Biometric Study of Acetoin Production in Hanseniaspora guilliermondii and Kloeckera apiculata

Gas chromatographic analysis by direct injection of samples yielded quantitative data on acetoin content. Ninety-six strains of Hanseniaspora guilliermondii and Kloeckera apiculata were investigated for the ability to produce acetoin in synthetic medium and in must. High-level production of acetoin was found to be a characteristic of both species. In synthetic medium, the two species were not significantly different with respect to sugar utilization and ethanol or acetoin production. In grape must, the two species were significantly different (P = 0.001) in acetoin production and K. apiculata exhibited a significantly negative correlation between acetoin production and either sugar consumption or ethanol production. Use of selected apiculate yeasts in mixed cultures with Saccharomyces cerevisiae seems promising for optimization of wine bouquet.     

Impact of preanalytical handling and timing for peripheral blood mononuclear cells isolation and RNA studies: the experience of the Interinstitutional Multidisciplinary BioBank (BioBIM)

Multicenter studies and biobanking projects require blood transportation from the participating center to a central collection or diagnostic laboratory. The impact of time delays between venous blood collection and peripheral blood mononuclear cells (PBMC) isolation prior to RNA extraction may affect the quality and quantity of isolated nucleic acids for genomic applications. Thus, standard operating procedure (SOP) optimization for the treatment of biological samples before RNA extraction is crucial in a biological repository. In order to define SOPs for whole blood preservation prior to RNA extraction, we sought to determine whether different blood storage times (0, 3, 6, 10, 24, and 30 hours) prior to PBMCs isolation and storage at -80°C, could affect the quality and quantity of extracted RNA. After spectrophotometric quantification, the quality and integrity of RNA were assessed by agarose gel electrophoresis, RNA integrity number and real time-PCR (RT-PCR).?Across the different time points we did not observe significant differences within the first 24 hours of blood storage at room temperature, while a significant loss in RNA yield and integrity was detected between 24 and 30 hours. We conclude that time delays before PBMCs isolation prior to RNA extraction may have a significant impact on downstream molecular biological applications.     

Specific protein changes contribute to the differential muscle mass loss during ageing

In the skeletal muscle, the ageing process is characterized by a loss of muscle mass and strength, coupled with a decline of mitochondrial function and a decrease of satellite cells. This profile is more pronounced in hindlimb than in forelimb muscles, both in humans and in rodents. Utilizing light and electron microscopy, myosin heavy chain isoform distribution, proteomic analysis by 2D‐DIGE, MALDI‐TOF MS and quantitative immunoblotting, this study analyzes the protein levels and the nuclear localization of specific molecules, which can contribute to a preferential muscle loss. Our results identify the molecular changes in the hindlimb (gastrocnemius) and forelimb (triceps) muscles during ageing in rats (3‐ and 22‐month‐old). Specifically, the oxidative metabolism contributes to tissue homeostasis in triceps, whereas respiratory chain disruption and oxidative‐stress‐induced damage imbalance the homeostasis in gastrocnemius muscle. High levels of dihydrolipoyllysine‐residue acetyltransferase (Dlat) and ATP synthase subunit alpha (Atp5a1) are detected in triceps and gastrocnemius, respectively. Interestingly, in triceps, both molecules are increased in the nucleus in aged rats and are associated to an increased protein acetylation and myoglobin availability. Furthermore, autophagy is retained in triceps whereas an enhanced fusion, decrement of mitophagy and of regenerative potential is observed in aged gastrocnemius muscle.     

The localization and rate of disappearance of a synaptic vesicle antigen following denervation

Summary A proteoglycan-specific antiserum has been used to monitor the effects of denervation in the electric organ of Torpedo marmorata. The antiserum was produced by injecting a highly purified synaptic vesicle fraction prepared from the electric organs of Torpedo marmorata. Following absorption the serum appears to be specific towards synaptic vesicles. The ultrastructural localization of the antigen determined by immuno-electron microscopy confirmed the specificity of the antiserum and showed that it did not crossreact with the proteoglycans of the basal lamina. The rate of disappearance of the vesicle proteoglycans following denervation was evaluated by means of the antiserum and was compared to the rate of disappearance of other vesicular and nerve terminal-associated markers. The results suggest that degeneration affects the vesicular constituents at varying rates resulting in a progressive disappearance of the entire functional capacity of the synaptic vesicles.     

Effects of sucrose on the internal dynamics of azurin

Sucrose is a natural osmolyte accumulated in cells of organisms as they adapt to environmental stresses. In vitro, sucrose increases protein stability and forces partially unfolded structures to refold. Its effects on the native fold structure and dynamics are not fully established. This study, utilizing Trp phosphorescence spectroscopy, examined the influence of molar concentrations of sucrose on the flexibility of metal-free azurin from Pseudomonas aeruginosa. In addition, by means of specific mutants of the test protein, namely I7S, F110S, and C3A/C26A, that altered its thermodynamic stability, its intrinsic flexibility, and the extent of internal hydration, this investigation sought to identify possible correlations between these features of protein structure and the influence of the osmolyte on protein dynamics. Alterations of structural fluctuations were assessed by both the intrinsic phosphorescence lifetime (tau), which reports on local structure about the triplet probe, and the acrylamide bimolecular quenching rate constant (k(q)) that is a measure of the average acrylamide diffusion coefficient through the macromolecule. From the modulation of tau and k(q) across a wide temperature range and up to a concentration of 2M sucrose, it is concluded that sucrose attenuates structural fluctuations principally when macromolecules are internally hydrated and thermally expanded. Preliminary tests with trehalose and xylitol suggest that the effects of sucrose are general of the polyol class of osmolytes.     

Different Tyrosine Autophosphorylation Requirements in Fibroblast Growth Factor Receptor-1 Mediate Urokinase-Type Plasminogen Activator Induction and Mitogenesis

Among the seven tyrosine autophosphorylation sites identified in the intracellular domain of tyrosine kinase fibroblast growth factor receptor-1 (FGFR1), five of them are dispensable for FGFR1-mediated mitogenic signaling. The possibility of dissociating the mitogenic activity of basic FGF (FGF2) from its urokinase-type plasminogen activator (uPA)-inducing capacity both at pharmacological and structural levels prompted us to evaluate the role of these autophosphorylation sites in transducing FGF2-mediated uPA upregulation. To this purpose, L6 myoblasts transfected with either wild-type (wt) or various FGFR1 mutants were evaluated for the capacity to upregulate uPA production by FGF2. uPA was induced in cells transfected with wt-FGFR1, FGFR1-Y463F, -Y585F, -Y730F, -Y766F, or -Y583/585F mutants. In contrast, uPA upregulation was prevented in L6 cells transfected with FGFR1-Y463/583/585/730F mutant (FGFR1–4F) or with FGFR1-Y463/583/585/730/766F mutant (FGFR1–5F) that retained instead a full mitogenic response to FGF2; however, preservation of residue Y730 in FGFR1-Y463/583/585F mutant (FGFR1–3F) and FGFR1-Y463/583/585/766F mutant (FGFR1–4Fbis) allows the receptor to transduce uPA upregulation. Wild-type FGFR1, FGFR1–3F, and FGFR1–4F similarly bind to a 90-kDa tyrosine-phosphorylated protein and activate Shc, extracellular signal-regulated kinase (ERK)₂, and JunD after stimulation with FGF2. These data, together with the capacity of the ERK kinase inhibitor PD 098059 to prevent ERK₂ activation and uPA upregulation in wt-FGFR1 cells, suggest that signaling through the Ras/Raf-1/ERK kinase/ERK/JunD pathway is necessary but not sufficient for uPA induction in L6 transfectants. Accordingly, FGF2 was able to stimulate ERK_1/2 phosphorylation and cell proliferation, but not uPA upregulation, in L6 cells transfected with the FGFR1-Y463/730F mutant, whereas the FGFR1-Y583/585/730F mutant was fully active. We conclude that different tyrosine autophosphorylation requirements in FGFR1 mediate cell proliferation and uPA upregulation induced by FGF2 in L6 cells. In particular, phosphorylation of either Y463 or Y730, dispensable for mitogenic signaling, represents an absolute requirement for FGF2-mediated uPA induction.