Genome sequence of a Japanese isolate of Radish mosaic virus: the first complete nucleotide sequence of a crucifer-infecting comovirus.

The complete nucleotide sequences of RNA1 and RNA2 of a Japanese isolate of Radish mosaic virus (RaMV-J), a crucifer-infecting comovirus, were determined. RNA1 is 6064 nucleotides long and encodes a 210-kDa polyprotein containing conserved motifs that are required for replication. RNA2 is 4020 nucleotides long and encodes a 123-kDa polyprotein containing the putative movement protein and two coat proteins. Comparisons of the encoded proteins confirmed that RaMV-J and a Czech RaMV isolate are isolates of the same species in the genus Comovirus. A phylogenetic analysis of RaMV-J and other comoviruses revealed that legume-infecting comoviruses constitute a single branch to which RaMV is distantly related.

Effects of the inhalation of diesel exhaust, Kanto loam dust, or diesel exhaust without particles on immune responses in mice exposed to Japanese cedar (Cryptomeria japonica) pollen.

To assess the potential enhancement by air-pollutants of immune responses in mice, especially with regard to allergen-specific immunoglobulin E (IgE) antibody production, female BDF(1) mice (60 mice in each group) were exposed to diesel exhaust (particles, 3.24 mg/m(3); nitrogen dioxide, 1.0 ppm: DE group), Kanto loam dust (particles, 3.29 mg/m(3); nitrogen dioxide, 0.01 ppm: KLD group), diesel exhaust without particles (particles, 0.01 mg/m(3); nitrogen dioxide, 1.1 ppm: DEG group), or clean air (pollen and control groups) for 16 h/day, 5 days/wk for 24 wk, as well as to Japanese cedar pollen (JCP) (around 550,000 grains of JCP/m(3)) for 2 days/wk in the same period. The control group was exposed to clean air alone throughout the experiment. The mean values for Japanese cedar pollen allergens (JCPAs)-specific immunoglobulin E (IgE) antibody titers in mice sera measured by enzyme-linked immunosorbent assay (ELISA) in the DE, KLD, and DEG groups were higher than that for the pollen alone group, but not significantly, after both 12 and 24 wk of exposure time. The percentages of animals expressing more than the minimum ELISA titer of JCPAs-specific IgE antibodies in each group were 22% (DE and pollen groups) and 27% (KLD and DEG groups) of the totals at wk 12, and no statistical differences were observed among the groups. However, at wk 24 in the DE, KLD, and DEG groups the responders comprised 73%, 63%, and 67%, respectively, significantly higher than the 33% for the pollen alone group. No significant differences were observed among the DE, KLD, and DEG groups. A slight dose-dependent increase of proliferative responses of mouse cervical lymph node cells to JCPAs in both DE and KLD groups was observed, but not in the DEG group. Remarkable decrease of interferon-gamma and significant increase of interleukin-4 in the nasal lavage fluid were apparent after DE or DEG exposure, but not in the KLD group. These results suggest that these air pollutants (DE, KLD, and DEG) enhance the production of IgE antibodies in mice, with similar adjuvant activities in each case. Furthermore, in the early phase of exposure in which sensitization occurred with exposure to pollen, the fine particles and gas components are considered to have exhibited different enhancing mechanisms in mice as follows: (1) The fine particles augmented production of IgE antibodies through activation of T lymphocytes, and (2) the gas components exhibited almost no action on T lymphocytes, but directly induced disorders of the cytokine network and augmented the production of IgE antibodies.

Increased basal levels of plasma nitric oxide in Type 2 diabetic subjects. Relationship to microvascular complications.

To assess the underlying mechanisms of decreased endothelial function and advanced vascular complications in patients with Type 2 diabetes, we determined basal levels of plasma nitric oxide (NO(x): NO(2)(-) and NO(3)(-)) using a newly developed high-performance liquid chromatography (HPLC)-Griess method in hospitalized 129 diabetic and 76 nondiabetic subjects, and examined their clinical characteristics. Serum lipid peroxide and advanced glycation end products (AGEs) as markers of oxidative stress were also measured, and intima-media thickness (IMT) of the carotid artery was evaluated as a marker of atherosclerosis. In diabetic subjects, microvascular complications were newly evaluated during their admission. There were no differences in age or sex between the diabetic and nondiabetic subjects. Although there was no difference in basal plasma NO(2)(-) levels between the two groups, the basal levels of plasma NO(3)(-) in diabetic subjects were significantly higher than those in nondiabetic subjects. Plasma NO(x) levels in neither diabetic nor nondiabetic subjects correlated with serum lipids, HbA1c, or IMT. In diabetic subjects, plasma NO(3)(-) levels were related not only to the presence of hypertension but also to advanced microvascular complications. Moreover, plasma NO(3)(-) levels were positively correlated with both serum lipid peroxide and AGEs. Multiple regression analysis revealed that serum AGEs level was strongly associated with plasma NO(3)(-) level. Thus, the findings are consistent with the hypothesis that decreased endothelium-dependent vasodilation in diabetic subjects is associated with the impaired action of NO secondary to its inactivation resulting from increased oxidative stress, rather than decreased NO production from vascular endothelium, and that abnormal NO metabolism is related to advanced diabetic microvascular complications.

NO(x) contamination in laboratory ware and effect of countermeasures.

Contamination of various types of laboratory wares with NO(x) (NO(-)(2) and NO(-)(3)) was assessed systematically and the effect of extensive washing as a countermeasure was evaluated. Mean NO(x) contamination arising from a model procedure for NO(x) determination in plasma was 0.93 microM (range, 0.35-1.49 microM). The major source of contamination included conical tubes (54.8%) and pipette tips used for transfer of solution (12.3-16.3%). Except for soft glassware, most NO(x) contamination could be washed out by pure water. Although NO(x) contamination in respective laboratory wares could be reduced below detection levels by extensive washing, summation of the contamination through the model procedure could not be completely abolished (but the effect of washing persisted at least 10 days). Heavy contamination was noted in glassware (especially soft glass) and ultrafiltration units, which was difficult to remove. Several types of vacuum blood sampling tubes contained various levels of NO(x). Our results indicated that a small but significant amount of contamination remained in laboratory ware even after extensive washing, and that it is advisable to avoid the use of glassware (soft glass), ultrafiltration units, and vacuum blood sampling tubes during the processing of clinical sampling for the measurement of NO(x).

The C677T mutation in the methylene tetrahydrofolate reductase gene increases serum uric acid in elderly men.

A common mutation, C677T, in the methylene tetrahydrofolate reductase gene (MTHFR) reduces the activity of MTHFR and increases total homocysteine levels in plasma. Increased homocysteine levels are reportedly associated with high serum uric acid levels. The relationship between the MTHFR mutation and uric acid metabolism remains unclear, however. To investigate whether the C677T MTHFR mutation is a risk factor for hyperuricemia, we performed MTHFR genotyping and clinical laboratory determinations, including serum uric acid, in 271 elderly Japanese men (age range, 40-79 years; mean, 52.6 years). The mean uric acid levels for the C/C, C/T, and T/T genotypes were 5.67, 6.00, and 6.39 mg/dl, respectively (P = 0.012). The T/T genotype was more frequent in subjects with high uric acid levels than in those with low uric acid levels (P = 0.038). These findings suggest that the C677T MTHFR mutation contributed to higher uric acid levels in subjects enrolled in this study. In conclusion, the mutation of the MTHFR gene may be a risk factor for hyperuricemia in elderly men.

Anorexia nervosa with severe liver dysfunction and subsequent critical complications.

A twenty-year-old woman with anorexia nervosa (body mass index=11) suffered from severe liver dysfunction (aspartate aminotransferase 5,000 IU/l, alanine aminotransferase 3,980 IU/l, prothrombin time 32%), hypoglycemia (serum glucose 27 mg/dl), and pancreatic dysfunction (amylase 820 IU/l, lipase 558 IU/l). She fell into a depressive state with irritability, which was not improved by intravenous glucose. Despite treatment with plasmapheresis for the liver dysfunction, she subsequently developed pulmonary edema, acute renal failure, gastrointestinal bleeding, and disseminated intravascular coagulation. Hemodialysis, mechanical ventilation and drug therapy including prednisolone, prostaglandin E1, and branched-chain amino acid, improved her critical condition. In this case, malnutrition may have been the cause for the liver dysfunction and subsequent complications.

Candida guilliermondii infection in SCID mice in association with the acceleration of the elimination of transfused human red blood cells.

An acceleration of the elimination of transfused human (Hu) red blood cells (RBC) was found in C.B-17 scid (SCID) mice that were kept in our facility. Yeast like organisms were isolated from their tap water just as a pure culture and the two isolates SW5 and SW6 were assigned to be Candida guilliermondii by analysing their generic small subunit ribosomal RNA sequences. To test whether the isolates are infectious in mice, we inoculated SCID and BALB/c mice orally with SW5 and observed them for 63 and 48 days, respectively. The yeasts were frequently recovered from oral swabs, feces and their tap water throughout the experiment. Although none of the mice developed clinical signs or histopathological changes, a positive sero-conversion was confirmed in 4 of 5 SW5-inoculated BALB/c mice. Moreover, a significant acceleration of Hu-RBC elimination in all of the SW5-infected SCID mice was demonstrated. We believe this to be the first report of an inapparent but significant outbreak of C. guilliermondii infection in mice.

Comparison of the effects of various fine particles on IgE antibody production in mice inhaling Japanese cedar pollen allergens.

The adjuvant effects of various fine particles [Kanto loam dust, fly ash, carbon black, diesel exhaust particles (DEP), and aluminum hydroxide (alum)] on immunoglobulin E (IgE) antibody production in female BDF1 mice were examined. In experiment 1, animals both received 25 micrograms of each particle intranasally and were exposed to aerosolized Japanese cedar pollen allergens (JCPA) for 30 min/d at 1-wk intervals for the first 8 wk. This was followed by exposure for 30 min every 3 wk for the next 9 wk. As parameters of allergic rhinitis, measurements were made of JCPA-specific IgE and IgG antibody titers, the protein-adsorbing capacity of each type of particle, and nasal rubbing movements. The increases in anti-JCPA IgE and IgG antibody production in mice treated with aerosolized JCPA plus respective particles were significantly greater than that found with aerosolized JCPA alone. This was associated with no marked differences in the other allergic rhinitis parameters. In experiment 2, after the administration of particles as in experiment 1, about 160,000 grains of Japanese cedar pollen (JCP, native dry pollen) were dropped onto the tip of the nose of mice twice a week for 16 wk. Six weeks after the first immunization, the anti-JCPA IgE antibody titers of groups treated with the respective particles were greater than 1:20, whereas those of mice treated with JCP alone were 1:10. No significant differences in the anti-JCPA IgE and IgG antibody productions, nasal rubbing counts, or histopathological changes were observed after 18 wk. These results suggested the nature of the particles, their capacity to adsorb antigens, and/or their size may not be related to enhancement of IgG antibody production nor symptoms of allergic rhinitis. However, IgE antibody production seemed to occur earlier in mice treated with particles than in mice immunized with allergens alone.

Short-term exposure to diesel exhaust induces nasal mucosal hyperresponsiveness to histamine in guinea pigs.

The increasing prevalence of allergic rhinitis in many countries is becoming a social problem. It is important to determine whether air pollutants are related to the increase in the prevalence rate of allergic rhinitis or not. In this respect, it is necessary to elucidate whether exposure to air pollutants affects the nasal mucosa and causes nasal mucosal hyperresponsiveness to chemical mediators released by antigen-antibody reactions. A previous study revealed that diesel exhaust particulates are potent in augmenting increases in nasal congestion and nasal secretion induced by histamine (T. Kobayashi and T. Ito, 1995, Fundam. Appl. Toxicol. 27, 195-202). In the present study, using a rhinitis model of guinea pigs, we investigated whether short-term (3-hr) exposure to diesel exhaust induces nasal mucosal hyperresponsiveness to histamine. Guinea pigs of each group were exposed to filtered air or to a low or high concentration of diesel exhaust (1 and 3.2 mg/m3 particulates in diluted diesel exhaust, respectively) for 3 hr. After diesel exhaust exposure, sneezing frequency, nasal secretion from the nostril, and intranasal airway resistance induced by histamine were measured as indices of sneezing response, rhinorrhea, and nasal congestion, respectively. Short-term exposure to a low or high concentration of diesel exhaust itself did not induce sneezing, nasal secretion, or nasal congestion. However, short-term exposure to a high concentration of diesel exhaust augmented sneezing and nasal secretion, but not nasal congestion, induced by histamine. In conclusion, short-term exposure to diesel exhaust potently induces nasal mucosal hyperresponsiveness.

Changes in body weight and organ weight of Ishibashi (IS) rats with growth.

Changes in body weight (25-175 days old, every 10 days) and weights of various organs (70, 105, 140 and 175 days old), i.e., cerebrum, cerebellum, pituitary gland, thyroid gland, thymus, heart, liver, spleen, adrenal gland, kidney, seminal vesicle, prostate, epididymis, testes, bulbourethral gland and ovary, of Ishibashi (IS) rats with growth, which are model animals for congenital vertebral malformation (spontaneous kyphoscoliosis) were examined as compared with Brown Norway (BN) rats, which are genetically irrelevant to IS rats, and also with hybrid rats (IBF, rats) which are between IS and BN rats. The experimental results showed that body weight and weights of various organs except cerebrum, cerebellum and thymus were greater in IS rats than in BN rats, and body weight and weights of various organs of IBF, rats were intermediate between the two strains of rats.

Translocation of bacteria from the gastrointestinal tract in immunodeficient mice.

Host defence mechanisms associated with the inhibition of translocation of bacteria from the gastrointestinal (GI) tract were investigated in SCID and beige mice after decontamination with oral antibiotics and colonization with Escherichia coli C25. SCID mice, which have impaired T and B cell function, tended to have a greater incidence of bacterial translocation from the GI tract up to 7 days after inoculation compared with controls. However, after 7 days both SCID and controls cleared the E. coli C25 from the liver, spleen, blood and peritoneal cavity. Beige mice, with impaired NK cell and polymorphonuclear leukocyte function, were not able to clear the inoculated bacteria from their liver by 14 days after inoculation although the controls were cleared by 7 days. Numbers of bacteria in the mesenteric lymph nodes (MLN) of beige mice did not decrease significantly by 14 days after inoculation, whereas numbers in SCID mice decreased markedly within 7 days. These results suggest that defence mechanisms other than T and B cell function are important in the inhibition of systemic infection from the GI tract.

Effect of iron as a new type of phosphate binder in hemodialysis patients.

Hyperphosphatemia is one of the major problems requiring management in the majority of hemodialysis patients and they require phosphate-binding agents to control the hyperphosphatemia. Aluminum hydroxide and calcium compounds are used currently as phosphate-binding agents to treat hyperphosphatemia, but these compounds can cause undesirable side effects. Therefore, the development of new phosphate-binding agents is imperative. It is well known that oral and intravenous administration of iron causes hypophosphatemia. We hypothesized that this side effect of iron may be beneficial for the treatment of hyperphosphatemia in hemodialysis patients. Consequently, we conducted a fundamental and clinical investigation of the effects of iron administration. Membrane permeability of phosphorus in a mixture of sodium ferrous citrate and dessicated aluminium hydroxide in the presence of hydrogenated lecithin as a phosporic compound was examined. Fifteen patients undergoing hemodialysis were treated with 150 mg of sodium ferrous citrate given orally for eight weeks. The permeability of the filtering membrane to phosphorus decreased in accordance with the dosage of sodium ferrous citrate and dessicated aluminum hydroxide. The degree of phosphate-binding effect of sodium ferrous citrate was larger than that of dessicated aluminum hydroxide. Serum phosphorus decreased significantly during the experiment. These results suggest that the oral administration of sodium ferrous citrate as a new phosphate binder is a useful therapeutic method for hemodialysis in patients with hyperphosphatemia.

Evidence for an increased intracellular free calcium concentration in platelets of bronchial asthma patients.

The pathogenesis of bronchial asthma is not yet fully understood. Recently much attention has been given to the hypothesis that intracellular free calcium ([Ca2+]i) metabolism is abnormal in various diseases. In this study we investigated whether [Ca2+]i exists abnormally in subjects with bronchial asthma. The [Ca2+]i in 32 treated or untreated subjects with bronchial asthma were compared with 63 normal subjects. Resting levels of [Ca2+]i were estimated by loading the fluorescent indicator Fura-2 in washed platelets. The [Ca2+]i level in the control subjects was 129.7 +/- 18.0 nM (mean +/- SD). However, in that of the bronchial asthma patients was 152.7 +/- 44.1 nM, significantly higher than that of the control subjects (p < 0.05). It is well recognized that an increase of [Ca2+]i in vascular smooth muscle involves contraction. The findings suggest that the same phenomenon is quite possible in the tracheal smooth muscle and that it plays an important role in the pathogenesis of bronchial asthma.

Spatial and temporal regulation of the rat calmodulin gene III directed by a 877-base promoter and 103-base leader segment in the mature and embryonal central nervous system of transgenic mice.

Three non-allelic rat calmodulin (CaM) genes CaMI, CaMII and CaMIII, which share no homology in their 5'-upstream regions, are coordinately expressed in neurons of the central nervous system (CNS). Deletion analysis of the CaMIII promoter showed that the upstream segments longer than 700 bases functioned as efficient promoters, and that the sequence from -133 to -65 was required for the activity of house-keeping type promoter in transient expression assays on a mouse glioma cell line C6. However, the transient expression seemed not to be cell type specific. To determine the temporal and spatial specificity of the promoter function, we produced transgenic mice carrying a fusion gene of the CaMIII segment from -877 to +103 and the lacZ reporter gene. In CNS of the adult transgenic mice, the localization of transgene expression was similar to that of endogenous CaMIII transcripts analyzed by in situ hybridization. The transgene was expressed prominently in pyramidal cells of the cerebral neocortex and the hippocampal regions CA1 to CA3, in Purkinje cells of the cerebellar cortex, and in neurons of the spinal cord, and moderately in granule cells of the dentate gyrus and the cerebellar cortex. In the developing CNS, the overall profiles of neuron-specific expression were also similar for both transgene and endogenous CaMIII that were expressed in the mantle layer and the dorsal root ganglia of the embryonal spinal cord. These results indicated that the neuron-specific expression of rat CaMIII was directed by this 877-base promoter sequence. The CaMIII segment used for the promoter of transgene contained a 29-bp sequence at -410, namely H3, which was conserved in the upstream regions of vertebrate CaMII and CaMIII. H3 seemed to play a pivotal role in the temporal and spatial expression of transgene in CNS, although the deletion of H3 did not decrease CAT activity in the transient expression. The transgene expression was not observed in the external granular cells of the developing cerebellum and in some neurons of the embryonic sensory ganglia in which the endogenous CaMIII was obviously expressed. Therefore, the other cis-acting element(s) located outside of this 877-bp segment seemed to be required for the temporal regulation of CaMIII in certain rudimentary neurons.

[A long-term hemodialysis patient with spontaneous deep vein thrombosis, showing high levels of coagulo-fibrinolytic markers].

A 70-year-old woman on maintenance hemodialysis for 3 years was admitted to our hospital because of deep vein thrombosis (DVT) in the right femoral vein. Seven days before admission, she suddenly noticed severe pain in her right inguinal region while she was walking on the street. A wide range of stenosis from the iliac to the distal femoral region was detected by both CT scanning and venography. Her hematocrit reading was 30% and her serum erythropoietin level was 10.5 MU/ml, which was within the normal range, on the day of admission. The results of routine coagulation tests, including prothrombin time, activated partial thrombin time and plasma fibrinogen values, were normal. Plasma anti-thrombin III and plasminogen were also normal. In contrast, beta-thromboglobulin, platelet factor IV, fibrinogen degradation product, D-dimer, thrombin-antithrombin III complex (TAT) and fibrinopeptide A were abnormally elevated. In the venous occlusion test which was performed in the forearm of the opposite side of the arterio-venous shunt, plasma tissue type plas-minogen activator values showed little response to occlusion indicating that the vessel endothelium may have been partially damaged. These data suggest that the DVT had been induced by imbalance of increased blood coagulation and decreased fibrinolytic activity. Damaged vascular wall may also have contributed to the production of DVT. Furthermore, it is surprising that the patient had elevated values of D-dimer and TAT for many years without recurrence of the DVT. Spontaneous DVT in an apparently healthy individual on maintenance hemodialysis seems to be rare, compared with arterial thrombosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical double-labeling study of gonadotropin-releasing hormone (GnRH)-immunoreactive cells and oxytocin-immunoreactive cells in the preoptic area of the dwarf gourami, Colisa lalia.

The distribution of gonadotropin-releasing hormone (GnRH)-immunoreactive (ir) cells in the preoptic area (POA) of the dwarf gourami, Colisa lalia, was immunohistochemically studied. These neurons form cell columns on both sides of the common ventricle, and their axons project to the pituitary gland by forming distinct bundles. Also examined was the distribution of isotocin (IST) cells in the POA by using an anti-oxytocin (OXT) serum which has been proven to crossreact with IST. These two kinds of immunoreactive cells were distributed quite similarly in the POA. However, by using an immunofluorescence double-labeling method on thinner sections we found that a population of small IST cells in the ventral POA were also immunoreactive to GnRH, but that large IST cells in the dorsal POA were not immunoreactive to GnRH, and small GnRH-ir cells in the most ventral POA were not immunoreactive to the OXT antiserum. In the pituitary gland, GnRH-ir fibers were found in both the neurohypophysis and proximal pars distalis, but IST fibers were found only in the neurohypophysis.

Cloning and nucleotide sequence of the alkaline protease gene from Fusarium sp. S-19-5 and expression in Saccharomyces cerevisiae.

We have cloned a genomic DNA encoding the alkaline protease (Alp) of Fusarium sp. S-19-5 from a genomic DNA library and sequenced the nucleotides. Complementary DNA encoding Alp was also isolated from the cDNA library after amplifying the gene by PCR using partial sequences of the Alp genomic DNA as primers. The Alp gene has an open reading frame of 1137 nucleotides containing three introns. A TATA box (TAAATA) was observed 112 base pairs upstream from the translation initiation codon in the 5'-non coding region. The Alp protein has a pre region consisting of 14 amino acids and a pro region of 85 amino acids preceding the mature region, which consists of 280 amino acids. The amino acid sequence of Fusarium Alp has 52% homology with that of Aspergillus oryzae and 51% homology with that of Acremonium chrysogenum. The entire cDNA encoding Fusarium Alp was introduced into Saccharomyces cerevisiae, which then secreted enzymatically active Alp into the culture medium.

Subchronic (12-week) inhalation toxicity study of methanol-fueled engine exhaust in rats.

To evaluate the inhalation toxicity to rats of exhaust at low concentration for longer periods, Fischer 344 rats were exposed to 3 concentrations of exhaust generated by an M85 methanol-fueled engine (methanol with 15% gasoline) without catalyst for 8 h/d, 6 d/wk for 4, 8, or 12 wk. Concentration- and time-dependent increase carboxyhemoglobin in the erythrocytes and decrease in cytochrome P-450 in the lungs were observed in all treated groups. Furthermore, significant increases in plasma formaldehyde were observed in all treated groups. Furthermore, significant increases in plasma formaldehyde were observed in the group exposed to the highest concentration of exhaust (carbon monoxide, 89.8 ppm; formaldehyde, 2.3 ppm; methanol, 8.1 ppm; nitrogen oxides, 22.9 ppm; nitrogen dioxide, 1.1 ppm) for 8 or 12 wk. No change of plasma folic acid was observed in any group, and no methanol or formic acid was detected in the plasma in any animals. Histopathologically, exposure-related changes were found only in the nasal cavity of the high-concentration group. Slight hyperplasia/squamous metaplasias of the respiratory epithelium lining the nasoturbinate and maxilloturbinate were observed after 4 wk of exposure, and the incidences and degrees of these lesions increased slightly with the exposure time. No changes were found in the olfactory epithelium of the nasal cavity. As judged by optical microscopy, the exhaust concentration with no effect on the nasal cavity under the experimental conditions was concluded to be the medium concentration level containing 0.55 ppm formaldehyde. In the present study, however, concentration- and time-dependent increase of carboxyhemoglobin in the erythrocytes and decrease of the lung P-450 level were observed. Therefore, further study on more long-term inhalation of lower concentrations of exhaust might be needed.