Functional immobilization of plant receptor-like kinase onto microbeads towards receptor array construction and receptor-based ligand fishing

Despite the large number of receptor-like kinases (RLKs) in plants, few of their specific ligands are known. Ligand fishing is one of the most challenging post-genomic technologies. Here, we report a strategy for functional immobilization of plant RLKs on microbeads via covalent linkage. Because of the high density of immobilized RLKs, ligand-receptor interaction can be visualized at high sensitivity using fluorescent-labeled ligands under the confocal laser scanning microscope. Moreover, using a receptor-based affinity chromatography system with RLK microbeads, the ligand of the receptor was directly retrieved at high purity from complex natural samples. Our RLK microbeads and receptor-based ligand fishing approach is a feasible alternative to conventional forward genetics and bioassay-based biochemical purification for identification of novel ligand-receptor pairs in plants.     

Effect of norflurazon on resorcinolic lipid metabolism in rye seedlings

Norflurazon is a selective pyridazinone herbicide excessively employed in the control of many annual grasses and broad-leaved weeds. This chemical causes plant bleaching due to the inhibition of the carotenoid pigment biogenesis as well as induces irreparable changes to chloroplasts, which are considered the organelles where the biosynthesis of resorcinolic lipids takes place. Resorcinolic lipids, a group of phenolic compounds, constitute not only an essential part of the plant antifungal defense system, but also are an important component of the human cereal diet. The aim of this study was to investigate the effect of norflurazon on the biosynthesis of resorcinolic lipids in 5-day-old rye plants (Secale cereale L.) that were grown at three different temperatures under light or dark conditions. At all tested temperatures, norflurazon decreased the fresh biomass of light-grown rye seedlings and increased the weight of plants grown in darkness. Compared with respective controls, this herbicide caused an increase in total content of alkylresorcinols in both green and etiolated plants with the exception of dark-grown norflurazon-treated rye at 29 degrees C. The general level of saturated homologues was markedly decreased by norflurazon in all etiolated plants and in light-grown seedlings at 15 degrees C. Independent of thermal and light conditions, in all norflurazon-treated samples two alkylresorcinol derivatives predominated: 1,3-dihydroxy-5-n-heptadecylbenzene and 1,3-dihydroxy-5-n-nonadecylbenzene. Thus, our results suggest that norflurazon affected the metabolism of alkylresorcinols in rye seedlings and its action was dependent on external stimuli.     

ABCG2 is a high-capacity urate transporter and its genetic impairment increases serum uric acid levels in humans

The ATP-binding cassette, subfamily G, member 2 (ABCG2/BCRP) gene encodes a well-known transporter, which exports various substrates including nucleotide analogs such as 3'-azido-3'-deoxythymidine (AZT). ABCG2 is also located in a gout-susceptibility locus (MIM 138900) on chromosome 4q, and has recently been identified by genome-wide association studies to relate to serum uric acid (SUA) and gout. Becuase urate is structurally similar to nucleotide analogs, we hypothesized that ABCG2 might be a urate exporter. To demonstrate our hypothesis, transport assays were performed with membrane vesicles prepared from ABCG2-overexpressing cells. Transport of estrone-3-sulfate (ES), a typical substrate of ABCG2, is inhibited by urate as well as AZT and ES. ATP-dependent transport of urate was then detected in ABCG2-expressing vesicles but not in control vesicles. Kinetic analysis revealed that ABCG2 is a high-capacity urate transporter that maintained its function even under high-urate concentration. The calculated parameters of ABCG2-mediated transport of urate were a Km of 8.24 ± 1.44 mM and a Vmax of 6.96 ± 0.89 nmol/min per mg of protein. Moreover, the quantitative trait locus (QTL) analysis performed in 739 Japanese individuals revealed that a dysfunctional variant of ABCG2 increased SUA as the number of minor alleles of the variant increased (p = 6.60 × 10(-5)). Because ABCG2 is expressed on the apical membrane in several tissues, including kidney, intestine, and liver, these findings indicate that ABCG2, a high-capacity urate exporter, has a physiological role of urate homeostasis in the human body through both renal and extrarenal urate excretion.     

Tryptophan auxotroph mutants suppress the superroot2 phenotypes,modulating IAA biosynthesis in arabidopsis

Plant phytohormone, Indole-3-acetic acid (IAA ), is synthesized by tryptophan (trp) dependent and independent pathway. Here we report that tryptophan auxotroph mutants completely suppressed the abnormalities of auxin over production mutant, superroot2. SUR2 is considered to modulate Trp dependent pathway, resulting IAA accumulation in Arabidopsis. Tryptophan auxotroph mutants showed hyper-sensitivity to the auxin polar transport inhibitor, NPA, on the phenotype of reduced gravitropism. These results together with the results of histochemical analyses, tryptophan auxotroph mutants seem to have a complete defect in Trp dependent IAA biosynthesis pathway, and it is also suggested that the Trp dependent pathway is responsible for the normal root gravitropism.Key words: Auxin, Trp dependent pathway, trp mutants, sur2, arabidopsis     

Multiple translocation of the AVR-Pita effector gene among chromosomes of the rice blast fungus Magnaporthe oryzae and related species

Magnaporthe oryzae is the causal agent of rice blast disease, a devastating problem worldwide. This fungus has caused breakdown of resistance conferred by newly developed commercial cultivars. To address how the rice blast fungus adapts itself to new resistance genes so quickly, we examined chromosomal locations of AVR-Pita, a subtelomeric gene family corresponding to the Pita resistance gene, in various isolates of M. oryzae (including wheat and millet pathogens) and its related species. We found that AVR-Pita (AVR-Pita1 and AVR-Pita2) is highly variable in its genome location, occurring in chromosomes 1, 3, 4, 5, 6, 7, and supernumerary chromosomes, particularly in rice-infecting isolates. When expressed in M. oryzae, most of the AVR-Pita homologs could elicit Pita-mediated resistance, even those from non-rice isolates. AVR-Pita was flanked by a retrotransposon, which presumably contributed to its multiple translocation across the genome. On the other hand, family member AVR-Pita3, which lacks avirulence activity, was stably located on chromosome 7 in a vast majority of isolates. These results suggest that the diversification in genome location of AVR-Pita in the rice isolates is a consequence of recognition by Pita in rice. We propose a model that the multiple translocation of AVR-Pita may be associated with its frequent loss and recovery mediated by its transfer among individuals in asexual populations. This model implies that the high mobility of AVR-Pita is a key mechanism accounting for the rapid adaptation toward Pita. Dynamic adaptation of some fungal plant pathogens may be achieved by deletion and recovery of avirulence genes using a population as a unit of adaptation.     

High levels of human recombinant cyclooxygenase-1 expression in mammalian cells using a novel gene amplification method

We report the expression of a high level of human cyclooxygenase-1 (hCOX-1) in mammalian cells using a novel gene amplification method known as the IR/MAR gene amplification system. IR/MAR-plasmids contain a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) and amplify autonomously without a specific induction process. In this study, the IR/MAR-plasmid pΔBN.AR1 was cotransfected with pCAG-COX1, which expresses hCOX-1, into human HEK293T cells, and G418 and blasticidin S double-resistant cells were obtained in about 1month. Real-time PCR and Western blotting revealed that the expressions of hCOX-1 mRNA and protein in both polyclonal and monoclonal cells were remarkably higher than those in only pCAG-COX1-transfected control cells. Southern blotting demonstrated the amplification of the hCOX-1 gene, and the copy number of clone #43 obtained by the cotransfection of pΔBN.AR1 and pCAG-COX1 was more than 20 copies per cell, though that of clone #14 obtained without using the IR/MAR plasmid pΔBN.AR1 was only two copies. These results indicate that a high level of hCOX-1 expression was achieved as a result of hCOX-1 gene amplification. Furthermore, the crude extract from clone #43 showed a strong COX-1 activity, and the activity was inhibited by the representative COX-1 inhibitor indomethacin, with an IC(50) value of 36nM. These results demonstrate that the IR/MAR gene amplification system is a simple but useful method for generating highly productive mammalian cells.     

Relationship of number of remaining teeth to health-related quality of life in community-dwelling elderly

Objective: The aim of this study was to evaluate the relationship between number of remaining teeth and health‐related quality of life in community‐dwelling elderly. Subjects: A total of 207 participants who were community‐dwelling, 85 years of age. Data were from a population‐based study of age‐related general and oral health in Fukuoka Prefecture, Japan. Measurements: The Japanese version of the Short Form 36 Health Survey (SF‐36). Results: The mental component score for the participants, from the SF‐36, was higher than the Japanese national norm for those aged ≥70 years. There were no significant differences in the mean of any scores on the SF‐36 by having spouse, living with family, or education level. The mean of the SF‐36 scores of physical functioning (PF) and of the physical component scores were significantly higher in the 85‐year‐old participants with ≥20 teeth than in those with ≤19 teeth (p < 0.05 and p < 0.01 respectively). In addition, a significant difference (p < 0.05) was observed between the mean of participants with ≥20 teeth and those with ≤19 teeth after adjustment for region where the participant lived, activities of daily living (ADL), and sex. The PF (p < 0.001), role‐physical (p < 0.005), bodily pain (p < 0.001), vitality (p < 0.001), social functioning (p < 0.05), and physical component (p < 0.001) scores were significantly higher in participants with a good activities of daily living (ADL) assessment. However, ADL was not associated with the number of teeth. Conclusions: The findings of the present study indicated that 85‐year‐old participants with ≥20 teeth had better subjective physical health than those with ≤19 teeth.     

Responses of the silkworm tyramine receptor to 2-phenylethylamines and 5-phenyloxazoles

Tyramine (TA), a biogenic amine, attenuates intracellular cAMP production by acting on its receptor in insects. Several non-biogenic amines were examined for their actions on native and heterologously expressed silkworm TA receptors. 5-(4-Hydroxyphenyl)oxazole, which showed an attenuating effect on cAMP production in silkworm-head membranes, did not attenuate forskolin-stimulated cAMP production in HEK-293 cells expressing the silkworm TA receptor, although the compound bound to the cloned receptor. 2-Phenylethylamines (2-PEAs), which showed positive and negative effects on cAMP production in silkworm-head membranes, inhibited [3H]TA binding to the cloned TA receptor. 2-Chloro-2-(4-chlorophenyl)ethylamine was the most potent inhibitor of [3H]TA binding among the 2-PEAs tested, with an IC50 of 30.4 nM. This compound acted as an antagonist and abolished TA-attenuation of forskolin-stimulated cAMP production in the cloned TA receptor. The discrepancy in the effects of the non-biogenic amines on the native and cloned TA receptors remains to be further examined. A newly synthesized 2-PEA, 2-chloro-2-(4-hydroxyphenyl)ethylamine, attenuated forskolin-stimulated cAMP production in the cloned TA receptor, indicating that the para-hydroxy group is important for the agonist action.     

Techniques of SNP genotyping and remote sensing applied for elucidation of the contribution of polygene and environmental factors to inter-individual variation in skin pigmentation phenotype

We present a conceptual framework for applying techniques of SNP genotyping as a molecular biological approach and remote sensing as an ecological approach to elucidation of the contribution of polygene and environmental factors to inter-individual variation in skin pigmentation phenotype. Additionally, we discuss the obstacles that frustrate our efforts to identify how the human genome encodes the complex phenotype and suggest the use of computational methods designed for knowledge discovery within hereditary database.