Influx of extracellular calcium is required for the membrane translocation of 5-lipoxygenase and leukotriene synthesis.

Our studies assessed the effects of increases in intracellular calcium concentrations [( Ca2+]i) on leukotriene synthesis and membrane translocation of 5-lipoxygenase (5LO). The calcium ionophore ionomycin and the tumor promoter thapsigargin stimulated leukotriene production and translocation of 5-lipoxygenase to the membrane. Both agents elicited prolonged rises in [Ca2+]i. Leukotriene C4 production associated with [Ca2+]i in cells stimulated with various concentrations of ionomycin and thapsigargin suggests that a threshold [Ca2+]i level of approximately 300-400 nM is required. In the absence of extracellular Ca2+, both the ionomycin- and thapsigargin-induced rises in [Ca2+]i were transient, indicating that the prolonged [Ca2+]i elevation is due to an influx of extracellular Ca2+. Addition of EGTA to the external medium before, or at different times during, the treatment with ionomycin or thapsigargin instantaneously inhibited 5LO translocation and leukotriene synthesis, indicating that Ca2+ influx plays an essential role in 5LO membrane translocation and leukotriene synthesis. No leukotriene production was detected when cells were stimulated by a physiological stimulus of leukotriene D4. The addition of 100 nM leukotriene D4 triggered peak rises in [Ca2+]i that were comparable to those achieved by the ionomycin and thapsigargin. However, the leukotriene D4 induced rise was transient and rapidly declined to a lower but still elevated steady-state level, which was attributed to Ca2+ influx. Stimulation with 100 nM leukotriene D4 for 15 s increased the cellular levels of 1,4,5-inositol triphosphate (IP3), 1,3,4-IP3, and 1,3,4,5-inositol tetraphosphate (IP4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrostatic contribution of surface charge residues to the stability of a thermophilic protein: benchmarking experimental and predicted pKa values

Optimization of the surface charges is a promising strategy for increasing thermostability of proteins. Electrostatic contribution of ionizable groups to the protein stability can be estimated from the differences between the pKa values in the folded and unfolded states of a protein. Using this pKa-shift approach, we experimentally measured the electrostatic contribution of all aspartate and glutamate residues to the stability of a thermophilic ribosomal protein L30e from Thermococcus celer. The pKa values in the unfolded state were found to be similar to model compound pKas. The pKa values in both the folded and unfolded states obtained at 298 and 333 K were similar, suggesting that electrostatic contribution of ionizable groups to the protein stability were insensitive to temperature changes. The experimental pKa values for the L30e protein in the folded state were used as a benchmark to test the robustness of pKa prediction by various computational methods such as H++, MCCE, MEAD, pKD, PropKa, and UHBD. Although the predicted pKa values were affected by crystal contacts that may alter the side-chain conformation of surface charged residues, most computational methods performed well, with correlation coefficients between experimental and calculated pKa values ranging from 0.49 to 0.91 (p<0.01). The changes in protein stability derived from the experimental pKa-shift approach correlate well (r = 0.81) with those obtained from stability measurements of charge-to-alanine substituted variants of the L30e protein. Our results demonstrate that the knowledge of the pKa values in the folded state provides sufficient rationale for the redesign of protein surface charges leading to improved protein stability.     

The association between leukocyte telomere length and cigarette smoking, dietary and physical variables, and risk of prostate cancer

Telomeres consist of nucleotide repeats and a protein complex at chromosome ends that are essential to maintaining chromosomal integrity. Several studies have suggested that subjects with shorter telomeres are at increased risk of bladder and lung cancer. In comparison to normal tissues, telomeres are shorter in high-grade intraepithelial neoplasia and prostate cancer. We examined prostate cancer risk associated with relative telomere length as determined by quantitative PCR on prediagnostic buffy coat DNA isolated from 612 advanced prostate cancer cases and 1049 age-matched, cancer-free controls from the PLCO Cancer Screening Trial. Telomere length was analyzed as both a continuous and a categorical variable with adjustment for potential confounders. Statistically significant inverse correlations between telomere length, age and smoking status were observed in cases and controls. Telomere length was not associated with prostate cancer risk (at the median, OR = 0.85, 95% CI: 0.67, 1.08); associations were similar when telomere length was evaluated as a continuous variable or by quartiles. The relationships between telomere length and inflammation-related factors, diet, exercise, body mass index, and other lifestyle variables were explored since many of these have previously been associated with shorter telomeres. Healthy lifestyle factors ( i.e. , lower BMI, more exercise, tobacco abstinence, diets high in fruit and vegetables) tended to be associated with greater telomere length. This study found no statistically significant association between leukocyte telomere length and advanced prostate cancer risk. However, correlations of telomere length with healthy lifestyles were noted, suggesting the role of these factors in telomere biology maintenance and potentially impacting overall health status.     

Minitags for small molecules: detecting targets of reactive small molecules in living plant tissues using 'click chemistry'

Small molecules offer unprecedented opportunities for plant research since plants respond to, metabolize, and react with a diverse range of endogenous and exogenous small molecules. Many of these small molecules become covalently attached to proteins. To display these small molecule targets in plants, we introduce a two-step labelling method for minitagged small molecules. Minitags are small chemical moieties (azide or alkyne) that are inert under biological conditions and have little influence on the membrane permeability and specificity of the small molecule. After labelling, proteomes are extracted under denaturing conditions and minitagged proteins are coupled to reporter tags through a 'click chemistry' reaction. We introduce this two-step labelling procedure in plants by studying the well-characterized targets of E-64, a small molecule cysteine protease inhibitor. In contrast to biotinylated E-64, minitagged E-64 efficiently labels vacuolar proteases in vivo . We displayed, purified and identified targets of a minitagged inhibitor that targets the proteasome and cysteine proteases in living plant cells. Chemical interference assays with inhibitors showed that MG132, a frequently used proteasome inhibitor, preferentially inhibits cysteine proteases in vivo . The two-step labelling procedure can be applied on detached leaves, cell cultures, seedlings and other living plant tissues and, when combined with photoreactive groups, can be used to identify targets of herbicides, phytohormones and reactive small molecules selected from chemical genetic screens.     

Synthesis and human NKT cell stimulating properties of 3-O-sulfo-alpha/beta-galactosylceramides

Two novel hybrid molecules 3-O-sulfo-alpha/beta-galactosylceramide 3 and 4, which are derived from an immunostimulatory agent alpha-GalCer 1 and self-glycolipid ligand sulfatide 2, were designed and synthesized. Compound 3 was shown to efficiently stimulate human NKT cells to secret IL-4 and IFN-gamma, with activities similar to 1, suggesting that modification of the 3'-OH position of the galactose moiety with sulfate has no significant effect on NKT cell stimulation. As a comparison, the beta-isomer 4 has no affinity to NKT cells, which demonstrates that the alpha-glycosidic bond of galactosylceramide is crucial to the NKT cells activation.     

Studies of H5N1 influenza virus infection of pigs by using viruses isolated in Vietnam and Thailand in 2004

To determine whether avian H5N1 influenza viruses associated with human infections in Vietnam had transmitted to pigs, we investigated serologic evidence of exposure to H5N1 influenza virus in Vietnamese pigs in 2004. Of the 3,175 pig sera tested, 8 (0.25%) were positive for avian H5N1 influenza viruses isolated in 2004 by virus neutralization assay and Western blot analysis. Experimental studies of replication and transmissibility of the 2004 Asian H5N1 viruses in pigs revealed that all viruses tested replicated in the swine respiratory tract but none were transmitted to contact pigs. Virus titers from nasal swabs peaked on day 2, and low titers were detected in the liver of two of the four pigs tested. Our findings indicate that pigs can be infected with highly lethal Asian H5N1 viruses but that these viruses are not readily transmitted between pigs under experimental conditions.