Genome-Wide Gene Expression Profiling Revealed a Critical Role for GATA3 in the Maintenance of the Th2 Cell Identity

Functionally polarized CD4+ T helper (Th) cells such as Th1, Th2 and Th17 cells are central to the regulation of acquired immunity. However, the molecular mechanisms governing the maintenance of the polarized functions of Th cells remain unclear. GATA3, a master regulator of Th2 cell differentiation, initiates the expressions of Th2 cytokine genes and other Th2-specific genes. GATA3 also plays important roles in maintaining Th2 cell function and in continuous chromatin remodeling of Th2 cytokine gene loci. However, it is unclear whether continuous expression of GATA3 is required to maintain the expression of various other Th2-specific genes. In this report, genome-wide DNA gene expression profiling revealed that GATA3 expression is critical for the expression of a certain set of Th2-specific genes. We demonstrated that GATA3 dependency is reduced for some Th2-specific genes in fully developed Th2 cells compared to that observed in effector Th2 cells, whereas it is unchanged for other genes. Moreover, effects of a loss of GATA3 expression in Th2 cells on the expression of cytokine and cytokine receptor genes were examined in detail. A critical role of GATA3 in the regulation of Th2-specific gene expression is confirmed in in vivo generated antigen-specific memory Th2 cells. Therefore, GATA3 is required for the continuous expression of the majority of Th2-specific genes involved in maintaining the Th2 cell identity.     

Risk Factors and Indices of Osteomyelitis of the Jaw in Osteoporosis Patients: Results from a Hospital-Based Cohort Study in Japan

Background

Several studies have reported osteomyelitis of the jaw (OMJ) as a side effect of bisphosphonates (BPs), and the risk of oral BPs has been recently clarified. However, other systemic risk factors of OMJ remain unclear. Importantly, the possibility of risk classification based on the clinical characteristics of patients has not been explored. Here, we clarified risk factors of OMJ and evaluate the predictive accuracy of risk indices in osteoporosis patients.

Methods

We performed sub-analysis using a database developed for a retrospective cohort study in patients taking medications for osteoporosis at Kyoto University Hospital. Risk indices for OMJ were constructed using logistic regression analysis, and odds ratios (OR) for OMJ cases and 95% confidence intervals (CI) were estimated. Potential risk factors included in the statistical analysis were age; sex; diabetes; use of oral BPs, corticosteroids, cancer chemotherapy, antirheumatic drugs, and biologic agents; and their interactions. Risk indices were calculated by the sum of potential risk factors of an individual patient multiplied by the regression coefficients. The discriminatory power of the risk indices was assessed by receiver operating characteristic (ROC) analysis.

Results

In analysis of all patients, oral BPs (OR: 4.98, 95% CIs: 1.94-12.75), age (OR: 1.28, 95% CI: 1.06-1.60) and sex-chemotherapy interaction (OR: 11.70, 95% CI: 1.46-93.64) were significant risk factors of OMJ. Areas under the ROC curves of these risk indices provided moderate sensitivity or specificity regardless of group (0.683 to 0.718).

Conclusions

Our data suggest that oral BP use, age, and sex-chemotherapy are predictors of OMJ in osteoporosis patients. The risk indices are moderately high, and allow the prediction of OMJ incidence.     

Rapid Detection of SNP (c.309T>G) in the MDM2 Gene by the Duplex SmartAmp Method

Background

Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP) c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans.

Methodology

In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named “Duplex SmartAmp,” which enabled us to simultaneously detect both 309T and 309G alleles in one tube. To develop this new method, we introduced new primers i.e., nBP and oBPs, as well as two different fluorescent dyes that separately detect those genetic polymorphisms.

Results and Conclusions

By the Duplex SmartAmp method, the genetic polymorphisms of the MDM2 gene were detected directly from a small amount of genomic DNA or blood samples. We used 96 genomic DNA and 24 blood samples to validate the Duplex SmartAmp by comparison with results of the conventional PCR-RFLP method; consequently, the Duplex SmartAmp results agreed totally with those of the PCR-RFLP method. Thus, the new SNP detection method is considered useful for detecting the SNP c.309T>G in the MDM2 gene so as to judge cancer susceptibility against some cellular stress in the clinical setting, and also to handle a large number of samples and enable rapid clinical diagnosis.     

Mastication and Risk for Diabetes in a Japanese Population: A Cross-Sectional Study

Background

Associations between mastication and insufficient nutrient intake, obesity, and glucose metabolism have been shown in previous studies. However, the association between mastication and diabetes has not been clarified. Our objective was to examine the association between mastication, namely masticatory performance or rate of eating, and diabetes in a population-based cohort.

Methods

We conducted a cross-sectional study of the association between mastication and diabetes in the Nagahama Prospective Cohort Study, an ongoing study which recruits citizens of Nagahama City in Shiga Prefecture, central Japan. 2,283 male and 4,544 female residents aged 40–74 years were enrolled from July 2009 to November 2010. Masticatory performance was evaluated by spectrophotometric measurement of color changes after masticating color-changeable chewing gum. Categorical rate of eating (fast, intermediate or slow) was self-assessed using a questionnaire.

Results

177 males (7.7%) and 112 (2.4%) females were diagnosed with diabetes. We divided participants into four groups by quartile of masticatory performance, namely Q1 (lowest), 2, and 3 and 4 (highest). Compared to the lowest performance group, the multivariable adjusted odds ratio (OR) of diabetes was 0.91 (95% confidence interval (CI), 0.58–1.4) in Q2, 0.77 (95% CI, 0.48–1.2) in Q3, and 0.53 (95% CI, 0.31–0.90) in the highest group in males, and 1.2 (95% CI, 0.73–2.0), 0.95 (95% CI, 0.54–1.6) and 0.56 (95% CI, 0.30–1.0) in females. We also estimated ORs of diabetes by rate of eating. Compared to the fast eating group, ORs in males were 0.87 (95% CI, 0.61–1.2) in the intermediate group and 0.38 (95% CI, 0.16–0.91) in the slow group, and ORs in females were 0.92 (95% CI, 0.59–1.4) and 1.5 (95% CI, 0.73–3.0).

Conclusions

These findings support the hypothesis that higher masticatory performance and slow eating prevent the occurrence of diabetes.     

Isolation and Identification of 3-(2-Furoyl)alanine and l-Pipecolic Acid from Green Gram Seeds

4-Hydroxy-3,7-dimethyl-2,6-octadienal (5-Hydroxycitral) (2) was prepared from 3,7-dimethyl-2,6-octatrienal (citral) (1) via bromoaldehyde (4) and acetoxyaldehyde (6). 3,7-Dimethyl-2,4,6-octadienal (7) was also obtained as a byproduct. 4-Hydroxy-3,7-dimethyl-2,6-octadienal (2) showed similar growth inhibition activity to that of 1 against Sarcoma 180.     

Generation of (2-Nitroethyl)benzene and related benzenoids from L-Phenylalanine; flower scents of the Japanese Loquat Eriobotrya japonica [Rosales: Rosaceae]

ABSTRACT

(2-Nitroethyl)benzene, methyl 4-methoxybenzoate and 4-methoxybenzaldehyde have been known as major scent components in flowers of the Japanese loquat Eriobotrya japonica [Rosales: rosaceae], together with 13 related benzenoids, including Z- and E-2-phenylacetaldoxime and benzyl alcohol. The scents air-trapped from a flowering panicle during 24 h incubation with d₈-L-phenylalanine were composed of 15 deuterium labeled compounds {d₆-styrene, d₅-benzaldehyde, d₇-2-phenylacetaldehyde, methyl d₅-benzoate, d_{7 ?}2-phenylethanol, d₇-2-phenylacetonitrile, d₄-1,4-dimethoxybenzene, d₇-Z-2-phenylacetaldoxime, d₄-4-methoxybenzaldehyde, d₇-E-2-phenylacetaldoxime, d₄-4-methoxybenzyl alcohol, d₇-(2-nitroethyl)benzene, methyl d₄-4-methoxybenzoate, methyl d₆-cinnamate and ethyl d₄-4-methoxybenzoate}. On the other hand, hexane extracts of the flower petal incubate with a mixture of d₅-Z- and d₅-E-2-phenylacetaldoxime after 24 h indicated generation of six d₅-labeld components {d₅-benzaldehyde, d₅-benzyl alcohol, d₅-2-phenylacetaldehyde, methyl d₅-benzoate, d₅-2-phenylethanol, and d₅-(2-nitroethyl)benzene}. By comparing those results, (2-nitroethyl)benzene was concluded as a product directly generated from a mixture of Z- and E-2-phenylacetaldoxime together with six minor benzenoids, while two major compounds (4-methoxybenzaldehyde and methyl 4-methoxybenzoate) together with three minors from L-phenylalanine, presumably via L-tyrosine. The other two minor components were derived from L-phenylalanine.     

Ect2, an Ortholog of <Emphasis Type="Italic">Drosophila’s</Emphasis><Emphasis Type="Italic">Pebble</Emphasis>, Negatively Regulates Neurite Outgrowth in Neuroblastoma × Glioma Hybrid NG108-15 Cells

To identify genes required for brain development, we previously performed in vivo RNA interference (RNAi) screening in Drosophila embryos. We identified pebble as a gene that disrupts development of the Drosophila nervous system. Although pebble has been shown to be involved in neuronal development of Drosophila in several screens, the involvement of Ect2, a mammalian ortholog of pebble, in mammalian neuronal development has not been addressed. To examine the role of Ect2 in neuronal differentiation, we performed Ect2 RNAi in the mouse neuroblastoma × rat glioma NG108-15 cell line. Depletion of Ect2 resulted in an increased proportion of binucleate cells and morphological differentiation of NG108-15 cells characterized by the outgrowth of neurites. These morphological changes were correlated with an increased level of acetylcholine esterase mRNA. In addition, expression of Ect2 was decreased in differentiated NG108-15 cells induced by dibutyryl cyclic AMP. These findings indicate that Ect2 negatively regulates the differentiation of NG108-15 cells and suggest that Ect2 may play a role in neuronal differentiation and brain development in vivo.     

Role of interaction of XPF with RPA in nucleotide excision repair

Nucleotide excision repair (NER) is a very important defense system against various types of DNA damage, and it is necessary for maintaining genomic stability. The molecular mechanism of NER has been studied in considerable detail, and it has been shown that proper protein-protein interactions among NER factors are critical for efficient repair. A structure-specific endonuclease, XPF-ERCC1, which makes the 5′ incision in NER, was shown to interact with a single-stranded DNA binding protein, RPA. However, the biological significance of this interaction was not studied in detail. We used the yeast two-hybrid assay to determine that XPF interacts with the p70 subunit of RPA. To further examine the role of this XPF-p70 interaction, we isolated a p70-interaction-deficient mutant form of XPF that contains a single amino acid substitution in the N-terminus of XPF by the reverse yeast two-hybrid assay using randomly mutagenized XPF. The biochemical properties of this RPA-interaction-deficient mutant XPF-ERCC1 are very similar to those of wild-type XPF-ERCC1 in vitro. Interestingly, expression of this mutated form of XPF in the XPF-deficient Chinese hamster ovary cell line, UV41, only partially restores NER activity and UV resistance in vivo compared to wild-type XPF. We discovered that the RPA-interaction-deficient XPF is not localized in nuclei and the mislocalization of XPF-ERCC1 prevents the complex from functioning in NER.     

Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli

1-Butanol, an important chemical feedstock and advanced biofuel, is produced by Clostridium species. Various efforts have been made to transfer the clostridial 1-butanol pathway into other microorganisms. However, in contrast to similar compounds, only limited titers of 1-butanol were attained. In this work, we constructed a modified clostridial 1-butanol pathway in Escherichia coli to provide an irreversible reaction catalyzed by trans-enoyl-coenzyme A (CoA) reductase (Ter) and created NADH and acetyl-CoA driving forces to direct the flux. We achieved high-titer (30 g/liter) and high-yield (70 to 88% of the theoretical) production of 1-butanol anaerobically, comparable to or exceeding the levels demonstrated by native producers. Without the NADH and acetyl-CoA driving forces, the Ter reaction alone only achieved about 1/10 the level of production. The engineered host platform also enables the selection of essential enzymes with better catalytic efficiency or expression by anaerobic growth rescue. These results demonstrate the importance of driving forces in the efficient production of nonnative products.