A Monitoring System for Green Fluorescence Protein Gene-transformed Fusarium oxysporum in Melon Seedlings

Using melon seedlings at the cotyledon stage and genetically marked fungi, a system for monitoring pathogenic and nonpathogenic Fusarium oxysporum was devised in the present study. Protoplasts were prepared from three formae speciales (melonis, radicis-lycopersici and fragariae )of F. oxysporum and transformed with a synthetic gene for green fluorescence protein. Transformants were primarily isolated in the presence of hygromycin B and then screened by the emission of bright green fluorescence. Roots of melon seedlings were inoculated with fluorescing microconidia of these fungi, and fungal infection behavior was traced. Using fluorescence microscopy, we directly observed not only the fungus at the root surface, but also the mycelia elongating in the trachea of roots. Both pathogenic and nonpathogenic fungi germinated and hyphae elongated superficially on the surface of root. Only pathogenic fungi caused root necrosis at the inoculation site. Hyphae grew within the stem to induce constriction or cracking of lower hypocotyls, then causing wilting of the seedlings. Infection behavior of genetically marked pathogenic and nonpathogenic F. oxysporum could be successfully monitored after inoculation of cotyledons of seedlings. Received 6 June 2001/ Accepted in revised form 3 August 2001     

Metabolic pathway of cyanidin 3-O-beta-D-glucopyranoside in rats

For better understanding of the physiological function of anthocyanins, the absorption and metabolism of cyanidin 3-O-beta-D-glucopyranoside (Cy3G), which is one of the major anthocyanins in colored food materials, were precisely investigated. Combining two modalities newly developed, that is, highly sensitive semi-micro-HPLC and vein cannulation, Cy3G and its four major metabolites (M1-M4) were detected in the blood plasma of rats after oral administration of Cy3G (100 mg/kg of body mass). The plasma concentration of Cy3G reached its maximum at 15 min after the ingestion. Metabolite 2 (M2) and metabolite 3 (M3) showed their maximum plasma levels at 15 and 30 min, respectively, whereas metabolite 1 (M1) and metabolite 4 (M4) showed their maximum levels at 60 and 120 min, respectively. The maximum plasma concentrations of the four metabolites were in the following order: M3 (21 nM) > M4 (20 nM) > M1 (8.5 nM) > M2 (5 nM). When Cy3G was directly injected into the neck vein, only M2 and M3 were detected in the plasma, indicating that both M1 and M4 were produced during absorption from the gastrointestinal tract. Tandem MS analysis of the metabolites showed that M2 and M3 were monomethylated Cy3G, while M1 and M4 were glucuronides of Cy and methylated Cy, respectively. M3 was assigned as peonidin 3-O-beta-D-glucopyranoside (Pn3G) from the comparison of the retention time of authentic Pn3G.     

Toll-like receptor 2 and 9 are expressed and functional in gut-associated lymphoid tissues of presuckling newborn swine

To clarify the crucial role of Toll-like receptor (TLR) 2 and TLR9 in immature gut-associated lymphoid tissues (GALT), we focused on the expression of TLR2 and TLR9 and the immune responses induced by their ligands in the GALT of presuckling newborn swine. Quantitative real-time PCR revealed that TLR2 and TLR9 mRNA were expressed at detectable levels in all tested tissues (heart, thymus, lung, spleen, liver, kidney, skeletal muscle, duodenum, jejunum, ileum, ileal Peyer patches (Pps), and mesenteric lymph nodes (MLN)). In particular, in immature intestinal tissues and GALT, TLR2 and TLR9 mRNA were expressed at higher levels in ileal Pps and MLN than in the duodenum, jejunum, and ileum. We confirmed that the TLR2 and TLR9 proteins were also highly expressed and that their ligands were preferentially recognized by TLR2- or TLR9-expressing cells in the MLN and ileal Pps. Zymosan, CpG2006, and lactic acid bacteria could promote mitogenesis and production of multiple cytokines by the MLN and ileal Pps. In addition, double immunostaining for cytokeratin 18 and either TLR2 or TLR9 revealed that both TLR2 and TLR9 are strongly expressed in the columnar membranous (M) cells. Interestingly, while the apical membrane of the columnar M cells strongly expressed TLR2 protein and preferentially recognized zymosan, both "TLR2 expression on the apical membrane" and "TLR2-mediated zymosan binding" were negligible in neighboring enterocytes. These results indicate that TLR2 and TLR9 allow MLN and ileal Pps to respond to a variety of bacterial components immediately after birth, thereby providing newborns with a host defense system.     

Deuterium isotope effects on the formation of mercapturic acids from lindane in rats

Metabolism experiments with rats showed that significant isotope effects ($\text{k}{}_{\mathrm{<mtext>H</mtext>}}\text{k}{}_{\mathrm{<mtext>D</mtext>}}\text{= 2.4}\text{to}\text{3.5}$) were associated with the in vivo formation of dichloro and trichlorophenylmercapturic acids from a 1:1 mixture of normal and hexadeuterated lindane. This is evidence that rate-determining dehydrogenation and dehydrochlorination, both of which proceed with significant isotope effects, are essential in the pathway of dichloro- and trichlorophenylmercapturic acid formation from lindane. No significant primary isotope effects were associated ($\text{k}{}_{\mathrm{<mtext>H</mtext>}}\text{k}{}_{\mathrm{<mtext>D</mtext>}}\text{= 1.31 ± 0.17}$) with the formation of monochlorophenylmercapturic acid. This suggests that the 1,2-dechlorination to tetrachlorocyclohexene followed by glutathione conjugation is the probable pathway that produces this metabolite from lindane.     

Benzpyrimoxan: Design,synthesis, and biological activity of a novel insecticide

Benzpyrimoxan (5-(1,3-dioxan-2-yl)-4-{[4-(trifluoromethyl)phenyl]methoxy}pyrimidine, NNI-1501) was discovered as a novel insecticide structurally characterized by a pyrimidine derivative substituted with 1,3-dioxanyl and 4-trifluoromethylbenzyloxy groups. The compound showed remarkable activity against nymphs of rice planthoppers, including strains resistant to existing insecticides. Furthermore, benzpyrimoxan had low adverse effects on pollinators and beneficial arthropods. Because of these features, benzpyrimoxan is expected to be a suitable part of an integrated pest management strategy. In this report, the history of the discovery to reach benzpyrimoxan and details of the structure–activity relationships are described.     

Fruit rot of Strawberry pear (pitaya) caused by <Emphasis Type="Italic">Bipolaris cactivora</Emphasis>

Strawberry pear (pitahaya, pitaya) [Hylocereus undatus (Haw.) Britt. and Rose] postharvest fruit rot was found at an agricultural products store in Itoman city, Okinawa Prefecture in 2006. The symptoms included depressed, water-soaked lesions with olive to black powdery spots coalescing into a soft rot. The causal fungus was identified as Bipolaris cactivora (Petrak) Alcorn. This is the first report of strawberry pear fruit rot caused by B. cactivora.     

Serum level of apoptosis inhibitor of macrophage in dogs with histiocytic sarcoma and its association with the disease

Histiocytic sarcoma (HS) is a rare neoplasm of macrophages or dendritic cells with a poor prognosis in dogs. As the apoptosis inhibitor of macrophage (AIM) is characteristically expressed in canine macrophages, we hypothesised that AIM is involved in the development or progression of HS in dogs. In this study, AIM expression in the tumour region and serum AIM levels in dogs with HS was assessed. Additionally, the effects of AIM overexpression on HS cell viability were investigated using a HS cell line that was selected from five validated HS cell lines. Immunohistochemistry showed that AIM expression was observed in the cytoplasm of the HS cells. CD36, a candidate AIM receptor, was also observed on the cell membrane of HS cells. When the serum AIM level was detected in 36 dogs with HS and 10 healthy dogs via western blot analysis, the AIM levels in the HS dogs were significantly higher than those in the controls. AIM mRNA expression in the 5 HS cell lines varied but was higher than that in the other tumour-derived lines. Among the five HS cell lines, DH82 originally had lower AIM and the highest CD36 expression. When AIM was overexpressed in DH82, therein cell growth speed and invasion, apoptosis inhibition and phagocytic activity were strongly upregulated. These data suggest that elevated intra-tumour expression of AIM could induce the progression of HS cells in dogs. Moreover, elevated serum AIM levels in dogs with HS could serve as a biomarker of HS.     

Complex vertebral malformation in a stillborn Holstein calf in Japan

A female stillborn Holstein calf with shortened cervical and thoracic regions, protrusion of the tongue, and bilateral symmetric flexural contraction of the anterior limbs was delivered on gestation day 281. Multiple hemivertebrae, fused and misshaped vertebrae, synostosis and scoliosis of cervical, thoracic and lumbar vertebral column were found in the affected calf by radiographic and computed tomographic (CT) analysis. Ten pairs of ribs were present and the sternum consisted of 9 sternebrae. Multiple morphologic abnormalities including fusion, malformation, and displacement, were found in the ribs and sternum. Cardiac anomalies, including atrial septal defect and hypertrophy of right ventricle, were observed. DNA-polymerase chain reaction (PCR) analysis demonstrated that amplified product from the liver DNA of the affected calf had identical pattern to that associated with complex vertebral malformation (CVM) of Holstein calves and that her dam was a heterozygous carrier of CVM. The affected calf was diagnosed as having CVM based on the DNA-PCR results and the characteristic findings, and was recorded as a first documentation of CVM confirmed in a Holstein calf in Japan.     

Examination of the failure behavior of wood with a short crack in the radial–longitudinal system by single-edge-notched bending test

The failure behavior of wood with a short crack was examined by conducting the single-edge-notched bending tests of a radial-longitudinal system on Agathis specimens. In the test, the mode I critical stress intensity factor was measured, and its validity was checked by the result from double cantilever beam testing method. The mode I critical stress intensity factor decreased when the crack length approached zero. With crack length correction, a constant critical stress intensity factor was obtained over a wide range of crack length including crack-free specimen.     

Stress hormone responses in ayu Plecoglossus altivelis in reaction to different catching methods: comparisons between electrofishing and cast netting

It is important to select the most appropriate method for fish sampling in streams and rivers; the sampling efficiency as well as any negative effect of the method of fish sampling on fishes such as stress, injury, and mortality must be considered. This study aimed to investigate stress hormone responses in ayu Plecoglossus altivelis in reaction to direct current (DC) electrofishing, alternating current (AC) electrofishing, and cast netting in artificial streams. The mortality, injury rate, and catch efficiency of these catching methods were also compared. It was found that immediately after capture, fish caught using a cast net exhibited significantly higher cortisol levels than did control fish that were caught rapidly by a hand net. Cortisol levels did not differ between electroshocked fish and the controls. Time required to catch 12 fish was shorter when DC was used (20 s) than when AC was used (45 s). The time required to catch fish greatly increased when a cast net was used (840 s). Cortisol levels in DC electroshocked and control treatment groups were at resting levels 24 and 48 h after capture. However, higher cortisol levels were found in fish captured using a cast net at both these time points. Cortisol levels in AC electroshocked fish returned to lower levels at 24 h, but increased again at 48 h. Furthermore, 48 h following capture, the fish caught by AC electrofishing exhibited higher mortality (7.5 %) than those captured by other methods (0 %). Spinal injury was also detected in one of the fish in this group. Considering these findings, DC electrofishing is the most effective and least damaging method for collecting P. altivelis in streams with respect to stress, physical damage, and efficiency.