In situ proteolysis for protein crystallization and structure determination

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.     

Transcriptional network analysis of the tryptophan-accumulating rice mutant during grain filling

In a previous study, we selected a high tryptophan (Trp)-accumulating rice (Oryza sativa L.) mutant line by in vitro mutagenesis using gamma rays. To obtain detailed information about the Trp biosynthetic pathway during the grain-filling in rice, we investigated the gene expression profiles in the wild-type (cv. Dongan) and the high-level Trp-accumulating mutant line (MRVII-33) at five different grain-filling stages using microarray analysis. The mutant line showed approximately 6.3-fold higher Trp content and 2.3-fold higher amino acids compared with the original cultivar at the final stage (stage V). The intensity of gene expression was analyzed and compared between the wild-type and mutant line at each of the five grain-filling stages using the Rice 4?×?44K oligo DNA microarray. Among the five stages, stage III showed the highest gene expression changes for both up- and down-regulated genes. Among the Trp biosynthesis-related genes, trpG showed high expression in the mutant line during stages I to IV and trpE showed higher at stage III. Gene clustering was performed based on the genes of KEGG's amino acid metabolism, and a total of 276 genes related to amino acid metabolism were placed into three clusters. The functional annotation enrichment analysis of the genes classified into the three clusters was also conducted using ClueGO. It was found that cluster 3 uniquely included biological processes related to aromatic amino acid metabolism. These results suggest that gene analysis based on microarray data is useful for elucidating the biological mechanisms of Trp accumulation in high Trp-accumulating mutants at each of the grain-filling stages.     

Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. vesicatoria

Biotic stress like pathogenic infection increases ethylene biosynthesis in plants and ethylene inhibitors are known to alleviate the severity of plant disease incidence. This study aimed to reduce the bacterial spot disease incidence in tomato plants caused by Xanthomonas campestris pv. vesicatoria (XCV) by modulating stress ethylene with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of Methylobacterium strains. Under greenhouse condition, Methylobacterium strains inoculated and pathogen challenged tomato plants had low ethylene emission compared to pathogen infected ones. ACC accumulation and ACC oxidase (ACO) activity with ACO related gene expression increased in XCV infected tomato plants over Methylobacterium strains inoculated plants. Among the Methylobacterium spp., CBMB12 resulted lowest ACO related gene expression (1.46 Normalized Fold Expression), whereas CBMB20 had high gene expression (3.42 Normalized Fold Expression) in pathogen challenged tomato. But a significant increase in ACO gene expression (7.09 Normalized Fold Expression) was observed in the bacterial pathogen infected plants. In contrast, Methylobacterium strains enhanced β-1,3-glucanase and phenylalanine ammonia-lyase (PAL) enzyme activities in pathogen challenged tomato plants. The respective increase in β-1,3-glucanase related gene expressions due to CBMB12, CBMB15, and CBMB20 strains were 66.3, 25.5 and 10.4% higher over pathogen infected plants. Similarly, PAL gene expression was high with 0.67 and 0.30 Normalized Fold Expression, in pathogen challenged tomato plants inoculated with CBMB12 and CBMB15 strains. The results suggest that ethylene is a crucial factor in bacterial spot disease incidence and that methylobacteria with ACC deaminase activity can reduce the disease severity with ultimate pathogenesis-related protein increase in tomato.     

Association of CXCL10 and CXCL13 levels with disease activity and cutaneous manifestation in active adult-onset Still’s disease

IntroductionC-X-C motif chemokine 10 (CXCL10) is produced in response to interferon-γ, and tumor necrosis factor-α (TNF-α) triggers the accumulation of activated lymphocytes. CXCL13 is constitutively expressed in secondary lymphoid tissues, and the expression is upregulated by TNF-α, via T cell stimulation. It appears that CXCL10 and CXCL13 could play a potential role in the pathogenesis of adult-onset Still’s disease (AOSD), therefore, we investigated the associations between CXCL10 and CXCL13 levels and clinical manifestations in patients with active AOSD.MethodsBlood samples were collected from 39 active AOSD patients, 32 rheumatoid arthritis (RA) patients and 40 healthy controls (HC). Of the AOSD patients, follow-up samples were collected from 15 9.6 ± 9.2 months later. Serum levels of CXCL10 and CXCL13 were determined using enzyme-linked immunosorbent assay. CXCL10, CXCL13, and C-X-C chemokine receptor type 3 (CXCR3) expression levels in biopsy specimens obtained from 26 AOSD patients with skin rashes were investigated via immunohistochemistry.ResultsThe CXCL10 levels in AOSD patients (1,031.3 ± 2,019.6 pg/mL) were higher than in RA (146.3 ± 91.4 pg/mL, p = 0.008) and HC (104.4 ± 47.9 pg/mL, p = 0.006). Also, the CXCL13 levels of AOSD patients (158.8 ± 151.2 pg/mL) were higher than those of RA (54.4 ± 61.1 pg/mL, p < 0.001) and HC (23.5 ± 18.1 pg/mL, p < 0.001). Serum CXCL10 levels correlated with ferritin and systemic scores. Serum CXCL13 levels correlated with those of hemoglobin, C-reactive protein, ferritin, and albumin, and systemic scores. In follow-up AOSD patients, the levels of CXCL10 and CXCL13 fell significantly (153.7 ± 130.1 pg/mL, p = 0.002, and 89.1 ± 117.4 pg/mL, p = 0.001, respectively). On immunohistochemistry, the percentages of inflammatory cells expressing CXCL10 ranged from 1 to 85 %, CXCL13 from 1 to 72 %, and CXCR3 from 2 to 65 %. The percentage of CXCL10-positive inflammatory cells was higher in skin biopsy samples exhibiting mucin deposition than in those that did not (p = 0.01). CXCL13 levels were correlated with those of CD4 and CD68.ConclusionsSerum CXCL10 and CXCL13 levels may serve as clinical markers for assessment of disease activity in AOSD. CXCL10/CXCR3 and CXCL13 may contribute to the inflammatory response, especially skin manifestations thereof, in AOSD.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0773-4) contains supplementary material, which is available to authorized users.     

Age- and Sex-Specific Relationships between Household Income,Education, and Diabetes Mellitus in Korean Adults: The Korea National Health and Nutrition Examination Survey, 2008-2010

Background

To investigate the effects of age and sex on the relationship between socioeconomic status (SES) and the prevalence and control status of diabetes mellitus (DM) in Korean adults.

Methods

Data came from 16,175 adults (6,951 men and 9,227 women) over the age of 30 who participated in the 2008-2010 Korea National Health and Nutrition Examination Survey. SES was measured by household income or education level. The adjusted odds ratios (ORs) and corresponding 95% confidence intervals (95% CI) for the prevalence or control status of diabetes were calculated using multiple logistic regression analyses across household income quartiles and education levels.

Results

The household income-DM and education level-DM relationships were significant in younger age groups for both men and women. The adjusted ORs and 95% CI for diabetes were 1.51 (0.97, 2.34) and 2.28 (1.29, 4.02) for the lowest vs. highest quartiles of household income and education level, respectively, in women younger than 65 years of age (both P for linear trend < 0.05 with Bonferroni adjustment). The adjusted OR and 95% CI for diabetes was 2.28 (1.53, 3.39) for the lowest vs. highest quartile of household income in men younger than 65 (P for linear trend < 0.05 with Bonferroni adjustment). However, in men and women older than 65, no associations were found between SES and the prevalence of DM. No significant association between SES and the status of glycemic control was detected.

Conclusions

We found age- and sex-specific differences in the relationship of household income and education with the prevalence of DM in Korea. DM preventive care is needed for groups with a low SES, particularly in young or middle-aged populations.