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Biomedical subjects

Jens R Coorssen

Publications and source records attributed to Jens R Coorssen.

16 recordsLinked to original sources
PubMed2006

Recent advances in basic neurosciences and brain disease: from synapses to behavior.

Understanding basic neuronal mechanisms hold the hope for future treatment of brain disease. The 1st international conference on synapse, memory, drug addiction and pain was held in beautiful downtown Toronto, Canada on August 21-23, 2006. Unlike other traditional conferences, this new meeting focused on three major aims: (1) to promote new and cutting edge research in neuroscience; (2) to encourage international information exchange and scientific collaborations; and (3) to provide a platform for active scientists to discuss new findings. Up to 64 investigators presented their recent discoveries, from basic synaptic mechanisms to genes related to human brain disease. This meeting was in part sponsored by Molecular Pain, together with University of Toronto (Faculty of Medicine, Department of Physiology as well as Center for the Study of Pain). Our goal for this meeting is to promote future active scientific collaborations and improve human health through fundamental basic neuroscience researches. The second international meeting on Neurons and Brain Disease will be held in Toronto (August 29-31, 2007).

Behavior↗
PubMed2006

Sphingomyelin-enriched microdomains define the efficiency of native Ca(2+)-triggered membrane fusion.

Membrane microdomains or ;rafts' are suggested to act as regulators of the exocytotic process and also appear to be the sites of Ca(2+)-triggered membrane fusion. Microdomains are postulated to maintain the localization of ;efficiency' factors, including Ca(2+) sensors and other protein and lipid components. Separation of the fundamental ability to fuse from the efficiency of the process has suggested dependence of efficiency factors on microdomain organization. Cholesterol, a key component of membrane microdomains, contributes to both the efficiency and the fundamental ability to fuse. However, testing for a selective effect of native microdomains on the efficiency of fusion, without affecting membrane cholesterol density, has not been assessed. Hydrolysis of sphingomyelin disrupts native raft domains on secretory vesicles. Disruption of microdomains enriched in sphingomyelin-cholesterol by treatment with sphingomyelinase selectively and dose dependently inhibited the Ca(2+) sensitivity and late kinetics of secretory vesicle fusion. As a native microdomain constituent, sphingomyelin is associated with Ca(2+) sensing through its interaction with other raft-bound lipid and/or protein factors, thereby supporting the physiological Ca(2+) sensitivity of membrane fusion. Furthermore, the sphingomyelinase-driven generation of ceramide, contributing to the total membrane negative curvature, preserves the ability to fuse despite extensive cholesterol removal. Membrane microdomain integrity thus underlies the efficiency of fusion but not the fundamental ability of native vesicles to undergo Ca(2+)-triggered membrane merger. The results are consistent with a fundamental fusion machine of intrinsically low Ca(2+) sensitivity that, supported by accessory ;efficiency' components, facilitates Ca(2+)-triggered bilayer merger under physiological conditions.

Animals↗
PubMed2006

An evaluation of in vitro protein-protein interaction techniques: assessing contaminating background proteins.

Determination of protein-protein interactions is an important component in assigning function and discerning the biological relevance of proteins within a broader cellular context. In vitro protein-protein interaction methodologies, including affinity chromatography, coimmunoprecipitation, and newer approaches such as protein chip arrays, hold much promise in the detection of protein interactions, particularly in well-characterized organisms with sequenced genomes. However, each of these approaches attracts certain background proteins that can thwart detection and identification of true interactors. In addition, recombinant proteins expressed in Escherichia coli are also extensively used to assess protein-protein interactions, and background proteins in these isolates can thus contaminate interaction studies. Rigorous validation of a true interaction thus requires not only that an interaction be found by alternate techniques, but more importantly that researchers be aware of and control for matrix/support dependence. Here, we evaluate these methods for proteins interacting with DmsD (an E. coli redox enzyme maturation protein chaperone), in vitro, using E. coli subcellular fractions as prey sources. We compare and contrast the various in vitro interaction methods to identify some of the background proteins and protein profiles that are inherent to each of the methods in an E. coli system.

Blotting, Far-Western↗
PubMed2006

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.

Animals↗
PubMed2006

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.

Anticoagulants↗
PubMed2006

Proteome resolution by two-dimensional gel electrophoresis varies with the commercial source of IPG strips.

By facilitating reproducible first dimension separations, commercial immobilized pH gradient (IPG) strips enable high throughput and high-resolution proteomic analyses using two-dimensional gel electrophoresis (2DE). Amersham, Biorad, Invitrogen, and Sigma all market linear pH 3-10 IPG strips. We have applied optimized 2DE protocols with both membrane and soluble brain protein extracts to critically evaluate all four products. Resolved protein spots were quantitatively evaluated after carrying out these protocols using IPG strips from the four companies. Biorad and Amersham IPG strips resolved a high number of membrane and soluble proteins, respectively. Furthermore, Amersham IPG strips eluted the largest amount of protein into the second dimension gels and had the most protein remaining in the strip after 2DE. Biorad and Amersham IPG strips maintained a consistent linear pH 3-10 gradient, whereas those from Invitrogen appeared nonlinear or "compressed" within the central pH region. The gradient range within Sigma IPG strips appeared to be slightly less than pH 3-10, due to one extended pH unit within the gradient. Overall, all four commercially available IPG strips have the ability to resolve both membrane and soluble brain proteomes. The difference is that Amersham and Biorad do so more consistently and with better spot resolution. It appears that the physical/chemical nature of commercially available IPG strips can vary considerably, leading to marked differences in subsequent protein resolution in 2DE. These differences likely reflect variations in the uptake of proteins into the strips, and differences in the focusing and elution of proteins from the first to the second dimension. These differences would appear, in part, to underlie some inter-lab variations in the effective resolution of proteomes.

Animals↗
PubMed2005

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.

Actin Depolymerizing Factors↗
PubMed2005

Cholesterol facilitates the native mechanism of Ca2+-triggered membrane fusion.

The process of regulated exocytosis is defined by the Ca2+-triggered fusion of two apposed membranes, enabling the release of vesicular contents. This fusion step involves a number of energetically complex steps and requires both protein and lipid membrane components. The role of cholesterol has been investigated using isolated release-ready native cortical secretory vesicles to analyze the Ca2+-triggered fusion step of exocytosis. Cholesterol is a major component of vesicle membranes and we show here that selective removal from membranes, selective sequestering within membranes, or enzymatic modification causes a significant inhibition of the extent, Ca2+ sensitivity and kinetics of fusion. Depending upon the amount incorporated, addition of exogenous cholesterol to cholesterol-depleted membranes consistently recovers the extent, but not the Ca2+ sensitivity or kinetics of fusion. Membrane components of comparable negative curvature selectively recover the ability to fuse, but are unable to recover the kinetics and Ca2+ sensitivity of vesicle fusion. This indicates at least two specific positive roles for cholesterol in the process of membrane fusion: as a local membrane organizer contributing to the efficiency of fusion, and, by virtue of its intrinsic negative curvature, as a specific molecule working in concert with protein factors to facilitate the minimal molecular machinery for fast Ca2+-triggered fusion.

Animals↗
PubMed2005

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.

Journal Article↗
PubMed2003

Revisiting the role of SNAREs in exocytosis and membrane fusion.

For over a decade SNARE hypotheses have been proposed to explain the mechanism of membrane fusion, yet the field still lacks sufficient evidence to conclusively identify the minimal components of native fusion. Consequently, debate concerning the postulated role(s) of SNAREs in membrane fusion continues. The focus of this review is to revisit original literature with a current perspective. Our analysis begins with the earliest studies of clostridial toxins, leading to various cellular and molecular approaches that have been used to test for the roles of SNAREs in exocytosis. We place much emphasis on distinguishing between specific effects on membrane fusion and effects on other critical steps in exocytosis. Although many systems can be used to study exocytosis, few permit selective access to specific steps in the pathway, such as membrane fusion. Thus, while SNARE proteins are essential to the physiology of exocytosis, assay limitations often prevent definitive conclusions concerning the molecular mechanism of membrane fusion. In all, the SNAREs are more likely to function upstream as modulators or priming factors of fusion.

Animals↗
PubMed2003

Calcium-triggered membrane fusion proceeds independently of specific presynaptic proteins.

Complexes of specific presynaptic proteins have been hypothesized to drive or catalyze the membrane fusion steps of exocytosis. Here we use a stage-specific preparation to test the roles of SNAREs, synaptotagmin, and SNARE-binding proteins in the mechanism of Ca2+-triggered membrane fusion. Excess exogenous proteins, sufficient to block SNARE interactions, did not inhibit either the Ca2+ sensitivity, extent, or kinetics of fusion. In contrast, despite a limited effect on SNARE and synaptotagmin densities, treatments with high doses of chymotrypsin markedly inhibited fusion. Conversely, low doses of chymotrypsin had no effect on the Ca2+ sensitivity or extent of fusion but did alter the kinetic profile, indicating a more direct involvement of other proteins in the triggered fusion pathway. SNAREs, synaptotagmin, and their immediate binding partners are critical to exocytosis at a stage other than membrane fusion, although they may still influence the triggered steps.

Animals↗
PubMed2003

Regulated secretion: SNARE density, vesicle fusion and calcium dependence.

SNAREs such as VAMP, SNAP-25 and syntaxin are essential for intracellular trafficking, but what are their exact molecular roles and how are their interactions with other proteins manifest? Capitalizing on the differential sensitivity of SNAREs to exogenous proteases, we quantified the selective removal of identified SNAREs from native secretory vesicles without loss of fusion competence. Using previously established fusion assays and a high sensitivity immunoblotting protocol, we analyzed the relationship between these SNARE proteins and Ca2+-triggered membrane fusion. Neither the extent of fusion nor the number of intermembrane fusion complexes per vesicle were correlated with the measured density of identified egg cortical vesicle (CV) SNAREs. Without syntaxin, CVs remained fusion competent. Surprisingly, for one (but not another) protease the Ca2+ dependence of fusion was correlated with CV SNARE density, suggesting a native protein complex that associates with SNAREs, the architecture of which ensures high Ca2+ sensitivity. As SNAREs may function during CV docking in vivo, and as further proteolysis after SNARE removal eventually ablates fusion, we hypothesize that the triggered steps of regulated fusion (Ca2+ sensitivity and the catalysis and execution of fusion) require additional proteins that function downstream of SNAREs.

Animals↗
PubMed2002

Quantitative femto- to attomole immunodetection of regulated secretory vesicle proteins critical to exocytosis.

Although immunoblotting (Western blotting) is widely used for the detection of specific proteins, it is often thought to be an inadequate technique for accurate and precise measurements of protein concentration. However, an accurate and precise technique is essential for quantitative testing of hypotheses, and thus for the analysis and understanding of proposed molecular mechanisms. The analysis of Ca(2+)-triggered exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents, requires such an unambiguous identification and a quantitative assessment of the membrane surface density of specific molecules. Newly refined immunoblotting and analysis approaches permit a quantitative analysis of the SNARE protein complement (VAMP, SNAP-25, and syntaxin) of functional secretory vesicles. The method illustrates the feasibility of the routine quantification of femtomole to attomole amounts of known proteins by immunoblotting. The results indicate that sea urchin egg secretory vesicles and synaptic vesicles have markedly similar SNARE densities.

Animals↗
PubMed2002

De novo sequencing of peptides using MALDI/TOF-TOF.

The recently developed MALDI TOF-TOF instrument yields relatively complex but interpretable fragmentation spectra. When coupled with a straightforward sequence extension algorithm, it is possible to develop complete peptide sequences de novo from the spectra. This approach has been applied to a set of peptides derived from typtic digestion of electrophoretically separated sea urchin egg membrane proteins. When directed to proteins that have been described previously, the results were in essential agreement with those obtained by conventional data base searching approaches, with certain important exceptions. The present method detected errors in published sequences and was able to develop sequences from peptides differing in mass by one dalton (Da). These results show both the power of the present approach and the need for using de novo methods more frequently than may be otherwise appreciated.

Algorithms↗
PubMedDate not supplied

Postfractionation for enhanced proteomic analyses: routine electrophoretic methods increase the resolution of standard 2D-PAGE.

Here we have addressed common issues of resolution in two-dimensional polyacrylamide gel electrophoresis (2DE) experiments including proteins 'stacked' at pH extremes, unresolved peptides migrating at the front of separation, and areas of the 2D gel obscured by high abundance proteins. Postfractionation, by selective application of well-established electrophoretic separations immediately following standard 2DE, yields markedly improved resolution in these traditional problem areas using no more specialized equipment or techniques than SDS-PAGE itself.

Animals↗