains in normal muscles and the hydrophilic moieties adjacent to a phenylene ring in diabetic muscles are related to the potentiating effect by beta-eudesmol on PMBA derivatives.

Animals↗
PubMed1992

Signal transduction cascade shared by epidermal growth factor and platelet-derived growth factor is a major pathway for oncogenic transformation in NRK cells.

We have isolated two recessive, mutually complementary NRK cell mutants that are refractory to transformation by epidermal growth factor (EGF) and transforming growth factor-beta. Both mutants are defective in a signal transduction cascade shared by EGF and platelet-derived growth factor (PDGF). Analysis of the mutants suggests that transformation of NRK cells by the v-fms, v-erbB, activated erbB-2, v-ras, v-fos, v-mos, v-fes, v-src, SV40 large T, polyomavirus middle T, and human papillomavirus type 16 E6,E7 oncogenes is mediated by the EGF/PDGF signal cascade. The data also suggest that the EGF/PDGF cascade branches into mitogenic and oncogenic signals, the latter of which is required for soft agar growth and focus formation.

Animals↗
PubMed1992

Oncogenic signal-induced ability to enter S phase in the absence of anchorage is the mechanism for the growth of transformed NRK cells in soft agar.

Upon neoplastic transformation, cells acquire the ability to grow in soft agar. We investigated how this occurs by cell cycle analysis of a rat cell line NRK-49F and its transformation-deficient mutants. Rapidly growing NRK and mutants arrest in G1 when deprived of anchorage by suspending in methylcellulose. Addition of epidermal growth factor (EGF) together with transforming growth factor-beta (TGF-beta), which is highly oncogenic to NRK, induces the rapid progression of G1-arrested NRK cells into S phase. The time course and the extent of synchronization are very similar to the cell cycle progression in the presence of anchorage. EGF alone, which is highly mitogenic but only slightly oncogenic, fails to induce such progression. Both mutants remain arrested in G1. These data indicate that oncogenic signals confer on NRK the ability to enter S phase in the absence of anchorage and that this is the principal mechanism for its ability to grow in soft agar.

Agar↗
PubMed1991

Mechanism of the blocking action of beta-eudesmol on the nicotinic acetylcholine receptor channel in mouse skeletal muscles.

beta-Eudesmol, an uncharged alcohol contained in Atractylodes lancea, blocks the neuromuscular junction. Atractylodes lancea is prescribed in a traditional Chinese medicine and plays a main role for "alleviation of pain in skeletal muscle". By using the cell-attached patch-clamp or conventional intracellular technique, the site of action of beta-eudesmol on the nicotinic acetylcholine (ACh) receptor (nAChR) channel in skeletal muscle of the adult mouse, was investigated and compared with that of different types of blockers of the nicotinic ACh receptor channel (bupivacaine, chlorpromazine and phencyclidine). beta-Eudesmol (200 microM) depressed completely the nerve-evoked twitch tension and reduced the amplitude and quantal size of endplate potentials but did not alter either the quantal content, resting membrane potential or action potential. beta-Eudesmol (100-200 microM) decreased the amplitude of ACh potentials and accelerated the slow decay of depolarization, induced by the continuous application of ACh. beta-Eudesmol (40 microM) and phencyclidine (10 microM) decreased both the open time and opening frequency, without affecting the single channel conductance. Bupivacaine (10 microM) decreased only the open time. Chlorpromazine (10 microM) decreased only the opening frequency. These results indicate that the blocking effect of beta-eudesmol on nerve-evoked contraction, was due to blockade of nicotinic ACh receptor channels at the neuromuscular junction. Like phencyclidine, beta-eudesmol blocked the nicotinic ACh receptor channel in both the open and closed conformations, and accelerated the desensitization of the nicotinic ACh receptor.

Animals↗
PubMed1991

External Ca2+ dependence of acetylcholine- and succinylcholine-induced changes in channel conductance, open time and frequency at endplates of single muscle cells of adult mice.

The involvement of external Ca2+ in channel activity of nicotinic acetylcholine (nACh) receptors was investigated in the dissociated skeletal muscle fibers of adult mice. Single cells were prepared from flexor digitorum brevis muscles by treatment with collagenase and trypsin. Acetylcholine receptor-channel currents were recorded at endplates in the cell-attached mode, using the patch-clamp technique. Opening frequency, opening pattern and channel conductance were measured at different concentrations of ACh and succinylcholine (SuCh), using a patch pipette. Bath-applied ACh (30 microM) and SuCh (3-30 microM) decreased the ACh (1 microM)-induced channel conductance (SuCh was more potent than ACh). Large concentrations of both ACh (100-1000 microM) and SuCh (30-300 microM), which were applied via a patch pipette, also decreased channel conductance in a concentration-dependent manner. Increasing the concentration of calcium [Ca2+]0 in the patch pipette from 0 to 10 mM CaCl2 reduced ACh (1 microM)- and SuCh (1 microM)-activated single channel conductances. The increase in [Ca2+]0 prolonged the mean open times by 34% for ACh and by 22% for SuCh and decreased the channel opening frequency by 50% and 60%, respectively. These results demonstrate that ACh receptor-channel properties are dependent on [Ca2+]0 and that the ACh- and SuCh-induced decrease in channel currents may be also related to intracellular concentration of Ca2+. The effect of SuCh was greater than that produced by ACh.

Acetylcholine↗
PubMed1991

Developmental regulation of calmodulin gene expression in rat brain and skeletal muscle.

Three different calmodulin genes that encode the identical protein have been identified in the rat (Nojima, 1989); however, calmodulin gene expression at the various stages of tissue differentiation and maturation has not been previously determined. We have quantitated the content of mRNAs encoding calmodulin in the developing brain and skeletal muscle using RNA blot analysis with three specific cDNA probes. Our results show that five species of calmodulin mRNAs: 4.0 and 1.7 kb for CaM I, 1.4 kb for CaM II, and 2.3 and 0.8 kb for CaM III are detectable at all ages in the brain as well as in skeletal muscle but exhibit a tissue-specific developmental pattern of expression. The comparison of the temporal pattern of calmodulin gene expression with both mitotic activity, as demonstrated by cyclin A mRNA levels, and differentiation and maturation of specific brain or muscle regions is consistent with calmodulin involvement in development.

Animals↗
PubMed1991

Phospholipase C genes display restriction fragment length polymorphisms between the genomes of normotensive and hypertensive rats.

The genomic loci of four distinct phospholipase C genes (PLC-beta, PLC-gamma I, PLC-delta and PLC-gamma II) were examined for restriction fragment length polymorphisms (RFLPs) between the genomes of three normotensive [Sprague-Dawley, Donryu and Wistar-Kyoto (WKY)] and two closely related hypertensive [spontaneously hypertensive (SHR) and SHR stroke-prone (SHR-SP)] rat strains. The RFLPs observed between SHR and WKY were classified into three types. Type I RFLPs are those observed at 4.3 kilobase (kb) and 1.9 kb by AvaI digestion for PLC-gamma probe and at 1.9 kb by AccI digestion for PLC-beta probe, where RFLP banding patterns are conserved in two hypertensive (SHR and SHR-SP) and one normotensive (Sprague-Dawley) strains. Type II RFLPs are those observed by AccI, BamHI, EcoRI and PstI digestions for PLC-beta probe, where RFLP pattern observed in SHR is shared by one normotensive (Sprague-Dawley) strain but not by SHR-SP, WKY or Donryu rats. Type III RFLPs are those detected at 6.3 kb band by Bg/II digestion for PLC-beta probe and at 1.0 kb by BamHI digestion for PLC-gamma II probe, where RFLP pattern observed in SHR is shared by two normotensive rats other than WKY. No RFLP was found for PLC-gamma I probe after testing 13 restriction enzymes. Since PLC plays a pivotal role in regulating the intracellular calcium concentration and the intracellular signal transduction, these RFLPs may offer a valuable tool for the analysis of genomic predisposition for hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗