Complete Genome Sequence Analysis of Two Pseudomonas plecoglossicida Phages,Potential Therapeutic Agents

Pseudomonas plecoglossicida is a lethal pathogen of ayu (Plecoglossus altivelis) in Japan and is responsible for substantial economic costs to ayu culture. Previously, we demonstrated the efficacy of phage therapy against P. plecoglossicida infection using two lytic phages (PPpW-3 and PPpW-4) (S. C. Park, I. Shimamura, M. Fukunaga, K. Mori, and T. Nakai, Appl Environ Microbiol 66:1416–1422, 2000, http://dx.doi.org/10.1128/AEM.66.4.1416-1422.2000; S. C. Park and T. Nakai, Dis Aquat Org 53:33–39, 2003, http://dx.doi.org/10.3354/dao053033). In the present study, the complete genome sequences of these therapeutic P. plecoglossicida phages were determined and analyzed for deleterious factors as therapeutic agents. The genome of PPpW-3 (myovirus) consisted of 43,564 bp with a GC content of 61.1% and 66 predicted open reading frames (ORFs). Approximately half of the genes were similar to the genes of the Escherichia coli phage vB_EcoM_ECO1230-10 (myovirus). The genome of PPpW-4 (podovirus) consisted of 41,386 bp with a GC content of 56.8% and 50 predicted ORFs. More than 70% of the genes were similar to the genes of Pseudomonas fluorescens phage ϕIBB-PF7A and Pseudomonas putida phage ϕ15 (podoviruses). The whole-genome analysis revealed that no known virulence genes were present in PPpW-3 and PPpW-4. An integrase gene was found in PPpW-3, but other factors used for lysogeny were not confirmed. The PCR detection of phage genes in phage-resistant variants provided no evidence of lysogenic activity in PPpW-3 and PPpW-4. We conclude that these two lytic phages qualify as therapeutic agents.     

Difference in Japanese flounder, Paralichthys olivaceus gene expression profile following hirame rhabdovirus (HIRRV) G and N protein DNA vaccination

The glycoprotein (G protein) gene, but not other genes, of fish rhabdoviruses, when used as a DNA vaccine was previously shown to be highly effective in inducing a protective immune response. In this study we used a DNA microarray to examine differential gene expression in Japanese flounder (Paralichthys olivaceus) in response to a DNA vaccine made from the genes of hirame rhabdovirus (Rhabdovirus olivaceus) (HIRRV) G protein (pHRV-G) and nucleocapsid (N) protein (pHRV-N). A high level of protection against HIRRV infection was observed following vaccination with the pHRV-G but no protection was observed following vaccination with the pHRV-N. Microarray analyses showed that the set of genes induced by pHRV-G was different from the set induced by pHRV-N. Specifically, five genes (Interferon-stimulated gene, 15kDa (ISG15), Interferon-stimulated gene, 56kDa (ISG56), Mx and two unknown genes) were strongly induced after injection by the pHRV-G but not pHRV-N and three of these genes are known as type I IFN-inducible genes. Poly I:C, a known inducer of type I interferon that elicits immune response similar to that elicited by a virus infection, also induced these five genes in kidney cells. These results suggest that in order to be effective and confer protection, vaccines against HIRRV and probably fish rhabdoviruses may need to stimulate the type I IFN system.     

The granulocyte colony-stimulating factors (CSF3s) of fish and chicken

Granulocyte colony-stimulating factor (CSF3) is a glycoprotein cytokine, which influences the hematopoiesis of the phagocytic neutrophils and its precursors and was used extensively in cancer therapy and for the treatment of neutropenia in mammals. However, CSF3 is yet to be identified in nonmammalian species mainly because of its rapid mutation. Here, we report the first CSF3 genes from three teleost fishes: Japanese flounder (Paralichthys olivaceus), fugu (Takifugu rubripes), and green-spotted pufferfish (Tetraodon nigroviridis) and present evidence that the chicken (Gallus gallus) myelomonocytic growth factor is in fact the chicken CSF3 orthologue. We support this by showing significant conservation of the CSF3 genes’ structure, domains, regulatory motifs, and synteny across species and by phylogenetic analysis. CSF3 orthologues are indeed evolving rapidly and appears to be undergoing purifying selection in mammals but positive selection in fish and chicken. Furthermore, the paralogous fugu and pufferfish CSF3-1s and CSF3-2s are shown to be the ancestral and duplicate genes, respectively. Finally, we demonstrate that the Japanese flounder CSF3 gene is at least involved in immunity based on its basal expression in immune-related tissues and its upregulation in kidney and peripheral blood leukocytes after in vitro stimulation with lipopolysaccharide and a combination of concanavalin A/phorbol myristate acetate.Electronic Supplementary Material Supplementary material is available for this article at ()     

Transverse elasticity of myofibrils of rabbit skeletal muscle studied by atomic force microscopy

Surface structure of myofibrils of rabbit skeletal muscle and their transverse elasticity were studied by atomic force microscopy. Images of myofibrils had a periodic structure characteristic of sarcomeres of skeletal muscle fibers. The transverse elasticity distribution in the sarcomere was determined based on force-distance curves measured at various loci of single myofibrils. The Z-line in rigor myofibrils was the most rigid in all the loci of myofibrils studied under various physiological conditions. The overall transverse elasticity of myofibrils decreased in the order in rigor solution > +AMPPNP solution > relaxing solution. The "apparent" transverse Young's modulus of myofibrils estimated at the overlap region between thin and thick filaments was 84.0 +/- 18.1, 37.5 +/- 14.0, and 11.5 +/- 3.5 kPa in rigor, +AMPPNP, and relaxing solution respectively.     

Effect of prolonged free-walking fatigue on gait and physiological rhythm

This study examined the ways in which gait patterns and physiological rhythms such as those of muscle activity (tibialis anterior (TA) and biceps femoris (BF)) and cardiac activity are affected by the fatigue induced by prolonged free walking. Twelve normal subjects who walked for 3 h at their preferred pace were divided into two groups according to whether their mean gait cycle time (reciprocal of stride rate) during the second 90 min was higher (Group A: n=8) or lower (Group B: n=4) than that during the first 90 min. For Group A, the level of subjective fatigue during the walking task was significantly higher and the heart rate at rest was significantly lower than Group B. In Group A, prolonged walking significantly decreased the mean power frequency of the electromyography from TA, increased the variability of gait rhythm, decreased the largest Lyapunov exponent of the vertical component of back-waist acceleration, and decreased the amplitude of the vertical component of back-waist acceleration. Taking the onset timings of these changes into account, we propose that subjects who tire easily during prolonged walking first show local muscle fatigue at TA followed by instability of gait rhythm and then they slow their gait rhythm to enhance local dynamic stability. For both groups we constructed a physical fatigue index described by linear regression of gait and physiological variables. When we compared the subjective fatigue level with the fatigue level predicted using the index, we obtained a relatively high correlation coefficient for both groups (r=0.77).     

MicroRNA-125b regulates the expression of aggrecanase-1 (ADAMTS-4) in human osteoarthritic chondrocytes

Introduction

Increased expression of aggrecanase-1 (ADAMTS-4) has emerged as an important factor in osteoarthritis (OA) and other joint diseases. This study aimed to determine whether the expression of ADAMTS-4 in human chondrocytes is regulated by miRNA.

Methods

MiRNA targets were identified using bioinformatics. Chondrocytes were isolated from knee cartilage and treated with interleukin-1 beta (IL-1β). Gene expression was quantified using TaqMan assays and protein production was determined by immunoblotting. Luciferase reporter assay was used to verify interaction between miRNA and target messenger RNA (mRNA).

Results

In silico analysis predicted putative target sequence of miR-125b on ADAMTS-4. MiR-125b was expressed in both normal and OA chondrocytes, with significantly lower expression in OA chondrocytes than in normal chondrocytes. Furthermore, IL-1β-induced upregulation of ADAMTS-4 was suppressed by overexpression of miR-125b in human OA chondrocytes. In the luciferase reporter assay, mutation of the putative miR-125b binding site in the ADAMTS-4 3''UTR abrogated the suppressive effect of miR125.

Conclusions

Our results indicate that miR-125b plays an important role in regulating the expression of ADAMTS-4 in human chondrocytes and this identifies miR-125b as a novel therapeutic target in OA.     

Biocontrol of black scurf on potato by seed tuber treatment with Pythium oligandrum

The biological control activity of Pythium oligandrum against black scurf of potato caused by Rhizoctonia solani AG-3 was evaluated in field experiments after treatment of potato seed tubers with P. oligandrum. Seed tubers infected with black scurf sclerotia were dipped for a few seconds in a suspension of 10³, 10⁴ or 10⁵ mL^?1 P. oligandrum oospores and were then air-dried. Each level of P. oligandrum-treatment significantly reduced the disease rates of stolon at a level similar to that achieved by chemical control. When P. oligandrum populations adherent to the surface of seed tubers were determined, oospore counts on tubers treated with 10⁴ or 10⁵ oospores mL^?1 were about 540/cm² or about 22,000/cm² just after dipping and decreased to about 170/cm² or 2900/cm² after a 3-week incubation, respectively. Confocal laser scanning microscopic observation with an immuno-enzymatic staining procedure showed that P. oligandrum hyphae had colonized the sclerotia and established close contact by coiling around the R. solani hyphae present on the surface of seed tubers, in a manner similar to that observed in the dual-culture test. Quantification of R. solani DNA by PCR indicated that the R. solani population was reduced on the seed tubers treated with P. oligandrum compared to untreated tubers. Furthermore, the ability of P. oligandrum to induce resistance against black scurf was determined using a potato tuber disk assay. Treatment of tuber disks with the cell wall protein fraction of P. oligandrum enhanced the expression of defense-related genes such as 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, lipoxygenase and basic PR-6 genes, and reduced disease severity upon challenge with R. solani compared with untreated controls. These results suggest that biocontrol mechanisms employed by P. oligandrum against black scurf involve both mycoparasitism and induced resistance.     

Molecular changes of organelle DNA sequences in rice through dedifferentiation,long-term culture,or the morphogenesis process

Callus-specific rearranged DNA in rice (Oryza sativa L.) was isolated by in-gel reassociation procedure. Southern hybridization experiments revealed that some clones were amplified significantly in primary callus induced from scutellum tissue. Rapid amplification of these clones was observed within 2 days after plating seeds onto callus-induction medium containing 2,4-D. NAA gave no significant effect on DNA amplification event. Colony formation process from isolated protoplasts and plant regeneration process from callus showed clone-specific and process-specific fluctuation patterns of copy number. Sequence analysis of the clones suggested that most of the clones were originated from organelle DNA. Comparison of copy number fluctuation pattern of organelle functional genes with that of the clones suggested multiformity and/or construction-specific amplification of organelle DNA.