Platelet mapping as part of modified thromboelastography (TEG®) in patients undergoing cardiac surgery and cardiopulmonary bypass.

The platelet-mapping assay of the thromboelastograph was used to measure platelet aggregation and to examine the effect of cardiopulmonary bypass on multiple platelet receptors and the role of altered receptor activity in postoperative bleeding. The percentage platelet aggregation for collagen, adenosine diphosphate and arachidonic acid was measured in 40 patients divided post-hoc into high- or low-bleeding groups depending on postoperative 24-h chest tube output. Platelet aggregation was lower after cardiopulmonary bypass compared to before it using collagen (mean (SD) 45 (25) vs 19 (12)%, p<0.001), adenosine diphosphate (76 (23) vs 35 (24)%, p<0.001), and arachidonic acid (61 (33) vs 31 (35)%, p<0.001). Only platelet aggregation as measured using collagen pre- and post-cardiopulmonary bypass was significantly less in the high- compared to the low-bleeding group. This finding was significantly correlated with the 24-h chest tube drainage, and it predicted postoperative bleeding with a sensitivity of 83% and a specificity of 68%. Therefore, platelet aggregation is reduced following cardiopulmonary bypass, and this may play a role in predicting postoperative blood loss.

Protein carbonylation in a murine model for early alcoholic liver disease.

Hepatic oxidative stress and subsequent lipid peroxidation are well-recognized consequences of sustained ethanol consumption. The covalent adduction of nucleophilic amino acid side-chains by lipid electrophiles is significantly increased in patients with alcoholic liver disease (ALD); a global assessment of in vivo protein targets and the consequences of these modifications, however, has not been conducted. In this article, we describe the identification of novel protein targets for covalent adduction in a 6-week murine model for ALD. Ethanol-fed mice displayed a 2-fold increase in hepatic TBARS, while immunohistochemical analysis for the reactive aldehydes 4-hydroxynonenal (4-HNE), 4-oxononenal (4-ONE), acrolein (ACR), and malondialdehyde (MDA) revealed a marked increase in the staining of modified proteins in the ethanol-treated mice. Increased protein carbonyl content was confirmed utilizing subcellular fractionation of liver homogenates followed by biotin-tagging through hydrazide chemistry, where approximately a 2-fold increase in modified proteins was observed in microsomal and cytosolic fractions. To determine targets of protein carbonylation, a secondary hydrazide method coupled to a highly sensitive 2-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS or MuDPIT) technique was utilized. Our results have identified 414 protein targets for modification by reactive aldehydes in ALD. The presence of novel in vivo sites of protein modification by 4-HNE (2), 4-ONE (4) and ACR (2) was also confirmed in our data set. While the precise impact of protein carbonylation in ALD remains unknown, a bioinformatic analysis of the data set has revealed key pathways associated with disease progression, including fatty acid metabolism, drug metabolism, oxidative phosphorylation, and the TCA cycle. These data suggest a major role for aldehyde adduction in the pathogenesis of ALD.

Reliability and validity of the Cocaine Selective Severity Assessment.

This article assesses the reliability and validity of the Cocaine Selective Severity Assessment (CSSA), a measure of cocaine abstinence signs and symptoms. Interrater reliability and scale internal consistency were high. Initial CSSA scores were significantly higher in cocaine-dependent subjects than in alcohol-dependent subjects. Initial CSSA scores were highly correlated with recent cocaine use and with severity measures from the Addiction Severity Index (ASI) including the interviewer severity rating and composite score in the drug section. Among cocaine-dependent subjects, initial CSSA scores were higher for those who failed to achieve abstinence or who subsequently dropped out of treatment. Further, CSSA scores showed consistent and marked declines over time for subjects who continued in treatment and remained abstinent. The CSSA appears to be a reliable and valid measure of cocaine abstinence symptoms and a useful predictor of negative outcomes in cocaine dependence treatment.

Functional reverse transcriptases encoded by A-type mouse LINE-1: defining the minimal domain by deletion analysis.

Long interspersed elements, or LINEs, are retrotransposons that move via an RNA intermediate. In mice, one polymorphic variant of L1 has amplified relatively recently, giving rise to the A-type subfamily in species belonging to the genus and subgenus Mus. Retrotransposition of LINE-1 (L1) requires the function of the L1-encoded reverse transcriptase that is produced from open reading frame 2 (ORF2). Here, we employ a convenient yeast genetic assay to determine the reverse transcriptase activity of the ORF2 obtained from three A-type L1 elements: one, a cDNA from the RNA in ribonucleoprotein particles; another with a purported inactivating mutation; and the third, a hypothetical ancestral construct. Because there are no examples of A-type elements that have transposed recently to inactivate a gene, this assay is the first step towards demonstrating the functional capability of mouse A-type LINE-1 elements. One of the three elements was believed to have been inactivated during evolution by the substitution of leucine for a highly conserved phenylalanine or tryptophan residue among known reverse transcriptases. This mutation did not inactivate the L1 reverse transcriptase in the yeast assay; thus, all three of the elements tested encoded reverse transcriptase activity. We further examined the minimal reverse transcriptase domain within ORF2 by creating a series of deletions. The results demonstrate that removal of the L1 endonuclease domain from the N-terminal region of ORF2 does not affect reverse transcriptase activity as determined by this assay, and that approximately half of the ORF2 coding sequence from mouse A-type L1 elements is required for functional reverse transcriptase.