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G Tsujimoto

Publications and source records attributed to G Tsujimoto.

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

Spatial and temporal regulation of protein expression by bldA within a Streptomyces lividans colony.

The bldA gene encodes the only tRNA for the UUA codon that, although dispensable in genes important for primary vegetative growth of Streptomyces spp., is important in genes that serve a regulatory purpose in the differentiation. To investigate this role further, the spatial and temporal expression profiles of the bldA-regulated and unregulated genes within a Streptomyces colony were examined using modified genes for the green fluorescent protein (gfp) as an expression-tag. A comparative study, based on computer-assisted quantitative analysis of the GFP fluorescence, revealed that the presence of TTA codons in gfp results in a temporal delay of translation and, consequently, changed the spatial pattern of the GFP expression within a colony, especially during early differentiation. The delay of GFP expression was undetectable at 60 h post-inoculation. These results provide the first extensive evidence that the bldA does indeed play a significant regulatory role during colony differentiation.

Amino Acid Sequence↗
PubMed1999

Interaction of the alpha(1B)-adrenergic receptor with gC1q-R, a multifunctional protein.

gC1q-R, a multifunctional protein, was found to bind with the carboxyl-terminal cytoplasmic domain of the alpha(1B)-adrenergic receptor (173 amino acids, amino acids 344-516) in a yeast two-hybrid screen of a cDNA library prepared from the rat liver. In a series of studies with deletion mutants in this region, the ten arginine-rich amino acids (amino acids 369-378) were identified as the site of interaction. The interaction was confirmed by specific co-immunoprecipitation of gC1q-R with full-length alpha(1B)-adrenergic receptors expressed on transfected COS-7 cells, as well as by fluorescence confocal laser scanning microscopy, which showed co-localization of these proteins in intact cells. Interestingly, the alpha(1B)-adrenergic receptors were exclusively localized to the region of the plasma membrane in COS-7 cells that expressed the alpha(1B)-adrenergic receptor alone, whereas gC1q-R was localized in the cytoplasm in COS-7 cells that expressed gC1q-R alone; however, in cells that co-expressed alpha(1B)-adrenergic receptors and gC1q-R, most of the alpha(1B)-adrenergic receptors were co-localized with gC1q-R in the intracellular region, and a remarkable down-regulation of receptor expression was observed. These observations suggest a new role for the previously identified complement regulatory molecule, gC1q-R, in regulating the cellular localization and expression of the alpha(1B)-adrenergic receptors.

Animals↗
PubMed1999

Identification of the sequence responsible for the nuclear localization of human Cdc6.

The Cdc6 is the essential protein for the initiation of DNA replication. Cdc6 is localized in the G1 nucleus, and abnormal nuclear localization of this protein induces irregular initiation of DNA replication. We identified here that amino acids K57 and R58 in the human Cdc6 protein play an important role in the nuclear localization of the protein. The fundamental features of the mechanism regulating the localization of Cdc6 seem to be maintained in yeast, Xenopus, and human, since the amino acid sequence surrounding K57 and R58, (S/T)PXKR(L/I), is conserved in these species. Substitution of amino acid residue S54 with E and not Q blocked partially the nuclear localization of the protein, implying that the phosphorylation at S54 is involved in the regulating mechanism of the cell cycle-dependent localization of Cdc6.

Amino Acid Sequence↗
PubMed1999

Semiquantitative evaluation of alpha1A-adrenoceptor subtype mRNA in human hypertrophied and non-hypertrophied prostates: regional comparison.

The main purpose of this study was to compare semiquantitatively the amount of alpha1a-adrenoceptor subtype mRNA in urethral, central and peripheral areas in benign hypertrophied prostates and non-hypertrophied prostate by the competitive reverse transcriptase polymerase chain reaction (RT-PCR) method. Prostates from twenty cases of men with benign prostatic hypertrophy (BPH) and 5 cases of bladder tumor without BPH were obtained for this study. The mRNA was extracted from the urethral, central and peripheral areas of each prostate, and quantitative RT-PCR was performed using the ratio of PCR product of alpha1a-adrenoceptor mRNA/beta2-microglobulin mRNA. The ratio for the central area of the hypertrophied prostate was significantly greater than that for the same area of the non-hypertrophied prostate (p < 0.05). In contrast, the urethral area showed no significant difference between the two prostate conditions. The central area of the hypertrophied prostate showed the tendency to have an increased alpha1a-adrenoceptor mRNA level compared with the urethral area, though no statistical difference was recognized because of the high standard error. Regional differences in the alpha1a-adrenoceptor mRNA in the non-hypertrophied prostate were rarely observed. The present finding demonstrates that the increased alpha1a-adrenoceptor mRNA content found in the hypertrophied prostate (1) was due to that in the central area.

Aged↗
PubMed1999

Effects of arginine-vasopressin fragment 4-9 on rodent cholinergic systems.

Arginine-vasopressin fragment 4-9 (AVP4-9) has been demonstrated in animal studies to facilitate learning and memory. To clarify the mechanisms of this facilitation, we focused on the effects of AVP4-9 on rodent cholinergic systems. AVP4-9 (0.1 microM) enhanced the basal and the high-potassium-evoked acetylcholine (ACh) release from rat hippocampal slices (122.4 and 120.0% of control, respectively) in the presence of 1.3 mM calcium (physiological level) at 60 min after the incubation at 37 degrees C. The AVP4-9-stimulated basal ACh release was inhibited by a V1-selective antagonist ([(beta-mercapto-beta,beta-cyclopentamethylene propionic acid)1, O-methyl-Tyr2, Arg8] vasopressin), but not by a V2-selective antagonist ([adamantaneacetyl1, O-ethyl-D-Tyr2, Val4, aminobutyryl6, Arg8,9]-vasopressin). In addition, AVP4-9 did not affect the basal ACh release under the calcium-free condition at 37 degrees C or in the presence of 1.3 mM calcium at 4 degrees C. However, AVP4-9 facilitated the passive-avoidance response of scopolamine (a cholinergic blocker)-induced memory-deficient mice. These findings demonstrate that AVP4-9 stimulates ACh release via mediation by V1-like vasopressin receptors, and shows dependence on calcium ion and temperature. The results also suggest that the mechanism of the facilitative effects of AVP4-9 on learning and memory consist of the observed stimulation of cholinergic systems and other parallel pathways that would not be inhibited by cholinergic blocking.

Acetylcholine↗
PubMed1999

Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter.

Primary systemic carnitine deficiency (SCD; OMIM 212140) is an autosomal recessive disorder characterized by progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia. SCD has also been linked to sudden infant death syndrome. Membrane-physiological studies have suggested a defect of the carnitine transport system in the plasma membrane in SCD patients and in the mouse model, juvenile visceral steatosis. Although the responsible loci have been mapped in both human and mouse, the underlying gene has not yet been identified. Recently, we cloned and analysed the function of a novel transporter protein termed OCTN2. Our observation that OCTN2 has the ability to transport carnitine in a sodium-dependent manner prompted us to search for mutations in the gene encoding OCTN2, SLC22A5. Initially, we analysed the mouse gene and found a missense mutation in Slc22a5 in jvs mice. Biochemical analysis revealed that this mutation abrogates carnitine transport. Subsequent analysis of the human gene identified four mutations in three SCD pedigrees. Affected individuals in one family were homozygous for the deletion of a 113-bp region containing the start codon. In the second pedigree, the affected individual was shown to be a compound heterozygote for two mutations that cause a frameshift and a premature stop codon, respectively. In an affected individual belonging to a third family, we found a homozygous splice-site mutation also resulting in a premature stop codon. These mutations provide the first evidence that loss of OCTN2 function causes SCD.

Amino Acid Sequence↗
PubMed1999

Characterization of alpha 1-adrenoceptors expressed in a novel vascular smooth muscle cell line cloned from p53 knockout mice, P53LMAC01 (AC01) cells.

1. We pharmacologically studied the alpha 1-adrenoceptor (AR) subtype(s) involved in receptor-mediated signalling in a novel vascular smooth muscle cell line cloned from p53 knockout mice, P53LMAC01 (AC01) cells. 2. Radioligand binding studies with [125I]-HEAT showed the existence of a homogeneous population of binding site with an affinity (Kd value) of 0.4 nM and a maximum number of binding sites (Bmax) of 100 fmol mg-1 protein. Catecholamines competed for [125I]-HEAT binding stereospecifically and with the characteristic alpha 1-AR potency series. 3. Displacement curves for BMY-7378 and KMD-3213 best fitted a one-site model with a pKi value (-log10 (equilibrium inhibition constant)) of 6.06 and 7.07, respectively. 4. Reverse transcription-polymerase chain reaction (RT-PCR) assay detected alpha 1B- and alpha 1D-AR, but not alpha 1A-AR transcript. 5. Chlorethylclonidine (CEC) treatment nearly abolished (-)noradrenaline (NA) (10 microM)-induced inositol[1,4,5]trisphosphate (IP3) production, and BMY-7378 inhibited the response with a Ki value of 0.3 nM, which value was similar to that obtained in the cells expressing alpha 1D-AR. In both AC01 cells and cells expressing alpha 1D-AR, BMY-7378 protected alpha 1-ARs from CEC alkylation while it had little protective effect on CEC alkylation and NA-induced IP3 production in cells expressing alpha 1B-AR. 6. The results indicate that AC01 cells contain predominantly alpha 1B-ARs and a small population of alpha 1D-ARs; however, phosphoinositide (PI)/Ca2+ signalling is mainly mediated through the minor population of alpha 1D-ARs, rather than the alpha 1B-ARs.

Alkylation↗
PubMed1999

Differential subcellular localization of DNA-dependent protein kinase components Ku and DNA-PKcs during mitosis.

The Ku protein is a complex of two subunits, Ku70 and Ku80. Ku plays an important role in DNA-PKcs-dependent double-strand break repair and V(D)J recombination, and in growth regulation, which is DNA-PKcs-independent. We studied the expression and the subcellular localization of Ku and DNA-PKcs throughout the cell cycle in several established human cell lines. Using immunofluorescence analysis and confocal laser scanning microscopy, we detected Ku70 and Ku80 in the nuclei in interphase cells. In mitotic cells (1) most of Ku protein was found diffused in the cytoplasm, (2) a fraction was detected at the periphery of condensed chromosomes, (3) no Ku protein was present in the chromosome interior. Association of Ku with isolated chromosomes was also observed. On the other hand, DNA-PKcs was detected in the nucleus in interphase cells and not at the periphery of condensed chromosomes during mitosis. Using indirect immunoprecipitation, we found that throughout the cell cycle, Ku70 and Ku80 were present as heterodimers, some in complex with DNA-PKcs. Our findings suggest that the localization of Ku at the periphery of metaphase chromosomes might be imperative for a novel function of Ku in the G(2)/M phase, which does not require DNA-PKcs.

Antigens, Nuclear↗
PubMed1999

Naftopidil, a novel alpha1-adrenoceptor antagonist, displays selective inhibition of canine prostatic pressure and high affinity binding to cloned human alpha1-adrenoceptors.

The pharmacological profiles of the alpha1-adrenoceptor antagonists naftopidil, tamsulosin and prazosin were studied in an anesthetized dog model that allowed the simultaneous assessment of their antagonist potency against phenylephrine-mediated increases in prostatic pressure and mean blood pressure. The intravenous administration of each of these compounds dose-dependently inhibited phenylephrine-induced increases in prostatic pressure and mean blood pressure. To further assess the ability of the three compounds to inhibit phenylephrine-induced responses, the doses required to produce a 50% inhibition of the phenylephrine-induced increases in prostatic and mean blood pressure and the selectivity index obtained from the ratio of those two doses were determined for each test compound. Forty minutes after the intravenous administration of naftopidil, the selectivity index was 3.76, and those of tamsulosin and prazosin were 1.23 and 0.61, respectively. These findings demonstrated that naftopidil selectively inhibited the phenylephrine-induced increase in prostatic pressure compared with mean blood pressure in the anesthetized dog model. The selectivity of naftopidil for prostatic pressure was the most potent among the test compounds. In addition, using cloned human alpha1-adrenoceptor subtypes, naftopidil was selective for the alpha1d-adrenoceptor with approximately 3- and 17-fold higher affinity than for the alpha1a- and alpha1b-adrenoceptor subtypes, respectively. The selectivity of naftopidil for prostatic pressure may be attributable to its high binding affinity for alpha1a- and alpha1d-adrenoceptor subtypes.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed1999

Characterization of the mouse alpha1D-adrenergic receptor gene.

Alpha1-adrenergic receptors (alpha1-ARs) play critical roles in the regulation of a variety of physiological processes. Increasing evidence suggests that multiple receptor subtypes of alpha1-ARs regulate these physiological processes. Molecular cloning has identified three distinct cDNAs encoding alpha1-AR subtypes (alpha1A, alpha1B and alpha1D) that are structurally homologous. Among the alpha1-AR subtypes, the function of the alpha1D-AR remains unclear. In order to examine the physiological role of alpha1D-AR, we cloned and characterized a gene for the mouse alpha1D-AR. Using a mouse alpha1D-AR cDNA as a probe, we isolated the gene for the mouse alpha1D-AR from a mouse genomic library. The alpha1D-AR consists of two exons and an intron that interrupts the coding region of the putative sixth transmembrane domain. The 5'-flanking region of exon 1 contains neither a TATA box nor a CAAT box but is high in GC content and contains several Sp1 binding sites (GC boxes). This pattern is similar to promoters described for other members of alpha1-ARs. The untranslated region also contains putative cyclic AMP response elements. Isolation of this gene will allow further investigation, via gene knock-outs and deletion mutants, of the mechanisms of transcriptional regulation and a greater understanding of the physiological role of alpha1D-AR.

Amino Acid Sequence↗
PubMed1999

Structure and sequence of the mouse V1a and V1b vasopressin receptor genes.

The structural organization and 5'-flanking region of the mouse V1a and V1b vasopressin receptor genes were determined. The mouse V1a receptor gene was located within an 8-kb XbaI fragment, and the mouse V1b receptor gene was located within a 14-kb EcoRV fragment. Both genes were comprised of two coding exons that were separated by a 2.3-kb and a 8.0-kb intron, respectively, located before the respective seventh transmembrane domain of the receptor sequence. The availability of these genes would allow us to study the functional role of V1a and V1b receptors by disrupting the gene in mice.

Animals↗
PubMed1999

In vivo and in vitro evidence of blood-brain barrier transport of a novel cationic arginine-vasopressin fragment 4-9 analog.

The blood-brain barrier (BBB) transport and metabolism of a novel arginine-vasopressin fragment 4-9 [AVP(4-9), isoelectric point; (pI) = 9.2] analog, that is, cationic AVP(4-9) (C-AVP(4-9), PI = 9.8), were examined in vivo and in vitro. At 45 min after an i.v. administration to mice, the cerebrum-to-plasma concentration ratios of (35)S-labeled AVP(4-9) and (125)I-labeled C-AVP(4-9) were 0.103 and 0.330 ml/g cerebrum, respectively, and the BBB permeation clearances were 1.47 x 10(-4) and 3.10 x 10(-4) ml/min/g cerebrum, respectively. In the in vitro study using mouse brain capillary endothelial cells immortalized by SV40 infection (MBEC4), the acid-resistant binding values of (35)S-labeled AVP(4-9) and (125)I-labeled C-AVP(4-9) to MBEC4 at 120 min were 0.93 and 1.95 microliter/mg protein (as the cell/medium ratios), respectively. (35)S-labeled AVP(4-9) showed two-phase saturable acid-resistant binding, and its half-saturation constants (K(D)) were 3.8 nM (high affinity) and 45.7 microM (low affinity). (125)I-labeled C-AVP(4-9) showed single-phase saturable acid-resistant binding, with a K(D) value of 16.4 microM. The acid-resistant binding of (125)I-labeled C-AVP(4-9) was significantly dependent on temperature and medium osmolarity. The acid-resistant binding of (125)I-labeled C-AVP(4-9) was inhibited by dancylcadaverine, phenylarsine oxide (endocytosis inhibitors), 2,4-dinitrophenol (a metabolic inhibitor), and AVP(4-9), poly(L-lysine), and protamine (cationic substances), but not by poly(L-glutamic acid) (an anionic peptide) and the V(1) and V(2) vasopressin receptor antagonists. In addition, the conversion of C-AVP(4-9) to AVP(4-9) in the cerebral homogenate was confirmed by HPLC and mass spectrometry. The present results demonstrate that C-AVP(4-9) is transported through the BBB more effectively than AVP(4-9), via absorptive-mediated endocytosis, and that C-AVP(4-9) is converted to the neuroactive parent peptide, AVP(4-9), in the cerebrum.

Animals↗
PubMed1998

Identification of a novel MCM3-associated protein that facilitates MCM3 nuclear localization.

MCM3 is essential for the initiation of DNA replication and also participates in controls that ensure DNA replication is initiated once per cell cycle. In a two-hybrid screen for proteins that interact with human MCM3, we identified and cloned a novel protein of which the calculated molecular weight is 80,291. A specific antibody against the protein identified a 80-kDa protein in HeLa cell extract, indicating the protein actually expressed in cells. The interaction of these proteins was confirmed by immunoprecipitation assay. Moreover, we clarified a nuclear localization signal of human MCM3, and we find that mutagenesis on the nuclear localization signal of MCM3 affected the binding of newly isolated MCM3-assosiated protein, Map80. Map80 was expressed in Escherichia coli as a fusion with His6 tag and purified with sequential column chromatographies. The addition of recombinant Map80 stimulated the amount of nuclear localized MCM3. These results suggest that Map80 is involved in the nuclear localization pathway of MCM3.

Acetyltransferases↗
PubMed1998

Effects of quinidine and verapamil on human cardiovascular alpha1-adrenoceptors.

BACKGROUND: The antiarrhythmic drugs quinidine and verapamil are known to block alpha1-adrenoceptors (alpha1ARs). Alpha1ARs are a heterogeneous family of three subtypes (alpha1A, alpha1B, and alpha1D), and little is known about the effects of quinidine and verapamil on the different human alpha1AR subtypes. METHODS AND RESULTS: Reverse transcriptase-polymerase chain reaction showed that all alpha1AR subtypes are expressed in both human heart (atrium and ventricle) and the mesenteric artery. Pharmacological profiles of quinidine and verapamil actions on the alpha1AR subtypes were characterized with Chinese hamster ovary cells stably expressing cloned human alpha1AR subtypes. Radioligand binding studies showed that quinidine and verapamil had high affinities for all alpha1AR subtypes. Also, both drugs synergistically inhibited alpha1AR-mediated inositol 1,4,5-triphosphate production at the clinical effective concentration range (1 micromol/L quinidine and 0.1 micromol/L verapamil). CONCLUSIONS: The results show that all alpha1AR subtypes are expressed in the human cardiovascular system and that quinidine and verapamil may have a potent, synergistic inhibitory effect on the alpha1ARs. Clinically observed hypotension after quinidine plus verapamil can be explained by their synergistic inhibitory effects on human alpha1ARs.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed1998

Pituitary adenylate cyclase-activating polypeptide rhythm in the rat pineal gland.

Pituitary adenylate cyclase-activating polypeptide (PACAP) was recently demonstrated to stimulate melatonin synthesis in the rat pineal gland. Circadian rhythms of melatonin concentration are well known. However, it has not been clarified whether PACAP contents in the pineal gland show circadian rhythm. In this study, we measured PACAP contents in the rat pineal gland throughout the day under 12:12 h light-dark cycle or constant dark conditions. A significant fluctuation was observed in the PACAP content under light-dark conditions but not under constant darkness. On the other hand, the pituitary gland showed no significant variation throughout the day under either conditions. These observations suggest that PACAP may participate in the modulation of melatonin synthesis depending on light conditions in the pineal gland.

Analysis of Variance↗
PubMed1998

Subtype-specific differences in subcellular localization and chlorethylclonidine inactivation of alpha1-adrenoceptors.

Chlorethylclonidine (CEC) inactivation has been used as one criterion to subclassify the alpha1-adrenoceptors (AR); however, the extent of CEC inactivation can vary depending on the CEC treatment. By constructing the FLAG-tagged (N-terminus) and green fluorescent protein (GFP)-fused (C-terminus) alpha1-ARs, we have determined the relationship between CEC sensitivity and the cellular localization of alpha1-AR subtypes using COS-7 cells. In GFP-expressing cells, flow cytometry analysis with anti-FLAG N-terminus antibody detected strong fluorescent signals in most of alpha1B-AR-expressing cells, but low signals in alpha1A-AR-expressing cells. Further examination with confocal microscopy showed that fluorescent signals densely localized intra-cellularly in alpha1A-AR-expressing cells, while most of alpha1B-AR localized on the cell surface. Furthermore, radioligand binding studies with [125I]HEAT showed that CEC (10 microM) treatment of intact cells inactivated approximately 30-40% of alpha1A-AR and >90% of alpha1B-AR, while the CEC treatment of membrane preparations resulted in >80% decrease in the alpha1A-AR density and >90% of alpha1B-AR density, respectively. The results showed that the hydrophilic alkylating agent CEC inactivated only alpha1-AR on the cell surface irrespective of its subtype, and that the subtype-specific sorting is a major determinant for CEC inactivation of alpha1-AR. Subtype-specific cellular localization suggests a new class of functional properties that may explain the signal and functional diversity of homologous alpha1-AR (as well as other G protein-coupled receptors) subtypes.

Adrenergic alpha-Antagonists↗
PubMed1998

Quantification and distribution of alpha1-adrenoceptor subtype mRNAs in human proximal urethra.

1. We performed RNase protection assays and in situ hybridization to investigate the ratio of the three alpha1-adrenoceptor subtype mRNAs, alpha1a, alpha1b and alpha1d, in human proximal urethra, and their localization in urethral cross-sections. As revealed by the RNase protection assays, alpha1a was the predominant subtype mRNA in both male and female urethral samples. Alpha1d mRNA was detected only in the female sample, and alpha1b mRNA was not detected in any of the samples tested. The ratio of the abundance of the subtype mRNAs, alpha1a:alpha1b:alpha1d, was 100:0:0 in the male urethra and 90:0:10 in the female urethra. 2. In situ hybridization studies showed no significant differences in the cross-sectional distribution of alpha1-adrenoceptor subtype mRNAs between male and female urethras. Intense alpha1a staining was observed in the smooth muscle of the urethra, but alpha1b and alpha1d staining was much less intense. 3. Of the three cloned alpha1 subtypes, alpha1a is the most likely to be responsible for the contraction of the human urethra. Owing to the side effects of nonselective alpha1 drugs, alpha1-selective drugs may be clinically superior to nonselective drugs for the treatment of urethral disorders.

Aged↗