Coomassie blue as a near-infrared fluorescent stain: a systematic comparison with Sypro Ruby for in-gel protein detection.

Quantitative proteome analyses suggest that the well-established stain colloidal Coomassie Blue, when used as an infrared dye, may provide sensitive, post-electrophoretic in-gel protein detection that can rival even Sypro Ruby. Considering the central role of two-dimensional gel electrophoresis in top-down proteomic analyses, a more cost effective alternative such as Coomassie Blue could prove an important tool in ongoing refinements of this important analytical technique. To date, no systematic characterization of Coomassie Blue infrared fluorescence detection relative to detection with SR has been reported. Here, seven commercial Coomassie stain reagents and seven stain formulations described in the literature were systematically compared. The selectivity, threshold sensitivity, inter-protein variability, and linear-dynamic range of Coomassie Blue infrared fluorescence detection were assessed in parallel with Sypro Ruby. Notably, several of the Coomassie stain formulations provided infrared fluorescence detection sensitivity to <1 ng of protein in-gel, slightly exceeding the performance of Sypro Ruby. The linear dynamic range of Coomassie Blue infrared fluorescence detection was found to significantly exceed that of Sypro Ruby. However, in two-dimensional gel analyses, because of a blunted fluorescence response, Sypro Ruby was able to detect a few additional protein spots, amounting to 0.6% of the detected proteome. Thus, although both detection methods have their advantages and disadvantages, differences between the two appear to be small. Coomassie Blue infrared fluorescence detection is thus a viable alternative for gel-based proteomics, offering detection comparable to Sypro Ruby, and more reliable quantitative assessments, but at a fraction of the cost.

Eastern Canadian Colorectal Cancer Consensus Conference: standards of care for the treatment of patients with rectal, pancreatic, and gastrointestinal stromal tumours and pancreatic neuroendocrine tumours.

The annual Eastern Canadian Colorectal Cancer Consensus Conference was held in Halifax, Nova Scotia, October 20-22, 2011. Health care professionals involved in the care of patients with colorectal cancer participated in presentation and discussion sessions for the purposes of developing the recommendations presented here. This consensus statement addresses current issues in the management of rectal cancer, including pathology reporting, neoadjuvant systemic and radiation therapy, surgical techniques, and palliative care of rectal cancer patients. Other topics discussed include multidisciplinary cancer conferences, treatment of gastrointestinal stromal tumours and pancreatic neuroendocrine tumours, the use of folfirinox in pancreatic cancer, and treatment of stage ii colon cancer.

Actin is not an essential component in the mechanism of calcium-triggered vesicle fusion.

Actin has been suggested as an essential component in the membrane fusion stage of exocytosis. In some model systems disruption of the actin filament network associated with exocytotic membranes results in a decrease in secretion. Here we analyze the fast Ca2+-triggered membrane fusion steps of regulated exocytosis using a stage-specific preparation of native secretory vesicles (SV) to directly test whether actin plays an essential role in this mechanism. Although present on secretory vesicles, selective pharmacological inhibition of actin did not affect the Ca2+-sensitivity, extent, or kinetics of membrane fusion, nor did the addition of exogenous actin or an anti-actin antibody. There was also no discernable affect on inter-vesicle contact (docking). Overall, the results do not support a direct role for actin in the fast, Ca2+-triggered steps of regulated membrane fusion. It would appear that actin acts elsewhere within the exocytotic cycle.

Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis.

BACKGROUND: The analysis of hydrophobic membrane proteins by two-dimensional gel electrophoresis has long been hampered by the concept of inherent difficulty due to solubility issues. We have optimized extraction protocols by varying the detergent composition of the solubilization buffer with a variety of commercially available non-ionic and zwitterionic detergents and detergent-like phospholipids. RESULTS: After initial analyses by one-dimensional SDS-PAGE, quantitative two-dimensional analyses of human erythrocyte membranes, mouse liver membranes, and mouse brain membranes, extracted with buffers that included the zwitterionic detergent MEGA 10 (decanoyl-N-methylglucamide) and the zwitterionic lipid LPC (1-lauroyl lysophosphatidylcholine), showed selective improvement over extraction with the common 2-DE detergent CHAPS (3 [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Mixtures of the three detergents showed additive improvements in spot number, density, and resolution. Substantial improvements in the analysis of a brain membrane proteome were observed. CONCLUSION: This study demonstrates that an optimized detergent mix, coupled with rigorous sample handling and electrophoretic protocols, enables simple and effective analysis of membrane proteomes using two-dimensional electrophoresis.

Collagen production and replication by cardiac fibroblasts is enhanced in response to diverse classes of growth factors.

The tissue distribution and cellular effects of platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta-1 (TGF beta 1) and insulin-like growth factor 1 (IGF-1) suggest a potential role for these factors in cardiovascular matrix deposition. The objective of this study was to assess the capacity of these growth factors to promote cardiac fibroblast collagen production and replication in vitro which will lead to studies identifying their role in vivo during cardiac development and disease. Fibroblasts were isolated from fetal rat hearts by explant culture, and their response to growth factors was assessed with respect to fibroblast replication and collagen synthesis. Fibroblast replication was stimulated by PDGF and by bFGF.IGF-1 and TGF beta 1 had no effect on fibroblast replication. Collagen production was stimulated by all of the growth factors tested in order of potency TGF beta 1 > PDGF, IGF > bFGF. None of the growth factors affected the proportion of newly synthesized collagen rapidly degraded. We have shown that TGF beta 1, PDGF, bFGF and IGF-1 are all capable of increasing collagen deposition by cardiac fibroblasts by either stimulating fibroblast replication or collagen synthesis or both. The sensitivity of cardiac fibroblasts to these factors is consistent with their playing a role in the rapid changes in cardiac collagen deposition seen during development and disease.

Endothelial cells produce a lipoxygenase derived chemo-repellent which influences platelet/endothelial cell interactions--effect of aspirin and salicylate.

We performed experiments to determine whether endothelial cells synthesize phospholipid metabolites via the lipoxygenase pathway and whether these metabolites influence platelet/vessel wall interactions. Monolayers of cultured human endothelial cells were incubated with 14C-arachidonic acid and their cyclo-oxygenase and lipoxygenase metabolites were extracted and identified by radioimmunoassay, thin layer chromatography and high performance liquid chromatography. We found that in addition to the membrane-associated production of PGI2, endothelial cells synthesized a cytosol-associated metabolite, LOX, which was presumably derived through the lipoxygenase pathway. Inhibition of LOX was associated with an increase in PGI2 production and inhibition of PGI2 with an increase in LOX production. Under either condition, platelet adhesion to cultured endothelial cells was significantly decreased. In contrast, when both PGI2 and LOX production were inhibited, platelet adhesion to endothelial cells was enhanced. Furthermore, when LOX was bound to a thrombogenic surface, platelet adhesion was significantly decreased whereas when arachidonic acid or 12-HETE was bound to the surface, platelet adhesion was increased. We conclude that endothelial cells produce not only a cyclo-oxygenase metabolite, but also a lipoxygenase metabolite, both of which influence platelet/endothelial cell interactions.

The sex-related differences in aspirin pharmacokinetics in rabbits and man and its relationship to antiplatelet effects.

There are a number of reports which suggest that the antithrombotic effect of aspirin is limited to males. It is unclear whether this effect is due to sex-related differences in the effect of aspirin on platelets, the vessel wall, or the pharmacokinetics of aspirin. To test these possibilities we examined the sex-related differences in (1) vessel wall PGI2 release and its inhibition by and recovery from aspirin in rabbits; (2) the effects of aspirin on platelet aggregation, thromboxane B2 and beta-thromboglobulin (BTG) release in man, and (3) the pharmacokinetic characteristics of aspirin, in both rabbits and man. Vascular wall PGI2 measured as 6-keto-PGF1 alpha, was not different in male and females rabbits, and was inhibited to a similar extent by identical concentrations of aspirin. The duration of this inhibitory effect was also the same in males and females. The pattern of inhibition of collagen-induced platelet aggregation, and collagen-induced thromboxane B2 and BTG release by aspirin were not different in either sex. There was, however, a sex-related difference in a number of pharmacokinetic characteristics of aspirin both in rabbits and man. Thus, aspirin was absorbed more rapidly, distributed in larger apparent volume and was hydrolysed more rapidly in females. These observations suggest that the sex-related differences in the antithrombotic effects of aspirin seen in clinical studies are not due to differences in the effects of aspirin on the inhibition of platelet function mediated by the inhibition of cyclo-oxygenase in either the platelet or the vessel wall. An effect of aspirin on platelet function independent of the inhibition of cyclo-oxygenase has been described and it is possible that this effect may be influenced by sex-related differences in the pharmacokinetics of aspirin.

Effect of suloctidil on platelet function in patients with shortened platelet survival time.

The effect of suloctidil (600 mg/day) on platelet survival time (PST) and plasma and urine betathromboglobulin (BTG) was studied in a double-blind, placebo-controlled six-week crossover trial in 13 patients with shortened PST (less than 110 hrs, exponential model). Mean PST after suloctidil (110.6 hrs) was significantly higher than in the placebo phase (94.5 hrs) (p = 0.04). Mean plasma BTG was significantly lower during the suloctidil phase (42.8 ng/ml) compared with the placebo phase (65.8 ng/ml) (p = 0.02), but there was no significant difference in urine BTG. These results suggest that suloctidil provides a platelet protective effect and therefore may be of benefit in reducing the frequency of platelet mediated thromboembolic events.

Effect of R and S enantiomers of naproxen on aggregation and thromboxane production in human platelets.

The effects of R and S enantiomers of naproxen [(+)-6-methoxy-alpha-methyl- 2-naphthaleneacetic acid] were studied on platelet aggregation and on the production of thromboxane B2 from collagen-stimulated human platelets in order to determine the effect of each enantiomer in terms of cyclooxygenase inhibition. S-Naproxen caused inhibition of platelet aggregation in platelet-rich plasma and washed human platelets in a concentration-related fashion in the range 1-80 micrograms/L. A similar concentration-related suppression was noted for R-naproxen, but this inhibition was significantly less than that induced by S-naproxen for all concentrations except 1 micrograms/L. Similarly, both R- and S-naproxen (1-80 micrograms/L) caused a concentration-dependent suppression of thromboxane B2 production from platelet-rich plasma. These values were significant at all concentrations of drug (10-80 micrograms/L) except at 1 micrograms/L. Significant differences in thromboxane B2 production from washed human platelets were noted at concentrations of 10 and 25 micrograms/L. The findings support previous studies reported in the literature that S-naproxen is more active than R-naproxen. Our findings that S-naproxen is more active than R-naproxen on collagen-stimulated platelet aggregation and prostaglandin production suggest that the findings of greater activity of S isomer over the R isomer in animal models of inflammation may be a direct expression of the differential action on prostaglandin synthesis.

Stereotactic biopsy of brain tumours.

Computerized tomography assisted Stereotactic biopsy technique using Leksell stereotactic frame was performed on 27 patients with small, multiple and deep seated brain tumours. There were 19 men and 8 women with an age range from 17 to 65 years. Histological diagnosis of 18 glial tumours, 9 non-glial tumours (5 colloid cysts, 4 metastatic lesions) was obtained. There was no mortality and minimal morbidity of 3.7%, histological diagnosis provided the information regarding differentiation from infectious and vascular lesions and grading of malignancy leading to logical guidance for therapeutic management of each lesion, confirming the value of stereotactic biopsy in brain tumours.

How soon is too soon? Addiction recovery and family reunification.

This article describes the addiction recovery process and its impact on parenting behaviors-information needed by child welfare workers involved in decisionmaking regarding family reunification. Two models of recovery-from alcoholism and from cocaine addiction-are reviewed, along with issues encountered in recovery, particularly for women. Case examples and discussion demonstrate how child welfare workers can apply these models in determining the appropriateness of reunification.

Alterations in protein and gene expression within the barrel cortices of ZnT3 knockout mice: experience-independent and dependent changes.

Much evidence exists for the involvement of vesicular zinc in neurotransmission and cortical plasticity. Recent studies have reported that mice deficient in zinc transporter-3 protein (ZnT3) and thus, vesicular zinc, have significant behavioural and biochemical deficits. Here, we examined whether phenotypic differences existed in the barrel cortices of ZnT3 KO mice using functional proteomics and quantitative PCR. Additionally, by manipulating whisker input, we also investigated experience-dependent changes in protein and gene expression, thereby assaying how cortical plasticity is different in the absence of vesicular zinc. The GABA metabolizing protein ABAT was observed in lower abundances consistently in KO mice. Several presynaptic proteins were identified that were abundant in differing amounts between the WT and KO groups in an experience-dependent manner. At baseline, we observed a decrease in the relative expression of Dlg4, Grin2a, Mt3, and Ntrkb genes in KO mice. The reduced expression of Nrtkb persisted with whisker plucking. These data demonstrate that fundamental changes in the expression of proteins and genes important in neurotransmission occur in the absence of vesicular zinc. Furthermore, the complement of experience-dependent changes were different between WT and KO mice, indicating that the lack of vesicular zinc affects the process of cortical plasticity.

Enabling coupled quantitative genomics and proteomics analyses from rat spinal cord samples.

Translational research is progressing toward combined genomics and proteomics analyses of small and precious samples. In our analyses of spinal cord material, we systematically evaluated disruption and extraction techniques to determine an optimum process for the coupled analysis of RNA and protein from a single 5-mm segment of tissue. Analyses of these distinct molecular species were performed using microarrays and high resolution two-dimensional gels, respectively. Comparison of standard homogenization with automated frozen disruption (AFD) identified negligible differences in the relative abundance of genes (44) with all genes identified by either process. Analysis on either the Affymetrix or Applied Biosystems Inc. gene array platforms provided good correlations between the extraction techniques. In contrast, the AFD technique enabled identification of more unique proteins from spinal cord tissue than did standard homogenization. Furthermore use of an optimized CHAPS/urea extraction provided better protein recovery, as shown by quantitative two-dimensional gel analyses, than did solvent precipitation during TRIzol-based RNA extraction. Thus, AFD of tissue samples followed by protein and RNA isolation from separate aliquots of the frozen powdered sample is the most effective route to ensure full, quantitative analyses of both molecular entities.

Assessing detection methods for gel-based proteomic analyses.

Proteomic analyses using two-dimensional gel electrophoresis (2DE) depend heavily upon the quality of protein stains for sensitive detection. Indeed, detection rather than protein resolution is likely a current limiting factor in 2DE. The recent development of fluorescent protein stains has dramatically improved the sensitivity of in-gel protein detection and has enabled more accurate protein quantification. Here, we have evaluated the overall quality and relative cost of five commercially available fluorescent stains, Krypton, Deep Purple, Rubeo, Flamingo, and the most commonly used stain, Sypro Ruby (SR). All stains were found to be statistically comparable with regard to number of protein spots detected, but SR was superior with regard to fluorophore stability (e.g., capacity for repeated use of the stain solution). Notably, colloidal Coomassie Blue was also found to be comparable to SR when detected using an infrared fluorescence imaging system rather than standard densitometry. Thus, depending on available equipment and operating budgets, there are at least two high-sensitivity alternatives to achieve the best currently available in-gel protein detection: Sypro Ruby or Coomassie Blue.

Pre-extraction sample handling by automated frozen disruption significantly improves subsequent proteomic analyses.

Here we quantitatively characterize two common homogenization strategies in the analysis of tissue proteomes: classical manual homogenization (MH) and an automated frozen disruption (AFD) technique. In a variety of tissues, many proteins were more efficiently extracted, resolved and detected, with high reproducibility after AFD, amounting to as much as 2% of the total resolved proteome. The benefits of AFD over MH are 2-fold: (1) AFD yields a much more thorough homogenate than MH; and (2) as a deep frozen alternative, AFD maintains a level of biological complexity that is not retained during MH. Thus, AFD coupled with refined 2DE protocols and Sypro Ruby staining yields quantitative proteomic analyses.

An initial proteomic analysis of human preterm labor: placental membranes.

Human preterm labor (PL) is the single most significant problem in modern Obstetrics and Gynecology, affecting approximately 10% of pregnancies worldwide, constituting the leading cause of perinatal mortality and morbidity, and contributing significantly to chronic childhood disease. Currently, our molecular understanding of PL remains staggeringly inadequate to reliably diagnose or rationally intervene in PL events; several molecular alterations have been implicated in PL, but these have proven of limited value as diagnostic/prognostic markers. The majority of PL events remain spontaneous and unpredictable: critical care emergencies. Here, we apply functional proteomics to dissect molecular mechanisms of human PL. Human placental tissue was collected in clearly differentiated cases of preterm and term labor. Highly refined two-dimensional gel electrophoresis (2DE) was used for protein separation, coupled with automated differential gel image analysis to compare the resulting proteomic maps. For this initial study, only the most important protein differences were selected for further analysis, that is, proteins that were unique to one sample, and absent from the other, with 100% reproducibility across the sample population. In total, 11 such proteins were identified by tandem mass spectrometry, falling into three distinct functional classes: structural/cytoskeletal components, ER lumenal proteins with enzymatic or chaperone functions, and proteins with anticoagulant properties. These expression changes form the groundwork for further molecular investigation of this devastating medical condition. This approach therefore holds the potential not only to define the underlying molecular components, but also to identify novel diagnostic tools and targets for rational drug intervention.