Smoking and COPD increase sputum levels of extracellular superoxide dismutase

Extracellular superoxide dismutase (ECSOD) is the major superoxide-scavenging enzyme in the lung. Certain ECSOD polymorphisms are protective against COPD. We postulated that smokers and COPD subjects would have altered levels of ECSOD in the lung, airway secretions, and/or plasma. Lung tissue ECSOD was evaluated from nonsmokers, smokers, and subjects with mild to very severe COPD by Western blot, immunohistochemistry, and ELISA. ECSOD levels in plasma, bronchoalveolar lavage fluid (BALF), and induced-sputum supernatants were analyzed by ELISA and correlated with smoking history and disease status. Immunohistochemistry identified ECSOD in extracellular matrix around bronchioles, arteries, and alveolar walls, with decreases seen in the interstitium and vessels of severe COPD subjects using digital image analysis. Plasma ECSOD did not differ between COPD subjects and controls nor based on smoking status. ECSOD levels in induced sputum supernatants were elevated in current smokers and especially in COPD subjects compared to nonsmokers, whereas corresponding changes could not be seen in the BALF. ECSOD expression was reduced around vessels and bronchioles in COPD lungs. Substantial increases in sputum ECSOD in smokers and COPD is interpreted as an adaptive response to increased oxidative stress and may be a useful biomarker of disease activity in COPD.     

High yield lipase-catalyzed synthesis of Engkabang fat esters for the cosmetic industry

Engkabang fat esters were produced via alcoholysis reaction between Engkabang fat and oleyl alcohol, catalyzed by Lipozyme RM IM. The reaction was carried out in a 500 ml Stirred tank reactor using heptane and hexane as solvents. Response surface methodology (RSM) based on a four-factor-five-level Central composite design (CCD) was applied to evaluate the effects of synthesis parameters, namely temperature, substrate molar ratio (oleyl alcohol: Engkabang fat), enzyme amount and impeller speed. The optimum yields of 96.2% and 91.4% were obtained for heptane and hexane at the optimum temperature of 53.9 °C, impeller speeds of 309.5 and 309.0 rpm, enzyme amounts of 4.82 and 5.65 g and substrate molar ratios of 2.94 and 3.39:1, respectively. The actual yields obtained compared well with the predicted values of 100.0% and 91.5%, respectively. Meanwhile, the properties of the esters show that they are suitable to be used as ingredient for cosmetic applications.     

Synthesis of methyl esters from palm (Elaeis guineensis) oil using cobalt doped MgO as solid oxide catalyst

The potential of Mg(x)Co(2-)(x)O(2) as heterogeneous reusable catalyst in transesterification of palm oil to methyl ester was investigated. The catalyst was prepared via co-precipitation of the metal hydroxides at different Mg-Co ratios. Mg(1.7)Co(0.3)O(2) catalyst was more active than Mg(0.3)Co(1.7)O(2) in the transesterification of palm oil with methanol. The catalysts calcined at temperature 300 °C for 4 h resulted in highly active oxides and the highest transesterification of 90% was achieved at methanol/oil molar ratio of 9:1, catalyst loading of 5.00 wt.%, reaction temperature of 150 °C and reaction time of 2 h. The catalyst could easily be removed from reaction mixture, but showed 50% decrease in activity when reused due to leaching of active sites.     

Cyclic presence and absence of conspecifics alters circadian clock phase but does not entrain the locomotor activity rhythm of the fruit fly Drosophila melanogaster

Circadian clocks use a wide range of environmental cues, including cycles of light, temperature, food, and social interactions, to fine-tune rhythms in behavior and physiology. Although social cues have been shown to influence circadian clocks of a variety of organisms including the fruit fly Drosophila melanogaster, their mechanism of action is still unclear. Here, the authors report the results of their study aimed at investigating if daily cycles of presence and absence (PA) of conspecific male visitors are able to entrain the circadian locomotor activity rhythm of male hosts living under constant darkness (DD). The results suggest that PA cycles may not be able to entrain circadian locomotor activity rhythms of Drosophila. The outcome does not change when male hosts are presented with female visitors, suggesting that PA cycles of either sex may not be effective in bringing about stable entrainment of circadian clocks in D. melanogaster. However, in hosts whose clock phase has already been set by light/dark (LD) cycles, daily PA cycles of visitors can cause measurable change in the phase of subsequent free-running rhythms, provided that their circadian clocks are labile. Thus, the findings of this study suggest that D. melanogaster males may not be using cyclic social cues as their primary zeitgeber (time cue) for entrainment of circadian clocks, although social cues are capable of altering the phase of their circadian rhythms.     

Simvastatin regulates CXC chemokine formation in streptococcal M1 protein-induced neutrophil infiltration in the lung

Streptococcus pyogenes of the M1 serotype can cause streptococcal toxic shock syndrome and acute lung injury. Statins exert beneficial effects in septic patients although the mechanisms remain elusive. This study examined effects of simvastatin on M1 protein-provoked pulmonary inflammation and tissue injury. Male C57BL/6 mice were pretreated with simvastatin or a CXCR2 antagonist before M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for determination of neutrophil infiltration, formation of edema, and CXC chemokines. Flow cytometry was used to determine Mac-1 expression on neutrophils. Gene expression of CXC chemokines was determined in alveolar macrophages by using quantitative RT-PCR. M1 protein challenge caused massive infiltration of neutrophils, edema formation, and production of CXC chemokines in the lung as well as upregulation of Mac-1 on circulating neutrophils. Simvastatin reduced M1 protein-induced infiltration of neutrophils and edema in the lung. In addition, M1 protein-induced Mac-1 expression on neutrophils was abolished by simvastatin. Furthermore, simvastatin markedly decreased pulmonary formation of CXC chemokines and gene expression of CXC chemokines in alveolar macrophages. Moreover, the CXCR2 antagonist reduced M1 protein-induced neutrophil expression of Mac-1 and accumulation of neutrophils as well as edema formation in the lung. These novel findings indicate that simvastatin is a powerful inhibitor of neutrophil infiltration in acute lung damage triggered by streptococcal M1 protein. The inhibitory effect of simvastatin on M1 protein-induced neutrophil recruitment appears related to reduced pulmonary generation of CXC chemokines. Thus, simvastatin may be a useful tool to ameliorate acute lung injury in streptococcal infections.     

Expression, purification and characterization of isoforms of peripheral stalk subunits of human V-ATPase

The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase.     

Methods for peptide and protein quantitation by liquid chromatography-multiple reaction monitoring mass spectrometry

Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and β-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633-641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20-30% for LRP and LF methods. Power calculations indicated that differences in measurement error between the methods have much less impact on measured protein expression differences than biological variation. All three methods detected significant (p < 0.05) differential expression of three endogenous proteins in a test set of 10 pairs of human lung tumor and control tissues. Further, the LRP and LF methods both detected significant differences (p < 0.05) in levels of seven biomarker candidates between tumors and controls in the same set of lung tissue samples. The data indicate that the LRP and LF methods provide cost-effective alternatives to SID for many quantitative liquid chromatography-multiple reaction monitoring mass spectrometry applications.     

The SUVR4 histone lysine methyltransferase binds ubiquitin and converts H3K9me1 to H3K9me3 on transposon chromatin in Arabidopsis

Chromatin structure and gene expression are regulated by posttranslational modifications (PTMs) on the N-terminal tails of histones. Mono-, di-, or trimethylation of lysine residues by histone lysine methyltransferases (HKMTases) can have activating or repressive functions depending on the position and context of the modified lysine. In Arabidopsis, trimethylation of lysine 9 on histone H3 (H3K9me3) is mainly associated with euchromatin and transcribed genes, although low levels of this mark are also detected at transposons and repeat sequences. Besides the evolutionarily conserved SET domain which is responsible for enzyme activity, most HKMTases also contain additional domains which enable them to respond to other PTMs or cellular signals. Here we show that the N-terminal WIYLD domain of the Arabidopsis SUVR4 HKMTase binds ubiquitin and that the SUVR4 product specificity shifts from di- to trimethylation in the presence of free ubiquitin, enabling conversion of H3K9me1 to H3K9me3 in vitro. Chromatin immunoprecipitation and immunocytological analysis showed that SUVR4 in vivo specifically converts H3K9me1 to H3K9me3 at transposons and pseudogenes and has a locus-specific repressive effect on the expression of such elements. Bisulfite sequencing indicates that this repression involves both DNA methylation-dependent and -independent mechanisms. Transcribed genes with high endogenous levels of H3K4me3, H3K9me3, and H2Bub1, but low H3K9me1, are generally unaffected by SUVR4 activity. Our results imply that SUVR4 is involved in the epigenetic defense mechanism by trimethylating H3K9 to suppress potentially harmful transposon activity.