Biomedical subjects

W R Burack

Publications and source records attributed to W R Burack.

11 recordsLinked to original sources

PubMed2001

The immunological synapse.

The adaptive immune response is initiated by the interaction of T cell antigen receptors with major histocompatibility complex molecule-peptide complexes in the nanometer scale gap between a T cell and an antigen-presenting cell, referred to as an immunological synapse. In this review we focus on the concept of immunological synapse formation as it relates to membrane structure, T cell polarity, signaling pathways, and the antigen-presenting cell. Membrane domains provide an organizational principle for compartmentalization within the immunological synapse. T cell polarization by chemokines increases T cell sensitivity to antigen. The current model is that signaling and formation of the immunological synapse are tightly interwoven in mature T cells. We also extend this model to natural killer cell activation, where the inhibitory NK synapse provides a striking example in which inhibition of signaling leaves the synapse in its nascent, inverted state. The APC may also play an active role in immunological synapse formation, particularly for activation of naïve T cells.

Animals↗

PubMed2000

Signal transduction: hanging on a scaffold.

Recent data concerning scaffolding proteins profoundly challenge our conceptions of multicomponent signal transduction systems. Recent studies of the phototransduction system in Drosophila suggest two points. First, scaffolding markedly limits the possibilities for signal amplification. Second, the methods generally available to study signal transduction may be too crude to assess the in vivo roles of scaffolds. Studies of the mitogen-activated protein kinase pathway scaffold, Ste5, indicate functions beyond that of a passive structural element. Finally, the identification of new mitogen-activated protein kinase pathway scaffolds suggests the existence of multiple 'signalosomes' or 'transducisomes'.

Adaptor Proteins, Signal Transducing↗

PubMed1997

The relationship between compositional phase separation and vesicle morphology: implications for the regulation of phospholipase A2 by membrane structure.

The action of phospholipase A2 (PLA2) on bilayer substrates causes the accumulation of reaction products, lyso-phospholipid and fatty acid. These reaction products and the phospholipid substrate generate compositional heterogeneities and then apparently phase separate when a critical mole fraction of reaction product accumulates in the membrane. This putative phase separation drives an abrupt morphologic rearrangement of the vesicle, which may be in turn responsible for modulating the activity of PLA2. Here we examine the thermotropic properties of the phase-separated lipid system formed upon hydrating colyophilized reaction products (1:1 palmitic acid:1-palmitoyl-2-lyso-phosphatidylcholine) and substrate, dipalmitoylphosphatidylcholine. The mixture forms structures which are not canonical spherical vesicles and appear to be disks in the gel-state. The main gel-liquid transition of these structures is hysteretic. This hysteresis is apparent using several techniques, each selected for its sensitivity to different aspects of a lipid aggregate's structure. The thermotropic hysteresis reflects the coupling between phase separation and changes in vesicle morphology.

Light↗

PubMed1997

Changes in vesicle morphology induced by lateral phase separation modulate phospholipase A2 activity.

The action of phospholipase A2 (PLA2) toward zwitterionic bilayers is modulated by lateral phase separation of reaction products and substrate. The experiments here address the mechanism of this modulation. PLA2 is particularly active toward lipid dispersions containing reaction products and substrates in which lateral phase separation has occurred. Here, we study PLA2 activity in two related model systems: first in a system in which lateral phase separation can be produced a priori, and second in a system in which the action of PLA2 produces sufficient reaction product in situ such that lateral phase separation occurs. The dispersions in which lateral phase separation occurs a priori form either disk micelles or disk vesicles, not canonical vesicles. When lateral phase separation occurs due to in situ PLA2 activity, there is an abrupt change in vesicle structure and a simultaneous profound increase in catalytic rate. This observation is surprising in light of several reports that vesicles remain intact even when the entire outer monolayer has been hydrolyzed. Membrane curvature and the associated structural defects and dynamic fluctuations in membrane structure have been proposed to modulate PLA2 activity. The mechanism by which lateral phase separation modulates PLA2 activity has been unclear. The data presented here indicate that lateral phase separation affects PLA2 activity by altering membrane curvature and/or inducing defects in the membrane structure.

Kinetics↗

PubMed1997

The activating dual phosphorylation of MAPK by MEK is nonprocessive.

Activation of mitogen-activated protein kinases (MAPKs), also known as extracellular-signal-regulated kinases (ERKs), by MAPK/extracellular protein kinase kinases (MEKs) requires phosphorylation at two sites. The first step in MAPK activation by MEK must be the formation of a MEK x MAPK enzyme-substrate complex, followed by phosphorylation producing monophosphorylated MAPK (pMAPK). Subsequently, one of two events may occur. (1) MEK catalyzes the second and fully activating phosphorylation of MAPK, producing ppMAPK (a processive mechanism). (2) The complex of MEK x pMAPK dissociates before the second phosphorylation occurs, full activation requiring a reassociation of pMAPK with MEK (a nonprocessive or distributive mechanism). Simulations indicate that these two mechanisms predict different kinetics of MAPK activation. Specifically, the nonprocessive mechanism predicts that there will be a paradoxical decrease in the rate of MAPK activation as the MAPK concentration is increased. The present study uses p42 MAPK, also known as ERK2, and MEK1 as representatives of their respective classes of enzymes. We find that increasing the ERK2 concentration decreases the rate of activation by a mechanism which does not involve inhibition of MEK1 function. The accumulation of the active, doubly phosphorylated ERK2 (ppERK2) was directly assessed using a phosphorylation-state-specific antibody. The rate of accumulation of ppERK2 is decreased by increasing the ERK2 concentration. Therefore, the mechanism of ERK2 activation by MEK1 in vitro is nonprocessive.

Animals↗

PubMed1995

Modulation of phospholipase A2: identification of an inactive membrane-bound state.

Phospholipase A2-catalyzed hydrolysis of vesicular phospholipid has been used to model the modulation of an enzyme's function by membrane properties. Phospholipase A2's (PLA2) kinetics toward large unilamellar vesicles (LUV) composed of dipalmitoylphosphatidylcholine (DPPC) are anomalous; these is a slow initial phase of catalysis (a lag) which ends abruptly with a sudden increase in the catalytic rate (a burst). The sudden increase in activity due to the accumulation of a critical mole fraction of reaction products and substrate undergoes compositional phase separation. In this work, we address the molecular details of the coupling between compositional phase separation and activation of PLA2. A prominent model for this coupling is that compositional phase separation leads to a surface for which PLA2 has increased affinity, resulting in the recruitment of PLA2 from solution to the surface. Here, we show that the bulk of PLA2 is associated with the membrane at a time well before the abrupt increase in catalytic rate. This finding indicates that there must be a relatively inactive, membrane-bound state. Furthermore, PLA2's kinetics are anomalous even when the substrate comprises a surface to which PLA2 is bound throughout the time course. With DPPC LUV as the substrate, detailed time courses show that the description of the time course as a lag and a burst is inadequate. Instead, the time course consists of multiple phases of acceleration and deceleration. The data presented here suggest that all these various changes in catalytic rate may be due to product-induced changes in membrane properties. In particular, we suggest that nonequilibrium, microheterogeneities of lipid composition may underlie these very complicated kinetics.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1994

Lipid bilayer heterogeneities and modulation of phospholipase A2 activity.

The regulation of phospholipase A2 (PLA2) activity toward synthetic vesicular substrates is a model for the modulation c enzyme function by biological membranes. PLA2's catalytic rate toward membrane phospholipids can be modified by several order of magnitude by altering the membrane's composition and structure. The physical basis of this sensitivity is the subject of thi report. The results described here imply that the salient features of membrane-structure which modulate PLA2 activity include compositional phase separation; membrane curvature and, possibly, curvature-associated defects; and dynamic product inhibition due to limitations imposed by the rate of lateral diffusion of lipid in the membrane. Furthermore, it is shown that the effects of membrane structure on the catalytic rate are not exerted merely by enhancing association of PLA2 with the membrane surface: a membrane-bound inactive state is spectroscopically identified. Finally, these results are discussed in the context of some published models for the role of membrane structure in the regulation of membrane-bound enzymes.

Enzyme Activation↗

PubMed1993

Role of lateral phase separation in the modulation of phospholipase A2 activity.

Phospholipase A2-catalyzed hydrolysis of phosphatidylcholine large unilamellar vesicles is characterized by a period of slow hydrolysis followed by a rapid increase in the rate of hydrolysis. The temporal relationship between the burst of PLA2 activity and the lateral distribution of substrate and product lipids was examined by simultaneously recording product accumulation and the fluorescence of 1-pyrenyldecanoate, a fatty acid derivative sensitive to lipid distribution and lateral diffusion. The excimer: monomer ratio of the probe changes slowly prior to the burst in activity and then abruptly at the time of the burst. A partial phase diagram for the ternary codispersion of substrate and products (dipalmitoylphosphatidylcholine and 1:1 monopalmitoylphosphatidylcholine/palmitic acid) was constructed by differential scanning calorimetry and suggests gel/gel immiscibility in this system. Thus, the changes in pyrene fluorescence during the time course of hydrolysis appear to be due to lateral phase separation. The critical mole fraction of product both for lateral phase separation in the gel state and for elimination of the lag phase is approximately 0.083. The simultaneous recordings of PLA2 activity and pyrene fluorescence show that the lateral rearrangement of lipids begins prior to and continues during the rapid activation process of PLA2. Two possible effects of lateral phase separation are that concentration of the protein in the product-rich regions promotes putative dimerization or that formation of phase interface regions promotes enzyme activation.

Enzyme Activation↗

PubMed1992

Expression of a group II phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus in Escherichia coli: recovery and renaturation from bacterial inclusion bodies.

A synthetic gene encoding the Group II phospholipase A2 (PLA2) from the venom of Agkistrodon piscivorus piscivorus has been constructed and expressed with high efficiency in Escherichia coli. No enzymatic activity was recovered when the polypeptide contained the initiator Met residue. Replacement of an Asn residue penultimate to the initiator Met with Ser or Gly permitted removal of the initiator Met by the endogenous methionine aminopeptidase. The amino-terminal serine (N-Ser) and amino-terminal glycine PLA2's were isolated from intracellular inclusion bodies and were renatured with 25% recovery. Automated Edman degradation confirmed the removal of the initiator Met and confirmed the sequence of the first 40 residues of N-Ser PLA2. The recombinant proteins were purified to apparent homogeneity and showed the same specific activity as the wild-type protein. N-Ser PLA2 demonstrated the same kinetics of activation as the wild type enzyme on large vesicles of zwitterionic lipid.

Amino Acid Sequence↗

PubMed1983

Cancer II. Distortions in standardized rates.

Age-adjustment (standardization), the statistical method most used by cancer epidemiologists to express incidence and mortality rates, makes use of an arbitrarily chosen "standard" population to calculate rates that are in reality only abstract index numbers. It is generally known that the "age" of any standard population affects the magnitude of the index, but it is not widely recognized that the use of U.S. Censuses as standards, possibly excepting the 1930 U.S. Census population, distorts the index number. The greater the disparity between the numbers of males and females in middle-aged groups and older, the greater is the distortion. Hence sex as well as age distribution in a standard must be considered if rates are to be expressed in terms of sex. Trends of cancer incidence or mortality will have different slopes depending on the census year chosen as a standard; yet no standard can arbitrarily be called the "correct" one. Age-, race-, and sex-specific rates are more precise and can be made to give considerably more meaningful description of cancer trends, albeit their use requires scrutiny of a greater assortment of numbers.

Actuarial Analysis↗

PubMedDate not supplied

Cancer: I. Analysis of recent new case incidence reports.

Provides an evaluation of recent government agency reports that allege an upward trend in cancer incidence. Visual and statistical analyses are performed to demonstrate that the data of two cancer surveys, TNCS and SEER, which cover discontinuous time periods and non-identical geographic populations, are incompatible. They may not, as they were, be pooled for time-trend analysis. There is misstatement of the population-at-risk in the use of TNCS data. In SEER, there is evidence that procedures have changed over time, that updating procedures have a sex bias, and that the findings are not yet sufficiently stable to provide a meaningful trend assessment. The alleged upward trend in cancer incidence is an illusion attributable to the overlooking of the discrepancies and inconsistencies between and within the two surveys. Neither survey individually shows an increase in cancer incidence.

Female↗