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Biomedical subjects

S Tsuda

Publications and source records attributed to S Tsuda.

At least 91 records · Page 5Linked to original sources
PubMed1997

Simple detection of in vivo genotoxicity of pyrimethamine in rodents by the modified alkaline single-cell gel electrophoresis assay.

We tested the genotoxicity of pyrimethamine in 5 mouse and rat organs (liver, lung, kidney, spleen, and bone marrow) using a modified alkaline single-cell gel electrophoresis (SCG) (Comet) assay. Mice and rats were sacrificed 1, 3, 6, and 24 h after oral administration of the drug at 50 and 120 mg/kg, respectively. Nuclei were isolated from each tissue and evaluated for DNA migration. Pyrimethamine induced DNA damage in cells of the liver, kidney, and lung in both species. For mice, DNA damage persisted in the liver for 24 h, while it peaked in the lung and kidney at 6 and 24 h, respectively. For rats, DNA damage in the liver peaked at 1 h and returned to almost control level at 24 h. Genotoxicity in the spleen was only observed in mice. Our results suggest that the SCG technique, using isolated nuclei can be applied to rats and mice and that the optimal sampling time is different for different organs and species.

Animals↗
PubMed1997

Detection of rodent liver carcinogen genotoxicity by the alkaline single-cell gel electrophoresis (Comet) assay in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow).

We have recently designed a simple method for applying the alkaline single-cell gel electrophoresis (SCG) assay to mouse organs. With this method, each organ is minced, suspended in chilled homogenizing buffer containing NaCl and Na2EDTA, gently homogenized using a Potter-type homogenizer set in ice, and then centrifuged nuclei are used for the alkaline SCG assay. In the present study, we used the method to assess the genotoxicity of 8 rodent hepatic carcinogens in 5 mouse organs (liver, lung, kidney, spleen, and bone marrow). The carcinogens we studied were p-aminoazobenzene, auramine, 2,4-diaminotoluene, p-dichlorobenzene, ethylene thiourea (ETU), styrene-7,8-oxide, phenobarbital sodium, and benzene-1,2,3,4,5,6-hexachloride (BHC); except for p-aminoazobenzene, they do not induce micronuclei in mouse bone marrow cells. Mice were sacrificed 3 and 24 h after the administration of each carcinogen. p-Aminoazobenzene, ETU, and styrene-7,8-oxide induced alkaline labile DNA lesions in all of the organs studied. Auramine, 2,4-diaminotoluene, p-dichlorobenzene, and phenobarbital sodium also produced lesions, but their effect was greatest in the liver. BHC, which is not genotoxic in in vitro tests, did not show any effects. We suggest that it may be possible to use the alkaline SCG assay to detect in vivo activity of chemicals whose genotoxicity is not expressed in bone marrow cells.

Animals↗
PubMed1997

Simple detection of chemical mutagens by the alkaline single-cell gel electrophoresis (Comet) assay in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow).

Recently, we designed a fast and simple method to obtain nuclei for the alkaline SCG assay and we tested it with mouse liver, lung, kidney, spleen, and bone marrow. Instead of isolating organ cells by trypsinization, we homogenized tissue and isolated the nuclei. Each organ was minced, and the mince was suspended in chilled homogenizing buffer containing NaCl and Na2EDTA, homogenized gently using a Potter-type homogenizer set in ice, and then centrifuged. The nuclei from the precipitate were used for the assay. To evaluate the validity of this method, we tested the genotoxicity in mouse organs of 11 chemical mutagens with different modes of action. Mice were sacrificed 3 and 24 h after administration of each mutagen. Treatment with three alkylating agents (MMS, EMS, and MNNG), a DNA crosslinking agent (MMC), two aromatic amines (2-AAF and phenacetin), a polycyclic aromatic hydrocarbon (B[a]P), and two inorganic chemicals (KBrO3 and K2CrO4) increased migration of the DNA from mouse organs. 5-FU (a base analog) and colchicine (a spindle poison) treatment produced negative results in all organ studied. Considering that the alkaline SCG assay detects genotoxicity as DNA fragments derived from DNA single-strand breaks and alkali-labile damage, our results showed that the SCG assay using our homogenization technique detected chemical mutagens as a function of their modes of action.

Alkylating Agents↗
PubMed1997

A hairpin-loop conformation in tandem repeat sequence of the ice nucleation protein revealed by NMR spectroscopy.

The 1H-NMR spectrum of a synthetic 24-residue peptide (A1-G-V-D-S-S-L-I-A-G-Y-G-S-T-Q-T-S-G-S-D-S-A-L-T24; INP24), comprising three repeats of the 8-residue consensus sequence of Pseudomonas syringae ice nucleation protein, was fully assigned using 2-dimensional (2D) NMR spectroscopy at 4 degrees C and 30 degrees C. Close proximity of the aliphatic protons between Leu7, Ile8, Ala9, and the ring-protons of Tyr11 was indicated from the observation of the inter-molecular nuclear Overhauser enhancement (NOE) effect. Hydrogen-bonding was strongly suggested for the NH group of Leu7 from its extremely low-temperature coefficient estimated from the temperature dependence of the chemical shift. These results indicate the formation of a hairpin-loop conformation constructed by a hexapeptide segment of INP24, -Leu7-Ile8-Ala9-Gly10-Tyr11-Gly12.

Amino Acid Sequence↗
PubMed1997

Modulation of [3H]dopamine release by neuropeptide Y in rat striatal slices.

Neuropeptide Y, a 36-amino-acid peptide, has a wide and specific distribution in the central nervous system. In this study we examined the regulatory mechanisms of neuropeptide Y on dopamine release in the rat central nervous system. The effects of neuropeptide Y on the electrically stimulated [3H]dopamine release were investigated in superfused striatal slices of Sprague-Dawley rats, spontaneously hypertensive rats and Wistar-Kyoto rats. Neuropeptide Y (1 x 10(-8) - 1 x 10(-7) mol/1) reduced the stimulation (1 Hz)-induced [3H]dopamine release by a comparable amount in Sprague-Dawley rats. The blockade of dopamine D2 receptors by the dopamine D2 receptor antagonist, sulpiride, diminished the inhibitory effects of neuropeptide Y on the stimulation-evoked [3H]dopamine release. Pretreatment of slices with pertussis toxin (a potent inhibitor of G1-proteins) attenuated the suppression of the stimulation-evoked [3H]dopamine release by neuropeptide Y. Unlabelled dopamine itself reduced the stimulation-evoked [3H]dopamine release, and the inhibitory effect was also attenuated in the pertussis toxin-pretreated slices. In spontaneously hypertensive rats, the inhibitory effect of neuropeptide Y on the stimulation-evoked [3H]dopamine release was more pronounced than that in Wistar-Kyoto rats. The results of the present study showed that neuropeptide Y inhibited the stimulation-evoked dopamine release partially mediated by dopamine D2 receptors and the pertussis toxin-sensitive G1-proteins in rat striatum. Furthermore, the greater effect of neuropeptide Y on dopamine release in spontaneously hypertensive rats suggests a possible involvement of the peptide in regulating the central dopaminergic nerve activity in hypertension.

Animals↗
PubMed1997

Detection of chemically induced DNA lesions in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow) using the alkaline single cell gel electrophoresis (Comet) assay.

The effect of 2 model chemical mutagens on DNA was evaluated with the alkaline single cell gel electrophoresis (SCG) (Comet) assay in 5 mouse organs--liver, lung, kidney, spleen and bone marrow. Mice were sacrificed 3 and 24 h after the administration of the direct mutagen ethyl nitrosourea (ENU) or the liver-targeting promutagen p-dimethylaminoazobenzene (DAB). Each organ was minced, suspended at a concentration of 1 g/ml in chilled homogenizing buffer (pH 7.5) containing 0.075 M NaCl and 0.024 M Na2EDTA, homogenized gently using a Potter-type homogenizer at 500-800 rpm set in ice, and then centrifuged nuclei were used for the alkaline SCG assay. ENU induced DNA damage in cells all of the organs studied DAB, on the other hand, produced a positive response in the liver only. We suggest that it may be possible to use the alkaline SCG assay using a homogenization technique to detect the genotoxicity of chemicals in vivo in their target organs.

Animals↗
PubMed1997

Effect of dietary zinc content on 65Zn metabolism in mice.

65Zn is one of the induced radioactive nuclides which are generated in power reactors. In the present experiment, several parameters of 65Zn metabolism were studied in mice maintained on diets with various zinc contents from 45 to 4,500 mg/kg to evaluate the efficacy of the dilution method for radiation protection against internal contamination with 65Zn. Gastrointestinal absorption of 65Zn was suppressed and its excretion accelerated as the dietary zinc content increased over a wide range. Clearance of 65Zn from tissues was generally accelerated by feeding mice a high-zinc diet, but that from the femurs was not affected by dietary zinc content. Zinc concentrations in tissues were regulated homeostatically up to a dietary zinc content of 1,350 mg/kg. Although a significant accumulation of zinc occurred in the liver, pancreas, kidneys, and femurs when mice were given 4,500 mg/kg diet, the concentrations except in the femurs recovered within a few days after switching to a normal-zinc diet. These results suggest that oral administration of zinc is effective for preventing the absorption and for enhancing the excretion of 65Zn to protect the body from internal radiation exposure with this isotope.

Animals↗
PubMed1997

Relationship between turnover of cesium-137 and dietary potassium content in potassium-restricted mice.

The biological half-life of 137Cs and its organ distribution were investigated in mice fed various potassium-deficient diets. The biological half-life, which was 6.1 days in mice receiving the normal level of potassium, became longer as the dietary potassium content decreased, and 137Cs was hardly excreted from the body when dietary potassium content was restricted to 200 mg/kg or less. The muscle showed the highest concentration of 137Cs in both mice that had sufficient amounts of potassium and those that were potassium-deficient. Clearance of 137Cs from tissues was generally suppressed when mice were fed a potassium-deficient diet, but the relative distribution pattern of 137Cs was not affected by dietary potassium content. These results suggest that dietary potassium intake, which may vary with eating habits, affects the biological half-life of 137Cs in humans.

Animals↗
PubMed1996

Factor XSt. Louis II. Identification of a glycine substitution at residue 7 and characterization of the recombinant protein.

A molecular defect in factor X (fX) results from a point mutation that causes glycine substitution for gamma-carboxylated glutamic acid at position 7. The variant (fXSt. Louis II) and wild type (fXWT) proteins were produced in a mammalian expression system and characterized. fXSt. Louis II has <1% and approximately 3% of normal clotting activity in modified prothrombin time and partial thromboplastin time assays, respectively. The rate of activation of fXSt. Louis II by factor VIIa and tissue factor is undetectable under conditions that result in complete activation of fXWT; activation by factors VIIIa and IXa is approximately 30% of normal activation. The X-activating protein from Russell's viper venom activates fXSt. Louis II completely but at a reduced rate. Thrombin generation on phoshopolipid vesicles or activated platelets is approximately 30% or approximately 5%, respectively. Membrane-dependent autolysis is markedly reduced for fXSt. Louis II. In reactions that are not surface-dependent, fXSt. Louis II is nearly identical to that of fXWT. The rate of inhibition by antithrombin is indistiguishable, as is the rate of thrombin formation in the absence of phospholipid, with or without factor Va.

Antithrombin III↗
PubMed1996

Effect of glimepiride (HOE 490) on insulin receptors of skeletal muscles from genetically diabetic KK-Ay mouse.

A new sulfonylurea, glimepiride (HOE 490), has been developed for the glycemic control in non-insulin-dependent diabetes mellitus. We examined the effect of glimepiride on glucose and insulin levels in KK-Ay mice, an animal model of non-insulin-dependent diabetes mellitus, which is characterized by hyperglycemia and hyperinsulinemia. Administration of glimepiride (0.5 mg/kg/day) for 8 weeks to KK-Ay mice resulted in decrease in glucose (297 +/- 36 to 250 +/- 51 mg/dl) and insulin (76 +/- 14 to 41 +/- 14 microU/ml) levels. To clarify the mechanism of the agent, we examined the effect of this new drug on insulin receptors in the skeletal muscles. There was no difference in insulin binding to the receptors from both glimepiride-treated and -untreated KK-Ay mice muscles. The insulin-stimulated autophosphorylation of insulin receptors from KK-Ay mice was decreased compared to that from normal mice (5 +/- 1 vs. 39 +/- 13% over basal). Glimepiride did not ameliorate impaired insulin-stimulated insulin receptor autophosphorylation. To determine the effect of glimepiride on post-insulin receptor signaling pathway, we measured 2-[3H]glycerol incorporation into diacylglycerol in the cultured rat fibroblast cell line overexpressing human insulin receptors. Glimepiride (100 microM) as well as insulin (10 nM) significantly stimulated diacylglycerol production. These results suggest that glimepiride has a potent extrapancreatic effect on glucose metabolism and may directly stimulate glucose transport activity through phospholipid signaling pathway, but not through insulin receptor kinase signaling pathway.

Animals↗
PubMed1996

Il-7 supports D-J but not V-DJ rearrangement of TCR-beta gene in fetal liver progenitor cells.

The rearrangement of TCR-beta gene, one of the earliest events in T cell development, consists of two consecutive steps: D-J rearrangement and V-DJ rearrangement. The present study examined the signals supporting D-J beta and V-DJ beta rearrangements during early T cell development from progenitor cells that reside in fetal liver. We have found that there is an interval of 1 to 2 days between D-J beta and V-DJ beta rearrangements during the early T cell development from fetal liver progenitor cells in deoxyguanosine-treated thymus lobes. We have also found that IL-7, a cytokine expressed in the subcapsular area of the thymus, can promote D-J beta rearrangement of fetal liver progenitor cells, and that anti-IL-7 and anti-IL-7R Abs inhibit the D-J beta rearrangement and further T cell development of fetal liver progenitor cells in the thymus environment. Interestingly, unlike the thymus environment, IL-7 alone was not capable of supporting V-DJ beta rearrangement in the fetal liver cell cultures. These results indicate that D-J beta rearrangement during fetal liver-derived early T cell development is supported in the thymus by IL-7. Furthermore, the present results demonstrate that IL-7, supporting D-J beta rearrangement, does not promote V-DJ beta rearrangement of fetal liver progenitor cells, suggesting that intrathymic molecules promoting V-DJ beta rearrangement are distinct from IL-7 that supports the D-J beta rearrangement.

Animals↗
PubMed1996

CD3-induced apoptosis of CD4+CD8+ thymocytes in the absence of clonotypic T cell antigen receptor.

Clonal selection of T cells mediated through the T cell antigen receptor (TCR) mostly occurs at the CD4+CD8+ double positive thymocyte stage. Immature CD4+CD8+ thymocytes expressing self-reactive TCR are induced to die upon clonotypic engagement of TCR by self antigens. CD3 engagement by antibody of the surface TCR-CD3 complex is known to induce apoptosis of CD4+CD8+ thymocytes, a process that is generally thought to represent antigen-induced negative selection in the thymus. The present study shows that the CD3-induced apoptosis of CD4+CD8+ thymocytes can occur even in TCR alpha- mutant mice which do not express the TCR alpha beta/CD3 antigen receptor. Anti-CD3 antibody induces death of CD4+CD8+ thymocytes in TCR alpha- mice either in cell cultures or upon administration in vivo. Interestingly, most surface CD3 chains expressed on CD4+CD8+ thymocytes from TCR alpha- mice are not associated with clonotypic TCR chains, including TCR beta. Thus, apoptosis of CD4+CD8+ thymocytes appear to be induced through the CD3 complex even in the absence of clonotypic antigen receptor chains. These results shed light on previously unknown functions of the clonotype-independent CD3 complex expressed on CD4+CD8+ thymocytes, and suggest its function as an apoptotic receptor inducing elimination of developing thymocytes.

Animals↗
PubMed1996

Nucleotide sequence of the capsid protein gene of papaya leaf-distortion mosaic potyvirus.

The DNA complementary to the 3'-terminal 1 404 nucleotides [excluding the poly(A) tail] of papaya leaf-distortion mosaic potyvirus (PLDMV) RNA was cloned and sequenced. The sequence starts within a long open reading frame (ORF) of 1 195 nucleotides and is followed by a 3' non-coding region of 209 nucleotides. Capsid protein (CP) is encoded at the 3' terminus of the ORF. The CP contains 293 residues and has a Mr of 33 277. The CP of PLDMV exhibits 49 to 59% sequence similarity at the amino acid level to the CPs of papaya ringspot potyvirus (PRSV) and other potyviruses. This result is consistent with the absence of a serological relationship between PLDMV and PRSV or other potyviruses. The results support the assignment of PLDMV as a distinct member of the genus Potyvirus.

Amino Acid Sequence↗
PubMed1996

Glutamatergic regulation of [3H]acetylcholine release in striatal slices of normotensive and spontaneously hypertensive rats.

It has been proposed that central cholinergic neurons may actively participate in blood pressure control and other cardiovascular regulations. The present study was performed to investigate the role of the glutamate receptors in the regulation of acetylcholine release in rat central nervous system in vitro. In the Mg2+-free condition, L-glutamate, an endogenous ligand for glutamate receptors, elicited [3H]acetylcholine release from striatal slices of Sprague-Dawley rats in a dose-related fashion. Glycine, an allosteric agonist for the N-methyl-D-aspartate type of glutamate receptor, significantly potentiated the increase in [3H]acetylcholine release evoked by L-glutamate. A non-competitive N-methyl-D-aspartate receptor antagonist, MK-801, blocked the L-glutamate-induced increase in [3H]acetylcholine release, although MK-801 had no effects on its own. In spontaneously hypertensive rats, the facilitatory effect of L-glutamate on [3H]acetylcholine release was significantly smaller than that in Wistar-Kyoto rats. Moreover, L-glutamate in combination with glycine increased the release of [3H]acetylcholine to a lesser extent in SHR than in WKY rats. These results show that L-glutamate increased acetylcholine release from rat striatum, which was highly dependent on the N-methyl-D-aspartate type of glutamate receptor. Furthermore, the lesser facilitation of acetylcholine release by L-glutamate in spontaneously hypertensive rats suggests that the excitatory amino acid may be, at least in part, involved in the regulation of central cholinergic nerve activity in hypertension.

Acetylcholine↗
PubMed1996

Heterogeneous expression of recombination activating genes and surface CD5 in CD3low CD4+ CD8+ thymocytes.

Clonal selection in the thymus occurs mostly in the CD4+ CD8+ (double positive; DP) stage. Within DP thymocytes, cells in the CD3low subset are believed to be involved in clonal selection, and this subset is generally considered as a homogeneous population. T-cell antigen-receptor (TCR) signals on DP thymocytes are known to (i) down-regulate recombination activating gene (RAG) expression; and (ii) up-regulate the expression of T-cell function-associated molecules, including the cell surface glycoprotein CD5. The present study examined the expression of RAG and CD5 molecules among the subpopulations of normal adult DP thymocytes. DP thymocytes were fractionated according to their cell surface expression levels of TCR-CD3 complex into CD3dim, CD3lo, CD3med, and CD3hi cells, since TCR expression is known to increase during thymocyte maturation. Down-regulation of RAG mRNA was located between the CD3low and CD3med DP populations. However, within the DP CD3low subpopulation, we found that CD5 varies from low to high expression levels. Upon fractionation of DP CD3low thymocytes into CD5low and CD5high subsets, we were able to detect down-regulation of RAG transcripts within the CD3low subpopulation. Thus, by the criteria of CD5 surface expression and RAG mRNA expression levels, DP CD3low thymocytes can be considered a heterogeneous thymocyte subpopulation.

Animals↗
PubMed1996

Secondary structure and Ca(2+)-binding property of the N-terminal half domain of calmodulin from yeast Saccharomyces cerevisiae as studied by NMR.

Using two- and three-dimensional NMR techniques, 1H and main-chain 15N resonances of the N-terminal half domain of yeast calmodulin (YCM0-N) in the presence of Mg2+ and Ca2+ (Mg(2+)-and Ca(2+)-forms) were assigned. The secondary structures of YCM0-N in both forms were determined. The NOESY and 15N-edited NOESY spectra of YCM0-N in each form indicate that there is a hydrophobic core and that two Ca(2+)-binding loops are connected by a short antiparallel beta-sheet. There are four helices (A, B, C, and D named from the N-terminus) for YCM0-N in the Mg(2+)-form. The B-helix is, however, not formed in the Ca(2+)-form. The Ca(2+)-binding of YCM0-N was monitored by (1H,15N)-HSQC at various Ca2+ concentrations. The observed spectral changes as a function of Ca(2+)-concentration can not readily be grouped into a small number of classes; each residue shows individual spectral change. There is no apparent relationship between the spectral change and the type or location of the amino acid concerned.

Amino Acid Sequence↗
PubMed1996

Serum levels of eosinophil cationic protein reflect the state of in vitro degranulation of blood hypodense eosinophils in atopic dermatitis.

In patients with atopic dermatitis (AD), serum levels of eosinophil cationic protein (ECP) have been shown to be a good reflector of disease severity. To elucidate what serum levels of ECP actually reflect, ECP levels in serum and plasma and cytological aspects of blood eosinophils were examined in AD patients (n = 27) and compared to healthy subjects (n = 12). Significantly elevated levels of serum ECP were noted in AD patients, while plasma ECP were uniformly recorded at nadir levels in both AD patients and normal subjects. In addition to blood eosinophilia, AD patients had significantly increased numbers of hypodense eosinophils (HEo) with morphological characteristics consistent with an activated state. Serum ECP levels strongly correlated with HEo numbers rather than with total eosinophil counts. These results indicate that elevated levels of serum ECP may be a consequence of in vitro degranulation of "activated" HEo, not of ECP supplementation from lesional skin. In addition, the dynamic correlations of eosinophil-associated parameters (total eosinophil counts, HEo numbers, and serum ECP levels) with AD severity suggest that inflammatory events in lesional skin may be involved in causing not only eosinophilopoiesis in bone marrow, but also development of HEo in the periphery, whose degree in turn may be mirrored in the levels of serum ECP in vitro.

Adult↗