[Cell cycle regulation and Ran/TC4 GTPase].
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Amino Acid Sequence↗
Biomedical subjects
T Nishimoto
Publications and source records attributed to T Nishimoto.
Enhancement of muscarinic receptor-coupled phosphatidyl inositol hydrolysis in diabetic bladder.
We previously have shown an increase in muscarinic receptor density in streptozotocin (STZ)-induced diabetic and sucrose-fed diuretic rat detrusor that correlates with an increase in the contractile response to muscarinic agonist (J Pharmacol Exp Ther 248:81, 1989; Diabetes 40: 265, 1991). To investigate the signal transduction pathway involved in this altered functional response, we examined muscarinic receptor-coupled phosphatidylinositol metabolism in STZ-diabetic, sucrose-fed diuretic and age-matched control rat bladders. [3H]myo-inositol uptake was similar in all groups, but incorporation of myo-inositol into phosphatidylinositol (PI) was significantly increased in the diabetic bladder compared to the sucrose-fed and control rat bladders. Carbachol-induced increase in inositol phosphate (IPs) production was higher in the diabetic bladder than in bladders from control and sucrose-fed animals although the EC50 values were similar for all groups. Enhanced inositol phosphate production after muscarinic agonist stimulation may be due not only to the upregulation of muscarinic receptors but also the increased incorporation of myo-inositol into PI in the STZ-induced diabetic bladder.
Animals↗
dad-1, an endogenous programmed cell death suppressor in Caenorhabditis elegans and vertebrates.
Programmed cell death (apoptosis) is a normally occurring process used to eliminate unnecessary or potentially harmful cells in multicellular organisms. Recent studies demonstrate that the molecular control of this process is conserved phylogenetically in animals. The dad-1 gene, which encodes a novel 113 amino acid protein, was originally identified in a mutant hamster cell line (tsBN7) that undergoes apoptosis at restrictive temperature. We have identified a dad-1 homologue in Caenorhabditis elegans (Ce-dad-1) whose predicted product is > 60% identical to vertebrate DAD-1. A search of the sequence databases indicated that DAD-1-like proteins are also expressed in two plant species. Expression of either human dad-1 or Ce-dad-1 under control of a C.elegans heat-shock-inducible promoter resulted in a reduction in the number of programmed cell death corpses visible in C.elegans embryos. Extra surviving cells were present in these animals, indicating that both the human and C.elegans dad-1 genes can suppress developmentally programmed cell death. Ce-dad-1 was found to rescue mutant tsBN7 hamster cells from apoptotic death as efficiently as the vertebrate genes. These results suggest that dad-1, which is necessary for cell survival in a mammalian cell line, is sufficient to suppress some programmed cell death in C.elegans.
Amino Acid Sequence↗
RanBP1, a Ras-like nuclear G protein binding to Ran/TC4, inhibits RCC1 via Ran/TC4.
A human protein that is 92% identical and 97% homologous at the amino acid level to RanBP1 from mouse was identified by the two-hybrid method, using two types of target cDNAs fused to sequences encoding the GAL4 DNA-binding domain. The target cDNAs encoded the human Ran/TC4 and human RCC1 proteins, respectively. An in vitro binding experiment showed that RanBP1 binds to RCC1 with the aid of Ran. Partially purified, GST-fused RanBP1 inhibited RCC1-stimulated guanine nucleotide release from Ran in vitro. Consistent with this in vitro finding, overproduction of human RanBP1 was detrimental to growth of tsBN2, a temperature-sensitive BHK21 hamster cell line defective in the RCC1 gene, and inhibited the growth of the Saccharomyces cerevisiae rcc1 mutants prp20, mtr1 and srm1. The specific effect of RanBP1 on rcc1- cells was confirmed by the finding that overproduction of RanBP1 induces significant levels of expression of a FUS1-lacZ gene and an increase in mating efficiencies in a ste3, pheromone receptor-deficient yeast mutant. This phenotype is similar to the srm1, a mutant isolated as a suppressor that restores mating to receptorless mutants. These findings indicate that RanBP1 negatively regulates RCC1.
Amino Acid Sequence↗
Expression of the genes for insecticidal crystal proteins in Bacillus thuringiensis: cryIVA, not cryIVB, is transcribed by RNA polymerase containing sigma H and that containing sigma E.
To investigate the mechanism of transcriptional regulation of cryIVA and cryIVB, encoding 130-kDa dipteran-active crystal proteins, in Bacillus thuringiensis subsp. israelensis, we introduced each gene into several sporulation mutants of Bacillus subtilis. A spoIIG mutation, the wild-type gene of which encodes sigma E precursor, completely blocked the cryIVB transcription. In contrast, low but detectable transcription of cryIVA was observed in the spoIIG mutant. In the wild-type B. subtilis, no transcription of cryIVB was detected before T2 (2 h after the onset of stationary phase), while the cryIVA transcription started at the late exponential phase at low levels. Furthermore, in a wild-type strain of B. thuringiensis subsp. israelensis, transcription of cryIVA began earlier than that of genes encoding other crystal components, cryIVB and cytA. A consensus sequence recognized by an RNA polymerase containing sigma H of B. subtilis was found upstream of the transcription start point of cryIVA, which overlapped with that recognized by sigma E.
Bacillus subtilis↗
Cloning and characterization of a Bacillus thuringiensis homolog of the spoIIID gene from Bacillus subtilis.
The SpoIIID protein of Bacillus subtilis (Bs) is a small DNA-binding protein that is essential for gene expression of the mother cell compartment during sporulation. We have cloned a DNA fragment from Bacillus thuringiensis (Bt) that showed a specific hybridization with the Bs spoIIID gene. Sequence analysis found an open reading frame encoding 90 amino acids (aa), which are 89% identical to the deduced aa sequence of Bs spoIIID. Upstream from the transcription start point (tsp), a nucleotide sequence highly homologous to the consensus sequence motif for the sigma 35-recognized promoters was found. Northern blot analysis has indicated that the expression of the gene is induced only at the midsporulation stage, and that the gene constitutes an operon with a downstream gene, mreB. The Bs strain carrying the spoIIID delta erm or spoIIID83 mutation completely restored sporulation ability upon introduction of the spoIIID homologous gene from Bt. These results strongly suggest that the gene we have cloned is a Bt homolog of spoIIID.
Amino Acid Sequence↗
Apoptosis is induced in BHK cells by the tsBN462/13 mutation in the CCG1/TAFII250 subunit of the TFIID basal transcription factor.
A temperature-sensitive (ts) mutant of the BHK21 cell line derived from golden hamsters, tsBN462 has a mutation in the gene encoding the largest subunit of the TFIID complex, TAFII250/p230/CCG1, and arrests in the G1 phase at the nonpermissive temperature, 39.5 degrees C. We found that tsBN462 cells underwent apoptosis following growth arrest at 39.5 degrees C, suggesting a role for CCG1 as a repressor of apoptosis. By electron microscopic observation, tsBN462 cells at 39.5 degrees C showed characteristic features of apoptosis. Apoptosis was not suppressed by expression of Bc1-2 or the adenovirus E1B 19 kDa protein. Cell death was suppressed completely by expression of wild-type CCG1 and partially by wild-type p53, a growth suppressor protein. Cell cycle arrest induced by p53 may help survival of tsBN462 cells at 39.5 degrees C. Apoptosis was accelerated in SV40 large T antigen-transformed tsBN462 cells at 39.5 degrees C where SV40 large T antigen formed a complex with p53, implying that the apoptosis of tsBN462 cells at 39.5 degrees C occurred in a p53-independent manner. Our results suggest that CCG1/TAFII250 is required for the expression of factors regulating apoptosis.
Adenovirus E1B Proteins↗
GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import.
Mediated import of proteins into the nucleus involves multiple cytosolic factors, including the small GTPase Ran. Whether Ran functions by interacting with other cytosolic proteins or components of the nuclear pore complex has been unclear. Furthermore, the precise transport step where Ran acts has not been determined. To address these questions, we have analyzed the binding interactions of Ran using permeabilized cells and isolated nuclear envelopes. By light and electron microscope immunolocalization, we have found that Ran accumulates specifically at the cytoplasmic surface of the nuclear pore complex when nuclear import in permeabilized cells is inhibited by nonhydrolyzable analogs of GTP. Ran associates with a peripheral pore complex region that is similar to the area where transport ligands accumulate by depletion of ATP, which arrests an early step of transport. Binding studies with isolated nuclear envelopes in the absence of added cytosol indicate that Ran-GTP directly interacts with a pore complex protein. Using blot overlay techniques, we detected a single prominent polypeptide of isolated nuclear envelopes that binds Ran-GTP. This corresponds to the 358-kD protein RanBP2, a Ran binding pore complex protein recently identified by two-hybrid screening. Thus, RanBP2 is likely to constitute the Ran-GTP-binding site detected at the cytoplasmic periphery of the pore complex. These data support a model in which initial ligand binding to the nuclear pore complex occurs at or near RanBP2, and that hydrolysis of GTP by Ran at this site serves to define commitment to the nuclear import pathway.
Adenosine Triphosphate↗
Age-dependent alterations in beta-adrenergic responsiveness of rat detrusor smooth muscle.
The relaxant effects of norepinephrine (NE, 10(-7) to 10(-4) M.) and isoproterenol (ISO, 10(-9) to 10(-4) M.) on maximal KCl-induced tonic contractions and the relaxant effects of ISO on contractions induced by electrical field stimulation (EFS) were measured in detrusor muscle strips obtained from 22-25 day, 90-95 day and 22-month-old male Fischer 344 rats. The maximum relaxant response to NE and ISO on KCl-induced tonic contractions decreased significantly with increasing age. The ED50 values for ISO, but not NE, increased with age. The maximum relaxant response to ISO on EFS-induced contractions also was reduced significantly in the old bladders. The relaxation effects of forskolin (10(-6) to 3 x 10(-5) M.), dibutyryl cyclic AMP (DBcAMP, 10(-4) to 3 x 10(-3) M.) and cholera toxin (10 micrograms/ml.) were examined on maximal KCl-induced contractions of the muscle strips obtained from the three age groups. The relaxant responses to forskolin decreased significantly with increasing age, whereas DBcAMP relaxed the muscle strips from the three age groups equally. Cholera toxin (10 micrograms) attenuated KCl-induced phasic contractions, and this effect was impaired in the aged rat detrusor. The density of beta-adrenergic receptors, as determined by radioligand binding with [125I]iodopindolol ([125I]-PIN) decreased with increasing age. These data demonstrate an age-related decrease in the responsiveness of the bladder detrusor to beta-adrenergic stimulation that may be related to the decreased density of beta-adrenergic receptors and decreased cyclic AMP (cAMP) production.
Aging↗
[Changes in the visco-elastic properties of the articular cartilage incubated in various kinds of liquid].
The purpose of our experiment was to investigate the way in which the visco-elastic properties of the articular cartilage of a loading joint responded to various liquid environments. Using articular cartilage extracted from the femoral head of a cow, we examined the viscoelasticity of the articular cartilage under a fixed load while it was immersed in distilled water, isotonic salt water, hypertonic salt water, and in hyaluronic acid. A stainless steel rod with a diameter of 1.5 mm was applied directly to the articular cartilage (weighing 25, 50 and 100 grams) and the resultant visco-elastic curves were measured. Results showed that the deformation change to the cartilage was greatest while it was immersed in hypertonic salt solution and smallest while immersed in distilled water. Secondly, when immersed in hyaluronic acid solution, the change in deformation proved to be greater when the solution was denser and the molecular weight heavier and the hyaluronic acid could not penetrate into the cartilage matrix and remained at the surface. These results demonstrated that the visco-elastic property of the articular cartilage was affected not only by the varying osmotic pressures on the internal parts of the cartilage, but also on the density of the solution surrounding the cartilage and on the different water retainability of that cartilage.
Animals↗
[Cell cycle analysis of temperature sensitive mutants for cell growth].
Cells proliferate through the cell cycle consisting of G1, S, G2 and M. Even in the case of animal cells, the temperature sensitive mutants for the cell growth are an important method to analyse the cell cycle.
Animals↗
A mutant form of the Ran/TC4 protein disrupts nuclear function in Xenopus laevis egg extracts by inhibiting the RCC1 protein, a regulator of chromosome condensation.
The Ran protein is a small GTPase that has been implicated in a large number of nuclear processes including transport. RNA processing and cell cycle checkpoint control. A similar spectrum of nuclear activities has been shown to require RCC1, the guanine nucleotide exchange factor (GEF) for Ran. We have used the Xenopus laevis egg extract system and in vitro assays of purified proteins to examine how Ran or RCC1 could be involved in these numerous processes. In these studies, we employed mutant Ran proteins to perturb nuclear assembly and function. The addition of a bacterially expressed mutant form of Ran (T24N-Ran), which was predicted to be primarily in the GDP-bound state, profoundly disrupted nuclear assembly and DNA replication in extracts. We further examined the molecular mechanism by which T24N-Ran disrupts normal nuclear activity and found that T24N-Ran binds tightly to the RCC1 protein within the extract, resulting in its inactivation as a GEF. The capacity of T24N-Ran-blocked interphase extracts to assemble nuclei from de-membranated sperm chromatin and to replicate their DNA could be restored by supplementing the extract with excess RCC1 and thereby providing excess GEF activity. Conversely, nuclear assembly and DNA replication were both rescued in extracts lacking RCC1 by the addition of high levels of wild-type GTP-bound Ran protein, indicating that RCC1 does not have an essential function beyond its role as a GEF in interphase Xenopus extracts.
Animals↗
Loss of RCC1 leads to suppression of nuclear protein import in living cells.
The role of RCC1-Ran/TC4 in nuclear protein import was examined in living cells using a temperature-sensitive RCC1 mutant cell line, tsBN2, and tsBN2 transformed with a RCC1 cDNA lacking the nuclear localization sequence domain, delta 8-29. Substrate, containing a small number of SV40 T antigen nuclear localization sequence peptides, injected into the cytoplasm of tsBN2 cells cultured at the non-permissive temperature of 39.5 degrees C did not accumulate efficiently in the nucleus. When the same substrate was injected into the cytoplasm of heterokaryons of tsBN2 and wild type BHK21 cells, import efficiency into the tsBN2 nuclei was not restored. Import into the BHK21 nuclei gradually decreased after fusion. In contrast, import efficiency into tsBN2 nuclei gradually recovered after fusion with tsBN2 cells transformed with delta 8-29 in which functional RCC1 was diffusely distributed in both the nuclei and cytoplasm. Substrate did not accumulate in the nuclei of digitonin-permeabilized tsBN2 cells cultured at 39.5 degrees C even in the presence of normal cytosol. These results suggest that loss of RCC1 function leads to the decline of import competence of the nucleus and accumulation of a factor in the cytoplasm that suppresses nuclear import. These results indicate that the RCC1-Ran/TC4 system may regulate nuclear import.
Amino Acid Sequence↗
Functional analysis of block 5, one of the highly conserved amino acid sequences in the 130-kDa CryIVA protein produced by Bacillus thuringiensis subsp. israelensis.
There are five amino acid sequences highly conserved among Bacillus thuringiensis delta-endotoxins. We have changed the amino acid residues in block 5, one of the conserved sequences, of CryIVA. When the amino acid residues with charged side chains were replaced by others, the amount of production of the altered CryIVA protein was markedly decreased. It is suggested that the decrease is caused by the unstable conformation of the altered CryIVA protein molecule, as judged by digestion with trypsin and thermolysin. On the other hand, the substitution of amino acid residues in block 5 did not affect the insecticidal activity of CryIVA. These results strongly suggest that block 5 of CryIVA is one of the stability-determining elements of the protoxin molecule.
Amino Acid Sequence↗
Structure of the human CCG1 gene: relationship between the exons/introns and functional domain/modules of the protein.
The human CCG1 gene, encoding CCG1/TAFII250/p250, was isolated by complementing tsBN462, a mutant BHK21 cell line that shows cell-cycle arrest at high temperature. Using the cDNA as a probe, the locations of exon-intron junctions were determined in the genomic DNA. Thirty-eight exons ranging from 68 to 219 bp in size were found. All the exon-intron junctions followed the GT-AG rule. Using a newly developed method, we performed a module analysis of the CCG1 protein. The functional domain previously predicted in CCG1 was further confirmed to be encoded in a single predicted module that is the minimal functional unit in the protein. The boundaries of the predicted modules show a close correlation to the intron/exon junction of CCG1. The entire gene, at least 110 kb long, has been recovered in a YAC, which provides a route to the further study of module function.
Base Sequence↗
The CCG1/TAFII250 gene is mutated in thermosensitive G1 mutants of the BHK21 cell line derived from golden hamster.
The CCG1 cDNA encoding a general transcription factor, TAFII250, complements a thermosensitive (ts) cell cycle mutant, tsBN462, of the BHK21 cell line, which arrests in the G1 phase at the restrictive temperature. In order to clarify whether the CCG1 is mutated in tsBN462 cells or a suppressor of tsBN462 mutation, CCG1 cDNAs isolated from parental wild-type (wt) BHK21 and tsBN462 cell lines were sequenced. Comparison of the nucleotide (nt) sequences showed a single transition: G-->A in the second base of codon 690 of the tsBN462 CCG1 cDNA, resulting in a Gly690-->Asp change. The BHK CCG1 cDNA, but not the tsBN462 CCG1 cDNA, complemented the tsBN462 mutation, proving that the CCG1 is mutated in the tsBN462 cell line. Thus, the defect of general transcription factor, TAFII250, is suggested to cause a G1 arrest in the cell cycle.
Amino Acid Sequence↗
Involvement of nitric oxide and cyclic GMP in rabbit urethral relaxation.
Soluble and particulate fractions from rabbit urethra converted [14C]arginine to [14C]citrulline, indicating the presence of nitric oxide synthase activity in these fractions. Both soluble and particulate nitric oxide synthase activities were NADPH dependent, and the soluble activity was Ca2+ dependent. Three nitric oxide synthase (NOS) inhibitors affected transmural nerve stimulation induced relaxation responses in the rabbit urethra and the activity of soluble nitric oxide synthase with the same rank order of potency, i.e., NG-nitro-L-arginine (NNA) > NG-methyl-L-arginine (NMA) > canavanine (CAN). The rank order of potency with respect to particulate NOS activity was CAN > NMA = NNA. The relaxation responses to electrical stimulation were accompanied by increases in cyclic GMP. These results suggest that NOS activity found in the soluble fraction of urethral homogenates produces nitric oxide that in turn increases cyclic GMP levels which mediates the relaxation responses induced by transmural nerve stimulation in the rabbit urethra.
Amino Acid Oxidoreductases↗
The human CHC1 gene encoding RCC1 (regulator of chromosome condensation) (CHC1) is localized to human chromosome 1p36.1.
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Animals↗