Hydrodynamic and circular dichroic analysis of mammary-derived growth inhibitor (MDGI)

The mammary-derived growth inhibitor exists in solution as a monomeric molecule with a molar mass of 14,500 +/- 400 g/mol. The largest diameter and the height of the polypeptide chain were estimated to be 3.75 +/- 0.25 nm and 2.01 +/- 0.13 nm respectively. This is in good agreement with the structurally related bovine peripheral myelin P2 protein (about 70% amino acid sequence homology). CD measurements have revealed MDGI to be a protein with about 50% beta structure and less than 20% alpha helix similarly as in fatty acid-binding proteins. Removal of endogenous long-chain fatty acid by lipidex or storage in the frozen state lead to a destabilization of the active MDGI conformation which is accompanied by a loss of its activity with regard to growth inhibition of Ehrlich Ascites cells.     

Establishment of reference values for the lysine acetylation marker Nɛ-acetyllysine in small volume human plasma samples by a multi-target LC–MS/MS method

Amino Acids - Cardiovascular disease (CVD) and chronic kidney disease (CKD) constitute substantial burdens for public health. The identification and validation of risk markers for CVD and CKD in...     

Stable isotope dilution assay for liquid chromatography-tandem mass spectrometric determination of L-homoarginine in human plasma

Nitric oxide (NO), the endogenous modulator of vascular tone and structure, originates from oxidation of L-arginine catalysed by NO synthase (NOS). The L-arginine derivative L-homoarginine serves as an alternative NOS substrate releasing NO, competing with L-arginine for NOS, arginase, and arginine transport. In the present article we report a liquid chromatography-tandem mass spectrometric (LC-tandem MS) method for the determination of L-homoarginine in human plasma by stable-isotope dilution. L-[(13)C(6)]-Homoarginine was used as internal standard. This method provides high sample throughput of 25-μl aliquots of plasma with an analysis time of 4 min using LC-tandem MS electrospray ionisation in the positive mode (ESI+). Specific transitions for L-homoarginine and L-[(13)C(6)]-homoarginine were m/z 245 → m/z 211 and m/z 251 → m/z 217, respectively. The mean intra- and interassay CVs were 7.4 ± 4.5% (±SD) for 0.1-50 μmol/L and 7.5 ± 2.0% for 2 and 5 μmol/L, respectively. Applying this method, a mean plasma concentration of L-homoarginine of 2.5 ± 1.0 μmol/L was determined in 136 healthy humans.     

In vivo evidence that Agxt2 can regulate plasma levels of dimethylarginines in mice

Elevated plasma concentrations of the asymmetric (ADMA) and symmetric (SDMA) dimethylarginine have repeatedly been linked to adverse cardiovascular clinical outcomes. Both dimethylarginines can be degraded by alanine–glyoxylate aminotransferase 2 (Agxt2), which is also the key enzyme responsible for the degradation of endogenously formed β-aminoisobutyrate (BAIB). In the present study we wanted to investigate the effect of BAIB on Agxt2 expression and Agxt2-mediated metabolism of dimethylarginines. We infused BAIB or saline intraperitoneally for 7 days in C57/BL6 mice via minipumps. Expression of Agxt2 was determined in liver and kidney. The concentrations of BAIB, dimethylarginines and the Agxt2-specific ADMA metabolite α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) was determined by LC–MS/MS in plasma and urine. As compared to controls systemic administration of BAIB increased plasma and urine BAIB levels by a factor of 26.5 (p < 0.001) and 25.8 (p < 0.01), respectively. BAIB infusion resulted in an increase of the plasma ADMA and SDMA concentrations of 27% and 31%, respectively, (both p < 0.05) and a 24% decrease of plasma DMGV levels (p < 0.05), while expression of Agxt2 was not different.Our data demonstrate that BAIB can inhibit Agxt2-mediated metabolism of dimethylarginines and show for the first time that endogenous Agxt2 is involved in the regulation of systemic ADMA, SDMA and DMGV levels. The effect of BAIB excess on endogenous dimethylarginine levels may have direct clinical implications for humans with the relatively common genetic trait of hyper-β-aminoisobutyric aciduria.     

A mammary-derived growth inhibitor (MDGI) related 70 kDa antigen identified in nuclei of mammary epithelial cells

The aim of the present study was to investigate the expression of the mammary-derived growth inhibitor (MDGI) and the subcellular localization of MDGI-related antigens in bovine mammary glands. Cell-free translation of poly(A+) = RNA, immunoprecipitation with rabbit anti-MDGI-antibodies, and estimation of the relative contents of MDGI by a radioimmunoassay in mammary tissue of different functional states revealed that the 13 kDa MDGI was dramatically increased in terminally differentiated mammary tissue compared with the proliferating tissue from pregnant animals. To address the question of tissue localization, polyclonal anti-MDGI antibodies and antibodies directed against a synthetic peptide corresponding to residues 69 to 78 of MDGI were used. Western blotting of tissue fractions revealed the cytosolic and microsomal localization of MDGI. Additionally, both types of antibodies detected a 70-kDa antigen in the nuclear fraction of differentiated mammary glands. Salt extraction and DNase I digestion of isolated nuclei, as well as chromatin purification, indicated an association of the 70-kDa antigen with the chromatin. By means of the immunogold technique, MDGI-related antigens were localized within euchromatic nuclear regions of epithelial cells in the intact differentiated mammary gland. The immunostaining was markedly diminished in the proliferating tissue. This finding raises the possibility that MDGI and the 70-kDa antigen influence cell proliferation by acting on gene expression within the nuclei of mammary glands.     

Cardiac autonomic balance in small-for-gestational-age neonates

The cardiac sympathetic nervous system is one putative key factor involved in the intrauterine programming of adult cardiovascular disease. We therefore analyzed cardiac autonomic system activity in small for gestational age (SGA) neonates. Heart rate variability (HRV) from 24-h ECG recordings were analyzed for time-domain and frequency-domain parameters in 27 SGA neonates [median 261 (240-283) days of gestation] compared with 27 appropriate for gestational age (AGA) neonates [median 270 (239-293) days of gestation]. In addition, salivary alpha-amylase levels were analyzed during resting conditions and in response to a pain-induced stress event in 18 SGA [median 266 (240-292) days of gestation] and 34 AGA [median 271 (240-294) days of gestation] neonates. Overall HRV was not significantly different in SGA neonates compared with AGA neonates (SD of all valid NN intervals: P = 0.14; triangular index: P = 0.29), and the sympathovagal balance [low frequency (LF)/high frequency (HF)] was similar (P = 0.62). Parameters mostly influenced by sympathetic activity did not reveal significant differences: (SD of the average of valid NN intervals: P = 0.27; average of the hourly means of SDs of all NN intervals: P = 0.66, LF: P = 0.83) as well as vagal tone-influenced parameters were unaltered (average of the hourly square root of the mean of the sum of the squares of differences between adjacent NN intervals: P = 0.59; proportion of pairs of adjacent NN intervals differing by >50 ms: P = 0.93; HF: P = 0.82). Median resting levels for alpha-amylase were not significantly different in SGA neonates (P = 0.13), and a neonatal stress stimulus revealed similar stress response patterns (P = 0.29). HRV and salivary alpha-amylase levels as indicators of cardiac autonomic activity were not altered in SGA neonates compared with AGA neonates. Thus, it appears that the intrauterine activation of the sympathetic system in SGA fetuses does not directly persist into postnatal life, and neonatal sympathovagal balance appears to be preserved.     

Evaluation of a positioning method for equine lateral stifle scintigrams

ABSTRACT: BACKGROUND: The current lack of a standardized protocol for positioning of the gamma camera relative to the horse limb in a lateral stifle scintigram, and thus the reliance on subjective positioning, may be a cause of diagnostic error and inter-operator variability due to variations of the view angle. The aims of this study were to develop a reliable method to obtain a lateral scintigram of the equine stifle based on fixed anatomical landmarks and measure the resulting foot to gamma camera angle on sequential measurements of the same horse and of different horses METHODS: 99mTechnetium filled capsules were glued on the skin on sites adjacent to the origin of the medial and lateral femorotibial collateral ligaments in 22 horses using ultrasound guidance. A lateral view of the stifle was defined as the image where the two radioactive point sources were aligned vertically (point sources guided method). Five sequential lateral acquisitions (one to five) of the stifle with the point sources vertically aligned were acquired in each horse, and the angle between the mid-sagittal foot-axis and the vertical axis of the gamma camera (FC angle) was measured for each of these acquisitions. Results: For acquisition group one to five, the mean of the means FC angle was 91.6 +/- 2degrees (2SD) and the coefficient of variation (COV) was 1.1%. In the 22 horses the 95% CI for the mean FC angles was 91.6degrees+/-12.1degrees (2SD) and the COV was 6.6%. Conclusions: The use of point sources to guide gamma camera position results in less variation in the lateral scintigram than if the distal limb is used as guidance due to a difference in FC angle between horses. The point source guided positioning method is considered suitable as a reference standard method to obtain lateral scintigrams of the equine stifle, and it will be of value in clinical scintigraphy and research. The use of alignment of specifically located point sources may also be applied in other regions to standardize scintigraphic views.