Comparative proteome profiling and functional analysis of chronic myelogenous leukemia cell lines

The aim of the present study was the molecular profiling of different Ph+ chronic myelogenous leukemia (CML) cell lines (LAMA84, K562, and KCL22) by a proteomic approach. By employing two-dimensional gel electrophoresis combined with mass spectrometry analysis, we have identified 191 protein spots corresponding to 142 different proteins. Among these, 63% were cancer-related proteins and 74% were described for the first time in leukemia cells. Multivariate analysis highlighted significant differences in the global proteomic profile of the three CML cell lines. In particular, the detailed analysis of 35 differentially expressed proteins revealed that LAMA84 cells preferentially expressed proteins associated with an invasive behavior, while K562 and KCL22 cells preferentially expressed proteins involved in drug resistance. These data demonstrate that these CML cell lines, although representing the same pathological phenotype, show characteristics in their protein expression profile that suggest different phenotypic leukemia subclasses. These data contribute a new potential characterization of the CML phenotype and may help to understand interpatient variability in the progression of disease and in the efficacy of a treatment.     

Coupling between high-resolution mass spectrometry and focalized data-analysis methods provides the identification of new putative glycosidic non-anthocyanic flavonoids in grape

Metabolomics - Much evidence for diabetes mellitus being associated with dysregulated lipid metabolism has been accrued from studies using blood plasma. However, the systemic dysregulation these...     

Lipid-Conjugated Rigidochromic Probe Discloses Membrane Alteration in Model Cells of Krabbe Disease

The plasma membrane of cells has a complex architecture based on the bidimensional liquid-crystalline bilayer arrangement of phospho- and sphingolipids, which in turn embeds several proteins and is connected to the cytoskeleton. Several studies highlight the spatial membrane organization into more ordered (L_o or lipid raft) and more disordered (L_d) domains. We here report on a fluorescent analog of the green fluorescent protein chromophore that, when conjugated to a phospholipid, enables the quantification of the L_o and L_d domains in living cells on account of its large fluorescence lifetime variation in the two phases. The domain composition is straightforwardly obtained by the phasor approach to confocal fluorescence lifetime imaging, a graphical method that does not require global fitting of the fluorescence decay in every spatial position of the sample. Our imaging strategy was applied to recover the domain composition in human oligodendrocytes at rest and under treatment with galactosylsphingosine (psychosine). Exogenous psychosine administration recapitulates many of the molecular fingerprints of a severe neurological disease, globoid cell leukodystrophy, better known as Krabbe disease. We found out that psychosine progressively destabilizes plasma membrane, as witnessed by a shrinking of the L_o fraction. The unchanged levels of galactosyl ceramidase, i.e., the enzyme lacking in Krabbe disease, upon psychosine treatment suggest that psychosine alters the plasma membrane structure by direct physical effect, as also recently demonstrated in model membranes.     

Regulation of skeletal muscle AMP deaminase: lysine residues are critical for the pH-dependent positive homotropic cooperativity behaviour of the rabbit enzyme

Reaction of rabbit skeletal muscle AMP deaminase with a low molar excess of trinitrobenzene sulfonic acid (TNBS) results in conversion of the enzyme into a species with about six trinitrophenylated lysine residues per molecule which no longer manifests positive homotropic cooperativity at pH 7.1 or at the optimal pH value of 6.5 in the presence of low K+ concentrations. Substitution of the reactive thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) does not protect the enzyme from the TNBS-induced changes of the catalytic properties, indicating that cysteine residues modification is not at the basis of the effects of TNBS treatment on AMP deaminase and strongly suggesting the obligatory participation of lysine residues to the constitution of a regulatory anionic site to which AMP must bind to stimulate the enzyme at alkaline pH. The TNBS-treated enzyme is also completely desensitized to inhibition by ATP, but not to inhibition by GTP and stimulation by ADP. This observation suggests a connection between the operation of the hypothesized anionic activating site, responsible for positive homotropic cooperativity, and the inhibition exerted by anionic compounds that compete for the same site, among them the most efficient metabolite being probably ATP.     

Essential role for ADAM19 in cardiovascular morphogenesis

Congenital heart disease is the most common form of human birth defects, yet much remains to be learned about its underlying causes. Here we report that mice lacking functional ADAM19 (mnemonic for a disintegrin and metalloprotease 19) exhibit severe defects in cardiac morphogenesis, including a ventricular septal defect (VSD), abnormal formation of the aortic and pulmonic valves, leading to valvular stenosis, and abnormalities of the cardiac vasculature. During mouse development, ADAM19 is highly expressed in the conotruncus and the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. ADAM19 is also highly expressed in osteoblast-like cells in the bone, yet it does not appear to be essential for bone growth and skeletal development. Most adam19(-/-) animals die perinatally, likely as a result of their cardiac defects. These findings raise the possibility that mutations in ADAM19 may contribute to human congenital heart valve and septal defects.     

Near infrared spectroscopy as a tool for the determination of eumelanin in human hair

Eumelanins are brown-black pigments present in the hair and in the epidermis which are acknowledged as protection factors against cell damage caused by ultraviolet radiation. The quantity of eumelanin present in hair has recently been put forward as a means of identifying subjects with a higher risk of skin tumours. For epidemiological studies, chromatographic methods of determining pyrrole-2,3,5-tricarboxylic acid (PTCA; the principal marker of eumelanin) are long, laborious and unsuitable for screening large populations. We suggest near infrared (NIR) spectroscopy as an alternative method of analysing eumelanin in hair samples. PCTA was determined on 93 samples of hair by means of oxidizing with hydrogen peroxide in a basic environment followed by chromatographic separation. The same 93 samples were then subjected to NIR spectrophotometric analysis. The spectra were obtained in reflectance mode on hair samples which had not undergone any preliminary treatment, but had simply been pressed and placed on the measuring window of the spectrophotometer. The PTCA values obtained by means of HPLC were correlated with the near infrared spectrum of the respective samples. A correlation between the PTCA values obtained by means of HPLC and the PTCA values obtained from an analysis of the spectra was obtained using the principal component regression (PCR) algorithm. The correlation obtained has a coefficient of regression (R(2)) of 0.89 and a standard error of prediction (SEP) of 13.8 for a mean value of 108.6 ng PTCA/mg hair. Some considerations about the accuracy of the obtained correlation and the main sources of error are made and some validation results are shown.     

Effect of L-carnitine administration on the seminal characteristics of oligoasthenospermic stallions

The effect of orally administered l-carnitine on the quality of semen obtained from stallions with different semen qualities was investigated. Four stallions with proven fertility (high motility group, HM) and with normal seminal characteristics (>50% progressive motility and > 80 x 10(6) spermatozoa/ml), and four questionable breeders (low motility group, LM) with <50% of sperm progressive motility and < 80 x 10(6) spermatozoa/ml, received p.o. 20 g of l-carnitine for 60 days. Blood and semen samples were collected before treatment (T0) and after 30 (T1) and 60 days (T2). Semen evaluation were performed on five consecutive daily ejaculates (n = 120 ejaculates) and conventional semen analysis was carried out on each ejaculate, both at collection and after refrigeration for 24, 48, and 72 h. Furthermore l-carnitine, acetylcarnitine, pyruvate, and lactate concentrations, and carnitine acetyltransferase activity (CAT) were determined both in raw semen and seminal plasma. There were an increase in progressive motile spermatozoa only in the LM group (26.8 +/- 12.9, 39.1 +/- 15.5, and 48.8 +/- 8.6 for T0, T1, and T2, respectively). Free seminal plasma carnitine concentration was higher in the LM group compared to the HM one. Both pyruvate and lactate were higher in the LM group. Raw semen and seminal plasma carnitine and acetylcarnitine levels correlate positively with both sperm concentration and progressive motility; moreover, acetylcarnitine content was positively correlated with total motile morphologically normal spermatozoa. In conclusion, oral administration of l-carnitine to stallions with questionable seminal characteristics may improve spermatozoa kinetics and morphological characteristics; whereas, it seem to be ineffective in normospermic animals.