Biomedical subjects

Y Azuma

Publications and source records attributed to Y Azuma.

At least 91 records · Page 5Linked to original sources

PubMed1997

Inhibition by anti-RCC1 monoclonal antibodies of RCC1-stimulated guanine nucleotide exchange on Ran GTPase.

Nine monoclonal antibodies to RCC1, the guanine nucleotide exchange factor on Ran GTPase, were obtained using recombinant RCC1 as the antigen. Epitopes of three monoclonal antibodies, which did not inhibit RCC1 function, were localized in the N-terminus outside the RCC1 repeat, while epitopes of the other 6 monoclonal antibodies were localized within the RCC1 repeat. Three of the latter 6 monoclonal antibodies, 2B6, 6C3, and 8D9, inhibited RCC1-stimulated nucleotide release. Two of them, 2B6 and 6C3, recognized the same amino acid residues in the N-terminus of the second RCC1 repeat, Tyr89, Ser90, Phe91, and Gly92, of which one, Gly92, is conserved in Saccharomyces cerevisiae and mutated in an rcc1(-) strain, mtr1-2. The monoclonal antibody 8D9 recognized two amino acid residues, Arg320 and Ala321, downstream of Gly319 in the N-terminus of the 6th RCC1 repeat, which corresponds to Gly92 in the second RCC1 repeat. The monoclonal antibodies which inhibited RCC1 function bound to RCC1 in homogeneous solution and stained cellular RCC1. We propose that the N-terminus of the RCC1 repeat is exposed at the surface of RCC1 on the coated plate or in fixed cells, and is involved in the RCC1-stimulated nucleotide exchange on the Ran GTPase.

Amino Acid Sequence↗

PubMed1997

A candidate case for lymphocytic infundibulo-neurohypophysitis mimicking a neurohypophysial tumor.

A 56-year-old Japanese man presented with a 2-month duration of polyuria and polydipsia. The diagnosis of diabetes insipidus was confirmed by water deprivation and vasopressin injection. The secretory function of the adenohypophysis was estimated as normal by a variety of provocative tests. Magnetic resonance imaging (MRI) displayed the loss of the hyperintense signal of the neurohypophysis and a tumor-like lesion confined to the neurohypophysis. The tissue specimen resected at transsphenoidal surgery showed diffuse lymphocytic infiltration. These findings suggest that this is a candidate case for lymphocytic infundibuloneurohypophysitis (LIN) that is not identical to classical lymphocytic hypophysitis. This patient will be followed up to determine whether this case simply represents an early stage of classical hypophysitis or a different clinical entity.

Diabetes Insipidus↗

PubMed1997

The effect of occlusal appliances and clenching on the temporomandibular joint space.

It has been suggested that stabilization appliances and mandibular anterior repositioning appliances work by decompressing the temporomandibular joint. To indirectly test this assumption, tomograms of right temporomandibular joints of seven subjects were taken during comfortable closure and maximum clenching in maximum intercuspation and on the two types of occlusal appliances. Outlines of the condyle and the temporal fossa were automatically determined by an edge detection protocol. Upon comfortable closure, the anterior joint space dimension was reduced with stabilization appliances and mandibular anterior repositioning appliances. Upon maximum clenching, the minimum joint space dimension on stabilization appliances was equivalent to that seen in maximum intercuspation, while that on mandibular anterior repositioning appliances was substantially less (P < .05). Findings do not indicate that these appliances induce an increase in joint space during clenching.

Adult↗

PubMed1997

Sequence-selective RNA scission by oligoamine--DNA conjugates (1).

Ethylenediamine and diethylenetriamine, which are active for RNA hydrolysis, are attached to the 5'-ends of synthetic oligonucleotides by urethane linkages. The resultant artificial ribonucleases selectively hydrolyze the substrate RNA at the target phosphodiester linkage. Acid/base cooperation in the oligoamines for the catalysis is confirmed in terms of kinetic evidences.

Base Sequence↗

PubMed1996

Dis3, implicated in mitotic control, binds directly to Ran and enhances the GEF activity of RCC1.

Using the two-hybrid method, we isolated a Saccharomyces cerevisiae cDNA encoding a protein homologous to Schizosaccharomyces pombe protein Dis3sp, using as bait, human GTPase Ran. The DIS3 gene is essential for viability and complements S.pombe mutant dis3-54 which is defective in mitosis. Although Dis3sc has no homology to RanBP1, it bound directly to Ran and the S.cerevisiae Ran homologue Cnr1, but not to the S.cerevisiae RCC1 homologue Srm1. Upon binding to Ran with a 1:1 molar ratio, Dis3sc enhanced a nucleotide-releasing activity of RCC1 on Ran. In the presence of Dis3sc, the K(m) of RCC1 on Ran decreased by half, while the kcat was unchanged. In vivo, Dis3sp was present as oligomers of M(r) 670-200 kDa as previously reported, and the 200 kDa oligomer of Dis3sp was found to include Spi1 and Pim1, the S.pombe homologues of Ran and RCC1, respectively. Although the biological function of the heterotrimeric oligomer consisting of Dis3, Spi1 and Pim1 is unknown, our results indicate that Dis3 is a component of the RCC1-Ran pathway.

Amino Acid Sequence↗

PubMed1996

Premature chromatin condensation induced by loss of RCC1 is inhibited by GTP- and GTPgammaS-Ran, but not GDP-Ran.

RCC1 is a guanine nucleotide exchanging factor acting on nuclear G protein Ran. Premature chromatin condensation occurs in the temperature-sensitive rcc1- mutant of the BHK21 cell line, tsBN2, at the restrictive temperature. This observation can be explained if the premature activation of MPF is normally inhibited by GTP-Ran. In the absence of RCC1, GDP-Ran predominates, resulting in MPF activation. However, experiments with Ran mutants to determine whether GTP- or GDP-Ran prevents activation of MPF have yielded conflicting results. In order to clarify this point, we have microinjected nucleotide-bound Ran, instead of mutated Ran, into the nuclei of tsBN2 cells treated to reduce RCC1-mediated guanine nucleotide exchange. GTP-Ran, GTPgammaS-Ran, and GDP-Ran all inhibited chromatin condensation. However, the inhibition of chromatin condensation by GDP-Ran could be completely abolished by co-injection with GDP, but not GTP. Thus, we conclude that GTP-Ran blocks the activation of MPF and that hydrolysis of GTP is not required to prevent MPF activation.

Animals↗

PubMed1996

Simultaneous determination of binding of a variety of radioligands related to ionotropic excitatory amino acid receptors in fetal and neonatal rat brains.

Expression of ionotropic excitatory amino acid receptors was assessed by membrane binding assays using a variety of radioligands in fetal and neonatal rat brains. In fetal rat brain, receptors sensitive to N-methyl-D-aspartate (NMDA) exhibited delayed onset of expression during the last 7 days before birth as compared with those insensitive to NMDA. In addition, developmental increases in agonist-preferring sites preceded those in antagonist-preferring sites within the first 7 postnatal days in particular brain structures with respect to each domain on the NMDA receptor complex. Growth of animals led to drastic increments of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK-801) binding to the NMDA channel in telencephalic regions until 21 to 28 days after birth, with concomitant desensitization to inhibition by protons of [3H]MK-801 binding in cortical membranes. By contrast, three different agonists were invariably effective in more potently potentiating [3H]MK-801 binding in cortical membranes of 14- and 28-day-old rats than in those of 5-day-old rats. These results suggest that the NMDA-sensitive subclass may play more critical roles in mechanisms underlying postnatal development of rat telencephalon than do the NMDA-insensitive subclasses.

Animals↗

PubMed1996

In vivo microautoradiography of [3H]1,24(OH)2D3 (tacalcitol) following topical application to normal rats and in vitro metabolism in human keratinocytes.

This study was conducted to investigate the mechanism of topical absorption of [3H]1,24(OH)2D3 (1,24-dihydroxyvitamin D3; tacalcitol) by applying an ointment containing 4 micrograms2/g [3H]1,24(OH)2D3 to the skin of rats using an occlusion method. Microautoradiography of the skin at the application site 1 h after topical treatment showed a high concentration of radiolabel in the stratum corneum, the epidermis and around the hair follicles. Radiolabel was also seen in the epidermis and hair follicle areas 8 h and 24 h after application. The radiolabel was distributed to a minor extent to the subcutaneous fat layer. Microautoradiography showed two routes of purcutaneous absorption of 1,24(OH)2D3: through the stratum corneum and epidermis into the microvessels, and through hair follicle areas into the bloodstream. After topical application of an ointment containing 4 micrograms/g or 40 micrograms/g [3H]1,24(OH)2D3 to the shaved neck skin of rats, the absorption rate, estimated by excretion in the urine and faeces, was about 30% of the total applied radioactivity. The main excretion route after topical application was in the faeces. Furthermore, 1,24(OH)2D3 added to human adult keratinocytes was not metabolized into other compounds, and only the unchanged compound was detected. These findings strongly suggest that 1,24(OH)2D3 distributed into the epidermis acts on epidermal keratinocytes. Topical application of 1,24(OH)2D3 appears to be a possible approach to the treatment of psoriasis and other skin diseases through its action on the 1,25(OH)2D3 receptor, which reportedly plays a very important role in the regulation of proliferation and differentiation of keratinocytes.

Absorption↗

PubMed1996

Differentiation by magnesium ions of affinities of nuclear proteins for consensus core nucleotide element of the transcription factor c-Myc in murine brain.

The addition of divalent cations such as Mg(2+) and Ca(2+) ions markedly reduced binding of a radiolabeled double stranded oligonucleotide probe for the transcription factor c-Myc in the presence of 100 mM KCl in nuclear extracts of the mouse whole brain. Irrespective of the addition of MgCl(2), binding was selectively competed with the unlabeled probe for c-Myc having a double stranded conformation. Treatment with V8 protease differentially modulated binding of the probe for c-Myc determined in the presence and absence of added MgCl(2). Introduction of irreversible covalent bonding between the radiolabeled probe and nuclear proteins led to retarded mobility of the radioactive probe/protein complex in the presence of MgCl(2) on sodium dodecyl sulfate electrophoresis regardless of treatment with DNase. However, an antibody against the c-Myc protein affected neither mobility nor intensity of the radioactive band on gel retardation electrophoresis. Moreover, regional distribution was different from each other in mouse brain when determined in the presence and absence of added MgCl(2). These results suggest that magnesium ions may have an ability to differentiate between nuclear c-Myc family proteins with different affinities for the consensus core nucleotide element CACGTG in murine brain.

Animals↗

PubMed1996

Measurement of intact rat osteocalcin in osteoblast (ROS17/2.8) cells and in ovariectomized rats with a sandwich enzyme immunoassay.

We developed a sandwich enzyme immunoassay system for intact rat osteocalcin to improve the region specificity for the detection of this molecule. We synthesized two peptides of N-terminal 20 residues and C-terminal 10 residues of rat osteocalcin. After conjugating these peptides with carrier protein, we obtained anti-N- and anti-C-terminal rat osteocalcin antibodies in rabbits raised against these two peptides, respectively. By using these antibodies, we measured intact rat osteocalcin levels in a two-site immunoassay manner. These antibodies did not show the cross-reactivity to human osteocalcin. The immunoreactive peak corresponding to the intact molecules was detected by our intact osteocalcin method after high-performance liquid chromatographic fractionation of osteocalcin fragments in plasma from uremic rats. Furthermore, the intact rat osteocalcin was stable over 8 hours at 25 degrees C. Intact rat osteocalcin levels extracellularly secreted from ROS 17/2.8 cells were measured by this method, showing time- and dose-dependent significant increases when administered 1,25(OH)2D3. The inhibition for the secretion of intact osteocalcin by actinomycin D was also detected quantitatively with this method. In ovariectomized rats, intact osteocalcin levels in plasma were acutely elevated after ovariectomy, and its elevation was significantly depressed by 17beta-estradiol administration. These data suggest that this sandwich method is able to measure the intact form of osteocalcin secreted by osteoblasts. As the antibodies identify the specific regions of osteocalcin molecule, this method would be useful for sensitive estimation of bone turnover for various experimental conditions in rats.

Animals↗

PubMed1996

Binding of double stranded oligonucleotide probes for particular transcription factors with leucine-zipper motifs in discrete brain structures of mice with acquired and inherent spontaneous seizures.

Nuclear extracts of mouse brain contained binding of radiolabeled oligonucleotide probes for particular transcription factors with leucine-zipper motifs including activator protein-1 (AP1), cyclic AMP response element binding protein (CREB) and c-Myc. An acute intraperitoneal injection of pentylenetetrazole (PTZ) at a convulsive dose significantly potentiated binding of the probe for AP1 in the cerebral cortex, hippocampus, striatum, hypothalamus and midbrain, without affecting that in the medulla-pons and cerebellum, 2 h after the administration. However, PTZ failed to affect binding of the probe for CREB under the similar experimental conditions. In contrast, PTZ induced a slight but statistically significant decrease in binding of the AP1 probe in the cerebellum, without altering that in the hippocampus, 14 h after the injection. On the other hand, repeated administration of PTZ at a subconvulsive dose led to spontaneous kindling seizures in animals, with a concomitant decrease in binding of the AP1 probe in both the hippocampus and cerebellum. In contrast to these animals with acquired spontaneous seizures, however, binding of the AP1 probe was significantly higher in three different telencephalic structures of inherently spontaneous epileptic El mice than that in the parent ddY mice, with binding of probes for CREB and c-Myc being unchanged. These results suggest that different molecular mechanisms may underlie the expression of being unchanged. These results suggest that different molecular mechanisms may underlie the expression of AP1 in discrete brain structures of mice with acquired and inherent spontaneous seizures.

Activating Transcription Factor 2↗

PubMed1996

Particular nuclear transcription factors responsive to systemic administration of kainic acid in murine brain.

Gel retardation electrophoresis revealed that binding of a radiolabeled double stranded oligonucleotide probe for the nuclear transcription factor activator protein-1 (AP1) was markedly potentiated 2 h after the intraperitoneal injection of kainic acid (KA) at a dose range of 10-40 mg/kg in a dose-dependent manner in the murine hippocampus. The potentiation was seen in a manner independent of the crisis of convulsive seizures following the administration of KA at different doses. At the highest dose employed, the systemic KA significantly potentiated the AP1 binding in most central discrete structures examined except the cerebellum. In contrast, KA significantly potentiated binding of a radiolabeled probe for cyclic AMP response element binding protein (CREB) in a dose-dependent fashion in the hippocampus, without altering that in other parts of murine brain. No significant alteration was detected in binding of a probe for c-Myc in any brain regions examined 2 h after the administration of KA at different doses. However, immunoblotting analysis demonstrated that KA was ineffective in altering endogenous levels of both CREB and CREB phosphorylated at serine133 in the hippocampus and cerebellum. These results suggest that in vivo systemic KA signals may be selectively transduced to nuclear AP1 in the hippocampus through a mechanism different from phosphorylation of CREB at serine133 in murine brain.

Animals↗

PubMed1996

Effects of sustained unilateral molar clenching on the temporomandibular joint space.

OBJECTIVE: To measure the effect of unilateral sustained clenching on the temporomandibular joints, changes in the minimum joint space dimension were assessed. STUDY DESIGN: Ten healthy subjects performed a sustained clench on a bite force transducer in the first molar region for 5 minutes with a constant force of 170 N. Three separate sagittal tomograms were bilaterally obtained with the transducer in place before clenching and during the beginning and at the end of the contraction. Changes were quantified with a computerized image analysis system. RESULTS: The minimum joint space of the contralateral temporomandibular joint was significantly reduced both at the beginning and at the end of the contraction task. Further the minimum joint space was also significantly less at the end than at the beginning of the contraction even though bite force level was identical. The ipsilateral condyle images showed no significant shift in the minimum joint space. CONCLUSIONS: These data suggest that unilateral molar clenching induces a significant reduction of the minimum joint space in the contralateral temporomandibular joint and a sustained condition remarkably increases this change.

Adult↗

PubMed1996

Conserved histidine residues of RCC1 are essential for nucleotide exchange on Ran.

Charged amino acid residues of human RCC1 were converted to alanine and mutants which were unable to complement tsBN2 cells (a temperature-sensitive rcc1- mutant of the hamster BHK21 cell line) were selected. These RCC1 mutants were analyzed for the ability to inhibit premature chromatin condensation by microinjection into tsBN2 cells, and their steady-state kinetic parameters for guanine nucleotide exchange reaction were measured. Examined RCC1 mutants were unstable in tsBN2 cells at the restrictive temperature, yet they significantly inhibited premature chromatin condensation. Mutants located on the N-terminus of the RCC1 repeat showed an increased K(m), while their kcat values were comparable to that of wild-type RCC1. In contrast, mutants containing the conserved histidine residues in the C-terminus of the RCC1 repeat showed a value of K(m) similar to that of wild-type RCC1, while the kcat values of these mutants were reduced, depending upon the RCC1 repeats on which the mutation was located. These steady-state kinetic parameters of mutants indicate that the N-terminus and the C-terminus of RCC1 repeats play different roles in guanine nucleotide exchange on Ran. The comparison of kcat among the histidine mutants suggests that those histidine residues which are conserved in the RCC1 repeats and also through evolution comprise the catalytic site for the guanine nucleotide exchange reaction.

Alanine↗

PubMed1996

Effects of alendronate on plasma calcium levels, urinary calcium excretion, and bone resorption markers in normal rats: comparison with elcatonin, synthetic eel calcitonin.

In normal rats given alendronate (0.01-6.25 mg/kg) or elcatonin (synthetic eel calcitonin; 0.32-8.0 U/kg), changes in urinary calcium (Ca), pyridinoline (Pyr), and deoxypyridinoline (D-Pyr) excretion during the hypocalcemic response were assessed. Although the lower doses (0.01-0.25 mg/kg) of alendronate did not influence plasma Ca, the high doses (1.25-6.25 mg/kg) significantly decreased plasma Ca by the third day after single iv administration. In these groups, urinary Ca excretion did not show any significant change, but urinary Pyr and D-Pyr excretion decreased significantly at high doses. The hypocalcemic effect lasted for only 1 or 2 days (2-3 days after injection) even at high doses of alendronate. In the group receiving elcatonin (8.0 U/kg, twice daily), a significant decrease in plasma Ca was evident as early as 1 day after the start of administration. This was accompanied by a marked increase in urinary Ca excretion in the early stage without a significant decrease in urinary Pyr or D-Pyr excretion, and the suppression of bone resorption was more pronounced in the late phase of treatment with elcatonin. These results suggest that alendronate decreases plasma Ca chiefly by suppressing bone resorption, whereas elcatonin decreases plasma Ca by inhibiting bone resorption and accelerating Ca excretion. The present data show that both alendronate and elcatonin inhibit bone resorption and exert an antihypercalcemic effect, but the mechanism of action is different in the two drugs.

Alendronate↗

PubMed1996

Effects of the mycelial extract of cultured Cordyceps sinensis on in vivo hepatic energy metabolism in the mouse.

Mice were given the extract of cultured Cordyceps sinensis (Cs) (200 mg/kg daily, p.o.) for 3 weeks. In vivo phosphorus-31 nuclear magnetic resonance (NMR) spectra of the liver were acquired at weekly intervals using a surface coil. From 1 to 3 weeks, a consistent increase in the ATP/inorganic phosphate ratio, which represents the high energy state, was observed in the Cs extract-treated mice. The intracellular pH of the Cs extract-treated mice was not significantly different from that of the control mice. No steatosis, necrosis, inflammation or fibrosis were observed in the liver specimens from Cs extract-treated mice.

Adenosine Triphosphate↗

PubMed1995

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↗

PubMed1995

Isolation of a yeast protein kinase that is activated by the protein encoded by SRP1 (Srp1p) and phosphorylates Srp1p complexed with nuclear localization signal peptides.

Srp1p, the protein encoded by SRP1 of Saccharomyces cerevisiae, is a nuclear-pore-associated protein. Its Xenopus homolog, importin, was recently shown to be an essential component required for nuclear localization signal (NLS)-dependent binding of karyophilic proteins to the nuclear envelope [Gorlich, D., Prehn, S., Laskey, R. A. & Hartman, E. (1994) Cell 79, 767-778]. We have discovered a protein kinase whose activity is stimulated by Srp1p (Srp1p fused to glutathione S-transferase and expressed in Escherichia coli) and is detected by phosphorylation of Srp1p and of a 36-kDa protein, a component of the protein kinase complex. The enzyme, called Srp1p kinase, is a protein-serine kinase and was found in extracts in two related complexes of approximately 180 kDa and 220 kDa. The second complex, when purified, contained four protein components including the 36-kDa protein. We observed that, upon purification of the kinase, phosphorylation of Srp1p became very weak, while activation of phosphorylation of the 36-kDa protein by Srp1p remained unaltered. Significantly, NLS peptides and the nuclear proteins we have tested greatly stimulated phosphorylation of Srp1p, suggesting that Srp1p, complexed with karyophilic proteins carrying an NLS, is the in vivo substrate of this protein kinase.

Amino Acid Sequence↗