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S Tsuda

Publications and source records attributed to S Tsuda.

At least 55 records · Page 3Linked to original sources
PubMed2000

Effects of beta-endorphin on norepinephrine release in hypertension.

Recent studies have suggested an involvement of the endogenous opioid system in blood pressure control. The purpose of the present study was to determine the role of beta-endorphin in the regulation of sympathetic nervous activity in the central nervous system of hypertension. The effects of beta-endorphin on the electrically evoked release of [3H]norepinephrine (NE) were investigated in superfused slices of rat medulla oblongata. Beta-endorphin inhibited the stimulation-evoked NE release in a dose-dependent manner in rat medulla oblongata. In the medulla oblongata of spontaneously hypertensive rats (SHR), the inhibitory effect of beta-endorphin on the stimulation-evoked NE release was significantly smaller than in the medulla oblongata of Wistar-Kyoto rats. These results showed that beta-endorphin might reduce NE release in rat medulla oblongata. Furthermore, the lesser inhibitory effect of beta-endorphin on NE release in SHR might suggest that the opioid peptide could be involved in the regulation of central sympathetic nervous activity in hypertension.

Animals↗
PubMed2000

Efficient translocation and processing with Xenopus egg extracts of proteins synthesized in rabbit reticulocyte lysate.

Cell-free translation/translocation systems are broadly applied to examine gene expression and characterize the structure-function relationship of gene products. We present the characterization of Xenopus egg extract (XEE) translocation and processing of proteins synthesized in rabbit reticulocyte lysate. The XEE was prepared from eggs laid by adult female frogs that received serial injections of gonadotropins. The eggs were then dejellied in 2% L-cysteine-HCl and the cytoplasm extracted by centrifugation at 10,000 rpm for 15 min. The in vitro translocation and processing of XEE was examined with a cell-free translation system containing reticulocyte lysate, and appropriate messenger ribonucleic acid (RNA) or complementary deoxyribonucleic acid plasmids with RNA polymerase. Cell-free production of the following proteins were used to assess posttranslational modifications: Escherichia coli beta-lactamase for signal sequence cleavage, Saccharomyces cerevisiae alpha-mating factor for translocation and N-linked glycosylation, the soluble protein luciferase for functional activity, and the membrane-bound human insulin receptor for translation efficiency. All translation products were identified by [35S]-methionine labeling, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The results demonstrate that (1) XEE produces near-complete signal sequence and N-glycosylation processing of proteins synthesized in reticulocyte lysate, (2) XEE contains endoplasmic reticulum-equivalent microsomes, which allows for protein translocation and protease protection, (3) the addition of XEE in the translation reaction does not affect synthesis and chemiluminescence activity of luciferase, (4) XEE is efficient in processing the nascent 160-kDa human insulin receptor precursor, a transmembrane protein, and (5) as compared to canine pancreatic microsomes, XEE translocation efficiency is minimally decreased with the addition of dimethylsulfoxide. These results are the first description of the combined use of XEE with reticulocyte lysate and clearly demonstrate a higher efficiency of translocation and processing compared to canine pancreatic microsomes. This method of cell-free translation and processing allows for more extensive in vitro examination of posttranslational modifications of secretory and membrane-bound proteins.

Animals↗
PubMed2000

Rheumatoid arthritis accompanied by colonic lesions.

A 69-year-old woman with a 6-year history of rheumatoid arthritis treated solely with an orally administered NSAID had slowly progressing persistent mild abdominal pain and diarrhea, accompanied with marked sing of inflammation as well as hypoproteinemia due to protein-losing gastroenteropathy. Examinations of the large intestine revealed variously shaped ulcerative lesions, centered around the left hemicolon, as well as luminal narrowing. The course of the disease and the shape of the lesions strongly suggested involvement of rheumatoid vasculitis; oral administration of prednisolone was effective.

Aged↗
PubMed1999

The in vitro synthesized and processed human insulin receptor precursor binds insulin.

The cell-free examination of the human insulin receptor during biogenesis may provide a greater understanding of the elements that contribute to the acquisition of receptor function. The insulin receptor precursor components were produced in a cell-free system and the insulin binding ability of the [35S]methionine-labeled translation products was determined. The processed proreceptor represented by a 190 kDa band was retained on insulin-linked biotin-streptavidin agarose or an insulin column. The insulin binding 190 kDa band migrated slower than the non-binding 190 kDa band on SDS-PAGE which suggests that covalent modifications account for these differences. The trypsin-digested product of the 190 kDa proreceptor was also retained on insulin-linked biotin-streptavidin agarose, however the alpha-subunit precursor was retained on insulin agarose to a much lesser degree. We conclude that a significant fraction of the processed, in vitro translated insulin proreceptor acquires insulin binding ability.

Animals↗
PubMed1999

Low-temperature-induced structural changes in the Apo regulatory domain of skeletal muscle troponin C.

Contractile activity of skeletal muscle is triggered by a Ca2+-induced "opening" of the regulatory N-domain of troponin C (apo-NTnC residues 1-90). This structural transition has become a paradigm for large-scale conformational changes that affect the interaction between proteins. The regulatory domain is comprised of two basic structural elements: one contributed by the N-, A-, and D-helices (NAD unit) and the other by the B- and C-helices (BC unit). The Ca2+-induced opening is characterized by a movement of the BC unit away from the NAD unit with a concomitant change in conformation at two hinges (Glu41 and Val65) of the BC unit. To examine the effect of low temperatures on this Ca2+-induced structural change and the implications for contractile regulation, we have examined nuclear magnetic resonance (NMR) spectral changes of apo-NTnC upon decreasing the temperature from 30 to 4 degrees C. In addition, we have determined the solution structure of apo-NTnC at 4 degrees C using multinuclear multidimensional NMR spectroscopy. Decreasing temperatures induce a decrease in the rates and amplitudes of pico to nanosecond time scale backbone dynamics and an increase in alpha-helical content for the terminal helices of apo-NTnC. In addition, chemical shift changes for the Halpha resonances of Val65 and Asp66, the hinge residues of the BC, unit were observed. Compared to the solution structure of apo-NTnC determined at 30 degrees C, the BC unit packs more tightly against the NAD unit in the solution structure determined at 4 degrees C. Concomitant with the tighter packing of the BC and NAD structural units, a decrease in the total exposed hydrophobic surface area is observed. The results have broad implications relative to structure determination of proteins in the presence of large domain movements, and help to elucidate the relevance of structures determined under different conditions of physical state and temperature, reflecting forces ranging from crystal packing to solution dynamics.

Animals↗
PubMed1999

The alkaline single cell gel electrophoresis assay with mouse multiple organs: results with 30 aromatic amines evaluated by the IARC and U.S. NTP.

The genotoxicity of 30 aromatic amines selected from IARC (International Agency for Research on Cancer) groups 1, 2A, 2B and 3 and from the U.S. NTP (National Toxicology Program) carcinogenicity database were evaluated using the alkaline single cell gel electrophoresis (SCG) (Comet) assay in mouse organs. We treated groups of four mice once orally at the maximum tolerated dose (MTD) and sampled stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow 3, 8 and 24 h after treatment. For the 20 aromatic amines that are rodent carcinogens, the assay was positive in at least one organ, suggesting a high predictive ability for the assay. For most of the SCG-positive aromatic amines, the organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Organ-specific genotoxicity, therefore, is necessary but not sufficient for the prediction of organ-specific carcinogenicity. For the 10 non-carcinogenic aromatic amines (eight were Ames test-positive and two were Ames test-negative), the assay was negative in all organs studied. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative non-genotoxic (Ames test-negative) carcinogens. The alkaline SCG assay, which detects DNA lesions, is not suitable for identifying non-genotoxic carcinogens. The present SCG study revealed a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic non-carcinogens. These results suggest that the alkaline SCG assay can be usefully used to evaluate the in vivo genotoxicity of chemicals in multiple organs, providing for a good assessment of potential carcinogenicity.

Amines↗
PubMed1999

Determination of the solution structure of the N-domain plus linker of Antarctic eel pout antifreeze protein RD3.

RD3, a new antifreeze protein (AFP) extracted from antarctic eel pout is a single polypeptide divided into homologous N-terminal (residues Asn(1)-Glu(64)) and C-terminal (residues Ser(74)-Glu(134)) domains, each of which has a high sequence identity with Type III AFP. A 9-residue linker (-D(65)GTTSPGLK(73)-) connects these two domains in tandem and is thought to play a significant role in defining the nature of the intact molecule. The present paper shows for the first time the solution structure and preliminary (15)N-NMR backbone dynamics data of the N-domain plus the linker of recombinant RD3 protein (RD3-Nl: residues 1-73) by employing homo- and heteronuclear multidimensional NMR spectroscopy. Forty converged structures of RD3-Nl were successfully calculated by using a total of 958 NMR-derived structural restraints. It was found that the N-domain of RD3-Nl has a globular form comprising six beta-strands, three type III turns, and several loops, which stabilize a flat, ice-binding site formed on one side of this domain. Further, the linker portion appears to have a definitive structure, which is independent of the globular N-domain. This definitive linker is roughly divided into two short strands, -D(65)GTTSP(70)- and -G(71)LK(73)-, which are bent around -T(67)TSPG(71)- at an angle of approximately 60 degrees. This bending motif of the linker may function to orient the two ice-binding sites of the N- and C-domains of RD3 in the same direction, leading to their simultaneous interactions with the ice crystal surface.

Amino Acid Sequence↗
PubMed1999

Prolonged incubation period of salmonellosis in an outbreak of Salmonella enteritidis infection.

An outbreak of Salmonella enteritidis infections occurred in Otaru, Japan, in September 1997. A total of 143 cases of salmonellosis were reported to the local Public Health Center. In this outbreak, one case had a 214-hr incubation period. We investigated 5 isolates including this case by phage typing and pulsed-field gel electrophoresis (PFGE) to determine the genetic heterogeneity of S. enteritidis. Five isolates were phage typed as reacted, but did not conform (RDNC) with identical reaction patterns and had quite similar PFGE patterns. Thus, the prolonged incubation period may not be attributed to genetic heterogeneity of the organism but rather to other factors.

Adult↗
PubMed1999

Recent progress in the neurotoxicology of natural drugs associated with dependence or addiction, their endogenous agonists and receptors.

Nicotine in tobacco, tetrahydrocannabinol (delta 9-THC) in marijuana and morphine in opium are well known as drugs associated with dependence or addiction. Endogenous active substances that mimic the effects of the natural drugs and their respective receptors have been found in the mammalian central nervous system (CNS). Such active substances and receptors include acetylcholine (ACh) and the nicotinic ACh receptor (nAChR) for nicotine, anandamide and CB1 for delta 9-THC, and endomorphins (1 and 2) and the mu (OP3) opioid receptor for morphine, respectively. Considerable progress has been made in studies on neurotoxicity, in terms of the habituation, dependence and withdrawal phenomena associated with these drugs and with respect to correlations with endogenous active substances and their receptors. In this article we shall review recent findings related to the neurotoxicity of tobacco, marijuana and opium, and their toxic ingredients, nicotine, delta 9-THC and morphine in relation to their respective endogenous agents and receptors in the CNS.

Acetylcholine↗
PubMed1998

Detection of in vivo genotoxicity of haloalkanes and haloalkenes carcinogenic to rodents by the alkaline single cell gel electrophoresis (comet) assay in multiple mouse organs.

The micronucleus test is widely used to assess in vivo clastogenicity because of its convenience, but it is not appropriate for some carcinogenic chemical classes. Halogenated compounds, for example, are inconsistent micronucleus inducers. We assessed the genotoxicity of 7 haloalkanes and haloalkenes carcinogenic to rodents in 7 mouse organs-stomach, liver, kidney, bladder, lung, brain, and bone marrow-using the alkaline single cell gel electrophoresis (SCG) assay. The carcinogens we studied were 1, 2-dibromo-3-chloropropane (DBCP), 1,3-dichloropropene (mixture of cis and trans) (DCP), 1,2-dibromoethane (EDB), 1,2-dichloroethane (EDC), vinyl bromide, dichloromethane, and carbon tetrachloride; only DBCP induces micronuclei in mouse bone marrow. Except for carbon tetrachloride, halocompounds studied are mutagenic to Salmonella typhimurium. Mice were sacrificed 3 or 24 h after carcinogen administration. DCP and EDC induced DNA damage in all of the organs studied. Vinyl bromide yielded DNA damage in all of the organs except for bone marrow. DBCP induced DNA damage in the stomach, liver, kidney, lung, and bone marrow; EDB in the stomach, liver, kidney, bladder, and lung; and dichloromethane in the liver and lung. Since no deaths, morbidity, clinical signs, organ pathology, or microscopic signs of necrosis were observed, the DNA damage was not attributable to cytotoxicity. On the other hand, the positive response in the liver induced by carbon tetrachloride, which was accompanied by necrosis, was considered to be a false positive response. We suggest that the alkaline SCG assay can be used in multiple organs to detect in vivo genotoxicity that is not expressed in bone marrow cells in mice given non-necrogenic doses of halocompounds.

Alkanes↗
PubMed1998

Detection of nivalenol genotoxicity in cultured cells and multiple mouse organs by the alkaline single-cell gel electrophoresis assay.

We tested the genotoxicity of nivalenol (NIV), a potent toxic trichothecene from Fusarium nivale, in cultured CHO cells and in several mouse organs and tissues (liver, kidney, thymus, bone marrow and mucosa of stomach, jejunum, and colon) using the alkaline single-cell gel electrophoresis (SCG, or Comet) assay. NIV at 50 and 100 micrograms/ml damaged the nuclear DNA of CHO cells in the absence of S9 mix, showing that NIV was a direct mutagen. In an in vivo study, mice were sacrificed 2, 4, and 8 h after either oral (20 mg/kg) or intraperitoneal (3.7 mg/kg) administration of NIV. DNA damage was measured by the SCG assay as modified by us. After oral dosing, DNA damage appeared in the kidney and bone marrow at 2 h (returning to almost control level within the following 2 h), and in the stomach, jejunum, and colon at 2, 4, and 8 h, respectively. Liver and thymus DNA were not damaged. After intraperitoneal injection, no DNA damage appeared in any of the organs or tissues tested except for the colon, where extensive DNA damage was observed, as in the oral study, at 8 h. For histopathological examination, mice were sacrificed 2, 4, and 8 h after oral (20 mg/kg) administration of NIV. No necrotic changes were detected in any of the organs where NIV yielded statistically significant DNA damage. To measure the effect of NIV on transport activity in mice, 10 ml/kg (same volume as NIV treatments) of 1% brilliant blue FCF (BB) was administered orally. Thirty minutes later, the BB reached the colon, and simultaneous oral administration of NIV (20 mg/kg, dissolved in 10 ml BB solution) did not affect the dye transport rate. Thus, the strong yet delayed damage to colon DNA may follow from a systemic absorption rather than a topical effect. As a direct mutagen, NIV showed organ specific genotoxicity in mice in time and intensity.

Alkalies↗
PubMed1998

Organ-specific genotoxicity of the potent rodent colon carcinogen 1,2-dimethylhydrazine and three hydrazine derivatives: difference between intraperitoneal and oral administration.

We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of four hydrazine derivatives--1,2-dimethylhydrazine (SDMH), 1,1-dimethylhydrazine (UDMH), hydrazine (HZ), and procarbazine (PCZ)--in mouse liver, lung, kidney, brain, and bone marrow, and in the mucosa of stomach, colon, and bladder. Mice were sacrificed 3 and 24 h after intra-peritoneal (i.p.) and oral (p.o.) administration. SDMH at 20 mg/kg i.p. yielded statistically significant DNA damage in all tested organs except for lung. In the gastrointestinal tract, SDMH was genotoxic in the stomach and the colon after i.p. treatment but only in the colon after 20 and 30 mg/kg p.o. treatment. UDMH at 50 mg/kg i.p. yielded DNA damage in the liver and lung at 3 h. PCZ at 200 mg/kg i.p. caused DNA damage in the liver, kidney, lung, brain, and bone marrow. UDMH and PCZ were positive in the stomach and colon p.o. but not by i.p. treatment. HZ at 100 mg/kg yielded DNA damage in the stomach, liver, and lung when given i.p. and in the brain when p.o. Thus, the administration route is important when evaluating organ-specific genotoxicity in multiple organs.

1,2-Dimethylhydrazine↗
PubMed1998

Detection of pyrimethamine-induced DNA damage in mouse embryo and maternal organs by the modified alkaline single cell gel electrophoresis assay.

We studied the embryonic and maternal genotoxicity of pyrimethamine (PYR), a potent teratogen and folate antagonist, using alkaline single cell gel electrophoresis (SCG, or Comet) assay as modified by us (we used isolated nuclei instead of isolated cells). ICR mice were treated on the 13th day of pregnancy with a single oral dose of 50 mg PYR/kg. Six maternal organs (liver, kidney, lung, brain, spleen, bone marrow), maternal and fetal placentas, and two embryos were taken 6 and 16 h after treatment; the embryos were divided into head and body portions. Each sample was minced, homogenized gently, and centrifuged. The nuclei from the precipitates were used. PYR induced DNA damage in all maternal organs except spleen and bone marrow 6 h after administration. The DNA damage in all the affected organs was less at 16 h than at 6 h, and that of the kidney and brain returned to control level at 16 h. PYR also induced DNA damage in maternal and fetal placentas and embryos that was detected at 6 and 16 h, with greater damage at 6 h. Co-treatment of folinic acid calcium salt (FNA, 10 mg/kg ip), a reduced active folate form, prevented the PYR-induced DNA damage in all target tissues examined 6 h after treatment. These data indicate that the observed embryonic and maternal DNA damage caused by PYR may be related to folate deficiency, and that the modified alkaline SCG assay can be used to predict fetal/embryonic genotoxicity in vivo, in addition to the organ-specific maternal genotoxicity.

Animals↗
PubMed1998

Colon-specific genotoxicity of heterocyclic amines detected by the modified alkaline single cell gel electrophoresis assay of multiple mouse organs.

The in vivo genotoxicity of five heterocyclic amines-Trp-P-2 (13 mg/kg), IQ (13 mg/kg), MeIQ (13 mg/kg), MeIQx (13 mg/kg), and PhIP (40 mg/kg)-in the mucosa of gastrointestinal and urinary tract organs (stomach, duodenum, jejunum, ileum, colon, and bladder) was studied by the alkaline single cell gel electrophoresis (SCG) (Comet) assay. Male CD-1 mice were sacrificed 1, 3, and 8 h after intraperitoneal injection. All the heterocyclic amines studied yielded statistically significant DNA damage in the colon but not the small intestine (duodenum, jejunum, and ileum) or urinary bladder. In this study, five heterocyclic amines were injected intraperitoneally to avoid the consequences of ingestion. Thus, the extensive damage to colon DNA was concluded to be due, at least in part, to a systemic effect.

Amines↗
PubMed1998

Backbone and methyl dynamics of the regulatory domain of troponin C: anisotropic rotational diffusion and contribution of conformational entropy to calcium affinity.

The N-terminal domain (residues 1 to 90) of chicken skeletal troponin C (NTnC) regulates muscle contraction upon the binding of a calcium ion to each of its two calcium binding loops. In order to characterize the backbone dynamics of NTnC in the apo state (NTnC-apo), we measured and carefully analyzed 15N NMR relaxation parameters T1, T2 and NOE at 1H NMR frequencies of 500 and 600 MHz. The overall rotational correlation time of NTnC-apo at 29.6 degrees C is 4.86 (+/-0.15) ns. The experimental data indicate that the rotational diffusion of NTnC-apo is anisotropic with a diffusion anisotropy, D parallel/D perpendicular, of 1.10. Additionally, the dynamic properties of side-chains having a methyl group were derived from 2H relaxation data of CH2D groups of a partially deuterated sample. Based on the dynamic characteristics of TnC, two different levels of "fine tuning" of the calcium affinity are presented. Significantly lower backbone order parameters (S2), were observed for calcium binding site I relative to site II and the contribution of the bond vector fluctuations to the conformational entropy of sites I and II was calculated. The conformational entropy loss due to calcium binding (DeltaDeltaSp) differs by 1 kcal/mol between sites I and II. This is consistent with the different dissociation constants previously measured for sites I and II of 16 microM and 1. 7 microM, respectively. In addition to the direct role of binding loop dynamics, the side-chain methyl group dynamics play an indirect role through the energetics of the calcium-induced structural change from a closed to an open state. Our results show that the side-chains which will be exposed upon calcium binding have reduced motion in the apo state, suggesting that conformational entropic contributions can be used to offset the free energy cost of exposing hydrophobic groups. It is clear from this work that a complete determination of their dynamic characteristics is necessary in order to fully understand how TnC and other proteins are fine tuned to appropriately carry out their function.

Animals↗
PubMed1998

Organ-specific genotoxicity of the potent rodent bladder carcinogens o-anisidine and p-cresidine.

We used a modification of the alkaline single-cell gel electrophoresis (SCG) (Comet) assay to evaluate the in vivo genotoxicity of two potent rodent bladder carcinogens, o-anisidine and p-cresidine, in mouse liver, lung, kidney, brain, and bone marrow, and in the mucosa of stomach, colon, and bladder. Male CD-1 mice (8 weeks old) were sacrificed 3 and 24 h after oral administration of o-anisidine at 690 mg/kg or p-cresidine at 595 mg/kg. Both chemicals were dissolved in olive oil. Both chemicals yielded statistically significant DNA damage in bladder mucosa 3 and 24 h after treatment. o-Anisidine yielded DNA damage in the colon at 3 h, but not at 24 h. No significant effects were observed in any other organs. Our results suggest the importance of the urinary bladder as a sentinel organ for evaluating chemical genotoxicity in rodents.

Aniline Compounds↗
PubMed1998

Long pedunculated colonic polyp composed of mucosa and submucosa: proposal of a new entity, colonic muco-submucosal elongated polyp.

We encountered 15 patients with colonic polyps showing histologic features that did not belong to any of the known categories. All polyps were elongated and drumstick-shaped, with lengths of 12 to 160 (mean, 29 mm) mm. Histologically, the polyps were covered with normal mucosa and consisted of edematous, loose, fibrous, connective tissues and dense, fibrous submucosal layers, often showing dilation of blood vessels and lymphatics. Although the mechanism of generation of such polyps remains unknown, their elongation may be caused by intestinal motion. Because this kind of polyp has not been described previously outside Japan, we here introduce a new type of polyp, which we have proposed calling the colonic muco-submucosal elongated polyp.

Adult↗