Comprehensive proteome analysis of lettuce latex using multidimensional protein-identification technology

Commercially, lettuce (Lactuca sativa) is one of the most important leafy vegetables. Lettuce produces a milky latex of variable chemical compositions within its laticifers. As a step toward understanding the main physiological roles of this latex in higher plants, we embarked on its proteomic analysis. We investigated 587 latex proteins that were identified from the lettuce latex using multidimensional protein-identification technology. A bioinformatics analysis showed that the most frequently encountered proteins in the latex were organellar proteins from plastids and mitochondria, followed by nucleic and cytoplasmic proteins. Functional classification of the identified proteins showed that proteins related to metabolism, cell rescue, defense, and virulence were the most abundant in lettuce latex. Furthermore, numerous resistance proteins of lettuce and viral proteins were present in the latex suggesting for the first time a possible function of the lettuce latex in defense or pathogenesis. To the knowledge of the authors, this is the first large-scale proteome analysis of lettuce latex.     

Effect of substrate loading on hydrogen production during anaerobic fermentation by Clostridium thermocellum 27405

We have investigated hydrogen (H₂) production by the cellulose-degrading anaerobic bacterium, Clostridium thermocellum. In the following experiments, batch-fermentations were carried out with cellobiose at three different substrate concentrations to observe the effects of carbon-limited or carbon-excess conditions on the carbon flow, H₂-production, and synthesis of other fermentation end products, such as ethanol and organic acids. Rates of cell growth were unaffected by different substrate concentrations. H₂, carbon dioxide (CO₂), acetate, and ethanol were the main products of fermentation. Other significant end products detected were formate and lactate. In cultures where cell growth was severely limited due to low initial substrate concentrations, hydrogen yields of 1 mol H₂/mol of glucose were obtained. In the cultures where growth ceased due to carbon depletion, lactate and formate represented a small fraction of the total end products produced, which consisted mainly of H₂, CO₂, acetate, and ethanol throughout growth. In cultures with high initial substrate concentrations, cellobiose consumption was incomplete and cell growth was limited by factors other than carbon availability. H₂-production continued even in stationary phase and H₂/CO₂ ratios were consistently greater than 1 with a maximum of 1.2 at the stationary phase. A maximum specific H₂ production rate of 14.6 mmol g dry cell⁻¹ h⁻¹ was observed. As cells entered stationary phase, extracellular pyruvate production was observed in high substrate concentration cultures and lactate became a major end product.     

Attenuated corticosterone response to chronic ACTH stimulation in hepatic lipase-deficient mice: evidence for a role for hepatic lipase in adrenal physiology

Hepatic lipase (HL), a liver-expressed lipolytic enzyme, hydrolyzes triglycerides and phospholipids in lipoproteins and promotes cholesterol delivery through receptor-mediated whole particle and selective cholesterol uptake. HL activity also occurs in the adrenal glands, which utilize lipoprotein cholesterol to synthesize glucocorticoids in response to pituitary ACTH. It is likely that the role of adrenal HL is to facilitate delivery of exogenous cholesterol for glucocorticoid synthesis. On this basis, we hypothesized that HL deficiency would blunt the glucocorticoid response to ACTH. Furthermore, because exogenous cholesterol also is derived from the LDL receptor (LDLR) pathway, we hypothesized that LDLR deficiency would blunt the response to ACTH. To test these hypotheses, we compared the corticosterone response to eight daily ACTH injections in HL-deficient (hl-/-), LDLR-deficient (Ldlr-/-), and HL- and LDLR-doubly deficient (Ldlr-/- hl-/-) mice with that in wild-type (WT) mice. Plasma corticosterone levels were measured on days 2, 5, and 8. Differences in plasma corticosterone levels between genotypes were analyzed by Kruskal-Wallis one-way ANOVA on ranks and pairwise multiple comparisons by Dunn's test. Our results demonstrate a trend toward reductions in plasma corticosterone levels on day 2 and significant reductions on day 5 and day 8 in the knockout models. Thus, on day 5, plasma corticosterone levels were reduced by 57, 70, and 73% (all P < 0.05) and on day 8 by 76, 59, and 63% (all P < 0.05) in hl-/-, Ldlr-/-, and Ldlr-/- hl-/- mice, respectively. These results demonstrate that HL deficiency, like LDLR deficiency, blunts the adrenal response to chronic ACTH stimulation and suggest a novel role for HL in adrenal physiology.     

Continuous hydrogen production during fermentation of alpha-cellulose by the thermophillic bacterium Clostridium thermocellum

Continuous hydrogen (H2) production during fermentation of alpha-cellulose was established using the thermophillic, anaerobic bacterium Clostridium thermocellum ATCC 27405. The objectives of this work were to characterize growth of C. thermocellum, quantify H2 production and determine soluble end-product synthesis patterns during fermentation of a cellulosic substrate under continuous culture conditions. A 5 L working volume fermentor was established and growth experiments were maintained for over 3,000 h. Substrate concentrations were varied from 1 to 4 g/L and the feed was introduced with continuous nitrogen gas sparging to prevent clogging of the feed-line. The pH and temperature of the reactor were maintained at 7.0 and 600 degrees C, respectively, throughout the study. At concentrations above 4 g/L, the delivery of alpha-cellulose was impaired due to feed-line clogging and it became difficult to maintain a homogenous suspension. The highest total gas (H2 plus CO2) production rate, 56.6 mL L(-1) h(-1), was observed at a dilution rate of 0.042 h(-1) and substrate concentration of 4 g/L. Under these conditions, the H2 production rate was 5.06 mmol h(-1). Acetate and ethanol were the major soluble end-products, while lactate and formate were greatly reduced compared to production in batch cultures. Concentrations of all metabolites increased with increasing substrate concentration, with the exception of lactate. Despite a number of short-term electrical and mechanical failures during the testing period, the system recovered quickly, exhibiting substantial robustness. A carbon balance was completed to ensure that all end-products were accounted for, with final results indicating near 100% carbon recovery. This study shows that long-term, stable H2 production can be achieved during direct fermentation of an insoluble cellulosic substrate under continuous culture conditions.     

Orai3 Constitutes a Native Store-Operated Calcium Entry That Regulates Non Small Cell Lung Adenocarcinoma Cell Proliferation

Orai channels have been associated with cell proliferation, survival and metastasis in several cancers. The present study investigates the expression and the role of Orai3 in cell proliferation in non-small cell lung cancer (NSCLC). We show that Orai3 is over-expressed in cancer tissues as compared to the non-tumoral ones. Furthermore, Orai3 staining is stronger in high grade tumors. Pharmacological inhibition or knockdown of Orai3 significantly reduced store operated calcium entry (SOCE), inhibited cell proliferation and arrested cells of two NSCLC cell lines in G0/G1 phase. These effects were concomitant with a down-regulation of cyclin D1, cyclin E, CDK4 and CDK2 expression. Moreover, Orai3 silencing decreased Akt phosphorylation levels. In conclusion, Orai3 constitutes a native SOCE pathway in NSCLC that controls cell proliferation and cell cycle progression likely via Akt pathway.     

Circulating IL-6 concentrations and associated anthropometric and metabolic parameters in South Asian men and women in comparison to European whites

ObjectiveTo determine any ethnic differences in circulating interleukin (IL)-6 concentrations among SAs and Europeans, and to assess their relationship with body composition and insulin resistance measures.MethodsBody composition was assessed among 80 SA and European men and women using anthropometry, dual-energy X-ray absorptiometry and abdominal CT scan. Oral glucose tolerance tests with insulin response were performed to assess insulin resistance measures. IL-6 levels were measured by high sensitivity ELISA.ResultsMedian IL-6 values were higher in SA compared with European women: 1.94 mg/l versus 1.51 mg/l, p = 0.041, but not so in men (1.56 mg/l versus 1.57 mg/l). Only measures of obesity, in particular percentage fat area (r = 0.6, p = 0.003), were positively correlated with IL-6 in SAs. Differences in body fat percentage (visceral and total) could explain up to 30% of the IL-6 difference between Asian and European women.ConclusionSA women have elevated circulating IL-6 levels, in part due to greater visceral and percent fat levels compared with European women. This observation may in part explain why Asians are at elevated cardiovascular disease risk. Future studies should address the effects of lifestyle factors (physical activity, diet) on plasma IL-6 concentrations in SA women.     

Characterization of the activities of the CpeY, CpeZ, and CpeS bilin lyases in phycoerythrin biosynthesis in Fremyella diplosiphon strain UTEX 481

When grown in green light, Fremyella diplosiphon strain UTEX 481 produces the red-colored protein phycoerythrin (PE) to maximize photosynthetic light harvesting. PE is composed of two subunits, CpeA and CpeB, which carry two and three phycoerythrobilin (PEB) chromophores, respectively, that are attached to specific Cys residues via thioether linkages. Specific bilin lyases are hypothesized to catalyze each PEB ligation. Using a heterologous, coexpression system in Escherichia coli, the PEB ligation activities of putative lyase subunits CpeY, CpeZ, and CpeS were tested on the CpeA and CpeB subunits from F. diplosiphon. Purified His(6)-tagged CpeA, obtained by coexpressing cpeA, cpeYZ, and the genes for PEB synthesis, had absorbance and fluorescence emission maxima at 566 and 574 nm, respectively. CpeY alone, but not CpeZ, could ligate PEB to CpeA, but the yield of CpeA-PEB was lower than achieved with CpeY and CpeZ together. Studies with site-specific variants of CpeA(C82S and C139S), together with mass spectrometric analysis of trypsin-digested CpeA-PEB, revealed that CpeY/CpeZ attached PEB at Cys(82) of CpeA. The CpeS bilin lyase ligated PEB at both Cys(82) and Cys(139) of CpeA but very inefficiently; the yield of PEB ligated at Cys(82) was much lower than observed with CpeY or CpeY/CpeZ. However, CpeS efficiently attached PEB to Cys(80) of CpeB but neither CpeY, CpeZ, nor CpeY/CpeZ could ligate PEB to CpeB.     

Retargeting of rat parvovirus H-1PV to cancer cells through genetic engineering of the viral capsid

The rat parvovirus H-1PV is a promising anticancer agent given its oncosuppressive properties and the absence of known side effects in humans. H-1PV replicates preferentially in transformed cells, but the virus can enter both normal and cancer cells. Uptake by normal cells sequesters a significant portion of the administered viral dose away from the tumor target. Hence, targeting H-1PV entry specifically to tumor cells is important to increase the efficacy of parvovirus-based treatments. In this study, we first found that sialic acid plays a key role in H-1PV entry. We then genetically engineered the H-1PV capsid to improve its affinity for human tumor cells. By analogy with the resolved crystal structure of the closely related parvovirus minute virus of mice, we developed an in silico three-dimensional (3D) model of the H-1PV wild-type capsid. Based on this model, we identified putative amino acids involved in cell membrane recognition and virus entry at the level of the 2-fold axis of symmetry of the capsid, within the so-called dimple region. In situ mutagenesis of these residues significantly reduced the binding and entry of H-1PV into permissive cells. We then engineered an entry-deficient viral capsid and inserted a cyclic RGD-4C peptide at the level of its 3-fold axis spike. This peptide binds α(v)β(3) and α(v)β(5) integrins, which are overexpressed in cancer cells and growing blood vessels. The insertion of the peptide rescued viral infectivity toward cells overexpressing α(v)β(5) integrins, resulting in the efficient killing of these cells by the reengineered virus. This work demonstrates that H-1PV can be genetically retargeted through the modification of its capsid, showing great promise for a more efficient use of this virus in cancer therapy.     

Conversion of olive pomace oil to cocoa butter-like fat in a packed-bed enzyme reactor

Refined olive pomace oil (ROPO) was utilized as a source oil for production of cocoa butter-like fat. Immobilized sn-1,3 specific lipase catalyzed acidolysis of ROPO with palmitic (PA) and stearic (SA) acids was performed in a laboratory scale packed-bed reactor. Effect of reactor conditions on product formation was studied at various substrate mole ratios (ROPO:PA:SA; 1:1:1, 1:1:3, 1:3:3, 1:2:6), enzyme loads (10%, 20%, 40%), substrate flow rates (1.5, 4.5, 7.5, 15 ml/min) and solvent amounts (150, 400 ml). The highest yield (10.9% POP, 19.7% POS and 11.2% SOS) was obtained at 40% enzyme load, 1:2:6 substrate mole ratio, 45 degrees C, 7.5 ml/min substrate flow rate, 150 ml solvent and 3h reaction time. The melting profile and SFC of the product were comparable to those of CB. Polarized light microscope (PLM) images showed no drastic changes in polymorphic behavior between CB and product.