G protein signaling events are activated at the leading edge of chemotactic cells.

Directional sensing by eukaryotic cells does not require polarization of chemoattractant receptors. The translocation of the PH domain-containing protein CRAC in D. discoideum to binding sites on the inner face of the plasma membrane reflects activation of the G protein-linked signaling system. Increments in chemoattractant elicit a uniform response around the cell periphery. Yet when cells are exposed to a gradient, the activation occurs selectively at the stimulated edge, even in immobilized cells. We propose that such localized activation, transmitted by the recruitment of cytosolic proteins, may be a general mechanism for gradient sensing by G protein-linked chemotactic systems including those involving chemotactic cytokines in leukocytes.

Over-draping: a practice question.

Traditional OR practices are under scrutiny today and perioperative practitioners are being asked to examine practices which do not affect the quality of patient care but potentially impact the cost of delivery. This article focuses on overdraping related to disposable products and proposes improved liaisons between supply and demand.

Structural studies of Manduca sexta lipid transfer particle with apolipoprotein-specific antibodies.

Studies have been conducted to characterize structural and functional properties of Manduca sexta lipid transfer particle (LTP). LTP is a high molecular weight complex of three apolipoproteins and lipid that facilitates the transfer of lipids between lipoproteins and tissues and among lipoproteins in insect hemolymph. Rabbit polyclonal antibodies were raised against each of the three LTP apolipoproteins isolated by preparative electrophoresis. Immunoblot experiments demonstrated that they are apolipoprotein-specific and that LTP apolipoproteins are immunologically distinct polypeptides. Antibody-capture enzyme-linked immunosorbent assay characterization of apolipoprotein-specific IgG demonstrated that each of the three antibodies recognizes native LTP and provided information on the apparent affinity of the antibodies for LTP. Apolipoprotein-specific IgG were then compared in lipid transfer assays to examine the effect of antibody binding on LTP-mediated lipid transfer. Although each of the antibodies inhibited transfer activity, anti-apoLTP-II was capable of nearly abolishing activity at low IgG concentrations (< 26.7 micrograms IgG/micrograms LTP). In contrast, anti-apoLTP-I and anti-apoLTP-III IgG inhibited LTP activity only at much higher concentrations (> 133.3 micrograms IgG/micrograms LTP). These results indicate that apoLTP-II is a catalytically important apolipoprotein. In immunoprecipitation experiments, using 125I-labeled LTP, anti-holoLTP, anti-apoLTP-I, and anti-apoLTP-II were each able to immunoprecipitate all three LTP apolipoproteins while anti-apoLTP-III was not.(ABSTRACT TRUNCATED AT 250 WORDS)

A turbidimetric assay of lipid transfer activity.

A novel method to assay insect plasma lipid transfer particle (LTP) activity has been developed that employs insect high density lipophorin (HDLp) and human low density lipoprotein (LDL) as donor/acceptor substrate particles. At a 3:1 or greater HDLp:LDL protein ratio, LTP-mediated net vectorial transfer of diacylglycerol from lipophorin to LDL produces destabilized LDL particles that aggregate, causing sample turbidity. Turbidity was measured spectrophotometrically as a function of absorbance at 340 nm. After an initial lag phase, lipoprotein sample turbidity increased as a function of reaction time and LTP concentration. Saturation was observed at longer times or higher LTP concentrations, indicating that a reaction end point had been reached. As the substrate HDLp concentration was increased relative to LDL, a saturable increase in LTP-induced lipoprotein sample turbidity was observed. When the LDL concentration was increased relative to HDLp, however, there was an initial production of turbidity but at higher concentrations the sample did not develop turbidity. Reaction progress was also dependent on temperature over the range 0-37 degrees C. Taken together the results are consistent with the concept that LTP-mediated diacylglycerol transfer from HDLp to LDL creates unstable product LDL particles that aggregate. The assay method is advantageous because it employs relatively abundant, natural lipoprotein substrates, does not require prelabeling of donor lipid particles with radioactive or fluorescent lipids, and does not require separation of donor and acceptor after incubation. This is the first description of a lipid transfer assay that can be measured spectrophotometrically.

Insect lipid transfer particle can facilitate net vectorial lipid transfer via a carrier-mediated mechanism.

The mechanism of facilitated lipid transfer by insect or mammalian plasma lipid transfer proteins has not been elucidated. Transfer catalysts may act as carriers of lipid between donor and acceptor lipoproteins or, alternatively, transfer may require formation of a ternary complex. This study was designed to determine if Manduca sexta hemolymph lipid transfer particle (LTP) can facilitate net vectorial transfer of lipid without concomitant contact between donor and acceptor lipoproteins and LTP. M. sexta [3H]diacylglycerol-high density lipophorin-larval ([3H]DAG-HDLp-L) and human low density lipoprotein (LDL) were covalently bound to Sepharose matrices and packed into separate columns. In incubations lacking LTP, greater than 98% of the recovered DAG remained associated with HDLp-L. An unrelated hemolymph storage protein, arylphorin, was unable to catalyze the transfer of DAG between solid-phase lipoproteins. Facilitated transfer of DAG from HDLp-L to LDL was observed when LTP was circulated between the columns. Under these conditions, facilitated transfer occurred at a rate of 2.24 ng of DAG/h (versus 0.16 microgram of DAG/h in the control), and after 16 h greater than 26% of recovered labeled DAG was transferred to LDL. This corresponds to a 14-fold rate enhancement induced by LTP. The LTP-specific transfer of DAG between physically separated lipoproteins demonstrates the ability of LTP to facilitate net lipid transfer via a carrier-mediated mechanism in the absence of a ternary complex involving donor, acceptor, and catalyst. In experiments aimed at assessing the relative contribution of ternary complex formation to DAG transfer, acceptor LDL was circulated with HDLp-L remaining immobilized. Under these conditions, LTP induced a 13-fold rate enhancement from 1.3 to 16.3 micrograms of DAG/h. The similar rate enhancements observed with both lipoproteins bound and only donor bound suggest the overall contribution of ternary complex formation to facilitated lipid transfer is insignificant. The described system should prove useful in mechanistic studies of other transfer proteins as well as studies of transfer of other lipids.