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ABSTRACTA number of epidemiological studies have shown human metapneumovirus (hMPV) to be one of the most important viral agents associated with acute respiratory infections in humans. However, due to the difficulty in growing the virus, all epidemiological studies of hMPV infection have been performed on the basis of the molecular method. Thus, the development of a cell line suitable for the isolation of hMPV from clinical specimens is a crucial step for further research. Using the Vero E6 cell line, which could be stably maintained for 1 month without passage or medium change, we succeeded in isolating 79 strains from 4,112 specimens obtained in Yamagata, Japan, in 2004 and 2005. The total isolation rate was 1.9% (79/4,112). The monthly distribution revealed that hMPV infections occurred between February and April in 2004 and throughout most of the year in 2005. Phylogenetic analysis indicated that subgenogroup B2 was predominant in 2004, whereas three subgenogroups, A2, B1, and B2, had cocirculated in 2005. Although multiple subgenogroups cocirculated in 2005, each individual subgenogroup strain was found to predominate at specific sites. An infectivity assay of hMPV strains also indicated that the infection efficiency in Vero E6 cells was better than that in LLC-MK2 cells. Finally, we found that Vero E6 cells are useful for the isolation of hMPVs and that this utility might aid further research into hMPVs beyond the epidemiological data shown in this study.

Dehydroepiandrosterone (DHEA) is the most abundant adrenal androgenic steroid in young adult humans. The physiological functions of DHEA in preventing human carcinogenesis are still controversial, but a lot of reports have shown that pharmacological doses of DHEA show chemopreventive and anti-proliferative effects on tumors in rodents. Although a therapeutic dose of DHEA has been reported to promote hepatocarcinogenesis in rats due to peroxisomal proliferation, it remains unclear whether DHEA is a peroxisome proliferator in human liver. The chemopreventive and anti-proliferative effects of DHEA are not explained by a single mechanism, and at least four mechanisms seem to contribute to these effects: 1) depletion of NADPH and ribose-5-phosphate due to the inhibition of glucose-6-phosphate dehydrogenase activity, 2) suppression of cholesterol biosynthetic pathway by inhibition of HMG-CoA reductase, 3) interference with cell proliferation signaling pathways, and 4) suppression of nitric oxide generation through down-regulation of nitric oxide synthase II. In addition to studies of the mechanisms underlying the anti-neoplastic effects, searches for more potent and less androgenic DHEA derivatives are ongoing. A small amount of DHEA is endogenously metabolized to 7-oxygenated DHEA, and this may represent a metabolic pathway to more potent steroid hormones. Androsterone, epiandrosterone and etiocholanolone have been considered to be merely inactive end products of DHEA, but may in fact be physiological effectors in their own right. In addition, DHEA analogs such as 3-methyl-5-androsten-17-one, 16-fluoro-5-androsten-17-one and 16-fluoro- 5-androstan-17-one have been synthesized and shown to be more effective inhibitors of tumor growth, compared with DHEA itself. However, to design potent and safe DHEA derivatives, identification of the DHEA receptor and clarification of the mechanisms of DHEA action are required.

ABSTRACTPhylogenetic analysis of 45 enterovirus 71 (EV71) isolates for 6 years in Yamagata, Japan, clarified that the annual outbreak of hand-foot-and-mouth disease was due to four genetically distinct subgenogroups, including a novel “B5.” Our results suggest that the importation of EV71 from surrounding countries has had a major epidemiological impact on the local community used in our study.

Summary. The aim of this study was to determine the effective and optimum dose of taurine for exercise performance and to maintain tissue taurine concentration. Rats received a respective daily dose of 0, 20, 100, and 500 mg/kg body weight of taurine (EC and ET-1, -2, -3 groups, respectively) for two weeks, and then, were subjected to treadmill until exhaustion. The running time to exhaustion was significantly prolonged by 25% and 50% in the ET-2 and -3 groups, respectively, compared to that in the EC group accompanied with maintenance of taurine tissue concentrations. Furthermore, the oxidative glutathione per total glutathione ratio in tissues was inhibited in the ET-2 and -3 groups whereas it was higher in the EC group than in both the no exercise and taurine-administered groups. Therefore the effective and optimal doses of oral taurine administration for two weeks on a transient exercise performance were between 100 and 500 mg/kg/day.

Experimental and analytical studies for freezing phenomena of ethylene glycol solution on a vertical cooled polyvinyl-chloride plate have been performed. It is found that the crystal ice formed on the vertical plate is removed from the plate surface due to buoyancy force acting on the crystal ice. It is shown that the crystal ice formed on the vertical plate slides along the plate surface due to buoyancy force and the crystal ice grows in a shape of sheet by joining with the neighbor ice. The number of the removed ice per unit of time is, therefore, decreased as compared to that for the horizontal plate. It is found that the onset of ice removal condition is related to the heat fluxes from the plate surface to the plate and from the plate surface to the solution. The ice removal occurs more easily for the vertical plate than for the horizontal one.

Between October 1999 and May 2000, a total of 28 strains of influenza C virus were isolated in four Japanese prefectures: Yamagata, Miyagi, Saitama and Hiroshima. Antigenic analysis showed that the 28 isolates were divided into three distinct antigenic groups, and viruses belonging to different antigenic groups were co-circulating in each of the four prefectures. Phylogenetic analysis of the seven protein genes demonstrated that the viruses having a similar genome composition spread in various areas of Japan during the same period. Furthermore, phylogenetic analysis showed that most of the influenza C viruses isolated in various areas of the world between the 1970s and 1980s were closely related to the contemporary Japanese viruses in all gene segments. These observations suggest that the influenza C viruses cause epidemics in some communities during the same season and that antigenically and genetically similar influenza C viruses spread throughout Japan and may be circulating worldwide.

ABSTRACTDuring the 3 years from January 1996 to December 1998, a total of 33 strains of influenza C virus were isolated from 10,726 throat swab specimens collected from children with acute respiratory illness who visited two pediatric clinics in Yamagata City, Japan. These 33 strains were isolated in clusters during two different periods, 20 strains in May to August 1996 and the remaining 13 in March to June 1998. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoprotein and phylogenetic analysis of seven RNA segments showed that the 33 influenza C viruses isolated were antigenically and genetically similar and that they were reassortant viruses which had obtained PB2, PB1, HE, M, and NS genes from a C/pig/Beijing/115/81-like virus and P3 and NP genes from a C/Mississippi/80-like virus. These observations suggest strongly that during the survey period of 3 years, two outbreaks of influenza C occurred in Yamagata City, both of which were caused by a reassortant virus having the genome composition described above.

Although numerous studies have shown that nm23-H1 gene product expression is inversely related to metastatic potential in some cancers, the expression in lymph nodes has not been studied in detail. An analysis of nm23-H1 gene product expression in mediastinal lymph nodes from lung cancer patients is reported.

Cerebrotendinous xanthomatosis (CTX) is a rare, recessively inherited lipid storage disease characterized by a markedly reduced production of chenodeoxycholic acid and an increased formation of 25-hydroxylated bile alcohols and cholestanol. Patients with this disease are known to have mutations in the sterol 27-hydroxylase (Cyp27) gene. However, one study showed that mice with a disrupted Cyp27 gene did not have any CTX-related clinical or biochemical abnormalities. To explore the reason, hepatic cholesterol, cholestanol, and 12 intermediates in bile acid biosynthetic pathways were quantified in 10 Cyp27(-/-) and 7 Cyp27(+/+) mice, two CTX patients (untreated and treated with chenodeoxycholic acid), and four human control subjects by high resolution gas chromatography-mass spectrometry. Mitochondrial 27-hydroxycholesterol and 5beta-cholestane-3alpha,7alpha,12alpha,27-tetrol were virtually absent in both Cyp27(-/-) mice and CTX patients. In Cyp27(-/-) mice, microsomal concentrations of intermediates in the early bile acid biosynthetic pathway (7alpha-hydroxycholesterol, 7alpha-hydroxy-4-cholesten-3-one, 7alpha,12alpha-dihydroxy-4-cholesten-3-one, and 5beta-cholestane-3alpha,7alpha,12alpha-triol), 25-hydroxylated bile alcohols (5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol, 5beta-cholestane-3alpha,7alpha,12alpha,23R,25-pentol, and 5beta-cholestane-3alpha,7alpha,12alpha,24R, 25-pentol), and cholestanol were all significantly elevated compared with those in Cyp27(+/+) mice, although the levels were lower than those in untreated CTX patients. The intermediate levels in early bile acid biosynthesis were more elevated in male (16;-86% of CTX) than in female Cyp27(-/-) mice (7-30% of CTX). In contrast, 25-hydroxylated bile alcohol concentrations were not significantly different between male and female Cyp27(-/-) mice and were considerably lower (less than 14%) than those in CTX patients.These results suggest that 1) in Cyp27(-/-) mice, especially in females, classic bile acid biosynthesis via 7alpha-hydroxycholesterol is not stimulated as much as in CTX patients; and 2) formed 25-hydroxylated bile alcohols are more efficiently metabolized in Cyp27(-/-) mice than in CTX patients.

In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5beta-cholestane-3alpha, 7alpha, 12alpha-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5beta-cholestane-3alpha, 7alpha, 12alpha-triol 25- and 27-hydroxylases and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography;-mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-beta-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13;-60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64;-142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178;-371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver.

Abstract: The treatment of choice for mediastinal paraganglioma is complete surgical resection. Its local recurrence, however, is very difficult to treat surgically because it usually involves important structures of the mediastinum, such as the aorta and trachea. We report a 64-year-old woman with aortico-pulmonary paraganglioma who developed local recurrence after complete resection. The recurrent tumor responded dramatically to single-agent chemotherapy with cyclophosphamide, whereas it did not respond to radiotherapy or combination chemotherapy with mitomycin C, vindesine, and cisplatin. We therefore consider that single-agent chemotherapy with cyclophosphamide could be an alternative treatment for unresectable recurrent paraganglioma.

The Smith-Lemli-Opitz syndrome (SLOS) is a congenital birth defect syndrome caused by a deficiency of 3beta-hydroxysterol Delta(7)-reductase, the final enzyme in the cholesterol biosynthetic pathway. The patients have reduced plasma and tissue cholesterol concentrations with the accumulation of 7-dehydrocholesterol and 8-dehydrocholesterol. Bile acid synthesis is reduced and unnatural cholenoic and cholestenoic acids have been identified in some SLOS patients. To explore the mechanism of the abnormal bile acid production, the activities of key enzymes in classic and alternative bile acid biosynthetic pathways (microsomal cholesterol 7alpha-hydroxylase and mitochondrial sterol 27-hydroxylase) were measured in liver biopsy specimens from two mildly affected SLOS patients. The effects of 7- and 8-dehydrocholesterols on these two enzyme activities were studied by using liver from SLOS model rats that were treated with the Delta(7)-reductase inhibitor (BM15.766) for 4 months and were comparable with more severe SLOS phenotype in plasma and hepatic sterol compositions. In the SLOS patients, cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase were not defective. In BM15.766-treated rats, both enzyme activities were lower than those in control rats and they were competitively inhibited by 7- and 8-dehydrocholesterols. Rat microsomal cholesterol 7alpha-hydroxylase did not transform 7-dehydrocholesterol or 8-dehydrocholesterol into 7alpha-hydroxylated sterols. In contrast, rat mitochondrial sterol 27-hydroxylase catalyzed 27-hydroxylation of 7- and 8-dehydrocholesterols, which were partially converted to 3beta-hydroxycholestadienoic acids. Addition of microsomes to the mitochondrial 27-hydroxylase assay mixture reduced 27-hydroxydehydrocholesterol concentrations, which suggested that 27-hydroxydehydrocholesterols were further metabolized by microsomal enzymes. These results suggest that reduced normal bile acid production is characteristic of severe SLOS phenotype and is caused not only by depletion of hepatic cholesterol but also by competitive inhibition of cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities by accumulated 7- and 8-dehydrocholesterols. Unnatural bile acids are synthesized mainly by the alternative pathway via mitochondrial sterol 27-hydroxylase in SLOS.

Objective. To determine whether elevations in serum leucine aminopeptidase (LAP) levels reflected the underlying evolution of active disease in systemic lupus erythematosus (SLE).Method. We studied serum LAP levels, other laboratory indicators, and SLE Disease Activity Index (SLEDAI) scores, in 46 consecutive patients with SLE admitted to Tokyo Metropolitan Komagome Hospital. LAP levels in 46 patients with rheumatoid arthritis were also measured.Results. Thirty-three SLE patients had elevated LAP levels. LAP levels correlated positively with levels of lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and γ-glutamyl transpeptidase, and negatively with the total serum haemolytic complement and leucocyte, neutrophil and lymphocyte counts, but showed no correlation with alkaline phosphatase, γ-globulin, β2-microglobulin or C-reactive protein levels, or platelet count. The SLEDAI score correlated positively with LAP levels. The LAP level in patients with rheumatoid arthritis was near normal.Conclusion. The serum LAP level may be a potential activity indicator for SLE.

Electronic structures of the two structural isomers of novel tetrasilyl-substituted ethylenes, 5,5,6,6,11,11,12,12-octaethyl-5,6,11,12-tetrasilanaphthacene (1) and 1,1‘-bis(3,4-benzo-2,2,5,5-tetraethyl-2,5-disilacyclopent-3-enylidene) (2), were studied from the viewpoint of electronic effect occurring from geometrical difference in the vicinity of the tetrasilylethene moiety. Characteristic weak bands at 350 (1) and 403 (2) nm of the absorption spectra in solution were attributed to the σ(HOMO) → π*(LUMO) transition on the basis of the quantum chemical configuration interaction (CI) calculations by the semiempirical INDO/S model and ab initio Hartree−Fock theory. Moreover, the large bathochromic shift of 53 nm between 1 and 2 could be ascribed to the raising of the HOMO of 2, as is supported by cyclic voltammetry measurements:  the small Si−C−Si bond angle of the tetrasilylethene moiety destabilizes the HOMO energy of 2 compared with that of 1.

The Fourier transform theory is applied to the analytical determination of the disturbance velocity potential and pressure acting on a two-dimensional plate, in order to reexamine those of previous analyses by other investigators. Simplified expressions of the generalized forces are presented for incompressible and nearly incompressible flows. As the Mach number tends to zero, the virtual mass induced by an oscillating fluid becomes infinitely large for a natural mode symmetric with respect to the midchord point. It is recommended to take into account a symmetric mode which changes no fluid volume contained in a control surface, when a coupled flutter oscillation at very low Mach numbers is analyzed. Incorrectness in the generalized forces of the previous analyses is pointed out by comparing with the present analysis.

A case of true pancreaticoduodenal artery (PDA) aneurysm is reported. A calcified lesion was initially detected by plain x-ray films, and an essential diagnosis was made before operation by intravenous digital subtraction angiography (IVDSA). Surgical resection of the aneurysm was performed successfully. Additionally, we reviewed a total of 82 cases with PDA aneurysm out of the 88 cases that had been reported in the English-language literature up to 1993. Fifty-three cases were accompanied by aneurysmal rupture (rupture group), and 29, including our case, were without rupture (nonrupture group). Because of the high mortality rate (49.1%) in the rupture group, a precise diagnosis and adequate treatment of PDA aneurysm before rupture is important. In the nonrupture group, a calcification on radiography appeared in 61.6% of the cases in which aneurysms were not found incidentally; this seems to be a significant indication for angiography. Moreover, intravenous digital subtraction angiography is quite useful for the screening of PDA aneurysm because it is an easy and noninvasive examination. (J VASC SURG 1995;22:161-6.)

Temperature dependencies of the optical rotation and size exclusion chromatography with low-angle laser light scattering (SEC−LALLS) were performed on the sonicated K⁺, κ-carrageenan in KCl salt solutions. The molecular weights were measured at the temperatures of the coil, helix, and coil−helix transition regions. The temperature at each region was determined by the optical rotation experiment. In a solution of low KCl salt concentration, the molecular weights did not change at any of the temperatures investigated. In this case, no hysteresis in the temperature dependence curve of optical rotation was observed. On the other hand, when the salt concentration increased, the molecular weight increased according to the transition from the coil to a helical conformation. In this case, a hysteresis was observed in the optical rotation curve. Moreover, the greater the increase in the KCl concentration, the greater the increase in molecular weight. These results indicate the existence of multiple conformational states for κ-carrageenan that depend upon the solution conditions. That is, κ-carrageenan makes a conformational transition from the coil to a single helical conformation at low KCl salt concentrations. However, κ-carrageenan takes on a helical conformation with a dimerized molecular weight when the salt concentration increases. These dimerized helical molecules further aggregate when the salt concentration increases further.

The effects of portal venous inoculation with donor splenocytes on lung allograft survival were evaluated in dogs. Within the dose range examined, portal venous inoculation with donor splenocytes alone did not affect the graft survival time. Although the mean graft survival time was prolonged by FK 506 treatment for 14 days, the transplanted lungs were rejected within 12 days after termination of FK 506 administration. However, when the recipients were given portal venous inoculation with donor splenocytes at the time of transplantation, the graft survival time after termination of FK 506 administration was significantly prolongated. In the recipients that received combined therapy of portal venous inoculation with donor splenocytes and FK 506 treatment, arterial oxygen tension and histologic architecture of the transplanted lungs remained within the normal range for a longer period than these measures did in control animals that received FK 506 treatment alone. Dose-response experiments revealed that 1 x 10^8 cells/recipient was most effective in causing prolonged graft survival after termination of FK 506 treatment. On the basis of these results, perioperative portal venous inoculation with donor splenocytes can enhance the immunosuppressive effect of FK 506 in the canine lung transplantation model. (J Thorac Cardiovasc Surg 1996;112:300-5)

This paper presents an extension of the previous analyses on the collapsible tubeflow problem using a simplified model based on a two-dimensional channel conveying a one-dimensional flow. The main objective of the paper is to exploit the static and dynamic behavior of the model, by comparing with available experimental data and examining the accuracy of calculated results obtained for different numerical resolutions. The main revision from the previous analyses is the incorporation of a universal “tube” law that is valid for a wide range of positive and negative transmural pressure. Most of the numerical results agree qualitatively with the experimental observations. Self-excited high-frequency oscillation with very small amplitude of the membrane wall is, however, predicted to occur in a flow range where the slope of the pressure drop curve is positive. It is seen that the high-frequency oscillation is associated with the motion of the separation point of the flow.

The effect of vitamin B12on learning disturbance was tested in rats. Rats were fed a choline-enriched, choline-deficient, and choline-deficient diet with vitamin B12. Concentrations of acetylcholine in the brain were significantly lower in rats fed a choline-deficient diet than rats fed a choline-enriched diet. Passive avoidance learning shows that rats on a choline-deficient diet showed significantly impaired learning compared to rats on a choline-enriched diet. However, there was no significant difference of acetylcholine in the brain or in the passive avoidance learning between rats fed a choline-enriched and a choline-deficient with vitamin B12diet. We, therefore, suggest that vitamin B12potentiates learning in an acetylcholine-deprived brain.

We describe a family with Liddle's disease caused by a novel mutation of the beta subunit of the human epithelial sodium channel (ENaC). A 15-year-old Japanese female was referred to our outclinic because of hypertension. The physical examination showed no abnormal findings except mild hypertension, but the laboratory data revealed low levels of plasma renin activity, plasma aldosterone and serum potassium. A comprehensive analysis of steroid hormones showed only high levels of urinary free cortisol and 17-hydroxycorticosteroids. During loading tests, blood pressure and serum potassium responded well to triamterene and slightly to spironolactone, but did not respond to dexamethasone. In addition, the normal ratio of tetrahydrocortisol plus 5alpha-tetrahydrocortisol to tetrahydrocortisone in a 24 h urinary excretion test strongly suggested a diagnosis of Liddle's disease rather than apparent mineralocorticoid excess syndrome. DNA sequence analysis of members of this family revealed a single cytosine base insertion at Arg-597 of the beta human ENaC in the proband and her mother, leading to a loss of the last 34 amino acids from the normally encoded protein as the result of a frameshift. We conclude that a de novo cytosine insertion into the final exon of the C-terminus of the beta human ENaC is responsible for Liddle's disease in this Japanese family.

This paper examines the dynamic behavior of a two-dimensional channel whose upper and lower walls deform symmetrically with respect to the center line of the channel. Unsteady fluid dynamic forces acting on the internal wall are analytically evaluated on the basis of a linearized compressible potential flow theory. The effects of distributed springs outside the channel and an internal pressure on the stability characteristics are studied by considering small disturbances about flat and buckled equilibrium configurations of the wall. The analytic methods indicate that no flutter of the flat or buckled wall is predicted when the Mach number is small and the viscous damping coefficient is positive. Numerical results by the Runge-Kutta-Gill method suggest that nonlinear effect of flow should be taken into account to fully examine the dynamic characteristics of the channel conveying a flow.