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Biomedical subjects

K Tsukamoto

Publications and source records attributed to K Tsukamoto.

At least 73 records · Page 4Linked to original sources
PubMed2000

Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains.

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.

Amino Acid Sequence↗
PubMed2000

Application of representational difference analysis to genomic fragments of Marek's disease virus.

A rapid and simple method for isolation of DNA fragments of Marek's disease virus (MDV) based on representational difference analysis (RDA) was developed. Multiple viral DNA fragments, the sizes of which were restricted to 0.3 to 3.5 kbp, were simultaneously amplified after subtraction of chicken DNA from BamHI-, BglII-, EcoRI-, HindIII-, or XhoI-digested DNA fragments of MDV-infected cells. Nucleotide sequence of two RDA-derived fragments coincided with the sequence determined from direct sequencing of the MDV genome. We detected an interstrain difference in the size of restriction enzyme-digested fragments on agarose gel. This method was used on a single feather pulp to generate sufficient MDV DNA for cloning.

Animals↗
PubMed2000

Avian nephritis virus (ANV) as a new member of the family Astroviridae and construction of infectious ANV cDNA.

The complete RNA genome of the avian nephritis virus (ANV) associated with acute nephritis in chickens has been molecularly cloned and sequenced. Excluding the poly(A) tail, the genome comprises 6,927 nucleotides and contains three sequential open reading frames (ORFs). The first ORF (ORF 1a) contains a sequence encoding a serine protease motif, and the second ORF (ORF 1b) has a sequence encoding an RNA-dependent RNA polymerase. ORF 1a may be linked to the second ORF by a ribosomal frameshifting mechanism. The third ORF (ORF 2) may encode the virion structural proteins as a polyprotein precursor. Two RNAs, probably genonic and subgenonic RNA (7.5 and 3.0 kb), were detected in the cytoplasm of ANV-infected cells. ANV and human astroviruses have the same genonic organization, and both are characterized by the presence of two RNA bands. The amino acid homologies of the products of ORF 1a, 1b, and 2 were 20.3, 41.9, and 25.8% to products of the corresponding ORFs of human astrovirus serotype 1 (A/88 Newcastle strain). We have constructed a genonic-length cDNA clone of ANV to test whether the in vitro transcript is infectious. When a chicken kidney cell culture was transfected with RNA transcribed in vitro and the cDNA clone, infectious virus was produced with cytopathic effects in the absence of trypsin. These observations suggested that the ANV (G-4260 strain) is a new genus of the family Astroviridae.

Animals↗
PubMed2000

Involvement of Epstein-Barr virus latent membrane protein 1 in disease progression in patients with idiopathic pulmonary fibrosis.

BACKGROUND: The role of Epstein-Barr virus (EBV) in idiopathic pulmonary fibrosis (IPF) is uncertain. A study was undertaken to detect the virus in IPF as well as to clarify the influence of EBV on the clinical features of the disease. METHODS: Twenty nine lung specimens were obtained from patients with IPF, as well as five specimens from patients with systemic sclerosis with pulmonary fibrosis (SSc) and 15 specimens from controls. EBV DNA and EBV latent membrane protein 1 (LMP1) were detected using the PCR method and immunohistochemical analysis, respectively. RESULTS: EBV DNA was detected in 24 of 25 patients with IPF (96%), in all five patients with SSc (100%), and in 10 of 14 controls (71%). The detection ratio was significantly higher in patients with IPF than in controls (p = 0.047, odds ratio (OR) = 9.60, 95% confidence interval (CI) 0.9 to 96.9). Immunohistochemical analysis revealed that cuboidal epithelial cells were positively stained with anti-LMP1 antibody in nine of the 29 lung specimens from IPF patients. In contrast, neither the patients with SSc nor the control subjects showed positive staining. In the follow up periods LMP1 positive patients with IPF died more frequently from respiratory failure than LMP1 negative patients (4/9 versus 1/20; p = 0.022, OR = 15.20, 95% CI 1. 3 to 168.0). CONCLUSIONS: EBV LMP1 positivity may be associated with more rapid disease progression in IPF.

Adult↗
PubMed2000

In vitro pharmacological profile of SK-896, a new human motilin analogue.

SK-896 ([Leu(13)]motilin-Hse) is a new human motilin analogue synthesized by Escherichia coli using a biotechnological method. We investigated the binding of SK-896 to motilin receptors and the contractile effect of SK-896 on smooth muscle preparations isolated from the gastrointestinal tract and various regional organs in order to clarify its in vitro pharmacological profile. SK-896 inhibited the binding of (125)I-human motilin to rabbit gastroduodenal motilin receptors with the same potency as unlabeled human motilin. The IC(50) values of SK-896 and human motilin were 3.5 +/- 1.5 and 3.1 +/- 1.8 nmol/l, respectively. The K(d) of human motilin was 3.0 +/- 1.5 nmol/l, and the Ki of SK-896 was 3.4 +/- 1.5 nmol/l. SK-896 induced contraction of smooth muscle preparations isolated from rabbit duodenum in a concentration-dependent manner. However, there was no effect of SK-896 on duodenal preparations isolated from the dog and the rat. SK-896 thus exhibited species specificity in its contractile effect. We next investigated the effect of SK-896 on various smooth muscle preparations isolated from rabbit gastrointestinal tract, trachea, bladder, gallbladder, uterus, vas deferens and artery. Results showed that SK-896 induced contraction of smooth muscle preparations isolated from gastrointestinal tract, with potencies in the order duodenum > gastric pylorus = jejunum = descending colon > ascending colon >/= ileum. However, there was no effect of SK-896 on smooth muscle preparations from gastric fundus and other regional organs. SK-896 thus exhibited regional specificity in its contractile effect. Moreover, the effects of SK-896 on smooth muscle preparations from rabbit duodenum were the same as those of human motilin, and were not inhibited by pretreatment with tetrodotoxin and atropine but were inhibited by verapamil. These findings indicate that SK-896 has the same pharmacological profile as human motilin. They suggest that SK-896 acts on gastrointestinal smooth muscle isolated from rabbit directly and specifically.

Animals↗
PubMed2000

Relationship between the serum level of leptin and life-style habits in Japanese men.

AIMS/METHODS: To investigate whether smoking affects the serum level of leptin, 708 male workers aged 25-65 years old were cross-sectionally surveyed. RESULTS: Multiple regression analysis indicated that among the various parameters examined, the level of leptin was positively associated with the body mass index and the levels of insulin, total cholesterol and uric acid, and was inversely associated with physical activity and the level of creatinine. The partial correlation coefficient of leptin was highest against the body mass index (r = 0.40), followed by insulin (0.29) and physical activity (-0.14), after adjustment for other leptin-related variables. However, no association was observed between the level of leptin and smoking (0.05), alcohol consumption (0.09) or age (0.09). CONCLUSIONS: The findings suggest that among life-style habits, physical activity, but not smoking or alcohol consumption, significantly affects the serum level of leptin in Japanese men.

Adult↗
PubMed2000

Excitatory amino acids in the rostral ventrolateral medulla support blood pressure in spontaneously hypertensive rats.

Injection of the excitatory amino acid (EAA) antagonist kynurenic acid (KYN) into the rostral ventrolateral medulla (RVLM) of anesthetized rats has no effect on arterial pressure. However, we recently reported that after inhibition of the caudal ventrolateral medulla, injection of KYN into the RVLM decreased arterial pressure to the same level as produced by complete inhibition of the RVLM. We have suggested that these results reflect tonically active EAA-mediated inputs to the RVLM producing both direct excitation of RVLM vasomotor neurons and indirect inhibition of these neurons. On the basis of this model, we hypothesize that the balance between these EAA-driven direct excitatory and indirect inhibitory influences on the RVLM may be altered in models of experimental hypertension. To begin to test this hypothesis, the effects of injecting KYN into the RVLM of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were compared. In chloralose-anesthetized WKY, bilateral injection of KYN into the RVLM did not alter arterial pressure, whereas similar injections in SHR reduced mean arterial pressure by approximately 40 mm Hg. After inhibition of the caudal ventrolateral medulla, which similarly increased arterial pressure in both strains, injection of KYN into the RVLM reduced mean arterial pressure to the same level as produced by autonomic blockade. These results suggest that the balance of excitatory and inhibitory influences on RVLM vasomotor neurons driven by tonically active EAA-mediated inputs to the RVLM is disrupted in SHR and may contribute to the hypertension in SHR.

Animals↗
PubMed2000

Characteristics factor structures of the Japanese version of the State-Trait Anxiety Inventory: coexistence of positive-negative and state-trait factor structures.

Previous factor studies of the State-Trait Anxiety Inventory (STAI; Spielberger, Gorsuch, & Lushene, 1970) have reported certain typical factors that are state-trait (S-T) 2-factor solutions and positively-negatively (P-N) worded item 2-factor solutions in addition to 4-factor solutions (positively and negatively worded state factors, positively and negatively worded trait factors). We explored the possibility that these factor structures are included in a factor space. Responses to the Japanese version of the STAI in a sample of 848 male workers were factor analyzed. The first-order factors obtained from principal-component analysis were almost equal to the previous 4 factors, except for a minor factor, and their second-order factors were the P-N factors. However, the S-T factors were also obtained from the same first-order factors by the oblique Procrustes rotation. Moreover, coexistence of these two 2-factor structures was determined in the same factor space by the orthogonal Procrustes rotation.

Adult↗
PubMed2000

Expression of leptin receptor in human endometrium and fluctuation during the menstrual cycle.

Leptin is secreted by adipocytes and regulates appetite through interaction with hypothalamic leptin receptors (OB-R). Accumulated evidence shows that leptin is involved in the stimulation of reproductive functions and that local expression of leptin and OB-R in the ovary, oocyte, embryo, and placenta plays a role in early development. To investigate the role of leptin in implantation, we examined the expression of OB-R and leptin in the human endometrium. Northern and Western blot analyses and RT-PCR showed that the long form of OB-R (OB-R(L)) messenger ribonucleic acid (mRNA) and protein were expressed. In contrast, leptin mRNA or protein was not detected. All of the splice variants of OB-R (OB-R(T)) and OB-R(L) transcripts were expressed in 90% and 84% of the cases, respectively. OB-R mRNA expression peaked in the early secretory phase. Decidual tissue of early gestation also expressed OB-R(T) and OB-R(L). Their incidence and abundance were comparable among endometria with benign uterine diseases and disease-free endometria and were not related to a body mass index within the normal range. The present results indicate that OB-R, but not leptin, is expressed in the human endometrium.

Carrier Proteins↗
PubMed2000

Progesterone induction of 17beta-hydroxysteroid dehydrogenase type 2 during the secretory phase occurs in the endometrium of estrogen-dependent benign diseases but not in normal endometrium.

In the human endometrium, inactivation of 17beta-estradiol to estrone is catalyzed by 17beta-hydroxysteroid dehydrogenase type 2 (17betaHSD2). Previous studies have shown that the 17betaHSD2 activity in the endometrium is elevated during the secretory phase, as compared with the level during the proliferative phase, and that the elevation is in response to progesterone via the progesterone receptors. Recently, it has been demonstrated that aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis, is not present in the endometrium obtained from normal menstruating women with cervical cancer in situ showing no other gynecological disease (defined as "disease free"), but present in the endometrium obtained from patients with endometriosis, adenomyosis, and/or leiomyomas (defined as "diseased"). However, the previous 17betaHSD studies have been performed without distinguishing between disease-free and diseased endometria. We, therefore, analyzed 17betaHSD2 distinguishing between disease-free and diseased endometria. During the proliferative phase, the abundance of messenger RNA (mRNA) and activity of 17betaHSD2 were comparable in both disease-free and diseased endometrium. However, during the secretory phase, while the abundance of mRNA and activity of 17betaHSD2 increased 4- to 6-fold in diseased endometrium, the 17betaHSD2 remained unchanged in the disease-free endometrium. Kinetic studies showed that the Km was identical among the four groups of endometria, suggesting that the elevation of 17betaHSD2 simply resulted from increased mRNA transcription. Organ culture of proliferative endometria in the presence of progestins resulted in the stimulation of 17betaHSD2 in diseased endometria via the progesterone receptors, whereas disease-free endometrium was not stimulated by progestins. These results suggest that the previous paradigm that 17betaHSD2 activity in the endometrium is elevated during the secretory phase is confined to diseased endometrium but not to disease-free endometrium and that the estrogen metabolism is altered in the endometria of the patients with estrogen-dependent benign diseases.

17-Hydroxysteroid Dehydrogenases↗
PubMed2000

Facilitation of acetylcholine release by SK-951, a benzofuran derivative, via the 5-hydroxytryptamine4 receptor in guinea pig stomach.

Facilitation of acetylcholine (ACh) release by SK-951 ((-)4-amino-N-[2-(1-azabicyclo[3.3.0] octan-5-yl)ethyl]-5-chloro-2,3-dihydro-2-methylbenzo[b]furan-7-carboxami de hemifumarate), a benzofuran derivative, via the 5-hydroxytryptamine (5-HT)4 receptor in guinea pig stomach was examined by in vitro receptor autoradiography and functional studies. [125I]SB207710 binding was detected in the myenteric plexus of the gastric corpus. High densities of binding sites were observed in the myenteric plexus and a moderate density in the muscle layer. SK-951 inhibited the binding of [125I]SB207710, a specific 5-HT4-receptor ligand, as in the case of SB204070, a specific 5-HT4-receptor antagonist, thus indicating the presence of 5-HT4 receptors in guinea pig stomach. SK-951 as well as 5-HT enhanced the electrically stimulated twitch contractions of gastric corpus strips, which were sensitive to tetrodotoxin and atropine, and enhanced electrically stimulated release of ACh from corporal strips, which was tetrodotoxin-sensitive and Ca2+-dependent. The enhancements of twitch contractions and ACh release by SK-951 were antagonized by GR113808, a selective 5-HT4-receptor antagonist. Thus, SK-951 binds to 5-HT4 receptors of the guinea pig gastric corpus and may accelerate gastric motility due to facilitation of ACh release.

Acetylcholine↗
PubMed2000

Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats 11). Testicular toxicity of 2- or 4-week repeated or single-dose administration of a novel platinum complex.

A novel platinum complex, compound C, developed as an antitumor agent, was intravenously administered at 10 mg/kg/day to 6-week-old and 8-week-old rats. After 4 weeks of administration to the former, testicular enlargement was observed at a similar incidence as testicular atrophy and dilatation or atrophy of the seminiferous tubules was observed. Degeneration/necrosis of the seminiferous epithelium, decrease of seminiferous epithelium, formation of multinucleated giant cells, and vacuolar degeneration of Sertoli cells were also seen. These lesions were more marked in seminiferous tubules with atrophy than in those without atrophy. After 2-week administration to 8-week-old rats, slight initial-phase findings such as dilatation of seminiferous tubules and degeneration/necrosis of the seminiferous epithelium were noted in all rats. In rats administered 20 mg/kg/day, for which the administration period was shortened to 1 week due to marked weight loss, very slight initial lesions were similarly observed. However, these lesions were not easy to detect in these rats. Following single administration of a sublethal dose (80 mg/kg), testicular lesions were clearly observed 14 days after administration, but the incidence and grade of lesions were very low at 7 days. In conclusion, testicular toxicity of compound C can be clearly detected after 2-week administration, although the progression of lesions differed from the case with 4-week administration.

Animals↗
PubMed2000

Markedly increased secretion of VLDL triglycerides induced by gene transfer of apolipoprotein E isoforms in apoE-deficient mice.

Apolipoprotein E (apoE) plays a key role in the receptor-mediated uptake of lipoproteins by the liver and therefore in regulating plasma levels of lipoproteins. ApoE may also facilitate hepatic secretion of very low density lipoprotein (VLDL) triglyceride (TG). We directly tested the hypothesis that reconstitution of hepatic apoE expression in adult apoE-deficient mice by gene transfer would acutely enhance VLDL-TG production and directly compared the three major human apoE isoforms using this approach. Second generation recombinant adenoviruses encoding the three major isoforms of human apoE (E2, E3, and E4) or a control virus were injected intravenously into apoE-deficient mice, resulting in acute expression of the apoE isoforms in the liver. Despite the expected decreases in total and VLDL cholesterol levels, apoE expression was associated with increased total and VLDL triglyceride levels (E2 > E4 > E3). The increase in TG levels significantly correlated with plasma apoE concentrations. In order to determine whether acute apoE expression influenced the rate of VLDL-TG production, additional experiments were performed. Three days after injection of adenoviruses, Triton WR1339 was injected to block lipolysis of TG-rich lipoproteins and VLDL-TG production rates were determined. Mice injected with control adenovirus had a mean VLDL-TG production rate of 74 +/- 7 micromol/h/kg. In contrast, VLDL-TG production rates in apoE-expressing mice were 363 +/- 162 micromol/h/kg, 286 +/- 175 micromol/h/kg, and 300 +/- 84 micromol/h/kg for apoE2, apoE3, and apoE4, respectively. The VLDL-TG production rates in apoE-expressing mice were all significantly greater than in control mice but were not significantly different from each other. In summary, acute expression of all three human apoE isoforms in livers of apoE-deficient mice markedly increased VLDL-TG production to a similar degree, consistent with the concept that apoE plays an important role in facilitating hepatic VLDL-TG production in an isoform-independent manner.

Adenoviridae↗
PubMed2000

Hepatic apolipoprotein E expression promotes very low density lipoprotein-apolipoprotein B production in vivo in mice.

In addition to its role in the uptake of apolipoprotein B (apoB)-containing lipoproteins, apoE promotes hepatic very low density lipoprotein-triglyceride (VLDL-TG) production in animal models. However, it is not known if apoE increases the amount of TG per VLDL particle or the number of VLDL particles secreted. VLDL-apoB production is a measure of the rate of VLDL particle secretion. We determined the effects of apoE deficiency and apoE overexpression on VLDL-apoB production in mice. [(35)S]methionine was injected into endogenously label VLDL-apoB and Triton WR-1339 was simultaneously injected to block the catabolism of VLDL. Compared with wild-type mice, the VLDL-apoB production rate was decreased by 33% in apoE-deficient mice. Conversely, VLDL-apoB production was increased by 48% in mice overexpressing apoE compared with controls. Nascent VLDL, obtained from post-Triton plasma, had a decreased, not increased, content of TG per apoB in the apoE-overexpressing group compared with the control group. This study demonstrates that hepatic apoE expression increases the output of VLDL triglyceride by increasing the production rate of VLDL-apoB, suggesting that hepatic apoE influences the number of VLDL particles secreted by the liver.

Animals↗
PubMed2000

[A case of tracheobronchomegaly].

We report a rare case of tracheobronchomegaly with crescent-type tracheobronchomalacia. A 77-year-old man with a chronic cough was referred to our hospital because of fever and dyspnea. Radiographic examination showed enlargement of the trachea and main bronchi. On chest radiography, the transverse diameter of the trachea was 31 mm, and consolidation shadows were seen in both upper lung fields. Tracheobronchomegaly with pneumonia was diagnosed. The pneumonia was improved by administration of PAPM/BP. On bronchoscopic examination, the trachea and main bronchi were extremely dilated on inspiration, and were collapsed on expiration. The biopsy specimen from the bronchial mucosa showed non-specific chronic inflammation.

Aged↗
PubMed1999

Regression of atherosclerosis induced by liver-directed gene transfer of apolipoprotein A-I in mice.

BACKGROUND: The ability of apolipoprotein (apo)A-I to induce regression of preexisting atherosclerotic lesions has not been determined, and a mouse model of atherosclerosis regression has not yet been reported. METHODS AND RESULTS: LDL receptor-deficient mice were fed a western-type diet for 5 weeks to induce atherosclerotic lesions. A second-generation recombinant adenovirus encoding human apoA-I or a control adenovirus were injected intravenously in order to express apoA-I in the liver. Three days after injection, total apoA-I levels in mice injected with the apoA-I-expressing adenovirus were 216+/-16.0 mg/dL, compared with 68.0+/-3.0 mg/dL in control virus-injected mice (P<0.001). HDL cholesterol levels in mice injected with the AdhapoA-I vector 7 days after injection were 189+/-21.0 mg/dL, compared with 123+/-8.0 mg/dL in control virus-injected mice (P<0.02). Total and non-HDL cholesterol levels did not differ between the 2 groups. Atherosclerotic lesion area was quantified by en face analysis of the aorta and cross-sectional analysis of the aortic root. Compared with baseline mice, atherosclerosis progressed in mice injected with the control adenovirus. In contrast, in mice expressing apoA-I compared with baseline mice, total en face aortic lesion area was reduced by 70% and aortic root lesion was reduced by 46%. Expression of apoA-I was associated with a significant reduction in the fraction of lesions occupied by macrophages and macrophage-derived foam cells. CONCLUSIONS: Liver-directed gene transfer of human apoA-I resulted in significant regression of preexisting atherosclerotic lesions in LDL receptor-deficient mice as assessed by 2 independent methods.

Animals↗
PubMed1999

Binding of salicylhydroxamic acid and several aromatic donor molecules to Arthromyces ramosus peroxidase, investigated by X-ray crystallography, optical difference spectroscopy, NMR relaxation, molecular dynamics, and kinetics.

The X-ray crystal structure of the complex of salicylhydroxamic acid (SHA) with Arthromyces ramosus peroxidase (ARP) has been determined at 1.9 A resolution. The position of SHA in the active site of ARP is similar to that of the complex of benzhydroxamic acid (BHA) with ARP [Itakura, H., et al. (1997) FEBS Lett. 412, 107-110]. The aromatic ring of SHA binds to a hydrophobic region at the opening of the distal pocket, and the hydroxamic acid moiety forms hydrogen bonds with the His56, Arg52, and Pro154 residues but is not asscoiated with the heme iron. X-ray analyses of ARP-resorcinol and ARP-p-cresol complexes failed to identify the aromatic donor molecules, most likely due to the very low affinities of these aromatic donors for ARP. Therefore, we examined the locations of these and other aromatic donors on ARP by the molecular dynamics method and found that the benzene rings are trapped similarly by hydrophobic interactions with the Ala92, Pro156, Leu192, and Phe230 residues at the entrance of the heme pocket, but the dihedral angles between the benzene rings and the heme plane vary from donor to donor. The distances between the heme iron and protons of SHA and resorcinol are similar to those obtained by NMR relaxation. Although SHA and BHA are usually considered potent inhibitors for peroxidase, they were found to reduce compound I and compound II of ARP and horseradish peroxidase C in the same manner as p-cresol and resorcinol. The aforementioned spatial relationships of these aromatic donors to the heme iron in ARP are discussed with respect to the quantum chemical mechanism of electron transfer in peroxidase reactions.

Binding Sites↗