A randomized placebo-controlled trial to investigate the effect of lactolycopene on semen quality in healthy males.

PURPOSE: Poor sperm quality is a major contributor to infertility in heterosexual couples, but at present there are few empirical therapies. Several studies have examined the role of dietary factors and data from randomized controlled trials suggest that oral antioxidant therapy can improve some sperm parameters. Health benefits of lycopene supplementation have been proposed for a variety of health conditions and here we examine whether it can help improve sperm quality. This study aimed to investigate the effect of 14 mg daily lactolycopene for 12 weeks on semen quality in healthy men. METHODS: Sixty healthy male participants were recruited and randomized to this double-blind, placebo-controlled parallel study and received either 14 mg/d lactolycopene or a placebo for 12 weeks. The primary endpoint was a change in motile sperm concentration. Secondary endpoints were all other aspects of sperm quality, including the level of sperm DNA damage. RESULTS: Fifty-six men completed the intervention and the level of plasma lycopene was significantly increased in the men randomized to receive lycopene supplementation. There was no significant change in the primary endpoint (motile sperm concentration) post-intervention (p = 0.058). However, the proportion of fast progressive sperm (p = 0.006) and sperm with normal morphology (p < 0.001) did improve significantly in response to lactolycopene intervention. CONCLUSIONS: Supplementation with 14 mg/d lactolycopene improves sperm motility and morphology in young healthy men. CLINICAL TRIAL REGISTRY NUMBER AND WEBSITE: ISRCTN33248724 http://www.isrctn.com/ISRCTN33248724.

Casein Hydrolysate with Glycemic Control Properties: Evidence from Cells, Animal Models, and Humans.

Evidence exists to support the role of dairy derived proteins whey and casein in glycemic management. The objective of the present study was to use a cell screening method to identify a suitable casein hydrolysate and to examine its ability to impact glycemia related parameters in an animal model and in humans. Following screening for the ability to stimulate insulin secretion in pancreatic beta cells, a casein hydrolysate was selected and further studied in the ob/ob mouse model. An acute postprandial study was performed in 62 overweight and obese adults. Acute and long-term supplementation with the casein hydrolysate in in vivo studies in mice revealed a glucose lowering effect and a lipid reducing effect of the hydrolysate (43% reduction in overall liver fat). The postprandial human study revealed a significant increase in insulin secretion ( p = 0.04) concomitant with a reduction in glucose ( p = 0.03). The area under the curve for the change in glucose decreased from 181.84 ± 14.6 to 153.87 ± 13.02 ( p = 0.009). Overall, the data supports further work on the hydrolysate to develop into a functional food product.

Protective effect of phosphorylated Hsp27 in coronary arteries through actin stabilization.

There is evidence for an inverse association between cellular expression of Hsp27 and vascular disease with carotid plaques, endarterectomy specimens, and cardiac biopsies investigated to date. Here we compare non-diseased coronary arteries from human heart transplant donors and patients with dilated cardiomyopathy (DCM) with no evidence of coronary artery disease, to coronary arteries from patients with ischemic heart disease (IHD) in order to determine abundance of phosphorylated Hsp27 (phospho-Hsp27) in plaque-free diseased vessels and elucidate how this protective effect is brought about through protein regulation. Western blotting identified phospho-Hsp27, phosphorylated on Ser82, Ser78, and Ser15, to be specifically decreased in IHD, but not DCM, compared to non-diseased vessels. Immunohistochemistry confirmed these results and revealed phospho-Hsp27 was located within both smooth muscle and endothelial cells. Disease-free coronary arteries and from patients with IHD were then subjected to 2-Dimensional Difference Gel Electrophoresis (2D-DIGE) analysis to detect proteins with altered abundance, which were subsequently identified by mass spectrometry. Hsp27 showed decreased abundance in ischemic vessels as expected. The expression of cytoskeletal proteins, namely vimentin was significantly reduced, while transgelin and tropomyosin showed significantly increased abundance in vessels with IHD. Immunohistochemistry studies suggested an increase in G-actin abundance to be present within IHD vessels. The results are consistent with the hypothesis that phospho-Hsp27 protects against vascular disease possibly by stabilizing the actin cytoskeleton within endothelial and/or smooth muscle cells.

Preserving the yeast proteome from sample degradation.

Sample degradation is a common problem in all types of proteomic analyses as it generates protein and peptide fragments that can interfere with analytical results. An important step in preventing such artefacts is to preserve the native, intact proteome as early as possible during sample preparation prior to proteomic analysis. Using the budding yeast Saccharomyces cerevisiae, we have evaluated the effects of trichloroacetic acid (TCA) and thermal treatments prior to protein extraction as a means to minimise proteolysis. TCA precipitation is commonly used to inactivate proteases; thermal stabilisation is used to heat samples to approximately 95 degrees C to inactivate enzyme activity. The efficacy of these methods was also compared with that of protease inhibitors and lyophilisation. Sample integrity was assessed by 2-D PAGE and a selection of spots was identified by MS/MS. The analysis showed that TCA or thermal treatment significantly reduced the degree of degradation and that these pre-treatment protocols were more effective than treatment with either protease inhibitors or lyophilisation. This study establishes standardised sample preparation methods for the reproducible analysis of protein patterns by 2-D PAGE in yeast, and may also be applicable to other proteomic analyses such as gel-free-based quantitation methods.

Assessing the use of thermal treatment to preserve the intact proteomes of post-mortem heart and brain tissue.

Protein degradation that occurs in tissue during post-mortem interval or sample preparation is problematic in quantitative analyses as confounding variables may arise. Ideally, such artefacts should be prevented by preserving the native proteome during sample preparation. We assessed the efficacy of thermal treatment (TT) to preserve the intact proteome of mouse heart and brain tissue in comparison to standard snap-freezing with liquid nitrogen (LN). Tissue samples were collected, either snap frozen (LN), subjected to TT, or snap frozen followed by thermal treatment, and subsequently analysed by 2-DE. In heart tissue, following quantitative image analysis, we observed 77 proteins that were significantly altered across the three treatment groups (ANOVA, p<0.05). Principal component and clustering analyses revealed LN and TT to be equally beneficial. These findings were confirmed by MS identification of the significantly altered proteins. In brain tissue, 189 proteins were significantly differentially expressed across the three treatment groups (ANOVA, p<0.05). Brain tissue appeared to be more responsive to TT than heart and distinct clusters of differentially expressed proteins were observed across treatments. Overall, TT of brain tissue appears to have beneficial effects on protein stabilisation during sample preparation with preservation of high-molecular-weight proteins and reduction in protein fragmentation.

Toll-like receptor agonist induced changes in clonal rat BRIN-BD11 beta-cell insulin secretion and signal transduction.

Evidence for involvement of toll-like receptors (TLRs) (e.g. TLR4 and TLR2, whose agonists include lipopolysaccharides (LPS) and saturated fatty acids) in altered patterns of signalling in adipose, liver and muscle from animal models of insulin resistance and obesity has been published. We have now extended this area of research and have determined the effects of LPS on cell viability, insulin secretion, insulin signalling and metabolism in a clonal beta-cell line. BRIN-BD11 beta-cells were treated for 24 h with increasing concentrations of LPS. Chronic (24 h) and acute (20 min) insulin secretion, insulin content and parameters of cell metabolism and insulin signalling were determined. Incubation of BRIN-BD11 cells for 24 h in the presence of increasing concentrations of the TLR4 ligand LPS significantly decreased chronic (24 h) insulin secretion from 1.09+/-0.19 to 0.76+/-0.18 microg insulin/mg protein in the presence of 100 ng/ml LPS (P<0.05). There was no change in acute (20 min) stimulated insulin secretion or insulin content. Cell metabolism was not changed. Insulin receptor-beta (IR beta) expression levels were increased significantly from 1+/-0.52 to 8.6+/-1.83 units (P<0.01), whereas calcineurin activity and Akt phosphorylation were significantly (P<0.01 and P<0.05 respectively) reduced in response to 24 h incubation in the presence of LPS. There was no change in IR substrate-1 protein expression or phosphorylation after 24 h. Further incubation for 24 h in the absence of LPS resulted in the recovery of chronic insulin secretion. The negative beta-cell effects of LPS may contribute to hyperglycaemia in vivo.

5th BSPR-EBI meeting, proteomics: from Technology to New Biology 8-10 July 2008, Wellcome Trust Conference Centre, Hinxton, Cambridge, UK.

This paper reports on the 5(th) joint British Society for Proteome Research (BSPR) and European Bioinformatics Institute (EBI) meeting which took place at the Wellcome Trust Conference Centre, Cambridge, UK, from the 8(th) to 10(th) July, 2008. As in previous years, the meeting attracted leading experts in the field who presented the latest cutting edge in proteomics. The meeting was entitled "Proteomics: From Technology to New Biology" taking into account the major transition proteomics has undergone in the past few years. In particular, the use of multiple reaction monitoring (MRM)-based targeted experiments for absolute quantification and validation of proteins was the hot topic of the meeting. Attended by some 250 delegates, the conference was extremely well organised and provided a great opportunity for discussion and initiation of new collaborations.

Bacitracin reveals a role for multiple thiol isomerases in platelet function.

The platelet-specific integrin alphaIIb beta3 has endogenous thiol isomerase activity associated with the CXXC motifs within the beta subunit. Using a highly purified form of bacitracin, a thiol isomerase inhibitor, we now provide further evidence of the functional significance of this enzymatic activity in integrin activation. In addition, we demonstrate a role for multiple thiol isomerases in platelet function. This bacitracin prevented platelet aggregation to thrombin and collagen, and directly inhibited alphaIIb beta3 activation, as detected by PAC-1 binding. In parallel, bacitracin inhibited the endogenous thiol isomerase activity of purified alphaIIb beta3 with a 50% inhibitory concentration of 15.5 micromol/l. In order to determine whether the effects of bacitracin are solely mediated by inhibition of integrin enzymatic activity, we examined integrin-independent indices of platelet activation. We found bacitracin inhibited both platelet secretion (CD62P and CD63) and thromboxane (TxA2) production, with complete inhibition at different concentrations. Thus, we demonstrated a role for multiple thiol isomerases in platelet function. Taken together, these studies support a role for the endogenous integrin thiol isomerase activity in activation of alphaIIb beta3 and highlight the novel regulation of platelet function by other, as yet undefined thiol isomerases.

Novel integrin-specific targets for anti-platelet therapies.

Extensive study in integrin research has seen the platelet specific receptor alpha(IIb)beta(3) (Glycoprotein GPIIb/IIIa) under much scrutiny, and provided vast information as to the workings of this integrin within the blood. Glanzmann's thrombasthenia, a rare autosomal recessive bleeding disorder, highlights the vital role played by this receptor in platelet function. Glanzmann's thrombasthenic platelets fail to aggregate due to a lack of surface expression of functional alpha(IIb) or beta(3) on the platelet surface. However, little is known about the precise molecular mechanisms involved in the operation of this receptor on the platelet surface. Clinical trials using intravenous antagonists to this receptor have shown them to be effective anti-thrombotics. However the recent observations that the oral alpha(IIb)beta(3)antagonists have failed to show benefits in the treatment of acute coronary syndromes, and in fact, increase mortality, underscores the necessity for a more complete understanding of alpha(IIb)beta(3) and its functions. A more profound knowledge of the precise nature of the platelet integrin-activation, along with an understanding of its interactions with cellular signaling proteins, will undoubtedly lead to the identification of novel strategies for the effective inhibition of platelet integrin function.