Pueruli of Panulirus longipes bispinosus (Crustacea, Decapoda, Palinuridae) stranded on the beach of Kuroshima Island, Ryukyu Archipelago, southern Japan

ABSTRACT: Stranded organisms were observed on the sandy beach of Kuroshima Island, Ryukyu Archipelago, in the early morning of 28 January 1993. Stranded organisms of 525 individuals were classified into 74 species. These organisms included 13 individuals of Panulirus pueruli, which were identical to P. longipes bispinosus. Kuroshima Island is located near Ishigakijima and Iriomote islands, Ryukyu Archipelago, where coral reefs develop. Coral reefs are common and develop in the Ryukyu Archipelago, southern Japan, with the exception of Yakushima and Tanegashima islands. Panulirus japonicus in Japanese waters has not been found in coral reef regions where P. longipes bispinosus are mainly found. The present study describes and identifies to species level puerulus specimens that were stranded on the beach of Kuroshima Island.     

Alternative Route of Infection for Bacterial Seedling Blight of Rice Caused by Burkholderia plantarii

Burkholderia plantarii , the pathogen of bacterial seedling blight of rice, was detected in paddy water. Its concentration rose in July and August. The bacterial concentration in the paddy water was always higher along levees than at distances more than 5 m from levees. Confirmed to be released into water when graminaceous weeds were immersed, B. plantarii survived for at least 4 days at 30°C. B. plantarii was splashed at least 30 cm upward by rain splash in the field. Harvested seeds, which had been sprayed with B. plantarii released from graminaceous weeds at the flowering stage, retained the bacteria. Bacterial seedling blight occurred when the seeds were then sown in nursery boxes. These results indicated that graminaceous weeds growing on levees of paddy fields are a source of infection of the disease and that rice seeds are infected through the paddy water. Received 23 May 2002/ Accepted in revised form 1 September 2002     

Lipid-rich bovine serum albumin improves the viability and hatching ability of porcine blastocysts produced in vitro

The effects of lipid-rich bovine serum albumin (LR-BSA) on the development of porcine blastocysts produced in vitro were examined. Addition of 0.5 to 5 mg/ml LR-BSA to porcine blastocyst medium (PBM) from Day 5 (Day 0 = in vitro fertilization) significantly increased the hatching rates of blastocysts on Day 7 and the total cell numbers in Day-7 blastocysts. When Day-5 blastocysts were cultured with PBM alone, PBM containing LR-BSA, recombinant human serum albumin or fatty acid-free BSA, addition of LR-BSA significantly enhanced hatching rates and the cell number in blastocysts that survived compared with other treatments. The diameter, ATP content and numbers of both inner cell mass and total cells in Day-6 and Day-7 blastocysts cultured with PBM containing LR-BSA were significantly higher than in blastocysts cultured with PBM alone, whereas LR-BSA had no effect on mitochondrial membrane potential. The mRNA levels of enzymes involved in fatty acid metabolism and β-oxidation (ACSL1, ACSL3, CPT1, CPT2 and KAT) in Day-7 blastocysts were significantly upregulated by the addition of LR-BSA. The results indicated that LR-BSA enhanced hatching ability and quality of porcine blastocysts produced in vitro, as determined by ATP content, blastocyst diameter and expression levels of the specific genes, suggesting that the stimulatory effects of LR-BSA arise from lipids bound to albumin.     

Comparison of dendritic cell-mediated immune responses among canine malignant cells

Dendritic cell (DC) vaccination is one of the most attractive immunotherapies for malignancies in dogs. To examine the differences in DC-mediated immune responses from different types of malignancies in dogs, we vaccinated dogs using autologous DCs pulsed with keyhole limpet hemocyanin (KLH) and cell lysate prepared from squamous cell carcinoma SCC2/88 (SCC-KLH-DC), histiocytic sarcoma CHS-5 (CHS-KLH-DC), or B cell leukemia GL-1 (GL-KLH-DC) in vitro. In vivo inductions of immune responses against these tumor cells were compared by the delayed-type hypersensitivity (DTH) skin test. The DTH response against SCC2/88 cells were observed in dogs vaccinated with autologous SCC-KLH-DC, while the response was undetectable against CHS-5 and GL-1 cells in dogs vaccinated with autologous CHS-KLH-DC and GL-KLH-DC. Skin biopsies taken from DTH challenge sites were then examined for immunohistochemistry, and recruitment of CD8 and CD4 T cells was detected at the site where SCC2/88 cells were inoculated in dogs vaccinated with SCC-KLH-DC. By contrast, neither CD8 nor CD4 T cell infiltration was found at the DTH challenge site in the dogs vaccinated with CHS-KLH-DC or GL-KLH-DC. These findings may reflect that the efficacy of immune induction by DC vaccination varies among tumor types and that immune responses could be inducible in squamous cell carcinoma. Our results encouraged further investigation of therapeutic vaccination for dogs with advanced squamous cell carcinoma in clinical trials.     

Isolation of Pathogenicity Mutants of Fusarium oxysporum f. sp. melonis by Insertional Mutagenesis

Restriction enzyme-mediated integration (REMI) mutagenesis was used to isolate mutants of Fusarium oxysporum f. sp. melonis impaired in pathogenicity. The race 2 strain Mel02010 was transformed with linearized pSH75, conferring resistance to hygromycin B, with or without the enzyme used to linearize the plasmid. Addition of restriction enzymes did not affect the transformation frequency. A total of 2929 REMI transformants were tested for pathogenicity to three melon cultivars, Amus, Ogon 9 and Ohi. The race 2 strains are pathogenic to Amus and Ogon 9, but not to Ohi. Of 43 transformants with reduced pathogenicity on susceptible melon cultivars, 12 mutants were examined in detail for pathogenicity, vegetative growth and integrative mode of pSH75. The levels of pathogenicity varied among these mutants. Two mutants (B48 and B137) almost completely lost pathogenicity to both susceptible cultivars, and the others had reduced pathogenicity. Mutants B48, B241, B886 and X36 were also impaired in vegetative growth. Mutant B809 was a biotin auxotroph. By DNA gel blot analysis, nine mutants were found to contain a single copy of the transformation vector. These mutants may thus be useful in isolating genes involved in pathogenicity. Received 22 December 2000/ Accepted in revised form 16 April 2001     

Rhizome-induction activity of chiral 2-α-methylbenzylamino-4-alkylamino-6-chloro-1, 3, 5-triazines in Cyperus serotinus Rottb.

A cytokinin-like effect of chiral 2-α-methylbenzylamino-4-alkylamino-6-chloro-1,3,5-triazines was found using a rhizome-inducing assay with Cyperus serotinus Rottb. tubers. C. serotinus tubers germinated in distilled water yielded plantlets with roots and leaves. Secondary rhizomes were normally not observed within the regular 14-day incubation time in water culture, whereas after increasing incubation periods a very short rhizome appeared (controls). 6-Benzylaminopurine (BA) significantly stimulated rhizome induction, while other plant hormones were inactive. The (R)-isomers of the 1, 3, 5-triazine compounds also stimulated induction of the rhizomes, whereas the (S)-isomers did not. The described rhizome induction system seems to be suitable as a cytokinin bioassay. The (R)-1, 3, 5-triazine compounds showing rhizome-inducing activity (RI activity) inhibited root formation and plant growth at high concentrations with symptoms which were very similar to those of BA. Therefore, the (R)-isomers appear to act as cytokinins in the rhizome induction assay.     

Evaluation of bovine embryos produced in high performance serum-free media

This review evaluates the quality of bovine embryos developed from in vitro-matured (IVM) and -fertilized (IVF) oocytes cultured in either serum-free or serum-containing media. Bovine embryos cultured in serum-supplemented medium contain numerous cytoplasmic lipid droplets and immature mitochondria compared to those cultured in serum-free medium. The accumulation of cytoplasmic lipids in embryos developed in serum-containing medium may be a result of incorporation of lipoproteins from the serum and may result in impaired function of mitochondria. The improved serum-free media (IVMD101 and IVD101) offer several advantages over culture in serum-containing medium, including increased rates of blastocyst formation and higher cell numbers. Additionally, the survival and hatching rates of embryos produced in serum-free media after post-thaw culture were superior to those of embryos produced in the serum-containing medium, suggesting that the abnormal accumulation of cytoplasmic lipids in embryos may have a negative effect on the sensitivity of embryos to chilling and freezing. These serum-free culture systems have proven to be beneficial for the production of good quality embryos from IVM-IVF bovine oocytes. Furthermore, recent studies have shown a correlation between mitochondrial function (oxygen consumption) and embryo quality. A new method using scanning electrochemical microscopy may be capable of assessing the viability and developmental potential of bovine embryos.     

Validation and application of real-time polymerase chain reaction assays for representative rumen bacteria

Real‐time polymerase chain reaction (PCR) assays for 11 representative rumen bacterial species were validated. The sensitivity was tested by using the serially diluted target 16S rDNA from respective bacterial species. The recovery of the target DNA and the assay reproducibility were determined using DNA from rumen fluid spiked with different quantities of the target. Minimum detection levels for the target were 10–100 copies in pure culture. The recovery of the added target ranged from 82.4 to 116.6%. The intra‐ and inter‐assay variations of each assay were <9.4 and <12.6%, respectively. Therefore, the real‐time PCR assays evaluated in the present study are considered to be sufficiently reliable for monitoring all 11 bacterial species in the rumen. The assays were then applied to the monitoring of the bacterial species attached to ruminally incubated rice straw. Among the monitored fibrolytic species, Fibrobacter succinogenes was found to be the most dominant, accounting for 2.61% of total bacteria after 24 h incubation. Selenomonas ruminantium and Streptococcus bovis, non‐fibrolytics, were detected on the rice straw at 8.96% and 1.16% of total bacteria, respectively. Such high levels of non‐fibrolytics on the plant fiber suggest a synergistic relationship between fibrolytics and non‐fibrolytics.     

Calcium ions are involved in egress of Babesia bovis merozoites from bovine erythrocytes

Bovine babesiosis is a livestock disease known to cause economic losses in endemic areas. The apicomplexan parasite Babesia bovis is able to invade and destroy the host’s erythrocytes leading to the serious pathologies of the disease, such as anemia and hemoglobinuria. Understanding the egress mechanisms of this parasite is therefore a key step to develop new therapeutic strategies. In this study, the possible involvement of Ca²⁺ in the egress of B. bovis merozoites from infected erythrocytes was investigated. Egress was artificially induced in vitro using calcium ionophore A23187 and thapsigargin to increase Ca²⁺ concentration in the cytosol of the parasite cells. The increased intracellular Ca²⁺ concentration following these treatments was confirmed using live cell Ca²⁺ imaging with confocal laser scanning microscopy. Based on our findings, we suggest a Ca²⁺ signalling pathway in the egress of B. bovis merozoites.     

Weak activity of UDP-glucuronosyltransferase toward Bisphenol analogs in mouse perinatal development

Bisphenol A (BPA) is a widely used industrial chemical that disrupts endocrine function. BPA is an endocrine disrupting chemical (EDC) that has been demonstrated to affect reproductive organ development, brain development, metabolic disease and post-natal behavior. Accordingly, Bisphenol analogs, Bisphenol F (BPF, bis (4-hydroxyphenyl) methane) and Bisphenol AF (BPAF, 4,4-hexafluoroisopropylidene) diphenol) are used as replacements for BPA. BPA is mainly metabolized by UDP-glucuronosyltransferase (UGT), UGT2B1, but this effective metabolizing system is weak in the fetus. In the present study, we demonstrated that hepatic UGT activity toward BPAF was very weak, in comparison with BPA and BPF, in the fetus, pups and dams. Conversely, hepatic UGT activity toward BPF was very weak in the fetus and newborn pups, and was increased to the same level as BPA post-partum. In conclusion, BPAF possibly tends to accumulate in the fetus, because of weak metabolism during the perinatal period, suggesting that the metabolism of individual Bisphenol analogs requires assessment to properly gauge their risks.