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Y Azuma

Publications and source records attributed to Y Azuma.

At least 127 records · Page 7Linked to original sources
PubMed1995

[Molecular mechanisms of apoptosis in the immune system].

Apoptosis is a controlled death process under regulation by multiple death and survival genes. In the immune systems, apoptosis has important roles in establishment of the mature immune system (embryological programmed cell death), in dynamic maintenance of functional cell number (physiological programmed cell death) and in elimination of unwanted cells (biochemical programmed cell death). The process of apoptosis is regulated by multiple genes and their products depending on different stimuli. The current knowledge on the molecular events of apoptotic cell death, particularly in thymocytes, is reviewed.

Apoptosis↗
PubMed1995

Staurosporine-induced thymocyte apoptosis is inhibited by herbimycin A, a specific inhibitor of protein tyrosine kinase.

When mouse thymocytes were incubated with staurosporine at low doses (1-100 nM), increased tyrosine phosphorylation of proteins with a molecular weight of 31-34 kD in the detergent-soluble fraction was observed, together with an increase in internucleosomal DNA fragmentation. Incubation with staurosporine for 6 h was enough to trigger and progress of apoptosis. Herbimycin A, a specific inhibitor of protein tyrosine kinases, and cycloheximide, an inhibitor of protein synthesis, reduced the amount of the phosphotyrosine proteins, this being followed by the inhibition of DNA fragmentation and cell death, suggesting a close relationship between protein tyrosine phosphorylation and the induction of apoptosis by staurosporine.

Alkaloids↗
PubMed1994

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↗
PubMed1994

Canine Lyme disease: clinical and serological evaluations in 21 dogs in Japan.

Of 21 dogs from Sapporo, Hokkaido that had been recognised as having been bitten by ticks, 16 were seropositive to Borrelia burgdorferi by ELISA. Thirteen of the seropositive dogs showed signs such as fever, astasia, convulsions, anorexia, fatigue, abnormal gait, nervous signs, diarrhoea, corneal opacity and conjunctivitis. These signs subsided as a result of antibiotic treatment within five days. The plasma concentrations of creatinine in the 21 dogs were higher than in control dogs. Seven ticks that were removed from seven of the dogs were Ixodes persulcatus, and B burgdorferi was isolated from the midgut of two of the ticks.

Animals↗
PubMed1994

Discrimination by added ions of ligands at ionotropic excitatory amino acid receptors insensitive to N-methyl-D-aspartate in rat brain using membrane binding techniques.

The addition of potassium thiocyanate almost quadrupled binding of [3H]DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) to an AMPA-sensitive subclass of brain excitatory amino acid receptors in rat brain synaptic membranes, treated with Triton X-100. Among several ligands tested, quisqualic acid (QA) was the most potent displacer of [3H]AMPA binding in the absence of added SCN- ions, followed by AMPA, 6,7-dinitroquinoxaline-2,3-dione (DNQX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), glutamic (Glu) and kainic (KA) acids in a rank order of decreasing potency. The addition of SCN- ions was effective in significantly reducing the potencies of antagonists such as DNQX and CNQX, without affecting those of agonists including QA, AMPA, Glu and KA. On the other, the addition of Ca2+ ions significantly inhibited [3H]KA binding in a concentration-dependent manner at concentrations of above 2.5 mM. Calcium ions were also effective in significantly potentiating potencies to displace [3H]KA binding of antagonists such as DNQX and CNQX, with concomitant reduction of those of agonists including KA, QA and Glu. However, N-methyl-D-aspartic acid (NMDA) did not affect binding of both radioligands at concentrations of below 0.1 mM. These results suggest that both SCN- and Ca2+ ions may be useful to discriminate agonists and antagonists among a variety of displacers of ligand binding to the non-NMDA receptors in the brain.

Animals↗
PubMed1994

Molecular dissection of influenza virus nucleoprotein: deletion mapping of the RNA binding domain.

Influenza virus nucleoprotein (NP) is associated with the genome RNA, forming ribonucleoprotein cores. To identify the amino acid sequence involved in RNA binding, we performed Northwestern blot analysis with a set of N- and C-terminal deletion mutants of NP produced in Escherichia coli. The RNA binding region has been mapped between amino acid residues 91 and 188, a stretch of residues that contains a sequence that is not only highly conserved among NPs from A-, B-, and C-type influenza viruses but also similar to the RNA binding domain of a plant virus movement protein.

Amino Acid Sequence↗
PubMed1994

Treatment system for nocturnal enuresis according to an original classification system.

Our department has reported recent studies for nocturnal enuresis. The morning bladder capacity in enuretic children was smaller in the 3- and 4-year-old age range but larger in the 7-year and over age range than that in nonenuretic children. 15% of nonenuretic children showed nocturnal polyuria with nocturnal urination after complete awakening more than once per week. It was not likely from this evidence that an immature bladder capacity or nocturnal polyuria was the primary cause of enuresis. Patients with enuresis were classified into three types based on overnight simultaneous monitoring by electroencephalography and cystometry. The pathogenesis of enuresis type I and type IIa was a disturbance of awakening, while that of type IIb was a disturbance of bladder function not shown in the daytime but only at night. An original systematic therapeutic plan was established with the development of an original therapeutic machine primarily for enuresis type I.

Child↗
PubMed1994

Alendronate modulates osteogenesis of human osteoblastic cells in vitro.

The bisphosphonates, which are carbon-substituted pyrophosphates, have been studied extensively both in vivo and in vitro to elucidate their effects on bone tissues and cells. However, because these agents were shown to have a potent inhibitory effect on bone resorption, the majority of studies have focused on only this aspect of bone metabolism. There appears to be less information regarding the direct effect of bisphosphonates on bone formation, so thus we undertook experiments to investigate the effects of bisphosphonates, especially alendronate, on the mineralization and matrix protein synthesis of human osteoblastic cells in vitro. The data show that the bisphosphonates, alendronate, etidronate and pamidronate, suppressed 1,25-dihydroxycholecalciferol (1,25(OH)2D3)-stimulated mineralization of human osteoblastic cells at high concentrations, while relatively lower concentrations of alendronate and etidronate potentiated mineralization of the cells in the presence of 1,25(OH)2D3. The potentiation of mineralization with alendronate was accompanied by increased synthesis of bone matrix proteins, osteocalcin and collagen, and the mRNA of pro alpha(I) collagen. These findings show that in addition to their well-known effects on bone resorption, bisphosphonates have significant and direct effects on osteogenesis in osteoblasts in vitro. The actual mechanism remains to be further investigated.

Alendronate↗
PubMed1994

A new immunohistochemical method for quantification of fast-myosin in frozen histologic sections of the rat skeletal muscles.

An immunohistochemical micromethod for quantification of fast-myosin in frozen histologic sections of rat skeletal muscles was developed. The principle of this method was enzyme-linked immunosorbent assay (ELISA). We used frozen tissue sections as models of the antigen-coated wells of ELISA plate. The intensity of immunoreactivity of the frozen section 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. Then, the same section was incubated in 0.01 M acetic acid solution to cleave antigen-antibody complexes, followed by colorimetric measurement to obtain the absolute value of total protein per section. Fast-myosin content in the frozen tissue section was expressed as mg of fast-myosin per g of total protein. In this micromethod, the minimum area and the optimum thickness of the section were 5 mm2 and 10 microns, respectively. Fast-myosin contents in the extensor digitorum longus and soleus muscles were 185.0 +/- 6.1 and 17.5 +/- 2.4 mg/g, respectively. The results obtained by this micromethod were in good agreement with those obtained by two conventional methods, immunohistochemical morphometry and biochemical determination. This micromethod is useful for a quantitative evaluation of the contractile function of the mammalian skeletal muscles.

Actins↗
PubMed1994

Phosphorus-31 nuclear magnetic resonance study of energy metabolism in intact slow- and fast-twitch muscles of rats.

The time course of the catabolism of phosphorylated metabolites was studied by phosphorus-31 NMR(31P NMR) spectroscopy over a 6-h period after the isolation of rat slow- and fast-twitch muscles, the soleus and the extensor digitorum longus (EDL), respectively, obtained without any muscle damage. In both muscles, rapid depletion of creatine phosphate was followed by a decrease in ATP. These high-energy phosphates disappeared earlier in the soleus than in the EDL. In both muscles, inorganic phosphate (Pi) largely increased in a biphasic pattern, and sugar phosphates (SP) also showed considerable increase. The NMR-visible total phosphates (NTP) increased significantly in the soleus during the latter stage of observation. This increase in NTP was attributed to an increase in Pi. Although the two muscles showed the same initial (7.21) and final (approximately 5.9) intracellular pH (pHi), the pattern of pHi decline differed between these muscles. The rapid fall of pHi in the soleus in the early stage suggests that nonlactic acid acidosis plays a significant role in postmortem changes.

Animals↗
PubMed1994

Lyme disease spirochetes in a wild fox (Vulpes vulpes schrencki) and in ticks.

Lyme disease spirochetes were demonstrated in a wild female fox (Vulpes vulpes schrencki) and in Ixodes persulcatus ticks collected from the fox on Sapporo, Hokkaido, Japan. Spirochetes were detected in I. persulcatus, as well as skin lesions, brain, heart, kidney, and liver of the fox. Five of seven isolates reacted with a monoclonal antibody against Borrelia afzelii specific Osp B. Deoxyribonucleic acid (DNA) relatedness of a brain isolate was 89% to B. afzelii, and ranged from 50 to 67% to three other species. Immunoglobulin G antibodies to B. afzelii, B. garinii and B. burgdorferi sensulato, when tested in an enzyme-linked immunosorbent assay (ELISA), were negative in the fox. There were no antibodies against seven serovars of Leptospira interrogans.

Animals↗
PubMed1994

bis(2,6-dioxopiperaxine) derivatives, topoisomerase II inhibitors which do not form a DNA cleavable complex, induce thymocyte apoptosis.

Internucleosomal DNA fragmentation and cell death were induced dose- and time-dependently by incubation of mouse thymocytes with bis(2,6-dioxopiperazine) derivatives, ICRF-154 and MST-16, inhibitors of topoisomerase II, which do not induce cleavable complex formation. The process was inhibited by actinomycin D and cycloheximide, indicating that the process was an active apoptotic process. Bis(2,6-dioxopiperazine) derivatives have been known to inhibit the etoposide-induced DNA cleavage, but ICRF-154 did not inhibit etoposide-induced apoptosis in thymocytes at 6 h incubation, suggesting that DNA cleavage is not essential for induction of apoptosis by topoisomerase II inhibitors. The alteration of DNA helicity induced by a subtle inhibition of topoisomerase II activity may have an important role in the induction of apoptosis in thymocytes, since topoisomerase II is a major component of the nuclear matrix that can regulate gene expression.

Animals↗
PubMed1993

Subunits of the Schizosaccharomyces pombe RNA polymerase II: enzyme purification and structure of the subunit 3 gene.

To improve our understanding of the structure and function of eukaryotic RNA polymerase II, we purified the enzyme from the fission yeast Schizosaccharomyces pombe. The highly purified RNA polymerase II contained more than eleven polypeptides. The sizes of the largest the second-, and the third-largest polypeptides as measured by SDS-polyacrylamide gel electrophoresis were about 210, 150, and 40 kilodaltons (kDa), respectively, and are similar to those of RPB1, 2, and 3 subunits of Saccharomyces cerevisiae RNA polymerase II. Using the degenerated primers designed after amino acid micro-sequencing of the 40 kDa third-largest polypeptide (subunit 3), we cloned the subunit 3 gene (rpb3) and determined its DNA sequence. Taken together with the sequence of parts of PCR-amplified cDNA, the predicted coding sequence of rpb3, interrupted by two introns, was found to encode a polypeptide of 297 amino acid residues in length with a molecular weight of 34 kDa. The S. pombe subunit 3 contains four structural domains conserved for the alpha-subunit family of RNA polymerase from both eukaryotes and prokaryotes. A putative leucine zipper motif was found to exist in the C-terminal proximal conserved region (domain D). Possible functions of the conserved domains are discussed.

Amino Acid Sequence↗
PubMed1993

Topoisomerase inhibitors induce apoptosis in thymocytes.

The effects of the inhibitors of topoisomerase I and II, camptothecin and etoposide, as well as novobiocin and adriamycin, on the DNA fragmentation and viability of mouse thymocytes in primary culture were examined. All inhibitors were shown to produce dose-dependent internucleosomal DNA cleavage by resolving isolated DNA by agarose-gel electrophoresis. The DNA fragmentation seemed to precede cell death, determined on the basis of LDH release, by a few hours. Etoposide-induced DNA fragmentation progressively increased after incubation and was enhanced by pretreatment with phorbol 12,13-dibutyrate, a phorbol ester capable of activating protein kinase C, whereas camptothecin-induced DNA fragmentation increased progressively after 12 h incubation and was unaffected by phorbol 12,13-dibutyrate-pretreatment. The process was also energy-dependent and required RNA and protein synthesis and protein phosphorylation, since it was inhibited by sodium azide, actinomycin D, cycloheximide and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine hydrochloride, a protein kinase inhibitor. DNA fragmentation was also inhibited by zinc ions, suggesting the involvement of a specific endonuclease in DNA cleavage. These phenomena are similar to those detected in thymocytes undergoing apoptosis following exposure to glucocorticoids (Cohen, J.J. and Duke, R.C. (1984) J. Immunol. 132, 38-42). Considering that topoisomerases function in cellular proliferation and differentiation by altering DNA topology, the results suggest that topoisomerases have important roles in T-lymphocyte ontogeny in the thymus and are in part involved in the elimination of autoreactive or harmful cells by an apoptotic process.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

An assay method for DNA topoisomerase activity based on separation of relaxed DNA from supercoiled DNA using high-performance liquid chromatography.

A method for assaying the activities of DNA topoisomerases I and II has been developed. The assay for type I and II enzymes is based on monitoring relaxation of supercoiled plasmid DNA in the absence or presence of ATP, respectively. The reaction product, relaxed DNA, was separated from the substrate, supercoiled pBR329 plasmid, by a linear gradient of NaCl using high-performance ion-exchange chromatography with a DEAENPR column. In this method, nanogram amounts of relaxed DNA are detectable within 30 min, indicating that a subtle change in the activity in the cells can be assayed precisely.

Adenosine Triphosphate↗
PubMed1993

Induction of mouse thymocyte apoptosis by inhibitors of tyrosine kinases is associated with dephosphorylation of nuclear proteins.

Incubation of mouse thymocytes with the protein tyrosine kinase inhibitors herbimycin A and methyl-2,5-dihydroxycinnamate induced a decreased and altered profile of nuclear phosphotyrosine proteins in parallel with an increase in internucleosomal DNA fragmentation and cell death dose-dependently. No change in the profile of cytoplasmic phosphotyrosine proteins was observed. DNA fragmentation was dependent on the synthesis of RNA and protein, suggesting that the inhibition of tyrosine phosphorylation of the nuclear proteins induces apoptosis. DNA fragmentation was enhanced by simultaneous incubation with phorbol esters capable of activating protein kinase C. Genistein, another inhibitor of protein tyrosine kinase, induced DNA fragmentation more rapidly than herbimycin A, but there was no predominant alteration of phosphotyrosine proteins in early incubation, suggesting that genistein may induce apoptosis by a mechanism other than direct inhibition of protein tyrosinekinase activity.

Animals↗
PubMed1993

Tumour necrosis factor-alpha enhances cAMP-induced programmed cell death in mouse thymocytes.

During T-lymphocyte differentiation in the thymus, the majority of thymocytes die by apoptosis in situ. This process is characterized by internucleosomal DNA fragmentation and is induced by a number of stimuli including glucocorticoids, calcium ionophore, cAMP and 12-o-tetradecanoylphorbol 13-acetate (TPA). In this study, the effect of cytokines tumour necrosis factor-alpha (TNF-alpha) and interferon gamma (IFN-gamma) on the programmed cell death of thymocytes was examined by measuring DNA fragmentation and LDH release. TNF-alpha and IFN-gamma had no effect on DNA fragmentation in control and TPA, or A23187-treated thymocytes. Both human and murine rTNF-alpha enhanced cAMP-induced programmed cell death dose-dependently, but IFN-gamma had no effect on the process. TNF-alpha did not stimulate cAMP accumulation in control or 2-chloroadenosine-treated thymocytes. TPA markedly stimulated cAMP-induced DNA fragmentation as a result of 6 h incubation, whereas TNF-alpha did not. Thus TNF-alpha did not appear to activate protein kinase C directly. The effect of TNF-alpha was observed in the cell preparations from which adherent cells had been removed, suggesting that cytokines secreted by adherent cells in response to TNF-alpha are not involved in the process. The enhancement of cAMP-induced DNA fragmentation was observed in CD4+CD(8+)-double positive cells, but not in CD4+CD(8-)-single positive cells. The results of the present study indicate that a physiological cytokine, TNF-alpha, may modulate programmed cell death in immature thymocytes in concert with cAMP.

2-Chloroadenosine↗