Development of an efficient method for recovery of Puumala and Puumala-related viruses by inoculation of Mongolian gerbils

Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10 (5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C.) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.     

Inhibition of growth hormone secretagogue receptor antagonist, [D-Lys-3]-GHRP-6, in adipogenesis of ovine and rat adipocytes

Ghrelin and growth hormone secretagogues receptor (GHS‐R or ghrelin receptor) have been reported as being one of the factors of adipogenesis in adipocytes. To investigate the involvement of ghrelin and GHS‐R in adipocytes, the effect of the GHS‐R antagonist, [D‐Lys‐3]‐GHRP‐6 (His‐D‐Trp‐D‐Lys‐Trp‐D‐Phe‐Lys‐NH₂), on the process of adipogenesis in ovine and rat adipocytes was evaluated. [D‐Lys‐3]‐GHRP‐6 (10^?7 mol/L) significantly inhibited adipogenic differentiation of ovine and rat preadipocytes prepared from adipose tissues. The level of peroxisome proliferator activated receptor (PPAR)‐γ2 mRNA, an adipogenic marker, was decreased during the differentiation of adipocytes treated with [D‐Lys‐3]‐GHRP‐6 for 10 days. Ghrelin stimulated adipogenesis, also causing an increment of glycerol‐3‐phosphate dehydrogenase and upregulation of PPAR‐γ2. Furthermore, the antilipolytic effect of ghrelin was attenuated by treatment with [D‐Lys‐3]‐GHRP‐6 in both types of isolated adipocytes. Overall, the results of the present study highlight that GHS‐R in adipogenesis can be blocked by treatment with [D‐Lys‐3]‐GHRP‐6.     

Colonization by two isolates of Plasmodiophora brassicae with differing pathogenicity on a clubroot-resistant cultivar of Chinese cabbage (Brassica rapa L. subsp. pekinensis)

A commercial clubroot-resistant F₁ cultivar of Chinese cabbage (Brassica rapa L. subsp. pekinensis), Kukai 70, is resistant to an isolate of populations of Plasmodiophora brassicae from Hagi (HG) city and is susceptible to another from Yamaguchi (YMG) city. The degree and frequency of primary and secondary cycle colonization by the isolates in the root hairs and root tissues of cv. Kukai 70 were compared. Seedlings of cv. Kukai 70 were grown in soils amended with inoculum of either HG or YMG and harvested 10 days after inoculation to observe the primary cycle (number of root-hair infections) and 20, 30, and 40 days after inoculation to observe of the secondary cycle (frequency of infected cells and degree of plasmodial development based on the number of nuclei in infected cells). Although more root hairs were infected in HG than in YMG, fewer cells in root tissues including the cortex and medullary rays were infected in HG than in YMG. In addition, YMG developed plasmodia with many nuclei and formed resting spores, whereas plasmodia remained immature with a small number of nuclei in HG and did not form resting spores even by 40 days after inoculation. These results suggest that suppression of plasmodial development during secondary colonization is associated with resistance mechanisms to HG in cv. Kukai 70. Starch did not accumulate (i.e., development of amyloplasts) in HG-infected cells. This may be involved in the suppression of secondary colonization of P. brassicae in the cultivar.     

Mutations in the acetolactate synthase genes of sulfonylurea-resistant biotypes of Lindernia spp.

Acetolactate synthase (ALS) is a key enzyme in the biosynthetic pathway of branched-chain amino acids. A mutation of the ALS gene causing amino acid substitution at the position of proline in Domain A makes ALS less sensitive to sulfonylureas, which are ALS-inhibiting herbicides. We cloned partial ALS genes from four Lindernia plants, L . dubia var. dubia , L . dubia var. major , L . micrantha and L . procumbens , for which biotypes resistant to sulfonylureas have been found in paddy fields. The clones were classified into two groups in each Lindernia plant: Als1 and Als2 . Sequencing of the clones and alignment of deduced amino acid sequences with previously reported ALS of other species suggested that the cloned region contains an intron in both Als1 and Als2 . Comparison of Als1 between resistant and susceptible biotypes showed that the proline of Domain A was replaced by alanine, serine or glutamine in all resistant biotypes of Lindernia plants, while it was conserved in all susceptible biotypes. This amino acid substitution in ALS encoded by Als1 is involved in the resistant mechanism of ALS to sulfonylurea in the four Lindernia plants.     

Starvation–refeeding causes cellular stress responses in the gut and liver of Masu salmon Oncorhynchus masou masou

Fisheries Science - Gene expression profiles during the transition from fasting to refeeding were investigated in the gut and liver of Masu salmon Oncorhynchus masou masou. Fish were starved for...     

Interannual variation in quantitative relationships among egg production and densities of larvae and juveniles of the Japanese mantis shrimp <Emphasis Type="Italic">Oratosquilla oratoria</Emphasis> in Tokyo Bay,Japan

To explore which lifestages affect the stock size of young-of-the-year mantis shrimp Oratosquilla oratoria in Tokyo Bay, Japan, we investigated interannual variations in the quantitative relationships among egg production, larval density, and juvenile density. We collected adult females, larvae, and juveniles during monthly field surveys from 2004 to 2007. The interannual trend for the juvenile density index differed from those for egg production and larval density; although indices of both egg production and larval density were high in 2004 and 2007, the juvenile density index was high only in 2007, suggesting high mortality during the pelagic larval stage or the early phase of the postsettlement juvenile stage in 2004. We found that larval settlement started at the end of August and peaked in October, although larvae from the early spawning season (May–June) should have settled in August or earlier. Juveniles were found throughout the bay except in areas where bottom hypoxia occurred, suggesting that hypoxia restricts the spatial distribution of juveniles. Our results suggest that mortality during the early life history fluctuates among years, probably because of changes in environmental conditions in the bay, resulting in interannual variation in the stock size of young-of-the-year juvenile O. oratoria.     

Bioremediation of organically enriched sediment deposited below fish farms with artificially mass-cultured colonies of a deposit-feeding polychaete <Emphasis Type="Italic">Capitella</Emphasis> sp. I

ABSTRACT:   For bioremediation of organically enriched sediment deposited below fish farms, the extremely high potential for population growth of a deposit-feeding polychaete, Capitella sp. I, in the organically enriched sediment, and the effect on decomposition of organic matter in the sediment, were examined. A mass-culturing technique was conducted for this species. Bioremediation experiments were conducted on the organically enriched sediment in a fish farm in Kusuura Bay, Japan in 2003–2006. Approximately 1.7 million individuals of the worms were placed on the sediment below one net pen in December 2003, 9.3 million individuals in November 2004, and 2.2 million individuals in November 2005. After the worms were spread on the sediment, they rapidly increased in number and reached the highest densities of approximately 134 000 inds/m² in February 2004, 527 000 inds/m² in March 2005 and 103 000 inds/m² in January 2006. In the process of rapid population growth, the decomposition of the organic matter of the sediment was enhanced markedly. Our results demonstrate that the promotion of population growth by spreading cultured colonies of Capitella can enhance the decomposition rate of organic matter markedly in organically enriched sediment below fish farms. This method is promising for minimization of the negative effects of fish farms.     

Successful introduction of modified dorsolumbar epidural anesthesia in a bovine referral center

This study describes the successful use of modified dorsolumbar epidural anesthesia with a fixed volume of anesthetic in a bovine referral center. Among the 130 Holstein cattle scheduled for flank surgery, 90 cattle received a mixed anesthetic consisting of 1 ml of xylazine hydrochloride and 3 ml of lidocaine hydrochloride by modified dorsolumbar epidural anesthesia. Eighteen cattle with dehydration and/or lameness received a mixed anesthetic containing 0.5 ml of xylazine and 3 ml of lidocaine. Infiltration anesthesia was performed in 22 cattle whose epidural space could not be reached in order to perform the flank surgery. The surgeries began about 12 min after the administration of the anesthetic and lasted for about 36 min. The modified method using a fixed volume of anesthetic was successfully introduced and effectively used in a bovine referral center. This modified method will allow veterinarians to save time and effort, thus lowering the cost of each surgery.     

Digestive enzyme activities of pancreas and intestinal digesta in streptozotocin-induced diabetic piglets

Six (Landrace × Large White) × Duroc crossbred, castrated weanling piglets were used to evaluate the effect of a streptozotocin (STZ) injection to induce diabetes on the activities of digestive enzymes derived from the pancreas (lipase, amylase, trypsin and chymotrypsin) in the pancreas and intestinal digesta. Fasting plasma glucose after the administration of STZ was maintained at the level of 302–491 mg/dL during this experiment, compared with the level of 89–125 mg/dL in the control group, and they were defined as the STZ‐induced diabetic piglets for about 7 weeks. Although their bodyweight increased in proportion to a quadratic curve (P < 0.0001) during 49 days after the administration of STZ, the growth of the STZ‐induced diabetic piglets was slower compared with the control. The STZ‐injection did not affect the percentage of the pancreas in bodyweight. The administration of STZ in the piglets tended to accelerate the activity of lipase (P = 0.06) and depress the activity of amylase (P = 0.15) or chymotrypsin (P = 0.18), as units/kg bodyweight, in the pancreas. In the case of measurement as units/kg bodyweight, the activities of intestinal digesta in the STZ group showed a tendency to be higher than those in control group, irrespective of the sort of enzymes. In conclusion, STZ‐induced diabetic piglets have a moderate digestive ability and the administration of exogenous digestive enzymes is not necessary when they are used as a diabetic animal model.     

In planta dynamic analysis of onion yellows phytoplasma using localized inoculation by insect transmission

ABSTRACT Due to the lack of a means to inoculate plants mechanically, the histological dynamics and in planta spread of phytoplasmas have been studied very little. We analyzed the dynamics of plant infection by phytoplasmas, using a technique to infect a limited area of a leaf, nested polymerase chain reaction (PCR), real-time PCR, and immunohistochemical visualization. Following localized inoculation of a leaf of garland chrysanthemum (Chrysanthemum coronarium) by the vector leafhopper Macrosteles striifrons, the onion yellows (OY) phytoplasma spread within the plant from the inoculated leaf to the main stem (1 day postinoculation [dpi]), to the roots and the top leaf (2 dpi), and to other leaves from top to bottom (from 7 to 21 dpi). The populations of the OY phytoplasmas in inoculated leaves and roots increased approximately sixfold each week from 14 to 28 dpi. At 14 dpi, the OY phytoplasmas colonized limited regions of the phloem tissue in both the root and stem and then spread throughout the phloem by 21 dpi. This information should form the basis for elucidating the mechanisms of phytoplasma multiplication and migration within a plant host.