Processing and transport of matrix gamma-carboxyglutamic acid protein and bone morphogenetic protein-2 in cultured human vascular smooth muscle cells: evidence for an uptake mechanism for serum fetuin

Matrix gamma-carboxyglutamic acid protein (MGP) is a member of the vitamin K-dependent protein family with unique structural and physical properties. MGP has been shown to be an inhibitor of arterial wall and cartilage calcification. One inhibitory mechanism is thought to be binding of bone morphogenetic protein-2. Binding has been shown to be dependent upon the vitamin K-dependent gamma-carboxylation modification of MGP. Since MGP is an insoluble matrix protein, this work has focused on intracellular processing and transport of MGP to become an extracellular binding protein for bone morphogenetic protein-2. Human vascular smooth muscle cells (VSMCs) were infected with an adenovirus carrying the MGP construct, which produced non-gamma-carboxylated MGP and fully gamma-carboxylated MGP. Both forms of MGP were found in the cytosolic and microsomal fractions obtained from the cells by differential centrifugation. The crude microsomal fraction was shown to contain an additional, more acidic Ser-phosphorylated form of MGP believed to be the product of Golgi casein kinase. The data suggest that phosphorylation of MGP dictates different transport routes for MGP in VSMCs. A proteomic approach failed to identify a larger soluble precursor of MGP or an intracellular carrier protein for MGP. Evidence is presented for a receptor-mediated uptake mechanism for fetuin by cultured human VSMCs. Fetuin, shown by mass spectrometry not to contain MGP, was found to be recognized by anti-MGP antibodies. Fetuin uptake and secretion by proliferating and differentiating cells at sites of calcification in the arterial wall may represent an additional protective mechanism against arterial calcification.     

The proteins encoded by the rbs operon of Escherichia coli: I. Overproduction, purification, characterization, and functional analysis of RbsA.

The nucleotide-binding component of the high-affinity ribose transport system of Escherichia coli, RbsA, was overproduced from a T7-7 expression vector, and the protein was purified. Biochemical analyses of the purified protein indicated that the ATP analogues, 5'-FSBA and 8-azido ATP, covalently labeled the protein, a reaction that was inhibited by ATP, but not by GTP or CTP. The pure protein exhibited low-level ATPase activity with a K(m) of about 140 microM. Analyses of bacterial strains carrying chromosomal deletions of rbsA and other rbs genes suggested that RbsA is important for the chemotaxis function, a surprising result that was not anticipated from previous studies. However, an inconsistency between the several results from deletion strains raises questions regarding the interpretations of the in vivo data.     

Expression of ACTH-induced corticosteroid biosynthesis in newborn rat adrenocortical cells cultured in serum-free and carrier protein-free medium containing a cholesterol-α-cyclodextrin complex

Newborn rat adrenocortical cells were successfully cultured in a serum free carrier protein free medium (SPFM) by using -cyclodextrin as a cholesterol carrier and have expressed corticosteroid biosynthesis in this medium. A stable inclusion complex of cholesterol--cyclodextrin with a molar ratio of almost 1 was obtained for a 5 × 10^–5 mol/1 -cyclodextrin concentration. Cell cultures incubated with [4-¹⁴C] cholesterol--cyclodextrin in SPFM produced, under ACTH stimulation, various ¹⁴C labeled steroids with a predominance of corticosterone and 18-hydroxy-11-deoxycorticosterone. As measured by gas chromatography and mass spectrometry, the ratio between corticosteroids (21-hydroxylated steroids) and 20-reduced steroids produced in SPFM with cholesterol--cyclodextrin was equal to 1.8. This corresponds to a value of 3.6 times higher than that found in the serum free medium with cholesterol-albumin. Consequently, the chemically defined SPFM with cholesterol--cyclodextrin used in this study is more suitable for corticosteroidogenesis by adrenal cells in culture than a serum free medium with cholesterol-albumin.Abbreviations -CD -cyclodextrin - ACTH adrenocorticotropic hormone - 20-dihydroprogesterone 20-hydroxy-4-pregnene-3-one - 11-hydroxy-20-dihydroprogesterone 11, 20-dihydroxy-4-pregnene-3-one - 11-hydroxyprogesterone 11-hydroxy-4-pregnene-3,20-dione - C--CD cholesterol--cyclodextrin complex - corticosterone 11,21-dihydroxy-4-pregnene-3,20-dione - deoxycorticosterone 21-hydroxy-4-pregnene-3,20-dione - 18-hydroxy-11-deoxycorticosterone 18,21-dihydroxy-4-pregnene-3,20-dione - 18-hydroxy-20-dihydroprogesterone 18-20-dihydroxy-4-pregnene-3-one - 18-hydroxyprogesterone 18-hydroxy4-pregnene-3,20-dione - progesterone 4-pregnene-3,20-dione - SFM-S serum-free medium - SPFM serum-free protein-free medium - SSM serum supplemented medium     

Intérêt de l’étude de l’oxydation de l’ADN des spermatozoïdes par marquage de la 8-oxo-guanine en cytométrie en flux chez l’homme infertile

Oxidative stress in semen is essentially due to excessive production of oxygen-reactive species (ORS) essentially derived from leukocytes. DNA oxidation is due to the direct action of ORS which produce several adducts the most extensively studied of which is 8-oxo-guanine. Integrity of DNA is essential for the fertility of sperm and is an important subject of research for scientists and clinicians all over the world. Although evaluation of the global integrity of sperm DNA has considerably developed over recent years, few tests are available to document oxidative DNA damage. This study was designed to review the various tests of sperm DNA integrity commonly used in the literature and to present the results of our study on DNA oxidation with 8-oxo-guanine labelling by flow cytometry in infertile men. This study was based on 15 semen samples that were submitted to sperm analysis according to WHO guidelines, with determination of the leukocyte concentration by a cytochemical method revealing peroxidase in cytoplasmic granulations. The DNA oxidation study was performed with 8-oxo-guanine labelling by flow cytometry. Linear regression analysis showed a strong correlation between DNA oxidation and leukocyte count in the semen (p = 0.006, r = 0.7). A leukocyte cut-off of 250,000/ml of semen was associated with a significant increase of DNA oxidation (p = 0.03). 8-oxo-guanine can therefore be considered to be a biological marker of the direct action of oxidative stress on sperm DNA which appears to be susceptible to relatively low levels of ORS produced by leukocytes present at concentrations well below the limit of leukospermia defined by WHO.     

Genetic diversity of SSR and ISSR markers in wild populations of Brachypodium distachyon and its close relatives B. stacei and B. hybridum (Poaceae)

The genetic diversity of seven wild populations of the grass Brachypodium distachyon (2n = 10), 4 of B. stacei (2n = 20) and 13 of B. hybridum (2n = 30) from the Mediterranean and southern areas of the Iberian Peninsula was studied via the analysis of microsatellite (SSR) and inter-microsatellite (ISSR) markers. The 11 SSR markers analysed provided a total of 156 polymorphic fragments. The B. hybridum populations returned more fragments (98) than the B. stacei populations (87), and more than twice the number recorded for the B. distachyon populations (44). Some fragments were specific to the B. distachyon (3.85 %), B. stacei (27.56 %) or B. hybridum populations (18.58 %). The analysis of 16 ISSR markers returned similar results: the B. hybridum populations returned more polymorphic fragments than the B. distachyon or B. stacei populations. The analysis of molecular variance, with distances between individuals, populations and species estimated on the basis of the presence/absence of the SSR and ISSR fragments, showed most of the variation (67 %) to occur among populations. This was followed by differences among individuals within populations (24 %), and finally among species (9 %). Grouping based on UPGMA and principal coordinate analysis showed a clear separation of three groups corresponding to the populations of the same species. Principal component analysis, involving chromosome number, low-molecular weight-glutenin subunits and the most influential climatic and geographic factors, was also performed. This revealed an obvious separation among the populations of B. distachyon and B. hybridum. The index of Mediterraneity and altitude explained 70.56 % of the total variation associated with the first axis. A trend was seen towards a greater presence of B. distachyon forms in areas of the Peninsular interior and higher altitude, with the B. hybridum forms more common in regions of greater summer rainfall and a lower index of Mediterraneity.     

Proteomic analysis of the metabolic adaptation of the biocontrol agent <Emphasis Type="Italic">Pseudozyma flocculosa</Emphasis> leading to glycolipid production

The yeast-like epiphytic fungus Pseudozyma flocculosa is known to antagonize powdery mildew fungi through proliferation on colonies presumably preceded by the release of an antifungal glycolipid (flocculosin). In culture conditions, P. flocculosa can be induced to produce or not flocculosin through manipulation of the culture medium nutrients. In order to characterize and understand the metabolic changes in P. flocculosa linked to glycolipid production, we conducted a 2-DE proteomic analysis and compared the proteomic profile of P. flocculosa growing under conditions favoring the development of the fungus (control) or conducive to flocculosin synthesis (stress). A large number of protein spots (771) were detected in protein extracts of the control treatment compared to only 435 matched protein spots in extracts of the stress cultures, which clearly suggests an important metabolic reorganization in slow-growing cells producing flocculosin. From the latter treatment, we were able to identify 21 protein spots that were either specific to the treatment or up-regulated significantly (2-fold increase). All of them were identified based on similarity between predicted ORF of the newly sequenced genome of P. flocculosa with Ustilago maydis' available annotated sequences. These proteins were associated with the carbon and fatty acid metabolism, and also with the filamentous change of the fungus leading to flocculosin production. This first look into the proteome of P. flocculosa suggests that flocculosin synthesis is elicited in response to specific stress or limiting conditions.     

l-glutamine is a key parameter in the immunosuppression phenomenon

The present work describes the influence of both vitamin C (VC) and mechanical stimulation on development of the extracellular matrix (ECM) and improvement in mechanical properties of a chondrocyte-agarose construct in a regenerating tissue disease model of hyaline cartilage. We used primary bovine chondrocytes and two types of VC, ascorbic acid (AsA) as an acidic form and ascorbic acid 2-phosphate (A2P) as a non-acidic form, and applied uniaxial compressive strain to the tissue model using a purpose-built bioreactor. When added to the medium in free-swelling culture conditions, A2P downregulated development of ECM and suppressed improvement of the tangent modulus more than AsA. By contrast, application of mechanical stimulation to the construct both increased the tangent modulus more than the free-swelling group containing A2P and enhanced the ECM network of inner tissue to levels nearly as high as the free-swelling group containing AsA. Thus, mechanical stimulation and strain appears to enhance the supply of nutrients and improve the synthesis of ECM via mechanotransduction pathways of chondrocytes. Therefore, we suggest that mechanical stimulation is necessary for homogenous development of ECM in a cell-associated construct with a view to implantation of a large-sized articular cartilage defect.     

Preablative stimulated thyroglobulin in predicting dynamic risk stratification after 1 year in patients with differentiated thyroid cancer

The aimThe aim of the study is to establish the utility of stimulated preablative stimulated thyroglobulin (ps-Tg) as a predictor of response to therapy and to determine a possible cut-off for ps-Tg as prognostic tool.Patients and methodsA total of 73 consecutive patients who underwent total thyroidectomy and remnant ablation with radioactive iodine therapy (RIT) were reviewed retrospectively. Patients were classified according to the dynamic risk stratification 1 year after initial treatment. The ps-Tg values were compared among the groups. ROC curve analysis was performed.ResultsThe mean age at diagnosis was 43.85 (range: 17–75) with a female-to-male ratio of 4.6. Ps-Tg value after total thyroidectomy and before RIT ranged from 0,1 to 256 ng/mL. When patients were restaged, 74% had excellent response to treatment, 12.3% indeterminate and 13.7% incomplete response 1 year after initial therapy. ROC curve analysis showed that the optimal cut-off for ps-Tg was 15 ng/mL with a sensivity of 61%; a specificity of 87%; PPV of 61% and NPV of 87%. Among the group of patients showing an excellent response to treatment after 1 year, 87% had ps-Tg < 15 ng/mL.ConclusionPs-Tg before RIT is associated with dynamic risk stratification at 1 year after therapy in patients with DTC. Higher ps-Tg levels were found in patients that had indeterminate, and particularly incomplete, response.