Characterization of a new viroid strain from hops: evidence for viroid speciation by isolation in different host species

A new viroid was detected in hops cultivated in Akita Prefecture, Japan where it is prevalent in many hops fields. In a survey of hop samples collected during the 1986–2002 growing seasons, the new viroid was present in the major Japanese hop-cultivating areas as early as the 1980s. A single-stranded circular RNA of 368–372 nucleotides that assume a highly basepaired, stable, rod-like secondary structure, shares 93%–98% sequence homology with Apple fruit crinkle viroid (AFCVd) isolated from apple and 85%–87% with Australian grapevine viroid (AGVd) isolated from grapevine. Taking into account the present concept of viroid species, we conclude that the viroid is AFCVd. Circumstantial evidence suggests that AFCVd from apples and hops were endemic in Japan only where cultivation of the two host plants overlapped, thereby strongly supporting the possibility that AFCVd (or an ancestral viroid) was transmitted across the species barrier from apples to hops or hops to apples somewhere in the region. Phylogenetic analysis of AFCVd from hops, AFCVd from apples, and AGVd together with the other members of the genus Apscaviroid revealed that the Akita isolates of AFCVd from hops (AFCVd-hop) formed a cluster that is distinct from AFCVd-apple and AGVd. Accumulation of host-specific sequence variation following their isolation in different host species may be leading to the formation of two viroid species from a common ancestor.     

Toxicity of wild juvenile “komonfugu” Takifugu flavipterus in the Bay of Sanriku Coast,Tohoku Area,Northern Japan

To clarify the mechanism of tetrodotoxin (TTX) accumulation in pufferfish, we compared the toxicity of two sets of wild juvenile “komonfugu” Takifugu flavipterus. The first set was sampled from Onisawa Fishing Port (FP) located in Okirai Bay, the Pacific Coast of Sanriku, Tohoku Area, Northern Japan. The second set was collected from the Onisawa FP and reared in an outdoor laboratory tank supplied with different seawater (Yoshihama Bay). The fish were sampled regularly and on the same days. The amount of TTX (mouse unit (MU)/fish) in the fish at Onisawa FP increased until 20 days and thereafter it did not change, while the amount of TTX in the fish in the laboratory tank remained low, and the TTX concentration (MU/g fish) decreased. Next, we compared the toxicity of wild juvenile T. flavipterus collected from Okirai Bay (Onisawa FP and Okirai FP) and Yoshihama Bay (Yoshihama FP). Large differences in TTX levels were observed among the fish from the three FPs. The amounts and concentrations of TTX in the fish at Onisawa FP were higher than those in the fish from the other two FPs. These results indicate that a large variation in toxic activity exists in the juvenile T. flavipterus in the bay of the Sanriku Coast.

     

Photic and circadian regulation of self-feeding activity in ayu

ABSTRACT:   The self-feeding rhythms of ayu Plecoglossus altivelis altivelis were examined. Individual ayu (mean body weight 40 g) were held in 60-L glass tanks equipped with self-feeders. Six of 14 fish learned self-feeding during the experiment. Under two different light–dark (LD) conditions (16 h:8 h and 8 h:16 h LD), self-feeding was synchronized to the LD cycle, and feeding occurred almost exclusively during the light phase. During exposure to constant light (LL), circadian feeding rhythms were observed. These results indicate self-feeding rhythms in ayu are restricted to the light phase under LD conditions and are controlled by the circadian clock under LL conditions.     

Gossypol inhibits LH‐induced steroidogenesis in bovine theca cells

Gossypol, a polyphenolic aldehyde found in cottonseed, has been shown to perturb steroidogenesis in granulosa and luteal cells of rats, pigs and cattle. However, little is known about the direct effect of gossypol on theca cell functions in any species. The present study was conducted to investigate the effect of gossypol on the steroidogenesis and the expression of genes involved in it in cultured bovine theca cells. Theca cells were isolated from healthy preovulatory follicles and were cultured in the presence of luteinizing hormone (LH) for up to 7 days. During the culture period, main steroid products of the theca cells shifted from androstenedione (A4) at day 1 to progesterone (P4) from day 2 onward. At days 1 and 7, theca cells were treated with gossypol (0‐25 μg/mL) for 24 h. Gossypol inhibited LH‐stimulated theca cell A4 and P4 production in a dose‐dependent manner at both occasions. The viability of theca cells was not affected by gossypol at any doses used. Gossypol down‐regulated expressions of steroidogenic enzymes CYP11A1, HSD3B1 and CYP17A1, but not that of LHR. These results indicate that gossypol inhibits thecal steroidogenesis through down‐regulating gene expressions of steroidogenic enzymes but without affecting cell viability in cattle.     

Purification and sensitivity of Clostridium chauvoei hemolysin to various erythrocytes

Using ammonium sulphate fractionation, the Clostridium chauvoei hemolysin was purified by cation exchange chromatography and sephacryl S-100 gel filtration. The molecular mass of the hemolysin, determined by SDS-PAGE was found to be approximately 27kDa. The activity of the hemolysin was determined in erythrocytes of various animals, with sensitivities observed in the order of cow, sheep, chicken, rabbit, rat, mouse, dog and horse. Temperature affected the sensitivity of erythrocytes to C. chauvoei hemolysin. These results may reflect distinct characteristics of the hemolytic activity of C. chauvoei hemolysin and that the hemolysin may be pore-forming.     

A case study of improving milking cow performance and milking system performance with using a flow simulator

Efforts to improve dairy performance have been focused on increasing milk productivity of cows through improved feeding systems and genetic potential. However, methods for evaluating milking system performance based on milk productivity have not yet been established. Milking system performance was evaluated by measuring the claw vacuum at five flow rates (1.9–8.7 kg/min) produced using a flow simulator for a single eight‐swing milking parlor with a high‐line system. Based on these results, a double eight‐parallel milking parlor with a low‐line system was installed and tested. Farmers can take data obtained from evaluations of milking system performance into account for future management decisions, such as renewing the milking system. By renewing the milking system, average milking productivity, somatic cell linear score (LS) of bulk milk, and LS of each cow were significantly improved in the year after installing the new system (p < .01). In addition to checking conventional milking systems, this novel diagnostic method using a flow simulator can be used for checking new installations and also for proposing renovations.     

Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.     

Development of a time-resolved fluoroimmunoassay for octopus gonadotropin-releasing hormone

We have developed a time-resolved fluoroimmunoassay (TR-FIA) for octopus gonadotropin-releasing hormone (oct-GnRH) to determine the profiles of oct-GnRH peptide levels in cephalopods. The sensitivity and the intra-assay and inter-assay coefficients of variation were 4.9 pg/well and 6.8 (n = 10) and 2.7% (n = 5), respectively. Anti-oct-GnRH antibody was tested on all known forms of GnRH and found to cross-react with lamprey GnRH-II (27.1%), annelid GnRH (3.36%), tunicate GnRH-I (0.92%), dogfish GnRH (0.51%), and scallop Patinopecten yessoensis GnRH (0.05%). The displacement curve obtained for serially diluted brain extracts of three cephalopods, the spear squid Loligo bleekeri, swordtip squid Loligo edulis, and North Pacific giant octopus Octopus dofleini, paralleled the oct-GnRH standard curve. The presence of oct-GnRH in the central nervous system (CNS) of these cephalopods was further examined by a combination of reverse-phase high performance liquid chromatography and oct-GnRH TR-FIA. CNS extracts from these cephalopods showed peaks with retention times similar to that of synthetic oct-GnRH. These results indicate that this novel oct-GnRH TR-FIA is widely applicable for oct-GnRH measurement in cephalopods.