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Biomedical subjects

Y Azuma

Publications and source records attributed to Y Azuma.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1995

Alendronate distributed on bone surfaces inhibits osteoclastic bone resorption in vitro and in experimental hypercalcemia models.

Alendronate is an aminobisphosphonate that acts as a potent inhibitor of osteoclastic bone resorption. To understand the mechanism of action of alendronate in vivo, in this study we investigated the relationship between distribution of [14C]-alendronate in rat bone and its effects on bone resorption in vitro or in rat hypercalcemic models. A single IV dose of 0.05 approximately 1.25 mg/kg inhibited the increase in plasma calcium level induced by bovine PTH or 1 alpha(OH)D3. The minimal effective dose of pamidronate (1.25 mg/kg) and etidronate (over 31.25 mg/kg) were at least 5 times and 25 times, respectively, higher than the dose of alendronate in the rat hypercalcemic model prepared by 1 alpha(OH)D3. The relative potencies of compounds in the hypercalcemic rat models reflected those of inhibitory effects on bone resorption in vitro. We conducted the ivory-slice assay under two conditions: (a) addition of a given bisphosphonate after adherence of the osteoclasts; and (b) preincubation of the ivory slices with a given bisphosphonate. The inhibitory IC50 values of alendronate under condition (b) were similar to those under condition (a). To evaluate the interaction between osteoclasts and alendronate in bone, we investigated the localization of [14C]-alendronate in the tibia of growing rats (4-day-old rats). Alendronate did not distribute uniformly in the tibia. At 1 day after injection (0.05 mg SC), dense labeling was seen primarily under osteoclasts. We injected 0.05 mg/kg of [14C]-alendronate (single i.v.) into rats [14C]-alendronate was rapidly eliminated from plasma, and mainly distributed to the bone in rats. These data suggest that alendronate which distributed on bone surface mainly contributed to the antihypercalcemic action in vivo.

Alendronate↗
PubMed1995

Isolation of thermolabile mutant RNA polymerase II from fission yeast Schizosaccharomyces pombe with mutations in the subunit 3 gene.

Subunit alpha of prokaryotic RNA polymerases plays key roles in protein-protein contacts for both subunit assembly and transcription activation. To gain an insight into the roles of subunit 3, the eukaryotic homologue of alpha, temperature-sensitive mutants of the fission yeast, Schizosaccharomyces pombe, have been isolated after transformation of the mutagenized rpb3 gene encoding mutant subunit 3 of RNA polymerase II. A total of 68 ts mutants were classified into two groups: mutants comprising one group ceased growing immediately after a temperature up-shift, while mutants comprising the other group exhibited delayed growth arrest at high temperatures. RNA polymerase II partially purified from Ts54, one of the group 2 mutants, was thermolabile in vitro, as measured by a non-specific transcription assay. This mutant carries double mutations in domain A of subunit 3, and thus can be used as a reference mutant of RNA polymerase II.

Cloning, Molecular↗
PubMed1995

Effects of protein tyrosine kinase inhibitors with different modes of action on topoisomerase activity and death of IL-2-dependent CTLL-2 cells.

We studied the effects of protein tyrosine kinase inhibitors with different modes of action on topoisomerase activity and cell death in CTLL-2 cells, whose growth is IL-2-dependent. The Flavonoids genistein, biochanin A, and apigenin inhibited topoisomerase II to the same extent as etoposide, a specific inhibitor of the enzyme. Methyl 2,5-dihydroxycinnamate (2,5-MeC) also inhibited topoisomerase II, but was less potent than genistein. Herbimycin A and staurosporine did not inhibit topoisomerase II. None of the inhibitors of protein tyrosine kinases examined inhibited topoisomerase I activity. All the inhibitors induced cell death with internucleosomal DNA fragmentation in the presence of IL-2. Genistein, biochanin A, and apigenin induced DNA fragmentation and cell death early in the incubation period and did not alter the profiles of phosphotyrosine proteins in either the lysate or pelleted fractions, indicating that the early cell death was induced by the inhibition of topoisomerase II activity rather than by the inhibition of protein tyrosine kinase activity. 2,5-MeC similarly induced early cell death and DNA fragmentation, but to a lesser extent than genistein presumably due to the inhibition of topoisomerase II activity. Herbimycin A induced a slow increase in DNA fragmentation and cell death, accompanied by a decrease in phosphotyrosine proteins in the pelleted fraction, suggesting that the inhibition of protein tyrosine phosphorylation, presumably of the nuclear proteins, is related to cell death and DNA fragmentation. Staurosporine-induced DNA fragmentation appeared to be due to mechanism(s) other than the inhibition of topoisomerases and protein tyrosine kinases, since it neither altered the profiles of phosphotyrosine proteins nor inhibited topoisomerase activity.

Animals↗
PubMed1995

Immunohistochemical microquantification of fast-myosin in frozen histological sections of mammalian skeletal muscles.

Fast-myosin in frozen histological sections was quantified by an immunohistochemical micromethod based on the ELISA. Frozen tissue sections mounted on glass slides were used analogously to the antigen-precoated wells of ELISA plates. The intensity of immunoreactivity of frozen sections to an anti-fast-myosin monoclonal antibody was quantified directly from the color developed with the second antibody coupled with peroxidase using phenol-4-aminoantipyrine as a substrate. Fast-myosin levels in the masseter muscles of pigs, rats, and rabbits were 185 +/- 6, 223 +/- 9, and 178 +/- 12 mg/g of total protein, respectively, and those in the gastrocnemius muscles from cows, pigs, goats, rats, and rabbits were 172 +/- 12, 211 +/- 7, 177 +/- 9, 211 +/- 10, and 205 +/- 10 mg/g, respectively. In the masseter of cows and goats, fast-myosin was not detected. The results obtained by this immunohistochemical micromethod were in good agreement with those obtained by histomorphometrical and biochemical analyses. This immunohistochemical micromethod could be used to quantitatively evaluate the muscle contractile characteristics that determine meat quality.

Animals↗
PubMed1995

[A comparative morphologic study of Carabelli cusp between Chinese and Japanese students.].

The study of teeth,dentition and occlusion is helpful to guide oral medical clinic and the study of anthropology.Our department of Osaka Dental University and had many items of research work about teeth,dentition and occlusion between Chinese and Japanese students.This article reports the study of Carabelli cusp.The results revealed:(1)the rate of Carabelli cusp is higher in Japanese than in Chinese and is higher in male than in female.(2)the rate of Carabelli cusp of the first maxillary molar of both side is very high.(3)The rate of Carabelli cusp is related to the height and the type of cusp numbers.These all illustrated the occurence of Carabeiil cusp were influenced by race, individual and the development of teeth.

English Abstract↗
PubMed1995

Biphasic effect of staurosporine on thymocyte apoptosis.

When mouse thymocytes were incubated with staurosporine at low doses (1-100 nM), apoptosis was induced dose- and time-dependently. Staurosporine-induced apoptosis was dependent on macromolecular synthesis, and it was also dependent on protein phosphorylation sensitive to 1-(5-isoquinolinesulfonyl-2-methylpiperazine dihydrochloride (H-7). Whereas, staurosporine at high doses (above 500 nM) did not induce significant DNA fragmentation, rather it inhibited the DNA fragmentation induced by 12-O-tetradecanoyl-13-acetate, A23187, and dibutyrylcyclic AMP, as H-7 did. K252a, a derivative of staurosporine, induced apoptosis, which was inhibited by H-7, even at high doses. These results indicate that staurosporine had a biphasic effect on thymocyte apoptosis, a stimulatory effect at low concentration, and an inhibitory effect at high concentration. K252a had only the former action.

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