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Biomedical subjects

S Azuma

Publications and source records attributed to S Azuma.

At least 37 records · Page 2Linked to original sources
PubMed1997

Vitamin B12 enhances GABA content but reduces glutamate content in the rat suprachiasmatic nucleus.

The present study attempted to elucidate effects of vitamin B12 (VB12) on the rat suprachiasmatic nucleus (SCN), which is the locus of the circadian clock in mammals, by evaluating the contents of its major neurotransmitters, gamma-aminobutyric acid (GABA) and glutamate (Glu). First, contents of these amino acids were analyzed from tissue samples prepared at three different circadian times (CTs) in free-running rats under constant dim illumination. The content of GABA was highest in the middle of the resting phase (CT 6), intermediate early in the active phase (CT 14), and lowest late in the active phase (CT 20), whereas Glu content indicated inverse circadian variations. Subsequently, effects of intracerebroventricularly infused VB12 were assayed at CT 20. Compared with the saline-infused control, the infusion of VB12 (30 nmol/30 microliters) at CT 12-15 significantly increased GABA content but decreased Glu content. The quantitative balance of these amino acids after VB12 infusion was similar to that at CT 6 in noninfused rats. These results suggest that infusion of VB12 to active rats shifts the ratio of SCN neurotransmitters toward that observed in resting rats.

Animals↗
PubMed1997

Germ-line contribution of embryonic stem cells in chimeric mice: influence of karyotype and in vitro differentiation ability.

The effect of the karyotype and the ability to differentiate in vitro upon germ-line transmission by A3-1 embryonic stem (ES) cells in chimeric mice were examined. Germ-line transmission was confirmed in ES cells exhibiting 38% and more of the normal karyotype, but no chimeric mice and/or germ-line transmitters were observed regardless of the karyotype when the cystic embryoid body (CEB) was formed on day 8 and later in the suspension culture. Germ-line transmission of the ES cells was not significantly influenced by formation of the simple embryoid body (SEB). Germ-line transmitters were preferentially observed in chimeras when the ES cell contribution to coat color was markedly increased, but this contribution to coat color varied regardless of the karyotype or in vitro differentiation ability. These results suggest that A3-1 ES cells which exhibit CEB at 7 days after suspension culture and approximately 40% of normal karyotype are capable of germ-line transmission in chimeric mice.

Animals↗
PubMed1996

Identification of TRAF6, a novel tumor necrosis factor receptor-associated factor protein that mediates signaling from an amino-terminal domain of the CD40 cytoplasmic region.

CD40 signalings play crucial roles in B-cell function. To identify molecules which transduce CD40 signalings, we have utilized the yeast two-hybrid system to clone cDNAs encoding proteins that bind the cytoplasmic tail of CD40. A cDNA encoding a putative signal transducer, designated TRAF6, has been molecularly cloned. TRAF6 has a tumor necrosis factor receptor (TNFR)-associated factor (TRAF) domain in its carboxyl terminus and has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other TRAF family proteins. TRAF6 does not associate with the cytoplasmic tails of TNFR2, CD30, lymphotoxin-beta receptor, and LMP1 of Epstein-Barr virus. Deletion analysis showed that residues 246-269 of CD40 which are required for its association with TRAF2, TRAF3, and TRAF5 are dispensable for its interaction with TRAF6, whereas residues 230-245 were required. Overexpression of TRAF6 activates transcription factor NFkappaB, and its TRAF-C domain suppresses NFkappaB activation triggered by CD40 lacking residues 246-277. These results suggest that TRAF6 could mediate the CD40 signal that is transduced by the amino-terminal domain (230-245) of the CD40 cytoplasmic region and appears to be independent of other known TRAF family proteins.

Amino Acid Sequence↗
PubMed1996

State-dependent changes of extracellular glutamate in the medial preoptic area in freely behaving rats.

Microdialysis technique was applied to the medial preoptic area (mPOA) of the hypothalamus, a crucial site for the regulation of sleep, in order to analyze the interrelationship between the extracellular level of glutamate (Glu) and the vigilance states. Dialysates from the mPOA were sampled at 5-min intervals for 2-h diurnal period with a perfusion rate of 2.0 microliters/min in freely moving rats, whose sleep-waking behaviors were polysomnographically monitored. Extracellular Glu increased during wakefulness, exhibiting a peak at the transition period from wakefulness to non-rapid-eye-movement sleep (NREMS) (18.0% above the average), whereas it decreased during NREMS (9.2% below the average). It is likely that Glu in the mPOA is dynamically involved in the alterations of the vigilance states.

Animals↗
PubMed1996

Effects of phosphate on in vitro 2-cell block of AKR/N mouse embryos based on changes in cdc2 kinase activity and phosphorylation states.

This study demonstrated the effects of phosphate on the 2-cell block of AKR/N mouse embryos at the molecular level and focused on changes in the kinase activity and the phosphorylation state of cdc2, which is shown to regulate the cell division cycle. Removal of phosphate from the culture medium dramatically increased developmental rates to the 4-cell (91.8%) and blastocyst (42.6%) stages compared with those of embryos cultured in 1.17 mM phosphate (3.3% and 0%, respectively). The rate of development to the 4-cell stage was significantly inhibited by 0.001 mM phosphate (p < 0.05), and no morula formation was observed at 1.0 mM. The patterns of cdc2 kinase activity during the first cell cycle in AKR/N embryos were similar to those of control MCH embryos, showing the highest activity at M phase and low activity during the interphase. The phosphorylated form of cdc2 increased during the interphase, indicating that the synthesis of cyclin B and accumulation of inactive pre-maturation-promoting factor (pre-MPF) as well as abrupt dephosphorylation of cdc2 at the first cleavage correlated with the activation of cdc2 kinase. When phosphate was absent, the activation pattern of cdc2 kinase during the second cell cycle in AKR/N embryos was similar to that in the first cell cycle. On the other hand, no dephosphorylation of cdc2 was observed and the kinase activity remained at a low level until 56 h after insemination in the presence of phosphate, although an increase in phosphorylated cdc2 was observed as in the phosphate-free group. Treatment of AKR/N embryos arrested at the 2-cell stage with okadaic acid resulted in the dephosphorylation and activation of cdc2, confirming the presence of a sufficient amount of pre-MPF. These results show that phosphate has a deteriorative effect on the in vitro development of AKR/N embryos and suggest that this effect was not on the synthesis of cyclin B but on the dephosphorylation of phosphorylated cdc2.

Animals↗
PubMed1996

Maintenance of pluripotency in mouse embryonic stem cells persistently infected with murine coronavirus.

A persistently coronavirus-infected embryonic stem (ES) cell line A3/MHV was established by infecting an ES cell line, A3-1, with mouse hepatitis virus type-2. Although almost all A3/MHV cells were found infected, both A3/MHV and A3-1 cells expressed comparable levels of cell surface differentiation markers. In addition, A3/MHV cells retained the ability to form embryoid bodies. These results suggest that persistent coronavirus infection does not affect the differentiation of ES cells.

Animals↗
PubMed1995

Gene transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector.

Replication-defective recombinant adenovirus, Adex4SRLacZL, was used as a vector for transferring exogenous genes in mouse zona pellucida-free eggs at the pronuclear stage. The vector contained the E. coli LacZ reporter gene under the control of the SR alpha promoter (SV40 early promoter-fused HTLV-I LTR), and the expression of the reporter gene was examined during preimplantation development in culture. Histochemical staining of the embryos for beta-galactosidase activity showed that the exogenous LacZ gene as expressed in 98% of the embryos at the morula-blastocyst stages. As in the microinjection method, the exogenous genes could be pursued from the 2-cell stage. Neither apparent morphological changes nor cytotoxic effects were observed. Both the percentages of embryos expressing reporter genes and the rate of development to the blastocyst stage were higher in the adenovirus vector-treated embryos than in the microinjected ones. These results suggest that the adenovirus vector system is a useful tool in investigating the genetic control of early mammalian development.

Adenoviridae↗
PubMed1995

Cryopreservation of wild mouse spermatozoa.

Spermatozoa of wild mice from China, Czechoslovakia, Denmark, India, Japan and Switzerland were frozen and stored at -196 degrees C. After thawing, intact oocytes were inseminated in vitro with relatively high motility frozen-thawed mouse spermatozoa from Czechoslovakia, Denmark and India, while oocytes with a partially dissected zona were inseminated with low motility frozen-thawed spermatozoa from China, Japan and Switzerland. Embryos developing to the 2-cell stage from oocytes fertilized with frozen-thawed spermatozoa were transferred to the oviducts of female recipients on the first day of pseudopregnancy (day when a vaginal plug was confirmed). Successful embryo development to the 2-cell stage was 46 to 67%. Offspring resulted from 17 to 51% of these transferred 2-cell embryos.

Journal Article↗
PubMed1995

Mice deficient for the IL-3/GM-CSF/IL-5 beta c receptor exhibit lung pathology and impaired immune response, while beta IL3 receptor-deficient mice are normal.

The receptors for IL-3, GM-CSF, and IL-5 share a common beta subunit (beta c), and mice have an additional IL-3 beta subunit (beta IL3). We have independently generated mice carrying null mutations of each molecule. beta c mutant bone marrow showed no response to GM-CSF or IL-5, whereas IL-3 stimulation of beta c or beta IL3 mutant bone marrow was normal. beta c mutant mice showed lung pathology consisting of lymphocytic infiltration and areas resembling alveolar proteinosis, and also exhibited low basal numbers of eosinophils. Infection of beta c mutant mice by Nippostrongylus brasiliensis resulted in the absence of blood and lung eosinophilia. Animals repopulated with beta c mutant bone marrow cells showed slower leukocyte recovery and reduced eosinophil numbers. These data define the role of beta c in vivo, and show a phenotype that is likely to be the cumulative effect of loss of GM-CSF and IL-5 stimulation.

Animals↗
PubMed1994

Sperm from mice carrying a targeted mutation of the acrosin gene can penetrate the oocyte zona pellucida and effect fertilization.

The physiological function of mammalian sperm acrosin has long been believed to be involved in the limited proteolysis of the oocyte zona pellucida, thus enabling the sperm to penetrate this extracellular matrix and to gain access to the oocyte plasma membrane. Here we show that male mice homozygous for a targeted mutation in the mouse acrosin gene are still fertile in spite of the complete absence of acrosin protease activity in the sperm. In vitro fertilization assays verified that sperm from the homozygous mutant mice penetrate the zona pellucida and effect fertilization. Therefore, acrosin is not essential for both sperm penetration of the zona pellucida and fertilization.

Acrosin↗
PubMed1993

PCR sexing and developmental rate differences in preimplantation mouse embryos fertilized and cultured in vitro.

A two-step polymerase chain reaction (PCR) assay was used to determine the sex of mouse preimplantation embryos obtained from oocytes fertilized and cultured in vitro, to investigate the differences in the developmental rates of mouse embryos according to the sex. All the in vitro developed embryos could be analyzed by this method. When the embryos were classified according to the time of morula to blastocyst transition as fast-intermediate- and slow-growing embryos, a significantly high percentage (78.0%) of the fast-developing embryos were identified as males; while a significantly lower percentage (42.5%) of slow-developing embryos were identified as males. The intermediate-developing embryos presented a sex ratio not significantly different from the total (57.5%). The deviation of sex ratio was further confirmed by embryo transfer experiment, where fast- and slow-developing embryos gave 76.2% and 25.7% male fetuses, respectively. We concluded that male mouse embryos fertilized and cultured in vitro develop faster than female embryos.

Animals↗
PubMed1993

[Immunological effects of locoregional immunochemotherapy for liver metastases of gastric cancer].

Nine gastric cancer patients with simultaneous liver metastases were given intermittent transarterial administration of chemotherapeutics (adriamycin, mitomycin C or 5-fluorouracil) and biological response modifiers (BRM; OK-432 and interleukin (IL) -2) after gastrectomy. In terms of direct antitumor effect on liver metastases, a partial response was observed in 4 of 9 cases (44%). In addition, the concentration of 3 kinds of cytokines such as IL-6, tumor necrosis factor (TNF) -alpha and interferon (IFN) -gamma in the peripheral blood sera was measured immediately before and after transarterial administration of agents. While the concentration of IL-6 increased by BRM, chemotherapeutics could not alter the level of IL-6. As for TNF-alpha and IFN-gamma, no particular changing pattern was observed after transarterial administration. Furthermore, natural killer (NK) activity of peripheral blood mononuclear cells was measured. Administration of either BRM or BRM in combination with chemotherapeutics caused a decrease in NK activity, whereas chemotherapeutics did not. Flow cytometric analysis using 3 kinds of monoclonal antibodies (Anti-CD16, CD56 and CD57) revealed that the proportion of subset of both highly activated NK cells (CD16+.CD56+.CD57-) and moderately activated NK cells (CD16+.CD56+.CD57+) reduced after administration of BRM.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1993

[A case of secondary leukemia induced by chemotherapy with a CDDP-based regimen for gastric cancer 5 years following radical resection].

Cisplatin, mitomycin C and 5-fluorouracil were given a 55-year-old woman for an unresectable gastric cancer, and successful radical gastrectomy was performed. Postoperative adjuvant immunochemotherapy using UFT and PSK was continued for about 4 years and 4 months. Pancytopenia was observed at 5 years after the treatment and then marked leucocytosis was noted. She also showed complications of general fatigue, appetite loss etc. A secondary acute leukemia associated with eosinophilia was diagnosed by peripheral blood examinations, showing WBC, 122,400: blast, 37.5 % and eosinophil, 41%. Results also showed atypia and pseudo-Pelger nuclear abnormality of eosinophil, high positive stain of cell myelogenic cell surface marker, many numeral and structural abnormalities of chromosomal analysis, etc. From the above results, it was suggested that the leukemia might be induced by previously performed chemotherapy. The patient died about 2 months following its onset.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1992

Tetraparental mice reveal complex cellular interactions of the mutant, autoimmunity-inducing lpr gene.

Lpr is a murine recessive autosomal mutant gene that, in conjunction with the background genes of different inbred mouse strains, produces varied syndromes of lymphadenopathy and autoantibody production. We have investigated the cellular basis of the lpr determined phenomena by constructing allophenic (tetraparental) chimeras of MRL/Mp-lpr/lpr combined with C57BL/6 and MRL/+ combined with C57BL/6-lpr/lpr. The appearance of lymphadenopathy, lpr atypical T cells, and autoantibodies was found to depend on the relative amounts +/+ and lpr tissue. The failure of these three abnormalities to correlate in certain animals suggested different pathways of lpr gene action. Furthermore, in the lpr predominant animals, both the lymphadenopathy and autoantibodies were essentially entirely contributed by the lpr donor. The expression of the lpr gene within the same cells expressing the MRL or B6 background determined the pathologic and serologic manifestations characteristic of the disease of that lpr strain, despite the presence of +/+ cells of the other background in the same individual. The lpr syndrome thus depends on the intracellular interaction of the lpr and background genes, and on positive signalling between different lpr cell populations.

Animals↗
PubMed1992

Effect of a partial deletion of Y chromosome on in vitro fertilizing ability of mouse spermatozoa.

The effect of a partial deletion of Y chromosome on sperm fertilizing ability was investigated through an in vitro fertilization technique. Epididymal spermatozoa of a congenic line, B10.BR-Ydel, which is characterized by a high incidence of abnormal spermatozoa, revealed a significantly lower in vitro fertilization rate (22%) than that (79%) of its control strain (B10.BR/SgSn), which has a normal-sized Y chromosome. Incidence of capacitated spermatozoa as determined by chlortetracycline fluorescence assay was significantly lower in B10.BR-Ydel than in B10.BR/SgSn spermatozoa. The fertilization rate was significantly improved when B10.BR-Ydel spermatozoa were separated from the supernatant of sperm suspension by Percoll gradient centrifugation. A reconstitution experiment revealed that the B10.BR-Ydel spermatozoa were more sensitive to the inhibitory effect of the supernatant than B10.BR/SgSn spermatozoa. Spermatozoa from F1 (C57BL/6N male x B10.BR-Ydel female) males showed higher fertilization rates than those from F1 (B10.BR.Ydel male x C57BL/6N female) males. These observations suggest that not only the morphology but also the fertilizing ability of spermatozoa is directly related to partial deletion of Y chromosome.

Animals↗
PubMed1992

C-fos expression in the rat brain after intraperitoneal injection of lithium chloride.

The distribution of evoked expression of the proto-oncogene c-fos was immunohistochemically examined in the rat brain after intraperitoneal injection of isotonic LiCl, which is commonly used to induce internal malaise in the conditioned taste aversion paradigm. C-fos-like immunoreactive neurones (c-fos neurones) were most densely observed in the central amygdaloid nucleus, external lateral subnucleus of the parabrachial nucleus (PBN), posteromedial and commissural parts of the nucleus of the tractus solitarius (NTS) and area postrema (AP). Experiments including vagotomy, intravenous injection of LiCl and lesions of the area postrema suggest that NTS neurones are activated via both sides of the vagus nerves, while AP neurones, humorally as well as neurally via the vagal nerve with a right side predominance. The activated NTS and AP neurones project mainly to the external lateral subnucleus of the PBN and lightly to the central lateral subnucleus of the PBN. These results are discussed in terms of the role of LiCl in the formation of conditioned taste aversion.

Animals↗
PubMed1992

Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry.

An Escherichia coli strain, B-62, that was isolated from a clinical source and was epidemiologically unrelated to E. coli K-12 was the source of chromosomal DNA for a sucrose utilization system (Scr+) in the construction of a plasmid, pST621. The cloned insert of a gene encoding Scr+ in pST621 conferred a sucrose-positive phenotype onto transformed cells of E. coli K-12 derivatives. Sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a vector plasmid pBR322, whereas the transformants' sucrose uptake activity was always lower than that of E. coli B-62. A region within an XhoI-SacI fragment (3.2 kb) of pBR322-glyA was replaced in the construction of another plasmid, pST5R7, by a fragment (about 2.6 kb) of pST622 containing the gene encoding Scr+. A genetically stable Scr+ derivative of E. coli K-12 was obtained by introducing the gene encoding Scr+ onto E. coli chromosome via homologous recombination between pST5R7 and the chromosome and subsequent plasmid segregation. The use of low-copy-number plasmid RP4 as a cloning vector was also effective for enhancing the stability of Scr+. Tryptophan producers E. coli SGIII1032S, in which the gene encoding Scr+ was cloned onto the chromosome, and E. coli SGIII1032, which carried Scr+ plasmid RP4.5R7, produced from 6% sucrose in shake flasks (33 degrees C, 96 h) 2.3 and 5.7 g of tryptophan per liter, respectively.

Biological Transport, Active↗