??4 Integrin Marks Interstitial Myogenic Progenitor Cells in Adult Murine Skeletal Muscle

Skeletal muscle growth and its regeneration following injury rely on myogenic progenitor cells, a heterogeneous population that includes the satellite cells and other interstitial progenitors. The present study demonstrates that surface expression of β4 integrin marks a population of vessel-associated interstitial muscle progenitor cells. Muscle β4 integrin–positive cells do not express myogenic markers upon isolation. However, they are capable of undergoing myogenic specification in vitro and in vivo: β4 integrin cells differentiate into multinucleated myotubes in culture dishes and contribute to muscle regeneration upon delivery into diseased mice. Subfractionation of β4 integrin–expressing cells based on CD31 expression does not further enrich for myogenic precursors. These findings support the expression of β4 integrin in interstitial, vessel-associated cells with myogenic activity within adult skeletal muscle.     

Candida zemplinina can reduce acetic acid produced by Saccharomyces cerevisiae in sweet wine fermentations

In this study we investigated the possibility of using Candida zemplinina, as a partner of Saccharomyces cerevisiae, in mixed fermentations of must with a high sugar content, in order to reduce its acetic acid production. Thirty-five C. zemplinina strains, which were isolated from different geographic regions, were molecularly characterized, and their fermentation performances were determined. Five genetically different strains were selected for mixed fermentations with S. cerevisiae. Two types of inoculation were carried out: coinoculation and sequential inoculation. A balance between the two species was generally observed for the first 6 days, after which the levels of C. zemplinina started to decrease. Relevant differences were observed concerning the consumption of sugars, the ethanol and glycerol content, and acetic acid production, depending on which strain was used and which type of inoculation was performed. Sequential inoculation led to the reduction of about half of the acetic acid content compared to the pure S. cerevisiae fermentation, but the ethanol and glycerol amounts were also low. A coinoculation with selected combinations of S. cerevisiae and C. zemplinina resulted in a decrease of ~0.3 g of acetic acid/liter, while maintaining high ethanol and glycerol levels. This study demonstrates that mixed S. cerevisiae and C. zemplinina fermentation could be applied in sweet wine fermentation to reduce the production of acetic acid, connected to the S. cerevisiae osmotic stress response.     

Effects of Greek legume plant extracts on xanthine oxidase,catalase and superoxide dismutase activities

Legumes are considered to have beneficial health implications, which have been attributed to their phytochemical content. Polyphenols are considered the most important phytochemical compounds extensively studied for their antioxidant properties. The aim of the present study was to examine the effects of potent antioxidant legume plant extracts on xanthine oxidase (XO), catalase (CAT) and superoxide dismutase (SOD) activities. XO exerts a dual role, as it is the major contributor of free radicals during exercise while it generates uric acid, the most potent antioxidant molecule in plasma. CAT and SOD are two of the main enzymes of the antioxidant defence of tissues. We demonstrate that the majority of the extracts inhibited XO activity, but they had no effect on CAT inhibition and SOD induction when used at low concentrations. These results imply that the tested extracts may be considered as possible source of novel XO inhibitors. However, we have shown that allopurinol administration, a known XO inhibitor, before exercise reduces performance and induces oxidative stress in rats. Considering the fact that the extracts examined had an inhibitory effect on XO activity, possibly posing a restriction in their characterization as antioxidants, phytochemical antioxidant administration before exercise should probably be reconsidered.     

Dimethyl Fumarate Ameliorates Lewis Rat Experimental Autoimmune Neuritis and Mediates Axonal Protection

BackgroundDimethyl fumarate is an immunomodulatory and neuroprotective drug, approved recently for the treatment of relapsing-remitting multiple sclerosis. In view of the limited therapeutic options for human acute and chronic polyneuritis, we used the animal model of experimental autoimmune neuritis in the Lewis rat to study the effects of dimethyl fumarate on autoimmune inflammation and neuroprotection in the peripheral nervous system.ConclusionsWe conclude that immunmodulatory and neuroprotective dimethyl fumarate may represent an innovative therapeutic option in human autoimmune neuropathies.     

Mutant forms of the extracellular domain of the human acetylcholine receptor gamma-subunit with improved solubility and enhanced antigenicity. The importance of the Cys-loop

The muscle nicotinic acetylcholine receptor (AChR) is the prototype of the ligand-gated ion channels (or Cys-loop receptors), formed by 5 homologous subunits (alpha(2)betagammadelta or alpha(2)betagammavarepsilon), and is the major autoantigen in the autoimmune disease, myasthenia gravis. Previously, we expressed the wild-type extracellular domain (ECD) of the gamma-subunit (gammaECD) of the AChR in yeast Pichia pastoris at 0.3-0.8 mg/L, in soluble but microaggregate form, to use as starting material for structural and antigenicity studies. To optimize these characteristics, we constructed and characterized four gammaECD variants: (a) mutants-1 (gammaC61S) and -2 (gammaC106S-C115S), where the non-conserved Cys of gammaECD were replaced by serines, (b) mutant-3 (gammaCysLoop), where the gamma Cys-loop region was substituted by the cognate region of the acetylcholine binding protein (AChBP) and (c) mutant-4 (gammaCysLoop-C106S-C115S), where both the C106S-C115S and Cys-loop mutations were combined. None of mutants-1 and -2 displayed any improvement, while mutant-3 and -4 were mostly in dimeric form and expressed at much higher levels (2.5 mg/L and 3.5 mg/L respectively). All four mutants and wild-type gammaECD were recognized by sera from myasthenic patients, but mutants-3 and -4 exhibited higher efficiency, compared to wild-type or mutants-1 and -2. These results suggest that the substitution of the Cys-loop region of any AChR ECD with the AChBP counterpart leads to AChR ECD of improved conformation, more suitable for structural and therapeutic studies.     

Culture-Dependent and -Independent Methods To Investigate the Microbial Ecology of Italian Fermented Sausages

In this study, the microbial ecology of three naturally fermented sausages produced in northeast Italy was studied by culture-dependent and -independent methods. By plating analysis, the predominance of lactic acid bacteria populations was pointed out, as well as the importance of coagulase-negative cocci. Also in the case of one fermentation, the fecal enterocci reached significant counts, highlighting their contribution to the particular transformation process. Yeast counts were higher than the detection limit (>100 CFU/g) in only one fermented sausage. Analysis of the denaturing gradient gel electrophoresis (DGGE) patterns and sequencing of the bands allowed profiling of the microbial populations present in the sausages during fermentation. The bacterial ecology was mainly characterized by the stable presence of Lactobacillus curvatus and Lactobacillus sakei, but Lactobacillus paracasei was also repeatedly detected. An important piece of evidence was the presence of Lactococcus garvieae, which clearly contributed in two fermentations. Several species of Staphylococcus were also detected. Regarding other bacterial groups, Bacillus sp., Ruminococcus sp., and Macrococcus caseolyticus were also identified at the beginning of the transformations. In addition, yeast species belonging to Debaryomyces hansenii, several Candida species, and Willopsis saturnus were observed in the DGGE gels. Finally, cluster analysis of the bacterial and yeast DGGE profiles highlighted the uniqueness of the fermentation processes studied.     

Acquisition of a bacterial RumA-type tRNA(uracil-54, C5)-methyltransferase by Archaea through an ancient horizontal gene transfer

The Pyrococcus abyssi genome displays two genes possibly coding for S-adenosyl-l-methionine-dependent RNA(uracil, C5)-methyltransferases (PAB0719 and PAB0760). Their amino acid sequences are more closely related to Escherichia coli RumA catalysing the formation of 5-methyluridine (m(5)U)-1939 in 23S rRNA than to E. coli TrmA (tRNA methyltransferase A) methylating uridine-54 in tRNA. Comparative genomic and phylogenetic analyses show that homologues of PAB0719 and PAB0760 occur only in a few Archaea, these genes having been acquired via a single horizontal gene transfer from a bacterial donor to the common ancestor of Thermococcales and Nanoarchaea. This transfer event was followed by a duplication event in Thermococcales leading to two closely related genes. None of the gene products of the two P. abyssi paralogues catalyses in vitro the formation of m(5)U in a P. abyssi rRNA fragment homologous to the bacterial RumA substrate. Instead, PAB0719 enzyme (renamed (Pab)TrmU54) displays an identical specificity to TrmA, as it catalyses the in vitro formation of m(5)U-54 in tRNA. Thus, during evolution, at least one of the two P. abyssi RumA-type enzymes has changed of target specificity. This functional shift probably occurred in an ancestor of all Thermococcales. This study also provides new evidence in favour of a close relationship between Thermococcales and Nanoarchaea.     

Use of novel solid-phase extraction sorbent materials for high-performance liquid chromatography quantitation of caffeine metabolism products methylxanthines and methyluric acids in samples of biological origin

An automated reversed-phase high-performance liquid chromatographic (RP-HPLC) method, using a linear gradient elution, is described for the simultaneous analysis of caffeine and metabolites according to their elution order: 7-methyluric acid, 1-methyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 1,3-dimethyluric acid, theobromine, 1,7-dimethyluric acid, paraxanthine and theophylline. The analytical column, an MZ Kromasil C₄, 250×4 mm, 5 μm, was operated at ambient temperature with back pressure values of 80–110 kg/cm². The mobile phase consisted of an acetate buffer (pH 3.5)–methanol (97:3, v/v) changing to 80:20 v/v in 20 min time, delivered at a flow-rate of 1 ml/min. Paracetamol was used as internal standard at a concentration of 6.18 ng/μl. Detection was performed with a variable wavelength UV–visible detector at 275 nm, resulting in detection limits of 0.3 ng per 10-μl injection, while linearity held up to 8 ng/μl for most of analytes, except for paraxanthine and theophylline, for which it was 12 ng/μl and for caffeine for which it was 20 ng/μl. The statistical evaluation of the method was examined performing intra-day (n=6) and inter-day calibration (n=7) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries from biological matrices: blood serum and urine ranging from 84.6 to 103.0%, were achieved using Nexus SPE cartridges with hydrophilic and lipophilic properties and methanol–acetate buffer (pH 3.5) (50:50, v/v) as eluent, requiring small volumes, 40 μl of blood serum and 100 μl of urine.