Effect of vitamin D on oxidative stress and serum inflammatory factors in the patients with type 2 diabetes.

The type 2 diabetes mellitus (T2DM) is an urgent global health problem. T2DM patients are in a state of high oxidative stress and inflammation. Vitamin D and glutathione (GSH) play crucial roles in antioxidation and anti-inflammation. However, T2DM patients have lower vitamin D and GSH levels than healthy persons. A randomized controlled trial was conducted to see the effect of the vitamin D supplementation on oxidative stress and inflammatory factors in T2DM patients. In this study, a total of 178 T2DM patients were randomly enrolled, 92 patients received regular treatment (T2DM group) and 86 patients in Vitamin D group received extra vitamin D 400 IU per day in addition to regular treatment. Serum vitamin D, GSH, GSH metabolic enzyme GCLC and GR, inflammatory factor MCP-1, and IL-8 levels were investigated. We found that the T2DM group has significantly higher concentrations of MCP-1 and IL-8 than those in the healthy donor group. After vitamin D supplementation for 90 days, T2DM patients had a 2-fold increase of GSH levels, from 2.72 ± 0.84 to 5.76 ± 3.19 µmol/ml, the concentration of MCP-1 decreased from 51.11 ± 20.86 to 25.42 ± 13.06 pg/ml, and IL-8 also decreased from 38.21 ± 21.76 to 16.05 ± 8.99 pg/ml. In conclusion, our study demonstrated that vitamin D could regulate the production of GSH, thereby reducing the serum levels of MCP-1 and IL-8, alleviating oxidative stress and inflammation, providing evidence of the necessity and feasibility of adjuvant vitamin D treatment among patients with T2DM. On the other hand, vitamin D and GSH levels have important diagnostic and prognostic values in T2DM patients.

"Fix and Click" for Assay of Sphingolipid Signaling in Single Primary Human Intestinal Epithelial Cells.

Capillary electrophoresis with fluorescence detection (CE-F) is a powerful method to measure enzyme activation in single cells. However, cellular enzymatic assays used in CE-F routinely utilize reporter substrates that possess a bulky fluorophore that may impact enzyme kinetics. To address these challenges, we describe a "fix and click" method utilizing an alkyne-terminated enzyme activation reporter, aldehyde-based fixation, and a click chemistry reaction to attach a fluorophore prior to analysis by single-cell CE-F. The "fix and click" strategy was utilized to investigate sphingolipid signaling in both immortalized cell lines and primary human colonic epithelial cells. When the sphingosine alkyne reporter was loaded into cells, this reporter was metabolized to ceramide (31.6 ± 3.3% peak area) without the production of sphingosine-1-phosphate. In contrast, when the reporter sphingosine fluorescein was introduced into cells, sphingosine fluorescein was converted to sphingosine-1-phosphate and downstream products (32.8 ± 5.7% peak area) without the formation of ceramide. Sphingolipid metabolism was measured in single cells from both differentiated and stem/proliferative human colonic epithelium using "fix and click" paired with CE-F to highlight the diversity of sphingosine metabolism in single cells from primary human colonic epithelium. This novel method will find widespread utility for the performance of single-cell enzyme assays by virtue of its ability to temporally and spatially separate cellular reactions with alkyne-terminated reporters, followed by the assay of enzyme activation at a later time and place.

Fluorogenic XY-69 in Lipid Vesicles for Measuring Activity of Phospholipase C Isozymes.

Mammalian phospholipase C (PLC) isozymes are major signaling nodes that regulate a wide range of cellular processes. Dysregulation of PLC activity has been associated with a growing list of human diseases such as cancer and Alzheimer's disease. However, methods to directly and continuously monitor PLC activity at membranes with high sensitivity and throughput are still lacking. We have developed XY-69, a fluorogenic PIP2 analog, which can be efficiently hydrolyzed by PLC isozymes either in solution or at membranes. Here, we describe the optimized assay conditions and protocol to measure the activity of PLC-γ1 (D1165H) with XY-69 in lipid vesicles. The described protocol also applies to other PLC isozymes.

A High-Throughput Assay to Identify Allosteric Inhibitors of the PLC-γ Isozymes Operating at Membranes.

The two phospholipase C-γ (PLC-γ) isozymes are major signaling hubs and emerging therapeutic targets for various diseases, yet there are no selective inhibitors for these enzymes. We have developed a high-throughput, liposome-based assay that features XY-69, a fluorogenic, membrane-associated reporter for mammalian PLC isozymes. The assay was validated using a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) in 384-well format; it is highly reproducible and has the potential to capture both orthosteric and allosteric inhibitors. Selected hit compounds were confirmed with secondary assays, and further profiling led to the interesting discovery that adenosine triphosphate potently inhibits the PLC-γ isozymes through noncompetitive inhibition, raising the intriguing possibility of endogenous, nucleotide-dependent regulation of these phospholipases. These results highlight the merit of the assay platform for large scale screening of chemical libraries to identify allosteric modulators of the PLC-γ isozymes as chemical probes and for drug discovery.

Effect of Isothermal Transformation Times below Ms and Tempering on Strength and Toughness of Low-Temperature Bainite in 0.53 C Bainitic Steel.

This study aims to investigate the microstructures, strength, and impact toughness of low-temperature bainite obtained by isothermal transformation at temperature below Ms (Martensite Starting temperature) for different times and tempering process in 0.53 C wt% bainitic steel. By using the optical microscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), electron back scatter diffraction (EBSD), and mechanical property test, it was found that the microstructures after heat treatment consist of small amounts of martensite, fine bainite, and film retained austenite. After tempered at 250 °C for 2 h, the volume fraction of retained austenite (10.9%) in the sample treated by isothermal transformation at 220 °C for three hours is almost the same as that of the sample without tempering. In addition, the retained austenite fraction decreases with the increase of holding times and is reduced to 6.8% after holding for 15 h. The ultimate tensile strength (1827 MPa), yield strength (1496 MPa), total elongations (16.1%), and impact toughness (up to 58 J/cm2) were obtained by isothermal transformation at 220 °C for three hours and tempered at 250 °C. Whereas, the impact toughness of sample without tempering is 28 J/cm2. After holding for 15 h, the impact toughness raises to 56 J/cm2, while the ductility and strength decreases. These results indicate that the tempering process is helpful to improve the impact toughness of low-temperature bainite.

Structural basis for the activation of PLC-γ isozymes by phosphorylation and cancer-associated mutations.

Direct activation of the human phospholipase C-γ isozymes (PLC-γ1, -γ2) by tyrosine phosphorylation is fundamental to the control of diverse biological processes, including chemotaxis, platelet aggregation, and adaptive immunity. In turn, aberrant activation of PLC-γ1 and PLC-γ2 is implicated in inflammation, autoimmunity, and cancer. Although structures of isolated domains from PLC-γ isozymes are available, these structures are insufficient to define how release of basal autoinhibition is coupled to phosphorylation-dependent enzyme activation. Here, we describe the first high-resolution structure of a full-length PLC-γ isozyme and use it to underpin a detailed model of their membrane-dependent regulation. Notably, an interlinked set of regulatory domains integrates basal autoinhibition, tyrosine kinase engagement, and additional scaffolding functions with the phosphorylation-dependent, allosteric control of phospholipase activation. The model also explains why mutant forms of the PLC-γ isozymes found in several cancers have a wide spectrum of activities, and highlights how these activities are tuned during disease.

Many enzymes are poised to receive signals from the surrounding environment and translate them into responses inside the cell. One such enzyme is phospholipase C-γ1 (PLC-γ1), which controls how cells grow, divide and migrate.When activating signals are absent, PLC-γ1 usually inhibits its own activity, a mechanism called autoinhibition. This prevents the enzyme from binding to its targets, which are fat molecules known as lipids. When activating signals are present, a phosphate group serves as a 'chemical tag' and is added onto PLC-γ1, allowing the enzyme to bind to lipids.Failure in the regulation of PLC-γ1 or other closely related enzymes may lead to conditions such as cancer, arthritis and Alzheimer's disease. However, it remains unclear how autoinhibition suppresses the activity of the enzyme, and how it is stopped by the addition of the phosphate group.Here, Hajicek et al. determine in great detail the three-dimensional structure of the autoinhibited form of the enzyme using a method known as X-ray crystallography. This reveals that PLC-γ1 has two major lobes: one contains the active site that modifies lipids, and the other sits on top of the active site to prevent lipids from reaching it. The findings suggest that when the phosphate group attaches to PLC-γ1, it triggers a large shape change that shifts the second lobe away from the active site to allow lipids to bind.The three-dimensional structure also helps to understand how mutations identified in certain cancers may activate PLC-γ1. In particular, these mutations disrupt the interactions between elements that usually hold the two lobes together, causing the enzyme to activate more easily.The work by Hajicek et al. provides a framework to understand how cells control PLC-γ1. It is a first step toward designing new drugs that alter the activity of this enzyme, which may ultimately be useful to treat cancer and other diseases.

A membrane-associated, fluorogenic reporter for mammalian phospholipase C isozymes.

A diverse group of cell-surface receptors, including many G protein-coupled receptors and receptor tyrosine kinases, activate phospholipase C (PLC) isozymes to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and 1,4,5-inositol trisphosphate. Consequently, PLCs control various cellular processes, and their aberrant regulation contributes to many diseases, including cancer, atherosclerosis, and rheumatoid arthritis. Despite the widespread importance of PLCs in human biology and disease, it has been impossible to directly monitor the real-time activation of these enzymes at membranes. To overcome this limitation, here we describe XY-69, a fluorogenic reporter that preferentially partitions into membranes and provides a selective tool for measuring the real-time activity of PLCs as either purified enzymes or in cellular lysates. Indeed, XY-69 faithfully reported the membrane-dependent activation of PLC-ß3 by Gαq Therefore, XY-69 can replace radioactive phosphatidylinositol 4,5-bisphosphate used in conventional PLC assays and will enable high-throughput screens to identify both orthosteric and allosteric PLC inhibitors. In the future, cell-permeable variants of XY-69 represent promising candidates for reporting the activation of PLCs in live cells with high spatiotemporal resolution.

Required hydrophobicity of fluorescent reporters for phosphatidylinositol family of lipid enzymes.

The phosphatidylinositol (PtdIns) family of lipids plays important roles in cell differentiation, proliferation, and migration. Abnormal expression, mutation, or regulation of their metabolic enzymes has been associated with various human diseases such as cancer, diabetes, and bipolar disorder. Recently, fluorescent derivatives have increasingly been used as chemical probes to monitor either lipid localization or enzymatic activity. However, the requirements of a good probe have not been well defined, particularly modifications on the diacylglycerol side chain partly due to challenges in generating PtdIns lipids. We have synthesized a series of fluorescent PtdIns(4,5)P2 (PIP2) and PtdIns (PI) derivatives with various lengths of side chains and tested their capacity as substrates for PI3KIα and PI4KIIα, respectively. Both capillary electrophoresis and thin-layer chromatography were used to analyze enzymatic reactions. For both enzymes, the fluorescent probe with a longer side chain functions as a better substrate than that with a shorter chain and works well in the presence of the endogenous lipid, highlighting the importance of hydrophobicity of side chains in fluorescent phosphoinositide reporters. This comparison is consistent with their interactions with lipid vesicles, suggesting that the binding of a fluorescent lipid with liposome serves as a standard for assessing its utility as a chemical probe for the corresponding endogenous lipid. These findings are likely applicable to other lipid enzymes where the catalysis takes place at the lipid-water interface.

[The relationship between abdominal fat volume and obstructive sleep apnea hypopnea syndrome in obesity people].

OBJECTIVE: To explore the relationship between abdominal fat volume and obstructive sleep apnea hypopnea syndrome in obesity people. METHODS: From July 2009 to July 2010, 50 patients with BMI > 25 were prospectively selected for study from the patients who complained of snoring in the Respiratory department. The patients were divided into OSAHS group and non-OSAHS group according to the result of sleep apnea monitoring. All the patients also received full abdominal CT and the whole abdominal fat volume was measured by 3-D CT reconstruction system. SPSS 13.0 was used for statistical analysis. RESULTS: The whole abdominal fat volume in the two groups was analyzed by T- test, which was significantly different between the two groups (P < 0.01). It showed that there was a statistical relationship between OSAHS and abdominal fat in obesity people. CONCLUSION: In obesity people, OSAHS has a close relationship with abdominal fat volume. The abdominal fat volume is markedly higher in OSAHS patients than that in non-OSAHS people.

Small molecule inhibitors of phospholipase C from a novel high-throughput screen.

Phospholipase C (PLC) isozymes are important signaling molecules, but few small molecule modulators are available to pharmacologically regulate their function. With the goal of developing a general approach for identification of novel PLC inhibitors, we developed a high-throughput assay based on the fluorogenic substrate reporter WH-15. The assay is highly sensitive and reproducible: screening a chemical library of 6280 compounds identified three novel PLC inhibitors that exhibited potent activities in two separate assay formats with purified PLC isozymes in vitro. Two of the three inhibitors also inhibited G protein-coupled receptor-stimulated PLC activity in intact cell systems. These results demonstrate the power of the high-throughput assay for screening large collections of small molecules to identify novel PLC modulators. Potent and selective modulators of PLCs will ultimately be useful for dissecting the roles of PLCs in cellular processes, as well as provide lead compounds for the development of drugs to treat diseases arising from aberrant phospholipase activity.

Isotope-coded, fluorous photoaffinity labeling reagents.

A pair of isotope-coded, fluorous photoaffinity labeling reagents has been developed and coupled with a peptide. The modified peptides form adducts with methanol upon light illumination, which show characteristic isotope labeling patterns in mass spectra and can be separated from other peptides through fluorous silica.

Kinetic analysis of PI3K reactions with fluorescent PIP2 derivatives.

Phosphatidylinositol 3-kinase (PI3K) signaling plays important roles in cell differentiation, proliferation, and migration. Increased mutations and expression levels of PI3K are hallmarks for the development of certain cancers. Pharmacological targeting of PI3K activity has also been actively pursued as a novel cancer therapeutic. Consequently, measurement of PI3K activity in different cell types or patient samples holds the promise as being a novel diagnostic tool. However, the direct measurement of cellular PI3K activity has been a challenging task. We report here the characterization of two fluorescent PIP(2) derivatives as reporters for PI3K enzymatic activity. The reporters are efficiently separated from their corresponding PI3K enzymatic products through either thin layer chromatography (TLC) or capillary electrophoresis (CE), and can be detected with high sensitivity by fluorescence. The biophysical and kinetic properties of the two probes are measured, and their suitability to characterize PI3K inhibitors is explored. Both probes show similar capacity as PI3K substrates for inhibitor characterization, yet also possess distinct properties that may suggest their different applications. These characterizations have laid the groundwork to systematically measure cellular PI3K activity, and have the potential to generate molecular fingerprints for diagnostic and therapeutic applications.

Factors associated with Taiwanese junior high school personnel advising students to quit smoking.

BACKGROUND: Most smokers in developing countries begin smoking before age 18, and smoking prevalence is rising among adolescents. School personnel represent a target group for tobacco-control efforts because they interact daily with students, are role models for students, teach about tobacco-use prevention in school curricula, and implement school tobacco-control policies. The prevalence of teenage smoking has been examined in numerous studies, but few have focused on the influence of school personnel and the characteristics of school personnel who enforce school nonsmoking policy. The purpose of this study was to determine the factors associated with junior high school personnel advising students to quit smoking. METHODS: School personnel (N = 7129) were recruited by cluster sampling from 60 junior high schools in Taiwan; of these, 5280 voluntarily returned self-administered, anonymous questionnaires (response rate = 74.06%) in 2004. RESULTS: Most personnel (70%) had advised students to quit smoking. School personnel who were older, male, responsible for teaching health, smokers, with positive attitude against tobacco, or with more knowledge of tobacco hazards were more likely to advise students to quit smoking. Personnel with more interest in and access to tobacco-related materials were more likely to advise students to quit smoking. Personnel who had received tobacco-prevention training were 2.41 times more likely to persuade students to quit smoking after adjusting for other factors. However, only half of the participants had ever had access to educational materials about tobacco use, and 8% had ever received training to prevent tobacco use. CONCLUSIONS: To reduce youth smoking prevalence, school tobacco-control programs should support tobacco-prevention training for school personnel.

A fluorogenic, small molecule reporter for mammalian phospholipase C isozymes.

Phospholipase C isozymes (PLCs) catalyze the conversion of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) into two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. This family of enzymes are key signaling proteins that regulate the physiological responses of many extracellular stimuli such as hormones, neurotransmitters, and growth factors. Aberrant regulation of PLCs has been implicated in various diseases including cancer and Alzheimer's disease. How, when, and where PLCs are activated under different cellular contexts are still largely unknown. We have developed a fluorogenic PLC reporter, WH-15, that can be cleaved in a cascade reaction to generate fluorescent 6-aminoquinoline. When applied in enzymatic assays with either pure PLCs or cell lysates, this reporter displays more than a 20-fold fluorescence enhancement in response to PLC activity. Under assay conditions, WH-15 has comparable K(m) and V(max) with the endogenous PIP(2). This novel reporter will likely find broad applications that vary from imaging PLC activity in live cells to high-throughput screening of PLC inhibitors.

Association between social climate for smoking and youth smoking behaviors in Taiwan: an ecological study.

BACKGROUND: Social acceptance of smoking is associated with smoking prevalence. Higher smoking rates and ETS exposure might be considered as important indicators for pro-tobacco social norms or social climate. Among studies indicating the association between youth smoking and adult smoking behaviors, most were from individual-level study designs. OBJECTIVES: An ecological study was conducted to determine the role of social climate, i.e., adult smoking behaviors and exposure to environmental tobacco smoke (ETS), on youth smoking behaviors. METHODS: Data on the smoking behavior and ETS of 16,688 Taiwanese adults were collected in 2004 by telephone administration of the Taiwan Adult Smoking Survey. Similar data on 22,339 junior high school students were collected in 2004 by school-based administration of the Taiwan Youth Tobacco Survey. City/county-level data were analyzed across 25 counties by descriptive statistics, Pearson's correlation, and hierarchical multiple regression. RESULTS: In both adult and youth populations, overall or gender-specific smoking prevalence and ETS varied widely across counties/cities. The current youth smoking rate within counties was significantly positively correlated with the current adult smoking prevalence as well as home ETS exposure in adults or youth. For male youth, a 1% increase in the rate of home ETS exposure reported by youth increased the current male youth smoking rate by 0.20% after controlling the female youth smoking rate and home ETS exposure reported by adults (p=0.0197). For female youth, male youth smoking prevalence was the only variable that contributed significantly (Beta=0.46, p<0.001). CONCLUSIONS: Community health nurses should develop tobacco-control interventions that are tailored to support smoke-free environments by decreasing the social acceptability of smoking.

Susceptibility to initiate smoking among junior and senior high school nonsmokers in Taiwan.

OBJECTIVES: Most smokers begin using tobacco in their teens and recent reports indicate that smoking prevalence is climbing among youth in Taiwan. The purpose of this paper was to determine the associated factors of susceptibility of youth smoking by different types of schools. METHODS: A total of 4689 junior high students and 3918 senior high students participated in a school-based survey to determine the associated factors of susceptibility of youth smoking through anonymous self-administered questionnaire in 2004-2005. RESULTS: Susceptibility to initiate smoking ranged from 11.3% for junior high to 12.7% for general senior high and 12.4% for vocational senior students. For all/male smoking-susceptible students, more junior high students had one or more parents or best friends who smoked than did general or vocational senior high students. For all/female smoking-susceptible students, significantly more junior high students experienced secondhand smoke in public places than did non-susceptible students. CONCLUSIONS: Developing tailored, comprehensive smoking-prevention programs for junior high students should involve establishing tobacco-free households and communities.

Prevalence of tobacco use among junior high and senior high school students in Taiwan.

BACKGROUND: Tobacco use is a major preventable cause of death in the world. This article describes and compares tobacco use prevalence for students attending junior high schools and senior high schools in Taiwan. METHODS: This report uses data from the Global Youth Tobacco Survey (GYTS) completed among 4689 junior high school students and 4426 senior high school students in Taiwan in 2004-2005. The GYTS uses a 2-stage sampling design to produce nationally representative data for junior and senior high students in general and vocational schools. RESULTS: Higher smoking prevalence was observed among senior high (10.1% general schools and 15.9% vocational schools) than junior high (5.5%) school students. Smoking prevalence of girls in junior high (3.2%) and senior high schools (4.6% general and 11.1% vocational) was almost as high or higher than adult females' (4.3%) smoking rates. The pattern of smoking intensity across school years and type of school shows that the percentage of smokers who were experimenters (47.1%) was higher in junior high school and the percentage of smokers who were regular/established smokers (over 50%) was higher in senior high school. CONCLUSIONS: Smoking prevalence described in this report shows that there are challenges facing the tobacco prevention and control program in Taiwan. The findings suggest that schools should increase their smoking initiation prevention efforts and make available cessation programs and counseling to help students quit smoking. If effective youth tobacco control programs are not developed and implemented in Taiwan, future morbidity and mortality attributed to tobacco will surely increase, especially among women.

Identification of 1-isopropylsulfonyl-2-amine benzimidazoles as a new class of inhibitors of hepatitis B virus.

A series of 1-isopropylsulfonyl-2-amine benzimidazole derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activity and cytotoxicity in the HepG2.2.15 cell line. In general, these derivatives are potent HBV inhibitors (IC(50)<4 microM) with high selectivity indices (SIs>40). Compounds 5b-e, g, j, and 9a were among the most prominent compounds, with IC(50)s of 0.70-2.0 microM and SIs of 41-274. The potent anti-HBV activity and safety profiles of the most promising compounds 5d and j (IC(50)s=0.70 microM, SIs>120) demonstrate the potential of this series of benzimidazoles for the development of new anti-HBV drugs.

Synthesis and anti-hepatitis B virus activity of novel benzimidazole derivatives.

A series of novel benzimidazole derivatives was synthesized and evaluated for their anti-hepatitis B virus (HBV) activity and cytotoxicity in vitro. Strong activity against HBV replication and low cytotoxicity were generally observed in these benzimidazoles. The most promising compounds were 12a and 12b, with similar high antiviral potency (IC50 = 0.9 and 0.7 microM, respectively) and remarkable selectivity indices (>1111 and 714, respectively). They were selected for further evaluation as novel HBV inhibitors.

Synthesis of miltirone analogues as inhibitors of Cdc25 phosphatases.

Miltirone analogues were synthesized and evaluated for inhibitory activity against Cdc25 and PTP1B. Most of the compounds demonstrated potent Cdc25 inhibitory activity, and several exhibited higher selectivity for Cdc25 than for PTP1B. In a cytotoxic assay, most of the compounds displayed cytotoxicity against the tumor cell lines A549 and HCT-116, producing IC(50) values in the micromolar range.