[A case of hepatocellular carcinoma associated with autoimmune hepatitis and increased scintigraphy of liver accumulation].
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Autoimmune Diseases↗
Biomedical subjects
T Seki
Publications and source records attributed to T Seki.
Dis3, implicated in mitotic control, binds directly to Ran and enhances the GEF activity of RCC1.
Using the two-hybrid method, we isolated a Saccharomyces cerevisiae cDNA encoding a protein homologous to Schizosaccharomyces pombe protein Dis3sp, using as bait, human GTPase Ran. The DIS3 gene is essential for viability and complements S.pombe mutant dis3-54 which is defective in mitosis. Although Dis3sc has no homology to RanBP1, it bound directly to Ran and the S.cerevisiae Ran homologue Cnr1, but not to the S.cerevisiae RCC1 homologue Srm1. Upon binding to Ran with a 1:1 molar ratio, Dis3sc enhanced a nucleotide-releasing activity of RCC1 on Ran. In the presence of Dis3sc, the K(m) of RCC1 on Ran decreased by half, while the kcat was unchanged. In vivo, Dis3sp was present as oligomers of M(r) 670-200 kDa as previously reported, and the 200 kDa oligomer of Dis3sp was found to include Spi1 and Pim1, the S.pombe homologues of Ran and RCC1, respectively. Although the biological function of the heterotrimeric oligomer consisting of Dis3, Spi1 and Pim1 is unknown, our results indicate that Dis3 is a component of the RCC1-Ran pathway.
Amino Acid Sequence↗
Evaluation of myocardial ischemia and infarction by signal-averaged electrocardiographic late potentials in children with Kawasaki disease.
We investigated myocardial ischemia and old myocardial infarction noninvasively using signal-averaged electrocardiographic late potentials (LPs) in patients with Kawasaki disease. Patients were divided into 4 groups: a noncoronary artery lesion group (n=136), a coronary artery lesion group (without myocardial ischemia and an old myocardial infarction; n=33), an ischemia group (n=16), and an old myocardial infarction group (n=13). Grouping was based on exercise thallium-201 myocardial scintigraphy, thallium-201 myocardial scintigraphy, exercise electrocardiography, coronary angiography, left ventriculography, and echocardiography. Signal-averaged electrocardiograms were recorded using a high-resolution system. Values of filtered QRS duration (f-QRSd), root-mean-square voltage, and duration of low-amplitude signal were judged using our own body surface area-related criteria (n=205) to determine positive rates of LPs and sensitivities and specificities to ischemia and infarction. These data were also interpreted using published criteria for adults and compared with those interpreted by our criteria. Positive rates by our criteria were 0% in the noncoronary artery lesion group, 9.1% in the coronary lesion group, 56.3% in the ischemia group, and 69.2% in the old myocardial infarction group. However, using the criteria for adults, these values were 0%, 3.0%, 25%, and 46.2%, respectively. Sensitivities to ischemia and infarction using our criteria were significantly higher (56.3% and 69.2%) than those using the criteria for adults (p < 0.05). Moreover, specificities to ischemia and infarction were very high (93.4% and 93.5%, respectively) using our criteria, and there were no significant differences from specificities using the criteria for adults. Also, we examined the reproducibility of values of LPs and LP parameters. The values of filtered QRS duration showed a high reproducibility in both LP-positive and -negative groups, followed by low-amplitude signal and then root-mean-square voltage. The results of LP presence or absence showed 100% reproducibility for both the LP-positive and -negative groups, supporting the utility of LPs for clinical applications. Thus, LPs provide useful information in a noninvasive manner for clarifying ischemia and infarction in patients with Kawasaki disease.
Action Potentials↗
Premature chromatin condensation induced by loss of RCC1 is inhibited by GTP- and GTPgammaS-Ran, but not GDP-Ran.
RCC1 is a guanine nucleotide exchanging factor acting on nuclear G protein Ran. Premature chromatin condensation occurs in the temperature-sensitive rcc1- mutant of the BHK21 cell line, tsBN2, at the restrictive temperature. This observation can be explained if the premature activation of MPF is normally inhibited by GTP-Ran. In the absence of RCC1, GDP-Ran predominates, resulting in MPF activation. However, experiments with Ran mutants to determine whether GTP- or GDP-Ran prevents activation of MPF have yielded conflicting results. In order to clarify this point, we have microinjected nucleotide-bound Ran, instead of mutated Ran, into the nuclei of tsBN2 cells treated to reduce RCC1-mediated guanine nucleotide exchange. GTP-Ran, GTPgammaS-Ran, and GDP-Ran all inhibited chromatin condensation. However, the inhibition of chromatin condensation by GDP-Ran could be completely abolished by co-injection with GDP, but not GTP. Thus, we conclude that GTP-Ran blocks the activation of MPF and that hydrolysis of GTP is not required to prevent MPF activation.
Animals↗
Simultaneous determination of uric acid and creatinine in biological fluids by column-switching liquid chromatography with ultraviolet detection.
A column-switching liquid chromatographic method for the simultaneous determination of uric acid and creatinine in human serum and urine was developed. Creatinine and uric acid were separated by size-exclusion chromatography on a hydrophilic gel column (C1) and creatinine eluted from C1 was separated from proteins by filtration through a longer hydrophilic gel column (C2). The creatinine fraction eluted from C2 was transferred to a weakly acidic cation-exchange column (C3) and then to a strongly acidic cation-exchange column (C4). Uric acid eluted from C1 after creatinine was transferred to an anion-exchange column (C5) and then to a hydrophilic gel column (C6). The mobile phase was a mixed buffer of pH 5.1 (propionic acid-succinic acid-NaOH, 60:15:60 mmol/l in water). Diluted serum and urine could be injected onto C1, and C1 was backflushed after the transfer of uric acid from C1 to C5. Creatinine and uric acid in the eluate were determined by measuring their ultraviolet absorption at 234 and 290 nm, respectively. The recovery of uric acid and creatinine added to diluted serum (20-fold dilution, concentration 20 and 5 mumol/l, respectively) was 98.9 +/- 0.56% and 100.9 +/- 1.29%, respectively. The recovery of uric acid and creatinine added to diluted urine (100-fold dilution, concentration 50 and 100 mumol/l, respectively) was 99.4 +/- 0.72% and 98.7 +/- 1.45%, respectively (mean +/- R.S.D., n = 6).
Chromatography, Gel↗
In situ identification of messenger RNA of endothelial type nitric oxide synthase in rat cardiac myocytes.
The endothelial type nitric oxide synthase (NOS) termed as NOS-III has been demonstrated in various nonendothelial cells. Although NOS-III was recently found in the cardiac tissues, identification of the cells expressing NOS-III gene in the heart remains unknown. In this study, we compared the expression of NOS-III mRNA in various tissues of rats by Northern blot analysis using a homologous cDNA probe and identified the cell type by in situ hybridization histochemistry with a cRNA probe. NOS-III mRNA was found more abundantly in atria and ventricle than in other various tissues examined. NOS-III mRNA was intensely and specifically visualized in the cardiac myocytes as well as in the endothelial cells of the heart. No staining was seen with a sense cRNA probe. These results provide the first in vivo evidence for NOS-III mRNA in the cardiac myocytes and suggest its role in the regulation of cardiac functions.
Animals↗
Angiotensin converting enzyme inhibitor normalizes vascular natriuretic peptide type A receptor gene expression via bradykinin-dependent mechanism in hypertensive rats.
We previously demonstrated that angiotensin converting enzyme (ACE) inhibitor normalizes the up-regulated gene expression of vascular natriuretic peptide type A (NP-A) receptor in hypertensive rats. To elucidate the mechanism, we examined the effect of angiotensin II receptor (AT1) antagonist (TCV-116) and bradykinin receptor (B2) antagonist (Hoe 140) on the NP-A receptor mRNA level in the aorta of genetically hypertensive rats (SHR-SP/Izm) using ribonuclease protection assay. The effect of ACE inhibitor on the NP-A receptor mRNA level was completely abolished by a concomitant administration of Hoe 140, while TCV-116 did not show any significant effect on the NP-A receptor mRNA level. These results suggest that bradykinin plays an important role in the regulation of the vascular NP-A receptor gene expression.
Angiotensin-Converting Enzyme Inhibitors↗
Interleukin-1 induction of type-1 plasminogen activator inhibitor (PAI-1) gene expression in the mouse hepatocyte line, AML 12.
Type-1 plasminogen activator inhibitor (PAI-1), the major regulator of fibrinolysis, is an important component of the acute phase (AP) response, the coordinated systemic reaction of an organism to tissue injury. As part of a combined in vivo and in vitro study of AP regulation of PAI-1 gene expression in murine hepatocytes, we have characterized the cytokine regulation of PAI-1 gene expression in AML 12 cells, an established line of normal hepatocytes derived from an adult transgenic mouse overexpressing transforming growth factor alpha. Interleukin (IL)-1 caused a rapid and transient 4-fold increase in PAI-1 mRNA that was maximal at 1 h. Half-maximal induction by IL-1 was obtained at 50 U/ml and maximal effects were seen at approximately 500 U/ml. Tumor necrosis factor alpha induced PAI-1 mRNA accumulation with the same magnitude and time course as IL-1, and was not additive with IL-1. IL-6 and dexamethasone alone had no effect on PAI-1 mRNA accumulation and did not enhance the effect of IL-1. Transforming growth factor beta caused a sustained 5- to 7-fold increase in the accumulation of PAI-1 mRNA that was maximal after 2 to 4 h. The IL-1 induction of PAI-1 was inhibited by actinomycin D, but not by cycloheximide. Nuclear run-on studies demonstrated that IL-1 induced a rapid and transient increase in PAI-1 gene transcription that was maximal at 30 min. IL-1 did not stabilize PAI-1 mRNA, and might, in fact, accelerate its rate of decay. These data demonstrate that IL-1, a potent mediator of AP response, induces the accumulation of PAI-1 mRNA in murine hepatocytes, at least in part, by rapidly and transiently increasing the rate of transcription of the PAI-1 gene.
Acute-Phase Reaction↗
Developmental change in expression of highly polysialylated neural cell adhesion molecule in C-cells in rat thyroid gland.
The expression of the neural cell adhesion molecule (NCAM), highly polysialylated NCAM, and E-cadherin was immunohistochemically studied in the calcitonin-producing cells (C-cells) of developing and adult rat thyroid glands of varying ages. In fetal and neonatal rat thyroids, almost all the C-cells displayed immunoreactivity for highly polysialylated NCAM, whereas most of the follicular cells were negative. The highly polysialylated NCAM-positive C-cells markedly decreased in number between 5 and 14 days after birth. From day 14 onward, immunoreactivity for highly polysialylated NCAM was almost negative in thyroid glands. On the other hand, the expression of immunoreactivity for NCAM peptide persisted in thyroidal C-cells throughout the life span. These results suggest that conversion of the highly polysialylated NCAM into a less sialylated form occurs in the thyroid C-cells between postnatal days 5 and 14. Intense immunoreactivity for E-cadherin was observed in the entire cell surfaces of all the C-cells and follicular cells in the rats of all ages tested. In the course of thyroid organogenesis, C-cells transiently form a cell mass, an ultimobranchial body, which is fated to disappear as the C-cells migrate diffusely into the thyroid. The duration of the polysialic acid expression in the C-cell surfaces appears to coincide with the period of C-cell migration. It is possible that the expression of highly polysialylated NCAM allows the C-cells to migrate into the thyroid by reducing the cell-to-cell adhesion of C-cells with adjacent C-cells and/or with the surrounding follicular cells.
Animals↗
The TraB protein, which mediates the intermycelial transfer of the Streptomyces plasmid pSN22, has functional NTP-binding motifs and is localized to the cytoplasmic membrane.
The traB gene on the Streptomyces conjugative plasmid pSN22 is required for intermycelial plasmid transfer and the mobilization of chromosomal markers (Cma). The predicted amino acid sequence of TraB contains one Walker type-A and two type-B NTP-binding motifs. Site-directed mutagenesis revealed that the type-A motif and one of the type-B motifs, 109 amino acid residues downstream of the type-A motif, were essential for both plasmid transfer and Cma. The second type-B sequence could be changed without any phenotypic effect. A modified traB gene was constructed, resulting in the production of a functional protein with an amino-terminal c-Myc epitope tag for immunological analysis. This protein was associated with the cytoplasmic membrane, suggesting that TraB is a membrane protein that uses energy from ATP hydrolysis to transport DNA between mycelia. The c-Myc tagging of TraB decreased the efficiency of intramycelial plasmid spread, suggesting that TraB is involved in both inter- and intramycelial transfer processes.
Amino Acid Sequence↗
Conserved histidine residues of RCC1 are essential for nucleotide exchange on Ran.
Charged amino acid residues of human RCC1 were converted to alanine and mutants which were unable to complement tsBN2 cells (a temperature-sensitive rcc1- mutant of the hamster BHK21 cell line) were selected. These RCC1 mutants were analyzed for the ability to inhibit premature chromatin condensation by microinjection into tsBN2 cells, and their steady-state kinetic parameters for guanine nucleotide exchange reaction were measured. Examined RCC1 mutants were unstable in tsBN2 cells at the restrictive temperature, yet they significantly inhibited premature chromatin condensation. Mutants located on the N-terminus of the RCC1 repeat showed an increased K(m), while their kcat values were comparable to that of wild-type RCC1. In contrast, mutants containing the conserved histidine residues in the C-terminus of the RCC1 repeat showed a value of K(m) similar to that of wild-type RCC1, while the kcat values of these mutants were reduced, depending upon the RCC1 repeats on which the mutation was located. These steady-state kinetic parameters of mutants indicate that the N-terminus and the C-terminus of RCC1 repeats play different roles in guanine nucleotide exchange on Ran. The comparison of kcat among the histidine mutants suggests that those histidine residues which are conserved in the RCC1 repeats and also through evolution comprise the catalytic site for the guanine nucleotide exchange reaction.
Alanine↗
RCC1 in the Ran pathway.
RCC1 is a chromosomal protein that functions as a GEF of the nuclear G protein Ran, which GTPase activity is enhanced by RNA1 located in the cytoplasm. RCC1 has no preference for GTP or GDP-bound Ran, so that GTP-Ran formation in vivo is regulated by relative concentrations of GTP/GDP and regulatory proteins interacting with RCC1, Ran, and RNA1. Proteins possessing the special Ran-binding motif have been found to be conserved in species ranging from yeasts to mammalians. The finding of RanBP2/NUP358 clearly indicates the involvement of the Ran pathway in the nuclear pore transport function, in agreement with the finding that both rcc1- and rna1- show defects in this process. However, loss of RCC1 induces premature initiation of mitosis, resulting in G1 arrest with the micronuclei possessing mitotic condensed chromosomes. How both the cell cycle and nucleocytoplasmic transport are regulated by the RCC1-Ran pathway is a major question.
Amino Acid Sequence↗
Parturition in six renal allograft recipients.
Between 1983 and 1994, we studied renal function and neonatal conditions for eight pregnancies and births to six women who had received renal transplants in order to assess the effect of an allograft on pregnancy and its outcome. The gestation period was 34 to 39 weeks (mean 36 weeks and 4 days), and four pregnancies ended before term. All eight babies were delivered by cesarean section. Intrauterine growth retardation (IUGR) was found in both babies of one woman who had been treated with conventional (without cyclosporin) immunosuppression. The serum creatinine level did not change during gestation in any of the women but was elevated after delivery in four. Four mothers suffered from proteinuria (25-364 mg/dl) during gestation, but the proteinuria disappeared after delivery in all but one case. The one exception, persistent proteinuria of 100-200 mg/dl, was assumed to result from the recurrence of the original renal disease (lgA nephropathy). The reduction of creatinine clearance and hydronephrosis of one graft noted during gestation were later reversed. None of the eight babies (four females and four males) was congenitally malformed, and their Apar scores were 6 to 9 (median 8). They are now 3 months to 11 years old, and seven of them are healthy and show good growth. One of the two IUGR babies has not grown well; her weight and height are more than 1 SD below the mean for her age, and she is mentally retarded and suffers from muscle weakness. Compared with dialysis patients, female renal allograft recipient have a better quality of life because they can safely deliver a child if they observe the criteria for pregnancy established for renal allogaft recipients.
Child↗
Development of a temperature-inducible expression system for Streptomyces spp.
PCR mutagenesis of a 0.9-kbp fragment, containing a repressor gene, traR, and its target promoter, Ptra, from Streptomyces nigrifaciens plasmid pSN22, produced Streptomyces lividans clones with temperature-inducible Ptra expression. Using the promoterless gene for the thermostable Thermus flavus malate dehydrogenase as an indicator, an induction of enzyme activity of as much as was observed in a temperature shift from 28 to 37 degrees C. Temperature downshift reestablished repression of Ptra, making these promoter cassettes very attractive for the temporally regulated expression of cloned genes in Streptomyces spp.
Bacterial Proteins↗
Surgical revascularization in children with renovascular hypertension.
Two boys, 11 and 10 years of age, respectively, having renovascular hypertension are reported. Diagnostically, captopril test and renal scintigraphy were useful as pharmacologic probes of the renal arterial stenosis and to determine its laterality, as well for examination of the operative outcome. Both children were rendered normotensive after renal revascularization, one by renal autotransplantation and the other by aortorenal bypass procedure. Renovascular hypertension in children is also discussed.
Aortography↗
Combination therapy of infantile spasms with high-dose pyridoxal phosphate and low-dose corticotropin.
A new combination therapy, high-dose pyridoxal phosphate (40 to 50 mg/kg daily) and low-dose corticotropin (0.01 mg [0.4 IU]/kg daily), was tried in 28 children with infantile spasms. Monotherapy with pyridoxal phosphate provided excellent seizure control in three (11%) of the 28 subjects. Corticotropin was subsequently added to the regimen of the remaining 25 patients. At 1 month after discontinuing corticotropin, 21 (84%) of the 25 patients experienced no seizures, and 22 (88%) of the 25 showed improvement in their electroencephalographic findings. The mean interval until achievement of seizure control was 4.1 days after the initiation of corticotropin. The outcome in the 21 patients has been followed for a mean period of 34.9 months (range, 2 to 81 months). Of these 21 patients, six (29%) have had relapses of infantile spasms, and 10 (48%) have experienced normal development. Transient increases in liver enzymes occurred in 14 (50%) of the 28 patients, but none of the patients developed more serious side effects. The investigators conclude that combination therapy with high-dose pyridoxal phosphate and low-dose corticotropin is a promising new therapy.
Adrenocorticotropic Hormone↗
Vascular action of circulating and local natriuretic peptide systems is potentiated in obese/hyperglycemic and hypertensive rats.
Hypertension is commonly associated with diabetes mellitus. The aim of the present study was to explore the pathophysiological significance of the natriuretic peptide (NP) system in hypertension associated with genetically obese/hyperglycemic Wistar fatty rats. The messenger RNA (mRNA) levels of the two biologically active NP receptors, NP-A receptor [more specific for atrial natriuretic peptide (ANP)] and NP-B receptor [more specific for C-type natriuretic peptide (CNP)], and CNP mRNA levels were determined in the aorta and kidney by ribonuclease protection assay. Plasma ANP levels were determined by RIA. Both NP-A and NP-B receptor mRNA levels in the aortae of Wistar fatty rats were double those in Wistar lean rats. Plasma ANP levels and CNP mRNA levels in the aorta of Wistar fatty rats were also significantly higher than those in Wistar lean rats. In contrast, there was no significant difference in renal levels of the mRNA for both NP receptors and CNP between the two strains. Administration of a NP-A and -B receptor antagonist, HS-142-1, to Wistar fatty rats resulted in a significant increase in systolic blood pressure and a larger decrease in plasma cGMP level than that in Wistar lean rats, with no difference in the extents of decrease in urine volume and urinary sodium excretion between the two strains. These results suggest that both the ANP/NP-A system and the CNP/NP-B system in vessels are up-regulated at the level of gene expression and may, thus, play an important role in counteracting the hypertension associated with diabetes mellitus.
Animals↗
Studies on agents with vasodilator and beta-blocking activities. IV.
A series of novel pyridazinone derivatives (II) having a phenoxypropanolamine moiety was synthesized. Their hypotensive and beta-blocking activities were evaluated after intravenous administration of the compounds to anesthetized rats. Among them, the 5-chloro-2-cyanophenoxy derivative (29) showed the promising dual activities and was selected for further studies.
Adrenergic beta-Antagonists↗