A plastidic glucose-6-phosphate dehydrogenase is responsible for hypersensitive response cell death and reactive oxygen species production

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in the supply of reduced nicotine amide cofactors for resistance to biotic and abiotic stresses. Here, we show participation of the plastidic P2 isoform of G6PDH in plant immunity. A cytosolic isoform (NbG6PDH-Cyto) and two plastidic isoforms (NbG6PDH-P1 and NbG6PDH-P2) cloned from Nicotiana benthamiana were localized in cytosol and chloroplasts, respectively. Hypersensitive response (HR) cell death and NADPH oxidase (RBOH; respiratory burst oxidase homolog)-dependent reactive oxygen species (ROS) production after recognition of INF1 elicitin, secreted by oomycete Phytophthora infestans, decreased in NbG6PDH-P2-silenced plants, but not in NbG6PDH-Cyto- and NbG6PDH-P1-silenced plants. Silencing of the cytosolic NAD kinase NbNADK1, which phosphorylates NADH to form NADPH, compromised HR cell death and ROS production, and concomitant silencing with NbG6PDH-P2 reduced HR cell death and ROS to levels near those in NbG6PDH-P2-silenced plants. Similarly, silencing NbG6PDH-P2 and NbNADK1 resulted in high susceptibility to P. infestans. These results suggest that NADPH produced by the P2 isoform of G6PDH in chloroplasts is responsible for HR cell death and ROS production mediated by RBOH and that NbNADK1 is involved in this pathway.     

High mobility group box 1 (HMGB1) protein is present in the cerebrospinal fluid of dogs with encephalitis

Many cases of encephalitic disease with unknown etiologies have been reported in specific breeds of small dogs. High mobility group box 1 (HMGB1) in neuronal cells was recently found to be a novel cytokine-like mediator that is a marker of neuronal necrosis and inflammation. The aim of this study was to determine whether HMGB1 levels are elevated in the cerebrospinal fluid (CSF) of dogs suspected of having encephalitis. CSF was obtained from 31 dogs that were diagnosed with an encephalitic disease by clinical examinations and magnetic resonance image (MRI) scanning. The CSF samples were analyzed via western blotting (WB) and an enzyme-linked immunosorbent assay (ELISA) with a polyclonal antibody against HMGB1. The mean HMGB1 concentration was significantly higher in the encephalitic dogs than that in the healthy controls. The concentrations of HMGB1 were correlated with the cell counts and total protein concentrations, which are known CSF indicators of the neuronal inflammation associated with encephalitis. These results suggest that HMGB1 protein in CSF confirms the presence of necrosis and inflammation in most cases of canine encephalitis and that HMGB1 will be a new indicator of encephalitis.     

Active expression of matrix metalloproteinase-13 mRNA in the granulation tissue of equine superficial digital flexor tendinitis

The DNA microarray analysis for matrix metalloproteinase (MMP)-related mRNA expression in equine superficial digital flexor tendinitis indicated that mRNA level of MMP-13 was apparently up-regulated in the tendinitis as compared to normal tendon. In situ hybridization also revealed that fibroblastic cells proliferated in the granulation tissue generated in the tendinitis actively expressed MMP-13 mRNA. On the other hand, in normal tendon, a few fibroblastic cells and vascular components lied in the endotenon barely expressed its mRNA, but other cellular components in the tendon bundle were not positively hybridized. As mentioned above, MMP-13 but not other collagenases or gelatinases, may play an important role in tendon injuries in the racehorses.     

Improvement of the material property of shark type I collagen by composing with pig type I collagen

Fibril reconstruction process, that is, the nucleation and growth of mixed type I collagen fibril of shark and pig, progressed faster than that of the individual collagen species of shark or pig. The reconstructed mixed collagen fibril had a greater resistance to return to the solution or to melt into gelatin in comparison with the counterpart consisting solely of shark collagen. The denaturation temperature of the mixed collagen gel was about 10 degrees C higher than that of shark, and about 5 degrees C lower than that of pig. By scanning electron microscopy, the diameter of mixed collagen fibril showed an intermediate range between shark and pig collagen fibril. The breaking strength of the mixed collagen gel was tougher than that of pig, but weaker than that of shark. Other physicochemical properties of the mixed type I collagen gel were observed to be at intermediate positions between those of shark and pig type I collagen gels.     

Characterization of local rhizobia in Thailand and distribution of malic enzymes

TWenty-six isolates were obtained from nodules of various legume plants (Glycine max, Vigna sinensis, Arachis hypogaea, Desmanthus virgatus, Acacia mangium, Centrosema pascuorum, Pterocarpus indicus, Xylia xylocarpa, and Sesbania rostrata) in Thailand. After confirming their nodulation and nitrogen-fixing abilities, they were identified by 16S rRNA gene analysis as Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium leguminosarum, Rhizobium gallicum, and Rhizobium galegae. Using these local isolates, the distribution of the activities of both NAD⁺-dependent (DME: EC 1.1.1.39) and NADP⁺-dependent (TME: EC 1.1.1.40) malic enzymes was surveyed. The malic enzyme activities were present in all the isolated rhizobia and in other 17 local Bradyrhizobium strains in Thailand. In almost all the rhizobia, the DME activity predominated whereas the TME activity predominated only in the Rhizobium gallicum strains that were major symbionts of Sesbania rostrata. Southern hybridization analysis was performed to survey the distribution of the malic enzyme genes among the local rhizobia, which are similar to those of B. japonicum. DNA probes (ME1 for DME and ME2 for TME) were prepared by polymerase chain reaction (PCR) using degenerated primers from conserved regions of the protein sequences of bacterial malic enzymes. Southern blot analysis with ME1 as a probe showed a single band in about half of the isolates, especially in B. japonicum and R. leguminosarum strains, suggesting the wide distribution of such DME genes among local rhizobia. In contrast, Southern blot analysis with ME2 as a probe detected a single band only in five B. japonicum strains, suggesting that the TME genes, which are similar to those of B. japonicum, would be unique in a group of B. japonicum.     

Estimation of buffering capacity of mangrove soils by using hydrophobic organic compounds,atrazine and linuron

Samples of two soils and two sediments collected at sites originating from mangrove forests in Thailand, were examined in terms of buffering capacity to organic compounds. Atrazine and linuron were used as representative hydrophobic organic compounds for estimating the buffering capacity by observing their adsorptive and desorptive behavior. The buffering capacity could be represented by the distribution of the adsorption ratio (AR) and desorption ratio (DR) as follows: AR (%) = (amount of herbicide adsorbed per unit weight of soil)/(initial amount of herbicide) x 100, and DR (%) = (amount of herbicide desorbed per unit weight of soil after herbicide desorption experiments) / (initial amount of adsorbed herbicide on soil) x 100. The soil under mangrove forests displayed a larger buffering capacity to atrazine and linuron. Compared with 42 soils from Japan, in terms of the adsorption proparty of atrazine and linuron, the mangrove soil ranked in a higher category on the classification of the Japanese soils. Thus, the importance of maintaining or recovering the mangrove forests to promote environmental conservation was emphasized.     

Carotenoid Profile of Edible Japanese Seaweeds: An Improved HPLC Method for Separation of Major Carotenoids

An improved multi-step gradient reverse phase high performance liquid chromatography (HPLC) method to simultaneously separate major carotenoids from natural as well as food samples was developed. Quantitative profiling of carotenoid compounds was carried out on three edible brown seaweeds (Sargassum horneri, Cystoseira hakodatensis, and Undaria pinnatifida) and three red seaweeds (Gracilaria vermiculophylla, Grateloupia asiatica, and Grateloupia livida). Fucoxanthin (Fx) was detected in all the brown seaweeds with quantities (mg g^?1 dry weight [dwt]) ranging from 1.3 ± 0.3 in C. hakodatensis to 2.4 ± 0.1 in S. horneri. U. pinnatifida, commonly known as wakame, had a Fx content of 2.3 ± 0.1 mg g^?1 dwt. In the case of red seaweeds, zeaxanthin (Zx) was the major carotenoid, and G. vermiculophylla had the highest Zx content (80.2 μg g^?1 dwt) among the red seaweeds apart from small amounts of Fx (9.1 μg g^?1 dwt). Similarly, the other two species of red seaweeds, G. asiatica and G. livida, contained (μg g^?1 dwt) lutein (Lut), Fx, and Zx as the major carotenoids (G. asiatica: Lut 10.4, Fx 1.5, Zx 1.1; G. livida: Lut 9.3, Fx 3.5, Zx 1.0). The results suggest the usefulness of edible varieties (barring wakame) of seaweeds as dietary sources of carotenoids.     

Determination of periods of sensitivity to low-salinity and low-temperature conditions during the early development of cultured Japanese eel Anguilla japonica larvae with respect to the rate of morphological deformity at completion of yolk resorption

The present study analyzed the rate of occurrence of deformities at completion of yolk resorption after Japanese eel larvae were exposed to low-salinity (29 psu) or low-water-temperature (20 °C) conditions (after they had been reared under suitable conditions: 34 psu, 25 °C) at various points between 0 and 8 days post-fertilization (dpf) in order to determine the period of sensitivity of the larvae to low-salinity or low-temperature conditions. No significant differences were observed between the rate of deformed larvae obtained in the groups reared under suitable conditions and the corresponding rates in groups that were exposed to low-salinity or low-temperature conditions at 4 dpf or later, but the frequency of normal larvae showed a downward trend in experimental groups that were subjected to low-salinity or low-temperature conditions at 3 dpf or earlier. Occurrence rates of pericardial edema were significantly higher in experimental groups that were exposed to low-salinity or low-temperature conditions at 2 dpf or earlier. Therefore, it was concluded that the rearing conditions for Japanese eel larvae should be maintained at 34 psu salinity and 25 °C until at least 4 dpf in order to reduce the occurrence of larval deformity at completion of yolk resorption.