Redox regulation of germline and vulval development in Caenorhabditis elegans

In vitro studies have indicated that reactive oxygen species (ROS) and the oxidation of signaling molecules are important mediators of signal transduction. We have identified two pathways by which the altered redox chemistry of the clk-1 mutants of Caenorhabditis elegans acts in vivo on germline development. One pathway depends on the oxidation of an analog of vertebrate low density lipoprotein (LDL) and acts on the germline through the Ack-related tyrosine kinase (ARK-1) kinase and inositol trisphosphate (IP3) signaling. The other pathway is the oncogenic ras signaling pathway, whose action on germline as well as vulval development appears to be modulated by cytoplasmic ROS.     

Detection of Mycoplasma hyopneumoniae in lung and nasal swab samples from pigs by nested PCR and culture methods

We examined nasal swab and lung homogenate samples collected from pigs experimentally and naturally infected with Mycoplasma hyopneumoniae for the detection of M. hyopneumoniae by the nested PCR (nPCR) and culture methods. In the 23 experimentally infected pigs, M. hyopneumoniae was commonly detected in nasal swabs by the nPCR and culture methods at 4 weeks after inoculation, and there was a significant correlation (P<0.01) between the titers of viable organisms in nasal swabs and in lung homogenates in the experimentally inoculated pigs. In the naturally infected pigs, on the other hand, discrepancies in detection were found between nasal swab and lung homogenate samples in 17 of 36 cases, although the presence of gross lung lesions correlated relatively well with the detection of organisms from the samples. Our results indicated that the diagnosis of mycoplasmal pneumonia by nPCR in individual pigs with nasal swabs is reliable under these experimental conditions. At present, nPCR with nasal swabs should only be used for monitoring the disease status at the herd level under field conditions.     

Effect of flutianil on the morphology and gene expression of powdery mildew

Flutianil, a fungicide effective only on powdery mildew, was previously reported to affect the host cell''s haustorial formation and nutrient absorption. Studies were conducted to investigate flutianil''s primary site of action on Blumeria graminis morphology using transmission electron microscope (TEM) observation and RNA sequencing (RAN-seq) techniques. TEM observation revealed that flutianil caused the extra-haustorial matrix and fungal cell wall to be obscured, without remarkable changes of other fungal organelles. RNA-seq analysis indicated that, unlike other powdery-mildew fungicides, flutianil did not significantly affect the constantly expressed genes for the survival of B. graminis. Genes whose expression is up- or downregulated by flutianil were found; these are the three sugar transporter genes and various effector genes, mainly expressed in haustoria. These findings indicate that the primary site of action of flutianil might be in the haustoria.     

Membrane proteins of the endoplasmic reticulum induce high-curvature tubules

The tubular structure of the endoplasmic reticulum (ER) appears to be generated by integral membrane proteins, the reticulons and a protein family consisting of DP1 in mammals and Yop1p in yeast. Here, individual members of these families were found to be sufficient to generate membrane tubules. When we purified yeast Yop1p and incorporated it into proteoliposomes, narrow tubules (approximately 15 to 17 nanometers in diameter) were generated. Tubule formation occurred with different lipids; required essentially only the central portion of the protein, including its two long hydrophobic segments; and was prevented by mutations that affected tubule formation in vivo. Tubules were also formed by reconstituted purified yeast Rtn1p. Tubules made in vitro were narrower than normal ER tubules, due to a higher concentration of tubule-inducing proteins. The shape and oligomerization of the "morphogenic" proteins could explain the formation of the tubular ER.     

Matrix metalloproteinases (MMPs) activity in cultured canine bone marrow stromal cells (BMSCs)

Autologous bone marrow stromal cells (BMSCs) infusion therapy improves the hepatic fibrosis. To investigate the mechanism of remission, we evaluated the matrix metalloproteinase (MMP)-2 and -9 activity in canine BMSCs and the effect of pro-inflammatory cytokines on their expression. The activity and the gene expression of MMPs were analyzed by gelatin zymography and quantitative RT-PCR, respectively. The specific gelatinase bands were indicative effect of MMP-2 and -9 in canine BMSCs. MMP-2 expression seemed to be increased by TNF-α and IL-1β while MMP-9 was enhanced by TNF-α and IL-6. These results suggested that remissive effect on liver fibrosis might be partly attributable to the MMP-2 and -9 activity in BMSCs under the inflammatory condition.     

Pharmacokinetics and effects on clinical and physiological parameters following a single bolus dose of propofol in common marmosets (Callithrix jacchus)

The objectives of this study were (a) to establish a population pharmacokinetic model and (b) to investigate the clinical and physiological effects of a single bolus dose of propofol in common marmosets. In Study 1, pharmacokinetic analysis was performed in six marmosets under sevoflurane anaesthesia. 8 mg/kg of propofol was administrated at a rate of 4 mg kg^?1 min^?1. Blood samples were collected 2, 5, 15, 30, 60, 90, 120 or 180 min after starting propofol administration. Plasma concentration was measured, and population pharmacokinetic modelling was performed. A two‐compartment model was selected as the final model. The population pharmacokinetic parameters were as follows: V₁ = 1.14 L, V₂ = 77.6 L, CL₁ = 0.00182 L/min, CL₂ = 0.0461 L/min. In Study 2, clinical and physiological parameters were assessed and recorded every 2 min after 12 mg/kg of propofol was administrated at a rate of 4 mg kg^?1 min^?1. Immobilization was sustained for 5 min following propofol administration without apparent bradycardia. While combination of propofol and sevoflurane caused apnoea in Study 1, apnoea was not observed following single administration of propofol in Study 2. These data provide bases for further investigation on intravenous anaesthesia using propofol in common marmosets.     

Quantitative nested real-time PCR detection of <Emphasis Type="Italic">Verticillium longisporum</Emphasis> and <Emphasis Type="Italic">V. dahliae</Emphasis> in the soil of cabbage fields

Verticillium longisporum and V. dahliae, causal agents of Verticillium wilt, are spreading through the cabbage fields of Gunma Prefecture. Using the V. longisporum-specific intron within the 18S rDNA and differences between ITS 5.8S rDNA sequences in Japanese isolates of V. longisporum and V. dahliae, we developed three quantitative nested real-time (QNRT) PCR assays. The QNRT-PCR quantification of V. longisporum or V. dahliae in cabbage field soil was consistent with the severity of Verticillium wilt disease in those fields. In field trials of resistant cultivar YR Ranpo grown for three seasons in soil infested with the pathogen, disease severity and pathogen density in the soil were significantly reduced in a field moderately contaminated by V. dahliae, but only slightly reduced in a highly contaminated field. These results suggest that continuous cultivation of a resistant cultivar is an effective way to reduce the pathogen population. QNRT-PCR assays provide a powerful analytical tool to evaluate the soil population dynamics of V. longisporum and V. dahliae for disease management.     

Synthesis and Structure–Activity Relationships of Miticidal 4,5-Dihydropyrazole-5-thiones

A series of novel 4,5-dihydropyrazole-5-thiones (DHPs) was synthesised by treating the corresponding dihydropyrazolones with ‘Lawesson’s reagent and evaluated for miticidal activity against two-spotted spider mites (Tetranychus urticae Koch). Of these, 3-(4-chlorophenyl)-4,4-dimethyl-1-phenyl-4,5-dihydropyrazole-5-thione, 3-(4-chlorophenyl)-4-ethyl-4-methyl-1-phenyl-4,5-dihydropyrazole-5-thione, 3-(4-chlorophenyl)-1-phenyl-4,5-dihydropyrazole-5-thione-4-spirocyclopentane and 4,4-dimethyl-1-phenyl-3-(4-trifluoromethyl-phenyl)-4,5-dihydropyrazole-5-thione were highly active (pEC₅₀>4·0) and were more effective than the miticide dicofol (pEC₅₀=3·879), which has traditionally been used for the control of phytophagous mites. Structure–activity relationship (SAR) studies were performed on each position of the pyrazole ring of DHPs. The results indicated that the unsubstituted phenyl, 4-substituted phenyl and thioxo groups on the 1-, 3- and 5-positions of DHPs respectively were required for activity. Quantitative SAR studies using physicochemical parameters of substituents and the capacity factor k′ as a hydrophobicity index suggested that: (a) the activities of all types of DHPs examined were mainly dominated by hydrophobicity, (b) the bulkiness of 4-substituents of the 3-phenyl ring favoured the activity and (c) the log k′ optimum for all DHPs was 1·675, equivalent to a log P_ow value of c. 5·0.     

The role of primary germ tubes in the life cycle of Blumeria graminis: The primary germ tube is responsible for the suppression of resistance induction of a host plant cell

When the conidia of Blumeria graminis f. sp. hordei (Bgh) are inoculated on barley coleoptile cells they produce short germ tubes called primary germ tubes (PGTs) about 2 h after inoculation. We evaluated the positive role of a PGT in inducing accessibility of the host cell under the germ tube. When an appressorium (APP) penetrated the same cell on which a PGT was present, the ratio of haustorium formation (penetration efficiency) was significantly higher than when an APP penetrated the cell adjacent to the one on which a PGT was present. When an APP penetrated the cell laterally adjacent to the one on which a PGT was present we killed the cell under the PGT by puncturing it with a microneedle and then investigated the penetration efficiency of the cell adjacent to the dead cell. As a control we killed the cell longitudinally adjacent to the one on which a PGT was present and investigated the penetration efficiency of the laterally adjacent cell. The results showed that the penetration efficiency of the former was significantly lower than that of the latter. This suggests that some accessibility factor might transfer from a cell on which a PGT is present to a laterally adjacent cell. The existence of a conidium body but not a PGT was not effective for induced accessibility of the host cell. Moreover, when a Bgh germling was removed 6 h after inoculation and another germling was transferred to the same cell, the penetration efficiency was significantly higher than that of control. As a control, a Bgh germling was transferred to a cell on which no germling was present. These results suggest that the existence of PGT is effective for induced accessibility of a host cell when penetrated by Bgh. However, it is unclear whether or not a PGT secretes some substance(s) which suppresses the resistance induction of a host cell.     

Catalog of Micro-Tom tomato responses to common fungal, bacterial, and viral pathogens

Lycopersicon esculentum cultivar Micro-Tom is a miniature tomato with many advantages for studies of the molecular biology and physiology of plants. To evaluate the suitability of Micro-Tom as a host plant for the study of pathogenesis, Micro-Tom plants were inoculated with 16 well-known fungal, bacterial, and viral pathogens of tomato. Athelia rolfsii, Botryotinia fuckeliana, Oidium sp., Phytophthora infestans, and Sclerotinia sclerotiorum caused typical symptoms and sporulated abundantly on Micro-Tom. Micro-Tom was resistant to Alternaria alternata, Corynespora cassiicola, and Fusarium oxysporum. When Micro-Tom was inoculated with 17 isolates of Ralstonia solanacearum, many isolates induced wilt symptoms. Agrobacterium tumefaciens also was pathogenic, causing crown galls on stem tissue after needle prick inoculation. In Micro-Tom sprayed with Pseudomonas syringae pv. tomato, P. s. pv. tabaci, or P. s. pv. glycinea, bacterial populations did not increase, and yellow lesions appeared only on leaves sprayed with P. s. pv. tomato. Tomato mosaic virus, Tomato aspermy virus, and Cucumber mosaic virus systemically infected Micro-Tom, which developed symptoms characteristic of other cultivars of tomato after infection with the respective virus. These results indicated that Micro-Tom was generally susceptible to most of the important tomato pathogens and developed typical symptoms, whereas certain pathogens were restricted by either hypersensitive resistance or nonhost resistance on Micro-Tom. Therefore, an assortment of Micro-Tom–pathogen systems should provide excellent models for studying the mechanism of susceptible and resistant interactions between plants and pathogens.