Three distinct groups of isolates of <Emphasis Type="Italic">Tomato yellow leaf curl virus</Emphasis> in Japan and construction of an infectious clone

Complete nucleotide sequences of eight Japanese isolates of Tomato yellow leaf curl virus (TYLCV) were determined and compared with four TYLCV isolates already reported. These isolates separated into three groups – Shizuoka (Sz), Aichi (Ai), Nagasaki (Ng) – and had 99% identities within the groups. Full-length molecules of DNA-A of group Sz consist of 2791nt and those of group Ai contain 2787nt. Both were closely related to TYLCV-Is.M, although those of group Ng had 2793nt and were more closely related to TYLCV-Is. Comparison of common sequences of isolates belonging to groups Sz and Ai had substitutions of 4nt in the intergenic region and nonsynonymous substitutions at open reading frames between the groups. None of the isolates tested had DNA molecules. Agroinfection of four plant species with a DNA-A dimeric infectious clone of TYLCV-SzY, a member of group Sz, resulted in systemic infection. Tomato plants then developed typical yellow leaf curl symptoms.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB116629, AB116630, AB116631, AB116632, AB116633, AB116634, AB116635, and AB116636     

Exploration of the antibacterial proteins in pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis> induced by bacterial inoculation

The aim of this research was to characterize immune-related antibacterial substances from pearl oyster Pinctada fucata induced by bacterial invasion. Bacteria inoculation was performed by injecting 0.1 ml of 1.0 × 10¹² colony-forming units/ml Vibrio parahaemolyticus into adductor muscle. Acidic extracts were prepared with 0.1% trifluoroacetic acid from different tissues after 8 h of injection, and antibacterial activity against V. parahaemolyticus was determined via the microdilution broth method. The acidic extracts from gills of inoculated oysters (AEg) showed stronger antibacterial activity than those from non-inoculated ones. Based on this result, antibacterial proteins were purified from AEg via two-step gel filtration chromatography, followed by high-performance liquid chromatography using a TSkgel G3000 column. Protein components were analyzed by both sodium dodecyl sulfate and native polyacrylamide gel electrophoresis. As a result, two antibacterial proteins, APg-1 (with a molecular mass of approximately 210 kDa) and APg-2 (of approximately 30 kDa), were obtained from AEg. Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis and partial amino acid sequences revealed that these proteins might be novel antibacterial proteins. These results indicate that antibacterial proteins are potentially upregulated in the gill of pearl oysters or released therefrom for defense against bacterial invasion.     

Evidence of a new Tomato yellow leaf curl virus in Japan and its detection using PCR

A new isolate of Tomato yellow leaf curl virus (TYLCV) has been identified from tomato plants in Kochi Prefecture in Japan and designated TYLCV-[Tosa]. The complete nucleotide sequence of the isolate was determined and found to consist of 2781 nt. In phylogenetic analyses of entire nucleotide sequences, TYLCV-[Tosa] was delineated as a single branch and was more closely related to TYLCV-[Almeria] than TYLCV isolates Ng, Sz, or Ai reported in Japan, which had spread since 1996. Isolate TYLCV-[Tosa] is suggested to be a newly introduced, novel isolate of TYLCV that dispersed into Kochi Prefecture. In addition, a rapid method using the polymerase chain reaction to separate TYLCV isolates into four genetic groups was established. This method would be useful for reliable diagnosis based on genetic differences among isolates of TYLCV.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB192965 and AB192966     

Obstructive nephropathy induced with DL-potassium hydrogen tartrate in F344 rats

We experienced obstructive nephropathy in F344 rats treated with DL-potassium hydrogen tartrate (PHT) in a 13-week oral repeated dose toxicity study. Six-week-old male and female F344/DuCrj rats were fed a diet containing up to 2.0% PHT for 13 weeks. Microscopical findings including irregular dilation of the distal tubule lumen, foreign body giant cells, inflammatory cell infiltration, and regeneration of renal tubules were observed focally or multifocally in the renal cortex and/or medulla in the 0.5% and higher dosage groups of both sexes. The severity of these lesions increased in a dose-dependent manner. In the urinalysis, an increase in protein and white blood cells or the concentration of tartaric acid was detected in the 0.5% PHT and higher dosage groups of both sexes or males, respectively, though conventional blood biochemical analysis did not indicate failure of renal function. These results indicate that the PHT induces obstructive nephropathy in rats. There were no other treatment-related changes in other organs.     

Determination of the geographic origin of onions between three main production areas in Japan and other countries by mineral composition

Onions (Allium cepa L.) are produced in many countries and are one of the most popular vegetables in the world, thus leading to an enormous amount of international trade. It is currently important that a scientific technique be developed for determining geographic origin as a means to detect fraudulent labeling. We have therefore developed a technique based on mineral analysis and linear discriminant analysis (LDA). The onion samples used in this study were from Hokkaido, Hyogo, and Saga, which are the primary onion-growing areas in Japan, and those from countries that export onions to Japan (China, the United States, New Zealand, Thailand, Australia, and Chile). Of 309 samples, 108 were from Hokkaido, 52 were from Saga, 77 were from Hyogo, and 72 were from abroad. Fourteen elements (Na, Mg, P, Mn, Co, Ni, Cu, Zn, Rb, Sr, Mo, Cd, Cs, and Ba) in the samples were determined by frame atomic adsorption spectrometry, inductively coupled plasma optical emission spectrometry, and inductively coupled plasma mass spectrometry. The models established by LDA were used to discriminate the geographic origin between Hokkaido and abroad, Hyogo and abroad, and Saga and abroad. Ten-fold cross-validations were conducted using these models. The discrimination accuracies obtained by cross-validation between Hokkaido and abroad were 100 and 86%, respectively. Those between Hyogo and abroad were 100 and 90%, respectively. Those between Saga and abroad were 98 and 90%, respectively. In addition, it was demonstrated that the fingerprint of an element pattern from a specific production area, which a crop receives, did not easily change by the variations of fertilization, crop year, variety, soil type, and production year if appropriate elements were chosen.     

Physiological effects of psyllid (Paratrioza cockerelli) on potato

Feeding by psyllid nymphs causes “Psyllid yellows,” a characteristic yellowing of shoots which results in a dramatic loss of tuber yield. If psyllid infestation is not controlled, the onset of shoot yellowing and growth reductions can occur within two weeks. When insecticides are applied after yellowing, recovery of plants often is not complete. In this study comparisons were made of the physiology and growth of tops and tubers of recovered and permanently injured potatoes of the cultivars ‘Denali,’ ‘Kennebec,’ and ‘Norgold Russet.’ Permanently injured plants senesced rapidly, developed less shoot growth, aerial tubers and shortened and thickened internodes. They had lower tuber yields than plants that recovered. Permanently injured ‘Denali’ developed shoots on old flowering stems. In permanently injured plants of all cultivars, the tubers were small and badly misshapen but numbers increased. In recovered ‘Kennebec’ plants tubers were also misshapen, but were of average size. Tubers of recovered ‘Denali’ and ‘Norgold Russet’ were of acceptable shape. In samplings made during tuber growth, dry weight percentages of total soluble sugars, sucrose and glucose of tubers from permanently injured plants were found to be the same as those of recovered plants. In all cultivars, starch percentages of tubers from permanently injured plants were higher than that of the recovered, but the pattern of carbohydrate content changes was similar for permanently injured and recovered cultivars.     

The First Case of Feline Infectious Peritonitis-like Pyogranuloma in a Ferret Infected by Coronavirus in Japan

A male ferret, which was purchased from abroad at 9 months of age, had shown significant weight loss starting at 13 months of age. The ferret subsequently showed decreasing motor activity and recumbency and was euthanized at 14 months of age. At necropsy, a white, quail egg-sized mass was found in the mesentery. Histopathologically, multifocal granulomas consisting of necrotic foci, macrophages, fibroblasts and plentiful fibrous connective tissues were observed in the mesenteric mass. Surrounding the granulomas, inflammatory cell infiltration consisting of neutrophils, lymphocytes and plasmacytes was observed diffusely and significantly. Immunohistochemistry revealed small numbers of macrophages around necrotic foci that were positively stained for anti-mouse feline coronavirus. Electron microscopically, the cytoplasm of the macrophages contained viral particles, which were identified as coronavirus. The histopathological features in this ferret were similar to those in cats with feline infectious peritonitis (FIP). This was the first case in ferrets in Japan.     

Effects of methoxychlor exposure during perinatal period on reproductive function after maturation in rats

Methoxychlor (MXC) is a non-steroidal pesticide that is known to possess estrogenic activity, and therefore may be potentially hazardous to the development and/or reproduction. The present study assessed the effects of perinatal exposure to MXC on reproductive function after maturation in both male and female rats. Pregnant rats were fed a phytoestrogen-free diet containing different doses of MXC (24, 240, and 1200 ppm) from day 15 of gestation to day 10 after parturition, and reproductive functions of offspring were examined after maturation. In males, MXC exposure during the perinatal period decreased serum LH and FSH, but not testosterone levels, but it did not affect copulatory behavior. In females, MXC exposure prolonged the days exhibiting cornified vaginal smears during the estrous cycle. In addition, both the lordosis reflex and preovulatory LH surge on the presumptive proestrous evening were suppressed in MXC-exposed females. These results suggest that perinatal exposure to MXC exerts permanent effects on several aspects of the reproductive function in both male and female rats.     

Mutations in the melanocortin 1 receptor, β-defensin103 and agouti signaling protein genes, and their association with coat color phenotypes in Akita-inu dogs

To identify factors that control coat color in Akita-inu dogs, we sequenced all the exons of the melanocortin 1 receptor (MC1R), β-defensin103 (CBD103) and agouti signaling protein (ASIP) genes of dogs with four distinct coat colors, namely, brindle, sesame, red and white. Then we examined correlations among specific alleles and coat color. In the case of the MC1R gene, all white dogs were homozygous for a nonsense mutation, R306ter, while brindle, sesame, and red dogs had at least one R306 allele. In the case of the CBD103 gene, all brindle dogs were heterozygous for the G23del mutation (deletion of codon 23, encoding glycine), while all sesame and red dogs were homozygous for G23. In the case of the ASIP gene, all dogs, regardless of coat color, had at least one S82 H83 allele. A missense mutation in the ASIP gene, P87L, was identified for the first time in some Akita-inu dogs but was not associated with any specific coloration. Our results indicate that the 2 key mutations, R306ter in the MC1R gene and G23del in the CBD103 gene, are associated with the phenotypic discriminations among brindle, red/sesame, and white coats, while no mutation that might potentially be associated with the discrimination of a sesame coat from a red coat is present in the coding sequences of these three genes.