Survey of Japanese encephalitis virus in pigs and wild boars on Ishigaki and Iriomote Islands in Okinawa, Japan.

SUMMARY We previously revealed that Japanese encephalitis virus (JEV) seroprevalence was 4.5% in pigs on Ishigaki Island from 2005 to 2007. However, a partial E gene sequence (151 bp) of the JEV genome (JEV/sw/Ishigaki/1/2005) was detected in one pig. Phylogenetic analysis showed that JEV/sw/Ishigaki/1/2005 belonged to genotype III and to the same lineages isolated in Taiwan from 2006 to 2008. Serum samples were collected from 128 pigs on Ishigaki from 2009 to 2010, 24 wild boars on Ishigaki from 2008 to 2010, and 117 wild boars on Iriomote Island from 2008 to 2010. Four (3.1%) pigs on Ishigaki were positive for JEV antibody, but all wild boars on the island were negative. Fifty-two (44.4%) wild boars on Iriomote were positive for JEV antibody, in contrast to a seroprevalence of 3.7% in 2000 and 2004. JEV on Iriomote and/or in Taiwan might be related to transmission on Ishigaki.

The pilot study: sphingomyelin-fortified milk has a positive association with the neurobehavioural development of very low birth weight infants during infancy, randomized control trial.

AIM: This study was a randomised control trial to examine the effects of sphingomyelin (SM), on the mental, motor and behavioural development of premature infants. PATIENTS AND METHODS: Randomised, double-blind controlled trial, enroling infants born with a birth weight of less than 1500 g between January 2004 and October 2007 at Juntendo University Hospital, with follow-up to 18 months. Twenty-four preterm babies were randomly assigned; 12 were assigned to a test group and fed SM-fortified milk (SM 20% of all phospholipids in milk) and 12 were assigned to a control group (SM 13% of all phospholipids in milk). We analysed the composition of the plasma phospholipids and red-cell-membrane fatty acids, after which VEP, Fagan, BSID-II, attention and memory tests were performed. RESULTS: The percentage of SM in the total phospholipids was significantly higher in the trial group than in the control group at 4, 6 and 8 weeks. The Behaviour Rating Scale of the BSID-II, the Fagan test scores, the latency of VEP, and sustained attention test scores at 18 months were all significantly better in the trial group than in the control group. CONCLUSION: This study is the first to report that nutritional intervention via administration of SM-fortified milk has a positive association with the neurobehavioural development of low-birth-weight infants. However, detailed studies on the effects of SM on longer-term development are required.

Greater fat oxidation with diacylglycerol oil consumption for 14 days compared with triacylglycerol oil consumption in overweight men and women.

BACKGROUND: Several studies have reported increased fat oxidation with diacylglycerol (DAG) oil consumption. However, the effects of long-term DAG oil consumption on energy metabolism remain to be investigated. OBJECTIVE: The objective of this study was to compare the effects of 14 days of either DAG or triacylglycerol (TAG) oil consumption on substrate oxidation, energy expenditure (EE) and dietary fat oxidation. DESIGN: Eight males and six females participated in this randomized, double-blind, crossover feeding study. Each patient consumed the 14-day controlled test diet containing either 10 g day(-1) of DAG or TAG oil for acclimatization before a respiratory chamber measurement, followed by a 2-week washout period between diet treatments. Substrate oxidation and EE were measured in the respiratory chamber at the end of each dietary treatment. The patients consumed test oil as 15% of total caloric intake in the respiratory chamber (mean test oil intake was 36.1+/-6.6 g day(-1)). RESULTS: Twenty-four hour fat oxidation was significantly greater with 14 days of DAG oil consumption compared with TAG oil consumption (78.6+/-19.6 and 72.6+/-14.9 g day(-1), respectively, P<0.05). There were no differences in body weight or body composition between diet treatments. Dietary fat oxidation was determined using the recovery rate of (13)CO(2) in breath, and was significantly enhanced with DAG oil consumption compared with TAG oil consumption, measured over 22 h after ingestion of (13)C-labelled triolein. Resting metabolic rate (RMR) was significantly greater with DAG oil consumption compared with TAG oil consumption (1766+/-337 and 1680+/-316 kcal day(-1), respectively, P<0.05). CONCLUSION: Consumption of DAG oil for 14 days stimulates both fat oxidation and RMR compared with TAG oil consumption, which may explain the greater loss of body weight and body fat with DAG oil consumption that has been observed in weight-loss studies.

Multicentre prospective study of perinatal depression in Japan: incidence and correlates of antenatal and postnatal depression.

A multicentre study on the epidemiology of perinatal depression was conducted among Japanese women expecting the first baby (N = 290). The incidence rate of the onset of the DSM-III-R Major Depressive Episode during pregnancy (antenatal depression) and within 3 months after delivery (postnatal depression) were 5.6% and 5.0%, respectively. Women with antenatal depression were characterised by young age and negative attitude towards the current pregnancy, whereas women with postnatal depression were characterised by poor accommodation, dissatisfaction with sex of the newborn baby and with the emotional undermining. Antenatal depression was a major risk factor for postnatal depression.

Simulation of exercise-dependent difference in metabolism with a mathematical model for analyses of measurements using near-infrared spectroscopy.

Near-infrared spectroscopy (NIRS) is a useful technique for noninvasive measurement of muscle oxygenation. However, analyses of the dynamic changes in muscle metabolism based only on experimental observations of NIRS are difficult. Therefore, we constructed a mathematical model of muscle metabolism, comprising of the ATP synthesizing systems and O2 diffusion system, to identify the mechanisms responsible for those observations. A customized NIRS instrument was used to measure the changes in muscle oxygenation of the forearm flexor muscles during intermittent and continuous isometric flexion exercises when healthy male subjects participated in exercises tests. The exercise-dependent difference in changes could be distinguished and the simulated results agreed well with that measured experimentally. Although the contraction intensity for both exercises was identical, the magnitude of energy needed to perform the respective exercises was different. This difference was reflected by the changes in the ATP synthesizing systems, in which the energy needed during the latter-half of continuous exercise was mostly supplied by anaerobic system, whereas that during intermittent exercise was supplied by the aerobic and anaerobic systems that operated synergistically. From the results, we conclude that the model could be a useful tool for the elucidation of the relationship between experimental observations of NIRS and muscle metabolism.

Molecular characterization of a putative protein disulfide isomerase from Babesia caballi.

We produced a mAb against the Babesia caballi extracellular merozoite termed mAb 2H2 and used it to screen a cDNA expression library prepared from B. caballi merozoite mRNA for highly expressed proteins. The complete nucleotide sequence of the cloned gene had 1547 nucleotides and contained a 36-nucleotide intron. The 1398 nucleotide open reading frame predicts a 51 kDa protein showing similarity to protein disulfide isomerase (PDI) from other species. The PDI gene had a predicted N-terminal signal sequence of 19 amino acids and a C-terminal tetrapeptide sequence (His-Thr-Glu-Leu; HTEL) for retention in lumen of the endoplasmic reticulum (ER). The recombinant protein expressed in baculovirus showed an apparent mass of 51 kDa, identical to that the native B. caballi protein. Moreover, the ER retention signal site (HTEL) of the recombinant protein retained its function in ER of insect cells. This 51 kDa protein was strongly expressed by extracelluar B. caballi merozoites in indirect immunofluorescence antibody tests, and was not expressed in the early phase of trophozoite development. Interestingly, detailed observation showed that the reaction of anti-P51 antibody and mAb 2H2 against pear-shaped forms was very erratic, some displaying one or two brightly fluorescent patterns.

Congenital dermatofibrosarcoma protuberans with fibrosarcomatous and myxoid change.

This report describes a case of congenital dermatofibrosarcoma protuberans (DFSP) with fibrosarcomatous (FS) and myxoid areas. Immunohistochemical results showed that tumour cells in ordinary DFSP areas were diffusely positive for CD34, whereas in the FS and myxoid areas, few tumour cells were positive for this antigen. Ki-67 positive tumour cell numbers were greater in the FS (11.8%) and myxoid areas (19.8%) relative to ordinary DFSP areas (2.2%). Reverse transcription polymerase chain reaction and sequence analysis showed the presence of an identical COL1A1-PDGFB fusion transcript in ordinary DFSP (plaque-like area), FS, and myxoid areas of DFSP. These results indicate that the three components of DFSP have a common histogenesis. This study documents the first application of gene analysis involving the myxoid area of DFSP.

Novel quantitative assessment of myocardial perfusion by harmonic power Doppler imaging during myocardial contrast echocardiography.

OBJECTIVE: To test the hypothesis that the power of the received signal of harmonic power Doppler imaging (HPDI) is proportional to the bubble concentration under conditions of constant applied acoustic pressure, and to determine whether a new quantitative method can overcome the acoustic field inhomogeneity during myocardial contrast echocardiography (MCE) and identify perfusion abnormalities caused by myocardial infarction. METHODS: The relation between Levovist concentration and contrast signal intensity (CI) of HPDI was investigated in vitro under conditions of constant acoustic pressure. MCE was performed during continuous infusion of Levovist with intermittent HPDI every sixth cardiac cycle in 11 healthy subjects and 25 patients with previous myocardial infarction. In the apical views myocardial CI (CI(myo)) was quantified in five myocardial segments. The CI from the left ventricular blood pool adjacent to the segment was also measured in dB and subtracted from the CI(myo) (relative CI (RelCI)). RESULTS: CI had a logarithmic correlation and the calculated signal power a strong linear correlation with Levovist concentration in vitro. Thus, a difference in CI of X dB indicates a microbubble concentration ratio of 10(X/10). In normal control subjects, CI(myo) differed between the five segments (p < 0.0001), with a lower CI(myo) in deeper segments. However, RelCI did not differ significantly between segments (p = 0.083). RelCI was lower (p < 0.0001) in the 39 infarct segments (mean (SD) -18.6 (2.8) dB) than in the 55 normal segments (mean (SD) -15.1 (1.6) dB). RelCI differed more than CI(myo) between groups. CONCLUSIONS: The new quantitative method described can overcome the acoustic field inhomogeneity in evaluation of myocardial perfusion during MCE. RelCI represents the ratio of myocardium to blood microbubble concentrations and may correctly reflect myocardial blood volume fraction.

Imaging of Regional Differences of Muscle Oxygenation during Exercise Using Spatially Resolved NIRS.

The development of imaging systems using near-infrared spectroscopy (NIRS) has enabled noninvasive measurement of regional changes in muscle oxygenation. A spatially resolved NIRS (SR-NIRS) imaging instrument was utilized for real-time measurement of spatial-temporal changes in muscle oxygenation during exercises. Changes in muscle oxygenation and localized O2consumption in the quadriceps muscle were measured during sustained isometric knee extension without and with leg-press to the ground exercises. In the former exercise, the level of tissue oxygen saturation (TOS) of the rectus femoris (RF) muscle was found to be lower than that of vastus lateralis (VL) and vastus medialis (VM) muscles. The highest localized O2consumption rate, VO2, reflecting the localization in distribution of muscle metabolism, was detected in the RF muscle at the initial stage of exercise. As exercise progressed, VO2of the RF muscle decreased whereas that of the VL and VM muscles increased. In contrast, TOS decreased to about the same level for the VL, RF and VM muscles in the latter exercise. Also, VO2of all three muscles decreased as exercise progressed. Initial results demonstrated that the SR-NIRS instrument enables measurement of regional differences in muscle oxygenation in the quadriceps muscle during different exercises.

Characterization of monoclonal antibodies against Gnathostoma nipponicum.

Monoclonal antibodies (mAbs) were produced against the proteins of advanced third-stage larvae (AdL3) of Gnathostoma nipponicum. Six mAbs (Gn2C3, Gn2H3, Gn4C3, Gn4E9, GnSH1, and Gn10B7) were obtained as determined by enzyme-linked immunosorbent assay (ELISA). Gn4E9 and GnSH1 seemed to be genus-specific, as they did not cross-react with Anisakis sp., Dirofilaria immitis, Gongylonema pulchrum, Toxocara canis, Trichinella sp., Trichuris vulpis, Metagonimus sp., or Spirometra erinaceieuropaei by ELISA. Immunohistochemistry showed that Gn2C3, Gn4E9, and Gn5H1 reacted strongly with the central esophagus; Gn2H3 reacted with cuticle,muscle, intestine, and the cervical sac; and Gn4C3 and Gn10B7 reacted with cuticle, muscle, esophagus, intestine, and the cervical sac of AdL3. In Western blotting analysis, Gn2C3, Gn4E9, and Gn5H1 reacted to 60-, 53-, 46-, and 41-kDa proteins; Gn4C3 reacted to the AdL3 protein of G. nipponicum (>42 kDa). Moreover, proteins purified using a mAb Gn4E9 immunoprecipitation method (sizes 60-, 53-, 46-, and 41-kDa) were used as antigens in ELISAs. A significant difference (P < 0.01) was shown between mouse sera infected with G. nipponicum and sera infected with Trichnella sp. or not infected. These results provide a rationale for evaluating esophageal proteins for the development of diagnostic methods for detecting G. nipponicum or Gnathostoma sp. infections.

Metabolic alterations by clofibric acid in the formation of molecular species of phosphatidylcholine in rat liver.

The mechanism by which p-chlorophenoxyisobutyric acid (clofibric acid) induces striking changes in the proportion of the molecular species of phosphatidylcholine (PC) in rat liver was studied. Treatment of rats with clofibric acid strikingly increased the content of 1-palmitoyl-2-oleoyl (16:0-18:1) PC, but decreased the contents of 1-palmitoyl-2-docosahexaenoyl (16:0-22:6), 1-stearoyl-2-arachidonoyl (18:0-20:4), and 1-stearoyl-2-linoleoyl (18:0-18:2) PC; the drug did not change the content of 1-palmitoyl-2-arachidonoyl (16:0-20:4) PC. The mechanism underlying these changes has been investigated with regard to the in vivo formation of the molecular species of PC by: (i) de novo synthesis, (ii) reacylation, and (iii) methylation of phosphatidylethanolamine (PE). We found that (i) the incorporation of [3H]glycerol, which was injected intravenously, into 16:0-18:1 diacylglycerol (DG) and 16:0-18:1 PC was increased markedly by clofibric acid feeding without changing the substrate specificity of CDP-choline:DG cholinephosphotransferase, (ii) the in vivo formation of 16:0-18:1 and 16:0-20:4 PC from 1-16:0-[3H]glycerophosphocholine (GPC), which was injected intraportally, was increased markedly by clofibric acid feeding, and (iii) the incorporation of [14C]ethanolamine, which was injected intravenously into 16:0-22:6, 18:0-22:6, and 18:0-20:4 PC, was decreased by clofibric acid feeding; the extent of the decrease in 16:0-20:4 PC was less than that of 18:0-20:4 PC. It was concluded, therefore, that (i) clofibric acid selectively increased the content and proportion of 16:0-18:1 PC by enhancing both the CDP-choline pathway and the remodeling of the pre-existing PC molecule, and (ii) the drug kept the content of 16:0-20:4 PC unchanged by stimulating the remodeling of the pre-existing PC molecule, whereas the formation of other more long chain, polyunsaturated molecular species, such as 16:0-22:6, 18:0-22:6, and 18:0-20:4, was decreased owing to the suppression of PE methylation.

Effects of dehydroepiandrosterone on oleic acid formation in the liver of rats, mice and guinea pigs.

The purpose of the present study is to answer the question of whether there is a species difference in the effects of a pharmacological dose of dehydroepiandrosterone (DHEA) on the enzymes that participate in oleic acid (18:1) formation in the liver. Feeding a diet containing 0.5% (w/w) DHEA for 14 days markedly increased the activities of acyl-coenzyme A (CoA) synthetase, palmitoyl-CoA chain elongase and stearoyl-CoA desaturase in the liver of rats and mice. These enzyme activities, however, were not changed by DHEA in guinea pigs. The treatments of rats and mice with DHEA markedly increased proportions of 18:1 in hepatic lipids, especially phosphatidylcholine (selectively at C-2 position), triacylglycerol and cholesterol ester. DHEA caused no significant changes in acyl compositions of hepatic lipids of guinea pigs. The levels of DHEA or dehydroepiandrosterone sulfate (DHEAS) were markedly increased in serum and livers by DHEA administration to rats, mice and guinea pigs. High correlations were observed between hepatic levels of DHEA or DHEAS and stearoyl-CoA desaturase activities in rats. These results indicate that there are species differences in the inducing effects of DHEA or DHEAS on hepatic formation of 18:1 and that guinea pigs lack the machinery to induce the enzymes.

The first host record of trichinosis in a red fox, Vulpes vulpes japonica, from Aomori Prefecture, northern Honshu, Japan.

A case of wildlife trichinosis was found in a red fox (Vulpes vulpes japonica) captured at Rokkasho, Aomori Prefecture on November 27, 1998. Trichinella larvae were obtained from almost all of the muscle tissues except for the masseter. The highest number of larvae per gram of tissue was found in the muscles of the gluteal region and throat. The lowest number was found in the diaphragm and tongue. Trichina cysts within the muscle fibers had groups of fatty cells at the poles, and minimal tissue reaction was observed around the cyst. No calcification was found in the cyst. These morphological findings suggested that the considerable time had elapsed since the invasion. This is the first case of trichinosis in a red fox in Japan.

Developmental changes in 5-hydroxytryptamine-induced rhythmic activity in the spinal cord of rat fetuses in vitro.

The roles played by glycine- and glutamate-mediated synaptic transmission in the generation of 5-hydroxytryptamine (5-HT)-induced rhythmic activity were examined in isolated spinal cord preparations from fetal rats. Bath application of 5-HT (0.1-30 microM) evoked rhythmic activity in lumbar ventral roots at and after E14.5. Bath application of strychnine (5 microM), a glycine-receptor antagonist, reduced the frequency of the rhythmic activity to 37% of control at E14.5. Although, kynurenate (4 mM), a glutamate-receptor antagonist, had little effect at this stage, it completely abolished the 5-HT-induced rhythmic activity at and after E18.5, when strychnine had little effect on the frequency. These results indicate that, at and shortly after its onset, the rhythmic activity is driven mainly by glycinergic rather than glutamatergic excitatory synaptic inputs, but that the latter become dominant later on.

Detection of cytokeratin 19 and 20 mRNA in peripheral and mesenteric blood from colorectal cancer patients and their prognosis.

To detect the presence of cancer cells in peripheral and mesenteric blood and to examine their relationship to prognosis in colorectal cancer patients undergoing curative surgery, we examined cytokeratin 19 and 20 mRNA in peripheral and mesenteric venous blood. Using reverse transcriptase polymerase chain reaction, cytokeratin 19 and 20 mRNA was amplified in peripheral and mesenteric blood samples obtained from 35 colorectal cancer patients who underwent curative surgery. Cytokeratin 19 or 20 mRNA in peripheral or mesenteric blood samples was detected in 18 of 35 cases (51%). There was no significant difference in Dukes' staging between the positive and negative groups. The median follow-up period was 56 months. In the positive group six patients (33%) showed recurrences. One patient (6%) showed recurrence in the negative group. The recurrence rate was significantly higher in the positive group than in the negative group. Five-year disease-free survival was significantly better in the negative group than in the positive group (94% vs. 65%). The detection of cytokeratin 19 or 20 mRNA in peripheral or mesenteric blood is thus associated with the prognosis for colorectal cancer patients undergoing curative surgery. Although the presence of these mRNAs in blood samples is a prognostic marker, the clinical utility of this assay is questionable because of the low recurrence rate in the positive group.

Comparison of the elimination between perfluorinated fatty acids with different carbon chain length in rats.

Elimination in urine and feces was compared between four perfluorinated fatty acids (PFCAs) with different carbon chain length. In male rats, perfluoroheptanoic acid (PFHA) was rapidly eliminated in urine with the proportion of 92% of the dose being eliminated within 120 h after an intraperitoneal injection. Perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) was eliminated in urine with the proportions of 55, 2.0 and 0.2% of the dose, respectively. By contrast, four PFCAs were eliminated in feces with the proportion of less than 5% of the dose within 120 h after an injection. In female rats, the proportions of PFOA and PFNA eliminated in urine within 120 h were 80% and 51% of the dose, respectively, which were significantly higher compared with those in male rats. There was the tendency that PFCA with longer carbon chain length is less eliminated in urine in both male and female rats. Fecal elimination of PFCAs was not different between PFCAs in female rats and comparable to those in male rats. The rates of biliary excretion of PFCAs in male rats were slower than those in female rats. Sex-related difference in urinary elimination of PFOA was abolished when male rats had been castrated. On the contrary, treatment with testosterone suppressed the elimination of PFOA in urine in both castrated male rats and female rats. The effect of testosterone was in a time- and dose-dependent manner. These results suggest that PFCAs are distinguished by their carbon chain length by a renal excretion system, which is regulated by testosterone.

Analysis of a growth-promoting factor for Babesia caballi cultivation.

Serum-free media were examined to culture Babesia caballi. Daigo's T (DT) basal medium supplemented with Daigo's GF21 (GF21) or GIT medium, which already contains GF21, supported the parasite propagation at 37 C in a humidified atmosphere under 5% CO2 in air. Growth of B. caballi was dependent of the suitable concentration (10-20%) of GF21. Therefore, GF21 was suggested as the growth-promoting factor for B. caballi. However, GIT medium did not support the growth of parasites from cryopreserved stabilates, and serum supplementation was essential for the retrieval of parasites.

Three-dimensional solution structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. japonica.

The three-dimensional structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. japonica, has been determined in solution at pH 6.8 and 25 degrees C by (1)H and (15)N NMR spectroscopy. The main body (Glu13-Asp97) of oryzacystatin-I is well-defined and consists of an alpha-helix and a five-stranded antiparallel beta-sheet, while the N- and C-terminal regions (Ser2-Val12 and Ala98-Ala102) are less defined. The helix-sheet architechture of oryzacystatin-I is stabilized by a hydrophobic cluster formed between the alpha-helix and the beta-sheet and is considerably similar to that of monellin, a sweet-tasting protein from an African berry, as well as those of the animal cystatins studied, e.g., chicken egg white cystatin and human stefins A and B (also referred to as human cystatins A and B). Detailed structural comparison indicates that oryzacystatin-I is more similar to chicken cystatin, which belongs to the type-2 animal cystatins, than to human stefins A and B, which belong to the type-1 animal cystatins, despite different loop length.