Biomedical subjects

Gabriel E Weinreb

Publications and source records attributed to Gabriel E Weinreb.

4 recordsLinked to original sources

PubMed2006

Analysis method for measuring submicroscopic distances with blinking quantum dots.

A method is described that takes advantage of the intermittency ("blinking") in the fluorescence of quantum dots (QDs) to measure absolute positions of closely spaced QDs. The concept is that even if two QDs are separated by only tens of nanometers, the position of each QD is resolvable if the point spread function of each can be imaged independently of the other. In the case of QDs, this is possible if each QD separately blinks completely on and off during a time-lapse sequence. To demonstrate the principle of this method, time-lapse sequences of single blinking QDs were acquired and the centroids of the point spread functions determined. Images of the blinking QDs were then overlapped in software, pixel by pixel, generating a range of submicroscopic distances between QD pairs. Methods were developed for analyzing the overlapped time sequences of the QD pairs so that the positions of the QDs and the distances between them could be determined without prior knowledge of the single QD positions. We subsequently used this method to measure the end-to-end length of a 122-basepair double-stranded DNA fragment.

Biotinylation↗

PubMed2006

Causal mapping as a tool to mechanistically interpret phenomena in cell motility: application to cortical oscillations in spreading cells.

Biological processes that occur at the cellular level and consist of large numbers of interacting elements are highly nonlinear and generally involve multiple time and spatial scales. The quantitative description of these complex systems is of great importance but presents large challenges. We outline a new systems biology approach, causal mapping (CMAP), which is a coarse-grained biological network tool that permits description of causal interactions between the elements of the network and overall system dynamics. On one hand, the CMAP is an intermediate between experiments and physical modeling, describing major requisite elements, their interactions and paths of causality propagation. On the other hand, the CMAP is an independent tool to explore the hierarchical organization of cell and the role of uncertainties in the system. It appears to be a promising easy-to-use technique for cell biologists to systematically probe verbally formulated qualitative hypotheses. We apply the CMAP to study the phenomenon of contractility oscillations in spreading cells in which microtubules have been depolymerized. The precise mechanism by which these oscillations are governed by a complex mechano-chemical system is not known but the data observed in experiments can be described by a CMAP. The CMAP suggests that the source of the oscillations results from the opposing effects of Rho activation leading to a decreased level of myosin light chain phosphatase and a cyclic calcium influx caused by increased membrane tension and leading to a periodically enhanced activation of myosin light chain kinase.

Animals↗

PubMed2005

Detecting microdomains in intact cell membranes.

Current models for cellular plasma membranes focus on spatial heterogeneity and how this heterogeneity relates to cell function. In particular, putative lipid raft membrane domains have been postulated to exist based in large part on the results that a significant fraction of the membrane is detergent insoluble and that molecules facilitating key membrane processes like signal transduction are often found in the detergent-resistant membrane fraction. Yet, the in vivo existence of lipid rafts remains extremely controversial because, despite being sought for more than a decade, evidence for their presence in intact cell membranes is inconclusive. In this review, a variety of experimental techniques that have been or might be used to look for lipid microdomains in intact cell membranes are described. Experimental results are highlighted and the strengths and limitations of different techniques for microdomain identification and characterization are assessed.

Biomarkers↗

PubMed2002

Cooperative roles of factor V(a) and phosphatidylserine-containing membranes as cofactors in prothrombin activation.

Activation of prothrombin, as catalyzed by the prothrombinase complex (factor X(a), enzyme; factor V(a) and phosphatidylserine (PS)-containing membranes, cofactors), involves production and subsequent proteolysis of two possible intermediates, meizothrombin (MzII(a)) and prethrombin 2 plus fragment 1.2 (Pre2 & F1.2). V(max), K(m), or V(max)/K(m) for all four proteolytic steps was determined as a function of membrane-phospholipid concentration. Proteolysis was monitored using a fluorescent thrombin inhibitor, a chromogenic substrate, and SDS-PAGE. The kinetic constants for the conversion of MzII(a) and Pre2 & F1.2 to thrombin were determined directly. Pre2 & F1.2 conversion was linear in substrate concentration up to 4 microm, whereas MzII(a) proteolysis was saturable. First order rate constants for formation of MzII(a) and Pre2 & F1.2 could not be determined directly and were determined from global fitting of the data to a parallel, sequential model, each step of which was treated by the Michaelis-Menten formalism. The rate of direct conversion to thrombin without release of intermediates from the membrane-V(a)-X(a) complex (i.e. "channeling") also was adjusted because both the membranes and factor V(a) have been shown to cause channeling. k(cat), K(m), or k(cat)/K(m) values were reported for one lipid concentration, for which all X(a) was likely incorporated into a X(a)-V(a) complex on a PS membrane. Comparing previous results, which were obtained either with factor V(a) (Boskovic, D. S., Bajzar, L. S., and Nesheim, M. E. (2001) J. Biol. Chem. 276, 28686-28693) or with membranes individually (Wu, J. R., Zhou, C., Majumder, R., Powers, D. D., Weinreb, G., and Lentz, B. R. (2002) Biochemistry 41, 935-949), with results presented here we conclude that both factor V(a) and PS-containing membranes induce similar rate increases and pathway changes. Moreover, we have determined: 1) factor V(a) has the greatest effect in enhancing rates of individual proteolytic events; 2) PS-containing membranes have the greatest role in increasing the preference for the MzII(a) versus Pre2 pathway; and 3) PS membranes cause approximately 50% of the substrate to be activated via channeling at 50 microm membrane concentration, but factor V(a) extends the range of efficient channeling to much lower or higher membrane concentrations.

Binding Sites↗