Differential proteomic profiling in human spermatozoa that did or did not result in pregnancy via IVF and AID.

PURPOSE: To identify biochemical markers in men with idiopathic infertility and normal sperm counts. EXPERIMENTAL DESIGN: We obtained proteomic profiling proteins in human spermatozoa following successful or unsuccessful pregnancy via assisted reproductive technology (ART) using 6-plex tandem mass tag (TMT) isobaric mass spectrometry. Our study design consisted of two groups: 1. The semen of 6 men whose sperm resulted in a clinical pregnancy following ART and 6 men whose semen did not result in a clinical pregnancy following ART. The results of differentiated mass spectrometry were validated by Western blotting. RESULTS AND DISCUSSION: A total of 2,045 proteins were detected in our cohort. 21 proteins were found to be differentially expressed (>1.2-fold) in men whose sperm resulted in a clinical pregnancy and those that did not. Using the results of bioinformatics analysis and Western Blotting, three proteins (A2LD1, ATP1B3 and FBXO2) were shown to have the same differential pattern (p<0.05) that was observed in the mass spectrometry analysis. CONCLUSIONS AND CLINICAL RELEVANCE: Proteomics may help identity a select cohort of men with abnormal semen parameters and aide infertility diagnoses.

Biomarker characteristics of alcohol use in the U.S. Army.

The objective of this study was to collect data providing information about the biomarker characteristics of alcohol use among a sample of military personnel in the U.S. Army. Military personnel enrolled in the Army Substance Abuse Program at the Walter Reed Army Medical Center in Washington, DC, received a comprehensive assessment that included a panel of direct and indirect biomarkers. A total of 80 records were reviewed to assess biomarker results. Higher Alcohol Use Disorders Identification Test scores correlated with higher gamma glutamyltransferase levels. All subjects tested negative on the initial breathalyzer. All subjects completed an initial ethyl glucuronide and approximately one-third received a positive report. A second positive ethyl glucuronide did correlate with a positive third and fourth result. Military personnel deployed to an area of combat operations reported tobacco use more frequently than military personnel not assigned to an area of combat operations. A broad range of assessment tools, including traditional interviews, standardized questionnaires, indirect, and direct biomarkers, provide clinicians the techniques to screen alcohol use disorders. Direct biomarkers are a valuable assessment tool but must be integrated with the other components of the diagnostic evaluation.

Spectrophotometric assays for L-lysine alpha-oxidase and gamma-glutamylamine cyclotransferase.

A new assay for l-lysine alpha-oxidase is described. In this assay, the oxidized product generated from l-lysine is reacted with semicarbazide to form alpha-keto-epsilon-aminocaproate semicarbazone. Formation of the alpha-keto acid semicarbazone is continuously monitored spectrophotometrically at 248 nm (epsilon 10,160 +/- 240 M(-1) cm(-1)). The method was adapted to provide a new assay for gamma-glutamylamine cyclotransferase. This enzyme catalyzes the conversion of many l-gamma-glutamylamines to 5-oxo-l-proline and free amine. A biologically important substrate is N(epsilon)-(gamma-l-glutamyl)-l-lysine, which is converted to 5-oxo-l-proline and l-lysine by the action of gamma-glutamylamine cyclotransferase. The l-lysine generated from N(epsilon)-(gamma-l-glutamyl)-l-lysine in an endpoint assay is converted to alpha-keto epsilon-aminocaproate semicarbazone in the presence of semicarbazide, excess l-lysine alpha-oxidase, and catalase. The methods were applied to the determination of gamma-glutamylamine cyclotransferase activity of partially purified preparations of the bovine kidney enzyme and to detect gamma-glutamylamine cyclotransferase activity in rat kidney and liver homogenates.

Identification of glycinin in vivo as a polyamine-conjugated protein via a gamma-glutamyl linkage.

To identify a polyamine-conjugated protein by the action of transglutaminase in the absence of radiolabelled polyamine, extracts prepared from the leaves and developing soybean seeds were investigated for the specific activity of transglutaminase and the content of free polyamines. We identified the major storage protein, glycinin, as a polyamine-conjugated protein. This was established by the following procedures: (1) immunolocalization with antibody against putrescine prepared in rabbit against putrescine-BSA conjugate; (2) immunocross-reactivity on nitrocellulose transblot of the purified glycinin subunits by using antibody against putrescine; (3) identification of polyamines in acid hydrolysates of purified glycinin; (4) release of polyamines in proteolytic digests through the catalytic action of gamma-glutamylamine cyclotransferase, an enzyme specific for the disassembly of gamma-glutamylamines. The activity of gamma-glutamylamine cyclotransferase was also identified in soybean seeds.

Amide bond cleavage monitored continuously through detection of a dansylcadaverine leaving group.

The transglutaminase-catalyzed incorporation of the fluorescent amine, dansylcadaverine, into casein derivatives, such as N,N-dimethylcasein, is accompanied by a large increase in intensity of emission (Lorand et al., Anal. Biochem. 44, 221-231, 1971). We have sought to make use of this sensitive detection device for the continuous, on-line monitoring of an amide-splitting reaction in which dansylcadaverine served as the leaving group. The transglutaminase-coupled test system comprised gamma-glutamyldansylcadaverine as the first substrate and gamma-glutamylamine cyclotransferase as the enzyme responsible for releasing dansylcadaverine from the gamma-amide. At close to saturating levels of transglutaminase, the measured rate of increase of fluorescence, i.e. the steady-state rate of dansylcadaverine incorporation into N,N-dimethylcasein, showed a near-linear relationship with the concentration of gamma-glutamylamine cyclotransferase present in the assay mixture. The general approach developed may be applicable to the assay of other amide cleaving enzymes.

A spectrophotometric method for the determination of gamma-glutamyl cyclotransferase with alanine dehydrogenase in the presence of anthglutin.

gamma-Glutamyl cyclotransferase activity is assayed in tissues by a colorimetric method using gamma-glutamyl alanine as a substrate coupled with alanine dehydrogenase from B. sphericus, to measure the formation of NADH. In order to avoid interference by the reaction catalyzed by gamma-glutamyl transpeptidase, anthglutin, a specific inhibitor of the transpeptidase was included in the reaction mixture. The Km value of rat kidney gamma-glutamyl cyclotransferase with respect to gamma-glutamyl alanine appeared to be the same when determined by either the colorimetric or the radiometric method. This assay presents a reliable alternative to the use of radiolabeled substrate and is used for the assay of gamma-glutamyl cyclotransferase in a variety of physiological and experimental samples.