Expression of hepatitis C virus core protein associated with malignant lymphoma in transgenic mice

Hepatitis C virus (HCV) is a major causative agent for chronic liver diseases leading to hepatocellular carcinoma (HCC) and has also been suggested to be a possible etiologic factor for different lymphoproliferative diseases, including mixed cryoglobulinemia (MC) and B-cell non-Hodgkin's lymphoma (NHL). To understand the roles of HCV core protein in the pathogenesis of HCV related diseases, we produced two lines of the transgenic mice (HC82310 and HC9053) that express the HCV core transgene. One of the lines, HC9053, developed malignant lymphoma (ML, follicular center cell type) with a high frequency (80%) at the ages over 20 months. Hepatocellular adenoma was also observed in this line of transgenic mouse. We demonstrated expression of HCV core protein and mRNA in the liver of transgenic mice, and also detected the core mRNA in the enlarged lymph nodes of the transgenic mice which developed ML. These results suggest that the core protein may play an important role in the development of ML, and that the HC9053 transgenic mice provide suitable models for understanding the mechanism of HCV-related lymphoproliferative diseases.     

Identification of a bisegmented double-stranded RNA virus (picobirnavirus) in calf faeces

To determine the incidence of rotavirus infection among dairy herds in the State of S?o Paulo, Brazil, 576 faecal samples obtained from calves aged 1-45 days with and without diarrhoea, reared on 63 dairy cattle farms, were analyzed. Polyacrylamide gel electrophoresis (PAGE) identified 28 samples positive for group A rotavirus, while four samples, two diarrhoeic and two non-diarrhoeic, showed a bisegmented genome with a typical picobirnavirus pattern. Electron microscopy revealed spherical virus particles with a diameter of 37 nm and without a defined surface structure. The present study is the first report of a bisegmented virus identified in cattle in Brazil.     

Positional effect of gene insertion on genetic stability of a clover yellow vein virus-based expression vector

The stability of the inserted genes in the viral expression vector varied depending on the sequence introduced and the position of insertion. Infectious cDNA to Clover yellow vein virus (pClYVV) was modified to insert a foreign gene at two independent sites: one, along with a polylinker, between the NIb and CP genes (pClYVV/CP/W) and the other between P1 and HC-Pro (pClYVV-Pst/CP). The green fluorescent protein (GFP) gene was inserted into either pClYVV/CP/W or pClYVV-Pst/CP. GFP gene was stably maintained and expressed in both vectors following serial passages in plants. Progeny viruses from both constructs accumulated in similar amounts and at rates of 70%–80% of that of the wild-type virus. On the other hand, progeny viruses carrying the human interferon- (hIFN) gene cloned in pClYVV-Pst/CP were genetically unstable owing to frequent deletions of the cloned gene during passage through plants. In contrast, the hIFN sequence cloned in pClYVV/CP/W was stably maintained in viruses after several passages in broad bean plants, and the progeny virus accumulated at the rate of about 50%–100% of that of the wild-type virus. The nucleotide sequence analyses indicated that the genetic instability of the inserted sequence results from homologous recombination of viral vector and inserted DNA sequences; it is not due to the inserted sequence alone.     

A Leafhopper (Hishimonus sellatus) Transmits Phylogenetically Distant Phytoplasmas: Rhus Yellows and Hovenia Witches' Broom Phytoplasma

Phytoplasmas causing a severe decline of three tree species, i.e., Rhus javanica, Hovenia tomentella and Zizyphus jujuba, in Japan were examined for their transmissibility by a leafhopper species Hishimonus sellatus, and for their phylogenetic relatedness. By H. sellatus, Rhus yellows (RhY) phytoplasma was transmissible to white clover and periwinkle seedlings, causing typical symptoms in these plants. Jujube witches' broom (JWB) phytoplasma was also transferred to the host plant, Z. jujuba, by the leafhopper. Because JWB phytoplasma was transmitted to Hovenia tomentella and caused the same symptoms as Hovenia witches' broom (HWB), JWB phytoplasma may be very closely related to HWB phytoplasma. RFLP analysis of the PCR products of 16S rDNA revealed that RhY phytoplasma belongs to the Aster yellows (AY) group, and JWB and HWB phytoplasmas belong to a different group (possibly Elm yellows group). Thus, we found that one species of leafhopper can carry phylogenetically distant phytoplasmas. Received 23 April 2001/ Accepted in revised form 29 October 2001     

Morphological analysis of corneal opacity in Shiba dog with GM1 gangliosidosis

GM1 gangliosidosis is one of the inherited metabolic lysosomal storage disorders characterized by neurological symptoms caused by beta-galactosidase deficiency and consequent accumulation of GM1 ganglioside in neuronal cells. Shiba dogs affected with GM1 gangliosidosis have been found to suffer from corneal opacity. In our morphological analysis, keratocyte enlargement was induced by abnormal intracellular accumulation of neutral carbohydrates, resulting in the loss of normal arrangement of collagen fibrils in the opaque cornea was found to be associated with the disorder. We therefore conclude that corneal opacity in this Shiba dog with GM1 gangliosidosis may be caused by neutral carbohydrate accumulation in lysosomes, swelling and dysfunction of keratocytes, and subsequent irregular arrangement of collagen fibrils in the corneal proper substance.     

Effect of volume of non-surgical embryo transfer medium on ability of porcine embryos to survive to term

Non-surgical embryo transfer is a promising method for improving efficiency in the pork industry and also for biotechnology applications, such as in vitro embryo production, transgenesis and cloning. Several groups have reported successful piglet production using an artificial insemination catheter or flexible catheter designed for this procedure; however, the efficiency of the technique is still low. The critical points that need to be addressed in order to improve this procedure are (1) the embryo deposition site and (2) volume of transfer medium associated with the embryos; however, the latter has not yet been examined systematically. In the present study, we evaluated the effect of the volume of non-surgical embryo transfer medium on the ability of porcine embryos to survive to term by using a recently produced flexible catheter. The catheter consists of a guide and an injector. Blastocysts 200-230 mum in diameter were collected from donor gilts and transferred to recipient gilts. The time required for the completion of embryo transfer using this catheter was 14.6 +/- 3.9 min. The tip of the injector was determined by laparotomy to be located in a uterine horn 20-30 cm anterior from the branching point of the uterus body. We transferred 17.0-17.3 embryos with different volumes of medium (1.6, 3.2 and 10 ml) into each of 5, 4 and 4 recipients, respectively, and pregnancy was confirmed in 4, 3 and 1 of these recipients, respectively. Three recipients in the 1.6 ml group farrowed a total of 19 piglets (4, 5 and 10 piglets, respectively). These results suggest that successful non-surgical embryo transfer is affected by the volume of transfer medium.     

Characterization of the Q.Ymym region on wheat chromosome 2D associated with wheat yellow mosaic virus resistance

Yellow mosaic disease, caused by wheat yellow mosaic virus (WYMV), is one of the most serious diseases of winter wheat in Japan and China. A single major QTL for WYMV resistance in the Japanese wheat variety 'Yumechikara', designated Q.Ymym, has been mapped on a 43.6 cM linkage block between the two markers Xcfd233 and Xgwm349 on chromosome 2D. We were able to obtain two recombinants within the block, which facilitated reducing the size of the linkage block. The pseudomolecule sequence of 'Chinese Spring' (CS) indicated that the original Q.Ymym region of 43.6 cM corresponded to 68.5 Mb and the narrowed Q.Ymym region represents a size of 27.3 Mb. The sequence features of the Q.Ymym region were unique in comparison with CS sequences, which may have led to the low recombination rate within the block. The Q.Ymym haplotype block was detected in other WYMV-resistant varieties but not in the susceptible varieties used in this study. The unique sequence structure of the Q.Ymym region allowed the development of co-dominant markers for use in marker-assisted selection.     

Experimental evidence on prevention of infection by the ectoparasitic protozoans Ichthyobodo salmonis and Trichodina truttae in juvenile chum salmon using ultraviolet disinfection of rearing water

In northern Japan, juvenile chum salmon Oncorhynchus keta (Walbaum) are released from hatcheries to enhance the fishery resource. Infections with ectoparasitic protozoans, particularly the flagellate Ichthyobodo salmonis and the ciliate Trichodina truttae, occasionally cause severe mortality among hatchery‐reared juveniles. This study examined the susceptibility of the two parasites to wide‐ranging UV irradiation (experiment 1) and then investigated whether UV disinfection of the rearing water using a commercial device was useful for preventing infections among juveniles in a small‐scale rearing system over a 28‐day period (experiment 2). In experiment 1, parasite mortality reached 100% with UV irradiation doses of ≥9.60 × 10⁵ μW s/cm² for I. salmonis and ≥8.40 × 10⁵ μW s/cm² for T. truttae. In experiment 2, disinfection of the rearing water at a UV irradiation dose of 2.2 × 10⁶ μW s/cm² succeeded in complete prevention of both parasites in the juvenile salmon. These results elucidate the minimum dose of UV irradiation for inactivation of I. salmonis and T. truttae, and demonstrate the usefulness of water disinfection using a commercial UV irradiation device to prevent infections by these parasites in hatchery‐reared juvenile chum salmon.     

Estimation of terminal molting probability of snow crab <Emphasis Type="Italic">Chionoecetes opilio</Emphasis> using instar- and state-structured model in the waters off the Pacific coast of northern Japan

Terminal molting probability (TMP) of snow crab Chionoecetes opilio was estimated using instar- and state-structured model incorporating the terminal molt in the waters off the Pacific coast of northern Honshu, Japan. TMP is defined as the probability that terminal molt occurs within a year in an instar and is an important parameter for stock assessment. TMP was estimated from the model and the rate of terminal molted crabs (RTM) for instars, which means carapace widths over 30 mm. Carapace widths at RTM of 0.5 were 81 mm for males and 66 mm for females. TMP was estimated as 0.23 and 0.29 for these carapace widths, respectively. Carapace widths at TMP of 0.5 were 94 mm for males and 71 mm for females, and for these carapace widths 57% of male and 50% of female crabs were considered as old-shell which had survived 1 year or more from terminal molt. Changing the fishing or natural mortality by ±0.1, the maximum difference of TMP was less than 0.1 from default estimates.