Direct RT-PCR method for detecting two chrysanthemum viroids using minimal amounts of plant tissue.

A direct reverse transcription-polymerase chain reaction (RT-PCR) method for detecting the chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd) to screen for a viroid-free chrysanthemum plant at a small plant size was established and named microtissue direct RT-PCR. A razor or syringe needle was used for RNA template preparations. Under a stereoscopic microscope, a razor or syringe needle was used to pierce, a tissue sample to a depth of 0.1-0.2mm, and the sample was directly transferred to the RT mixtures. Methods using razors or needles for the preparation of templates could detect CSVd and CChMVd with a high sensitivity. The most sensitive method used a razor or syringe needle to acquire template from the shoot tips. Using the microtissue direct RT-PCR method, both viroids could be detected from the high- and low-viroid-concentration plants. The microtissue direct RT-PCR method was more sensitive than a conventional template preparation method. Using the microtissue direct RT-PCR method established in this study, the laborious subculture step could be omitted because detecting viroids and screening for viroid-free plants even at a small plant size before the subculture could be possible. In addition, the microtissue direct RT-PCR method could also be a powerful tool for clarifing the viroid distribution among microtissues, such as shoot apical meristems.

Elimination of chrysanthemum stunt viroid from an infected chrysanthemum cultivar by shoot regeneration from a leaf primordium-free shoot apical meristem dome attached to a root tip.

In this research we eliminated chrysanthemum stunt viroid (CSVd) from a highly infected chrysanthemum cultivar using a newly established method. 'Piato' is one of the most difficult cultivars in which to obtain CSVd-free plants by conventional methods. Leaf primordium-free shoot apical meristems (LP-free SAMs) of 'Piato' plants were dissected and attached to CSVd-free chrysanthemum or cabbage root tips. As shown by nested-PCR, CSVd was not detected in some shoots regenerated on both types of root tip. The production rates of CSVd-free plants using chrysanthemum and cabbage root tips were 14% and 3%, respectively. Regeneration of plants from LP-free SAMs of chrysanthemum plants by attaching these SAMs to root tips is an efficient method of generating CSVd-free chrysanthemum plants.

Rescue of shoot apical meristems of chrysanthemum by culturing on root tips.

A new method to regenerate plants from leaf primordia-free shoot apical meristem domes (LP-free SAMs) was developed by establishing the meristem dome on the cut surface of root tips. Ten days after culture, the viable rate of LP-free SAMs of chrysanthemum 'Piato' attached to chrysanthemum root tips was >40%. Shoot regeneration was not observed from LP-free SAMs without the root tips. When LP-free SAMs of chrysanthemum were transferred to root tips of either petunia, cabbage, or carnation, the highest shoot regeneration rate was observed with cabbage root tips. Microscopic observation documented that the LP-free SAM temporarily adhered to the cut surface of the root tip of cabbage.

Adenoviral gizzard erosion in commercial broiler chickens.

Pathologic and immunohistochemical changes caused by group I of the fowl adenovirus (FAV) serotype-1 99ZH strain, isolated from broiler chickens exhibiting gizzard erosion, were investigated in commercial broiler chickens. One hundred twenty-two chickens were inoculated with the strain by both oral and ocular routes at 1, 3, or 5 weeks of age and euthanatized for necropsy within 4-18 days of inoculation. Focal gizzard erosions were observed in the inoculated chickens of each age group. A histologically degenerative koilin layer, necrotic mucosa, intranuclear inclusion bodies in the glandular epithelial cells, inflammatory cell infiltrations in the lamina propria, submucosa, and a muscle layer were seen in the gizzards. Immunohistochemical staining showed evidence of FAV antigens in the intranuclear inclusion bodies. These findings were recognized regardless of their maternal antibody levels for FAV serotype-1. Gizzard lesions appeared later in the lower-dose-inoculated chickens than in the higher-dose-inoculated chickens. Numerous CD3-positive cells and IgY-positive plasma cells were seen in the gizzard lesions. In 5-week-old chickens the heterophil infiltrations in the lesions were milder than in younger chickens. Intranuclear inclusion bodies also were observed in the epithelial cells of the ileum or cecal tonsils of some chickens. Thus, this study shows that FAV-99ZH causes adenoviral gizzard erosion in broiler chickens without hepatic or pancreatic lesions and that cell infiltration is more severe than in dietary gizzard erosions.

TRPV1 activation and induction of nociceptive response by a non-pungent capsaicin-like compound, capsiate.

Capsiate is a capsaicin-like ingredient of a non-pungent cultivar of red pepper, CH-19 sweet. To elucidate the mechanisms underlying the non-pungency of capsiate, we investigated whether capsiate activates the cloned capsaicin receptor, TRPV1 (VR1). In patch-clamp experiments, capsiate was found to activate TRPV1 expressed transiently in HEK293 cells with a similar potency as capsaicin. Capsiate induced nociceptive responses in mice when injected subcutaneously into their hindpaws with a similar dose dependency as capsaicin. These data indicate that the non-pungent capsiate is an agonist for TRPV1 and could excite peripheral nociceptors. In contrast to this, capsiate did not induce any significant responses when applied to the skin surface, eye or oral cavity of mice, suggesting that capsiate requires direct access to nerve endings to exhibit its effects. Capsiate was proved to have high lipophilicity and to be easily broken down in normal aqueous conditions, leading to less accessibility to nociceptors. Another highly lipophilic capsaicin analogue, olvanil, was similar to capsiate in that it did not produce irritant responses when applied to the skin surface, although it could activate TRPV1. Taken together, high lipophilicity and instability might be critical determinants for pungency and so help in understanding the effects of capsaicin-related compounds.

Experimental dual infection of specific pathogen-free pigs with porcine reproductive and respiratory syndrome virus and pseudorabies virus.

Twenty 6-week-old specific pathogen-free pigs were divided into four groups. On day 0 of the experiment, PRRSV-PRV (n = 6) and PRRSV (n = 4) groups were intranasally inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) (10(5.6) TCID50). On day 7, the PRRSV-PRV and PRV (n = 6) groups were intranasally inoculated with pseudorabies virus (PRV) (10(3.6) TCID50). Control pigs (n = 4) were kept as uninoculated negative controls. Half of the pigs in each group were euthanized and necropsied on day 14 or 21. Clinical signs such as depression and anorexia were observed in the PRRSV-PRV and PRV groups after inoculation with PRV. Although febrile response was observed after virus inoculations, the duration of that response was prolonged in the PRRSV-PRV group compared with the other groups. The lungs in the PRRSV-PRV group failed to collapse and were mottled or diffusely tan and red, whereas the lungs of the pigs in the other groups were grossly normal. Histopathologically, interstitial pneumonia was present in all PRRSV-inoculated pigs, but the pneumonic lesions were more severe in the PRRSV-PRV group. Mean PRRSV titres of tonsil and lung in the PRRSV-PRV group were significantly (P < 0.05) higher than that in the PRRSV group on day 21. These results indicate that dual infection with PRRSV and PRV increased clinical signs and pneumonic lesions in pigs infected with both viruses, as compared to pigs infected with PRRSV or PRV only, at least in the present experimental conditions.

Experimental reproduction of postweaning multisystemic wasting syndrome in cesarean-derived, colostrum-deprived piglets inoculated with porcine circovirus type 2 (PCV2): investigation of quantitative PCV2 distribution and antibody responses.

Sixteen cesarean-derived, colostrum-deprived piglets were inoculated intranasally with porcine circovirus type 2 (PCV2), originally isolated from a pig affected with postweaning multisystemic wasting syndrome (PMWS). At 1 day postinoculation (PI), 3 of the 5 piglets in the uninoculated control group were moved to the room of inoculated piglets for contact exposure. Porcine circovirus type 2 was detected by polymerase chain reaction (PCR) in swabs from inoculated piglets from 1 day PI and from contact piglets from 2 days after cohabitation. Porcine circovirus type 2 was also detected in all serum samples but not in control piglets 7 days PI. Until the end of study, PCV2 was detected in swabs and serum samples by PCR but not in the control piglets. One inoculated piglet died suddenly without clinical signs 19 days PI. Beginning at 14 days PI, 5 piglets, including 1 contact piglet, had clinical signs of depression, anorexia, and icterus, and 1 inoculated piglet died 21 days PI. Most of the piglets exhibiting the above clinical signs became moribund and were necropsied 21 and 28 days PI. In the piglets that showed clinical signs, gross lesions, including icterus of liver and hemorrhage in stomach, and typical histopathological lesions of PMWS, such as lymphoid depletion and basophilic intracytoplasmic inclusion bodies in lymph nodes and other tissues, were observed. Porcine circovirus type 2 was detected by PCR in all tissue samples except in those of the control piglets. Porcine circovirus type 2 was recovered from several tissue samples of the piglets necropsied until 35 days PI. In particular, PCV2 was recovered in high titer from most of the tissue samples of the piglets exhibiting clinical signs. Serum antibody against PCV2 was mostly detected in inoculated piglets and in contact piglets 14 and 21 days PI by an indirect fluorescence antibody test but was not detected in the piglets exhibiting clinical signs until 28 days PI. These results indicate that PCV2 was able to induce clinical PMWS in the absence of other swine pathogens and that there were significant differences in both the quantitative PCV2 distribution in tissues and the antibody response between the piglets that were infected and developed PMWS and those that were infected but remained healthy.

Outbreaks of adenoviral gizzard erosion in slaughtered broiler chickens in Japan.

Gizzard erosion in broiler chickens was investigated at 18 slaughterhouses in Japan. The condition was observed in 13 of them, and adenoviral gizzard erosion (AGE) was diagnosed histologically, immunohistochemically and virologically in the eroded gizzards from nine of these 13. The antigen-positive intranuclear inclusion body of group 1 fowl adenovirus was observed in the epithelial cells of the affected gizzards, and fowl adenoviruses were isolated from the lesions. In two of the slaughterhouses the total weights of the gizzards disposed of in three years were 3590 kg (0.40 per cent of the gizzards inspected) and 2880 kg (0.19 per cent). Sixteen of the 19 outbreaks of gizzard erosion on 15 farms that were confirmed in three of the slaughterhouses, including the previous two slaughterhouses, were diagnosed as AGE, and the condition was suspected in the other three outbreaks. Most of the adenoviruses isolated were identified as serotype-1 by PCR-restriction fragment length polymorphism. No apparent clinical signs were observed in any of the affected flocks.

CH-19 sweet, a non-pungent cultivar of red pepper, increased body temperature and oxygen consumption in humans.

We investigated the effect of CH-19 Sweet, a non-pungent cultivar of red pepper, on body temperature and oxygen consumption in humans. CH-19 Sweet was given to 11 healthy volunteers, and core body temperature, body surface temperature and oxygen consumption were measured. The control group ingested California-Wandar, which contained neither capsaicin nor capsiate. The core body temperature in the CH-19 Sweet group was significantly higher than that in the control group (P<0.01). The forehead temperature measured by infrared thermography in the CH-19 Sweet group was significantly higher than that in the control group. The body surface temperature was increased for about 20 min after consumption of CH-19 Sweet intake, and the neck temperature was significantly higher (P<0.001) than when the subjects consumed California-Wandar. We also measured respiratory gas by indirect calorimetry while subjects wore a face mask. A significant difference was detected in oxygen consumption between the two groups, and the value was significantly higher in the CH-19 Sweet group (P<0.03). These results suggest that CH-19 Sweet increased thermogenesis and energy consumption.

Functional mapping of human medial frontal motor areas. The combined use of functional magnetic resonance imaging and cortical stimulation.

Two functional brain-mapping techniques, functional magnetic resonance imaging (fMRI) and cortical stimulation by chronically implanted subdural electrodes, were used in combination for presurgical evaluation of three patients with intractable, partial motor seizures. Brain mapping was focused on characterizing motor-related areas in the medial frontal cortex, where all patients had organic lesions. Behavioral tasks for fMRI involved simple finger and foot movements in all patients and mental calculations in one of them. These tasks allowed us to discriminate several medial frontal motor areas: the presupplementary motor areas (pre-SMA), the somatotopically organized SMA proper, and the foot representation of the primary motor cortex. All patients subsequently underwent cortical stimulation through subdural electrodes placed onto the medial hemispheric wall. In each patient, the cortical stimulation map was mostly consistent with that patient's brain map by fMRI. By integrating different lines of information, the combined fMRI and cortical stimulation map will contribute not only to safe and effective surgery but also to further understanding of human functional neuroanatomy.

Bilateral paramedian thalamo-midbrain infarction showing electroencephalographic alpha activity.

A 57-year-old man became unresponsive and mute with bilateral ophthalmoplegia and quadriplegia. Magnetic resonance imaging (MRI) showed bilateral infarctions at the ventral midbrain and the dorsomedial nucleus of the thalamus. Serial studies with MR and vertebral angiography disclosed hypoperfusion and spontaneous reperfusion of the bilateral posterior cerebral arteries at their origin from the basilar artery. Electroencephalographically, a posteriorly distributed alpha rhythm was clearly recorded and it was reactive in response to external stimuli. The findings seen in the present patient suggest that the ventral midbrain and medial dorsal thalamus are not necessary to produce posterior electroencephalographic alpha activity.

Role of FK506-binding protein 12 in development of the chick embryonic heart.

A cDNA encoding chicken FK506-binding protein 12 (FKBP12) was isolated and sequenced. The predicted amino acid sequence of the chicken protein shows high homology to those of FKBP12 proteins of other species ranging from human to frog. The possible role of FKBP12 in chick embryonic cardiac development was examined. Northern blot analysis revealed that FKBP12 mRNA is distributed widely in chick embryos, being especially abundant in the heart; the amount of FKBP12 mRNA in the embryonic heart decreased with time. Administration of FK506 to chick embryos at 7 to 9 days resulted in marked cardiac enlargement. FK506 also reduced the expression of myosin, induced a more elongated cell morphology, and impaired network formation in cultured chick embryonic cardiomyocytes. These results suggest that FKBP12 is important in the regulation of contractile function and phenotypic expression in chick cardiomyocytes during embryonic development.

Experimental infection of specific-pathogen-free chickens with serotype-1 fowl adenovirus isolated from a broiler chicken with gizzard erosions.

Gizzard lesions were formed in specific-pathogen-free (SPF) white leghorn chickens inoculated with fowl adenovirus (FAV). The virus, serotype 1 FAV 99ZH strain (FAV-99ZH), was originally isolated from the gizzard mucosa of commercial broiler chickens exhibiting gizzard erosion with intranuclear inclusion bodies. Five-day-old and 53-day-old SPF white leghorn chickens were inoculated with FAV-99ZH by both oral and ocular routes and then examined at necropsy on days 3, 5, 7, 10, 14, and 21 postinoculation (PI). There were no clinical signs in any of the chickens after the inoculation. Focal gizzard lesions occurred macroscopically, however, in inoculated chickens at several experimental periods. FAV was recovered from tissue samples of the proventriculus, gizzard, pancreas, and rectum by day 10 or 7 PI but was not recovered from liver samples of any of the chickens. These results indicate that FAV isolated from gizzard erosion is able to reproduce gizzard lesions as necrosis and erosion in SPF white leghorn chickens and that it may have a greater degree of tissue tropism in gizzards and other digestive organs than in the liver.

Epizootic outbreaks of gizzard erosion associated with adenovirus infection in chickens.

Two outbreaks of gizzard erosion in slaughtered broiler chickens in Japan were examined pathologically and microbiologically. The prevalences of such lesions were 9%-11% and 4%-50% in the affected flocks. Affected chickens had no clinical signs. Group I fowl adenovirus (FAV) serotype 1 was isolated from gizzard lesions. Histologically, gizzard mucosa were necrotic. Intranuclear inclusion bodies were seen in the enlarged nuclei of degenerating epithelial cells of the gizzard. The keratinoid layer in the erosion was edematous and desquamated and contained degenerative cells. Moderate to marked inflammatory cell infiltration was observed in the lamina propria and perivascular connective tissue in the submucosa and muscle layer. Immunohistochemical staining showed evidence of FAV antigens in the intranuclear inclusion bodies within degenerating epithelial cells. Ultrastructurally, numerous viral particles were demonstrated in the inclusions.

Development of severe longitudinal atrophy of thoracic spinal cord following lupus-related myelitis.

A 26-year-old woman suffered from acute myelitis at Th 6 level associated with systemic lupus erythematosus. Methyl-prednisolone pulse therapy, intravenous high-dose immunoglobulin administration and plasmapheresis were not effective. Her neurological signs had persisted in spite of subsequent administration of oral prednisolone and azathiopurine. Magnetic resonance imaging (MRI) of spinal cord at the onset showed a marked swelling with intramedullary high intensity signals on T2WI along the whole thoracic cord. Three years later, MRI demonstrated a severe longitudinal and segmental atrophy of the mid to low thoracic cord which resulted in transverse spinal signs.

Molecular analysis of plasma alpha 1,3-fucosyltransferase deficiency and development of the methods for its genotyping.

Four patients with mental illness were found to be deficient in plasma alpha1,3-fucosyltransferase for the first time in Japan [Exp Clin Immunogenet 1999;16:125-130]. Complete sequencing of FUT6 genes in these individuals revealed the presence of two point mutations, i.e., G739 to A (Glu-->247 to Lys) and C945 to A (Tyr-->315 to stop). In addition to two reported alleles having G739 to A (pf1) and G739 to A and C945 to A (pf3), a new mutated allele having C945 to A (pf2) was found to be present and all the individuals who lack alpha1,3-fucosyltransferase activity in plasma were found to possess pf genes homozygously (pf/pf). In order to detect such lethal mutations in FUT6 genes easily, PCR-RFLP methods have also been developed and applied for the screening of FUT6 deficiency in a large number of samples which resulted in the demonstration of three additional FUT6-deficient individuals. The absence of alpha1,3-fucosylated molecules on alpha(1)-acid glycoprotein in plasma from all the 7 individuals was confirmed to result from the plasma alpha1,3-fucosyltransferase deficiency.

Administration of capsiate, a non-pungent capsaicin analog, promotes energy metabolism and suppresses body fat accumulation in mice.

We investigated the effects of a single oral administration of capsiate, which is found in the fruits of a non-pungent cultivar of pepper, CH-19 Sweet, and has the same structure as capsaicin except for replacement of NH by O in the alkyl chain, on the thermogenesis and fat accumulation in mice. The oxygen consumption and serum adrenalin concentration were higher in both the capsaicin (10 mg/kg-body weight) and capsiate (10 mg/kg-body weight) groups than those in the control group. We also examined the effects of 2 weeks of administration of capsaicin and capsiate on body fat accumulation. Eevery day for 2 weeks administration of capsiate (10, 50 mg/kg-body weight/day) markedly suppressed body fat accumulation as well as capsaicin (10 mg/kg-body weight/day). These results suggest that capsiate promotes energy metabolism and suppresses body fat accumulation as does capsaicin.

Fatal thoracic aortic aneurysm in a patient with childhood-onset vasculo-Behçet's disease: an autopsy report.

A 33-year-old man died following sudden unexpected rupture of a saccular thoracic aortic aneurysm. The patient had been treated as suspicious Behçet's disease (BD) for 9 years, however, the medication was discontinued at the age of 24. Autopsy disclosed multiple aneurysms at the descending thoracic and abdominal aorta. Histopathologically, infiltration of inflammatory cells and destruction with loss of elastic and muscle fibers of the aortic medial wall were seen. Taking the clinical course into account, all the postmortem findings were compatible with those of vasculo-BD. It is important that medical follow-up should be implemented even for childhood-onset patients who do not satisfy criteria of BD.

CH-19 sweet, nonpungent cultivar of red pepper, increased body temperature in mice with vanilloid receptors stimulation by capsiate.

We investigated the effect of CH-19 Sweet, a nonpungent cultivar of red pepper, and capsiate, a nonpungent capsaicin analog found in CH-19 Sweet on body temperature in mice. The body temperature was recorded from conscious and unrestrained mice by use of a telemetry system. The body temperature in the mice administered CH-19 Sweet was higher than in the mice administered California-Wandar, which contains no capsiate or capsaicin. The body temperature in the mice administered capsiate was higher than in the mice administered the vehicle. Furthermore, we injected capsazepine, a specific antagonist of vanilloid receptors, into the peritoneal cavity and orally administered capsiate via a stomach tube to mice. The body temperature in the mice pretreated with capsazepine was lower than in the mice injected with the vehicle. This result suggested that capsazepine suppressed the rise in body temperature induced by capsiate administration. In conclusion, CH-19 Sweet increased body temperature, and this effect may be induced by the vanilloid receptors' stimulation of capsiate.