Genome-wide expression analysis of a rice mutant line under salt stress.

Salinity is a major environmental stress to plants. In this study, the ability of plants to tolerate salt was investigated by studying growth, physiological characteristics, and expression levels of genes related to the salt-stress response in the salt-tolerant rice mutant (Till-II-877), which was derived from γ-ray irradiation. Compared to plants grown under normal conditions, the height and root length of wild type (WT) were reduced by approximately 40 and 29% following exposure to salt stress for 3 weeks, whereas Till-II-877 line showed 29 and 23% reductions in plant height and root length, respectively. No significant changes were observed in total chlorophyll content, and the malondialdehyde content of the mutant increased less than that of the WT under salt treatment. Gene expression was compared between the WT and mutant lines using microarray analysis. An unbiased analysis of the gene expression datasets allowed us to identify the pathways involved in salt-stress responses. Among the most significantly affected pathways, changes in gene expression were observed in α-linolenic acid and linoleic acid metabolism (in lipid metabolism), fructose and mannose metabolism and glycolysis-gluconeogenesis (in carbohydrate metabolism), cysteine and methionine metabolism (in amino acid metabolism), and carbon fixation (in the energy metabolism of photosynthetic organisms) under salt stress. These results show that the differential response of plants subjected to salt stress was due to changes in multiple metabolic pathways. These findings increase our understanding of the effects of salt stress in rice and may aid in the development of salt-tolerant rice cultivars.

Development of a transposon-based marker system for mutation breeding in sorghum (Sorghum bicolor L.).

Under certain circumstances, transposable elements (TE) can create or reverse mutations and alter the genome size of a cell. Sorghum (Sorghum bicolor L.) is promising for plant transposon tagging due to its small genome size and its low content of repetitive DNA. We developed a marker system based on targeted region amplification polymorphisms (TE-TRAP) that uses the terminal inverted repeats (TIRs) of transposons. A total of 3816 class 2 transposons belonging to the PIF/Harbinger family were identified from the whole sorghum genome that produced five primers, including eight types of TIRs. To define the applicability and utilization of TE-TRAP, we used 21 individuals that had been bred after ɤ-ray irradiation. In total, 31 TE-TRAP, 16 TD, and 21 AFLP primer combinations generated 1133, 223, and 555 amplicons, respectively. The percent polymorphic marker was 62.8, 51.1, and 59.3% for the TE-TRAP, TD, and AFLP markers, respectively. Phylogenetic and principal component analyses revealed that TE-TRAP divided the 21 individuals into three groups. Analysis of molecular variance suggested that TE-TRAP had a higher level of genetic diversity than the other two marker systems. After verifying the efficiency of TE-TRAP, 189 sorghum individuals were used to investigate the associations between the markers and the ɤ-ray doses. Two significant associations were found among the polymorphic markers. This TE-based method provides a useful marker resource for mutation breeding research.

Relationship of maternal grain intake and serum triglyceride levels with infant birth weight: Mothers and Children's Environmental Health (MOCEH) study.

BACKGROUND/OBJECTIVES: Maternal serum triglyceride (TG) level is known to be associated with neonatal birth weight. Although Koreans traditionally consume relatively high amounts of grain and grain products, mainly in the form of white rice, and the consumption is positively associated with serum TG levels, no study has investigated the relationship between dietary grain intake, serum TG levels and neonatal birth weight in pregnant women. This study was conducted to identify the association between infant birth weight and maternal intake of grain, as well as serum TG levels. SUBJECTS/METHODS: Subjects were 1011 pregnant women at 12-28 weeks' gestational age and their offspring. Maternal serum TG levels, dietary intake and infant birth weight were measured. RESULTS: Serum TG levels were positively related to neonatal birth weight both at mid-pregnancy (P=0.0015) and at late pregnancy (P<0.0001). Such an association only existed in subjects with the highest tertile of grain intake at mid-pregnancy (P=0.0055) but was observed in all tertiles at late pregnancy (1st P=0.0186, 2nd P=0.0146, 3rd P=0.0099). CONCLUSIONS: The relationship between maternal TG levels and infant birth weight may depend on dietary grain intake and stages of pregnancy in Korean pregnant women.

An important role of tumor necrosis factor receptor-2 on natural killer T cells on the development of dsRNA-enhanced Th2 cell response to inhaled allergens.

BACKGROUND: Recent evidence indicates that TNF-α is a key mediator of the development of dsRNA-enhanced Th2 cell response to inhaled allergens. Natural killer T (NKT) cells may be a candidate source of Th2-polarizing cytokines. OBJECTIVE: The objective of this study was to evaluate the role of lung NKT cells on the development of TNF-α-mediated Th2 cell response. METHODS: A virus-associated asthma mouse model was generated by the administration of ovalbumin (OVA, 75 µg) and poly[I:C] (0.1 µg). Role of NKT and type I NKT cells was evaluated using CD1d- and Jα18-deficient mice. TNF-α receptors (TNFRs) were antagonized by using TNFR blocking peptides. RESULTS: The number of infiltrated NKT cells was increased in a virus-associated asthma mouse model. Increase in Th2 and Th17 cytokine levels in wild-type mice were abolished in both CD1d- and Jα18-deficient mice. In vitro co-culture experiments with alveolar macrophages and NKT cells showed that TNF-α produced by macrophages in the presence of poly[I:C] acts on NKT cells, inducing production of Th2-polarizing cytokines. Moreover, the induction of Th2-polarizing cytokines by poly[I:C] or recombinant TNF-α was impaired in both CD1d- and Jα18-deficient mice and that the above effect was reversed by a TNF-α receptor-2 (TNFR2) blocking peptide, but not by a TNFR1 blocker. CONCLUSIONS: These findings suggest that NKT cells play a key role in the development of Th2 cell response to inhaled allergens and that TNF-α produced by alveolar macrophages induces Th2 cell response, via TNFR2 on NKT cells.

ADP-ribosylation factors 1 and 6 regulate Wnt/ß-catenin signaling via control of LRP6 phosphorylation.

It has been shown that inhibition of GTPase-activating protein of ADP-ribosylation factor (Arf), ArfGAP, with a small molecule (QS11) results in synergistic activation of Wnt/ß-catenin signaling. However, the role of Arf in Wnt/ß-catenin signaling has not yet been elucidated. Here, we show that activation of Arf is essential for Wnt/ß-catenin signaling. The level of the active form of Arf (Arf-GTP) transiently increased in the presence of Wnt, and this induction event was abrogated by blocking the interaction between Wnt and Frizzled (Fzd). In addition, knockdown of Fzds, Dvls or LRP6 blocked the Wnt-mediated activation of Arf. Consistently, depletion of Arf led to inhibition of Wnt-mediated membrane PtdIns (4,5)P2 (phosphatidylinositol 4, 5-bisphosphate) synthesis and LRP6 phosphorylation. Overall, our data suggest that transient activation of Arf modulates LRP6 phosphorylation for the transduction of Wnt/ß-catenin signaling.

Hair greying is associated with active hair growth.

BACKGROUND: Hair greying is an obvious sign of ageing in humans. White (nonpigmented) hair is thicker than black (pigmented) hair. The growth rate of white hair is also significantly higher than that of black hair. However, the mechanism underlying this is largely unknown. OBJECTIVES: To examine the association between hair greying and hair growth patterns by evaluating expression of the genes or proteins related to hair growth in white and black hairs. METHODS: Morphological characteristics were observed in eyebrow and scalp hairs. The differential expression of genes was analysed in black and white hairs from human scalp by a microarray analysis. Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry for genes and proteins related to hair growth were performed in black and white hairs. RESULTS: Keratin and keratin-associated protein (KRTAP) genes in white hair were upregulated at least two-fold in comparison with black hair in a microarray analysis. Upregulation of selected keratin genes and KRTAP4 isoform genes in white hair was validated by RT-PCR. Immunoreactivity for KRT6, KRT14/16 and KRT25 was increased in the hair follicle of white hair compared with black hair. Gene expression of fibroblast growth factor 5 (FGF5) was downregulated in white hair compared with black hair. However, gene expression of FGF7 was upregulated in white hair compared with black hair. CONCLUSIONS: Expression of genes and proteins associated with active hair growth is upregulated in white (nonpigmented) hair compared with black (pigmented) hair. These results suggest that hair greying is associated with active hair growth.

Callus formation is associated with hyperproliferation and incomplete differentiation of keratinocytes, and increased expression of adhesion molecules.

BACKGROUND: A callus is a local thickening of skin, characterized by accelerated keratinization and a reduced rate of desquamation. However, the mechanism of callus formation is not fully understood. OBJECTIVES: To evaluate the expression patterns, in callused skin, of genes that are implicated in keratinization and adhesion/desquamation. METHODS: Samples of skin from the dorsum of the foot (DF), centre of the plantar arch (CP) and anterior aspect of the heel (AH) were obtained from fresh cadavers, and protein and gene expression were determined by immunohistochemistry and reverse transcription-polymerase chain reaction, respectively. RESULTS: The stratum corneum in the DF showed a splitting phenotype by conventional haematoxylin and eosin staining, while the stratum corneum was normal in the AH. Cells of the stratum corneum in the AH were nonsquamous. Expression of cornification-related molecules including involucrin, filaggrin, caspase 14 and calcium-sensing receptor was higher in the AH. Similarly, expression of adhesive proteins such as corneodesmosin, desmoglein 1 and desmocollin 1 was increased in the AH. However, protease-activated receptor 2 expression was reduced in the stratum granulosum in the AH. The number of proliferating cells in the stratum basale was significantly increased in the AH, compared with the DF and CP. CONCLUSIONS: Our data suggest that calluses form as a result of hyperproliferation and incomplete differentiation of epidermal keratinocytes, and increased expression of adhesion molecules.

The complete genome sequence of freesia mosaic virus and its relationship to other potyviruses.

We have completed the genomic sequence of a potyvirus, freesia mosaic virus (FreMV), and compared it to those of other known potyviruses. The full-length genome sequence of FreMV consists of 9,489 nucleotides. The large protein contains 3,077 amino acids, with an AUG start codon and UAA stop codon, containing one open reading frame typical of a potyvirus polyprotein. The polyprotein of FreMV-Kr gives rise to eleven proteins (P1, HC-pro, P3, PIPO, 6K1, CI, 6K2, VPg, NIa, NIb and CP), and putative cleavage sites of each protein were identified by sequence comparison to those of other known potyviruses. Phylogenetic analysis of the polyprotein revealed that FreMV-Kr was most closely related to PeMoV and was related to BtMV, BaRMV and PeLMV, which belong to the BCMV subgroup. This is the first information on the complete genome structure of FreMV, and the sequence information clearly supports the status of FreMV as a member of a distinct species in the genus Potyvirus.

Placenta hominis protects osteoporosis in ovariectomized rats.

In China, Japan, and Korea, placenta hominis extracts (PHEs) are used clinically for the treatment of osteoporosis. The anti-osteoporotic effect of PHEs was studied. The trabecular bone area and thickness in OVX rats decreased by 50% from those in sham-operated rats; these decreases were completely inhibited by administration of PHEs for 7 weeks. Osteoclast numbers and the osteoblast surface were enhanced in OVX rats, but PHEs had no effect on these phenomena. Serum phosphorus and alkaline phosphatase in OVX rats increased compared to those in sham-operated rats, but the increases were not affected by the administration of PHEs. Thyroxine (T4) level was stimulated in OVX rats. The extracts inhibited the T4 level in the OVX rats. These results strongly suggest that PHEs be effective in preventing the development of bone loss induced by OVX in rats.

p38 MAPK and NF-kappaB on IL-6 release in human gingival fibroblasts.

The induction of interleukin-6 (IL-6) using a proinflammatory cytokine (IL-1beta) was studied in human gingival fibroblasts (HGFs) in relation to p38 MAPK and NF-kappaB transcription factor. When added to HGFs, IL-1beta had a stimulatory effect on the production of IL-6, and this effect was significantly reduced by SB203580, a specific p38 MAPK inhibitor. In addition, the stimulation of IL-6 release also was reduced by the addition of pyrrolidine dithiocarbamate or NF-kappaB SN50, which has been reported as potent NF-kappaB inhibitor. Both the NF-kappaB inhibitors in the presence of SB203580 had more inhibitory effect on IL-6 release. IL-13 stimulated NF-kappaB binding affinity as well as p38 MAP kinase activation, leading to the release of IL-6. However, a specific inhibitor of p38 MAPK, SB203580, had no effect on the NF-kappaB activation, and both the NF-kappaB inhibitors failed to reduce the p38 MAPK activation in the IL-1beta-stimulated HGFs. These results strongly suggest that both p38 MAPK and NF-kappaB are required in IL-1beta-induced IL-6 synthesis and that these two IL-1beta-activated pathways can be primarily dissociated.

Purification and biochemical properties of an alkaline pullulanase from alkalophilic Bacillus sp. S-1.

A novel extracellular pullulanase (PUL-E, pullulan 6-glucanohydrolase, EC 3.2.1.41) has been purified from the alkalophilic Bacillus sp. S-1. The purified enzyme had a molecular mass of about 140 kDa on denaturated and natural conditions. The pI was 5.5. The pullulanase, when resolved by SDS-PAGE, was negative for Schiff staining, suggesting that the enzyme is not a glycoprotein. The N-terminal amino acid sequence of the enzyme was Phe-Leu-Asn-Met-Ser-(Trp-Phe). The enzyme displayed a temperature optimum of around 60 degrees C and a pH optimum of around pH 9.0. The enzyme was stable to incubation from pH 4.0 to pH 11.0 at 4 degrees C for 24 h. The presence of pullulan protected the enzyme from heat inactivation, the extent depending upon the substrate concentration. The activity of the enzyme was stimulated by Mn2+ ions. Ca2+ ions and EDTA did not inhibit the enzyme activity. The enzyme hydrolyzed the alpha-1,6-linkages of amylopectin, glycogens, alpha,beta-limited dextrin, and pullulan. The enzyme had an apparent Km of 7.92 mg/ml for pullulan, a Km of 1.63 mg/ml for amylopectin, and a Km of 3.1 mg/ml for alpha,beta-limited dextrin, when measured at pH 9.0 and 50 degrees C. The enzyme caused the complete hydrolysis of pullulan to maltotriose. The activity was not inhibited by alpha, beta, or gamma-cyclodextrins. The western blotting analysis with mouse anti-serum against PUL-E showed that PUL-E is produced as a single enzyme form during bacterial cultivation.