Overexpression of csc1-1. A plausible strategy to obtain wine yeast strains undergoing accelerated autolysis

The potential of several alternative genetic engineering based strategies in order to accelerate Saccharomyces cerevisiae autolysis for wine production has been studied. Both constitutively autophagic and defective in autophagy strains have been studied. Although both alternatives lead to impaired survival under starvation conditions, only constitutively autophagic strains, carrying a multicopy plasmid with the csc1-1 allele under the control of the TDH3 promoter, undergo accelerated autolysis in the experimental conditions tested. Fermentation performance is impaired in the autolytic strains, but industrial strains carrying the above-mentioned construction are still able to complete second fermentation of a model base wine. We suggest the construction of industrial yeasts showing a constitutive autophagic phenotype as a way to obtain second fermentation yeast strains undergoing accelerated autolysis.     

Poly(β-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus

Polyhydroxyalkanoates (PHAs) comprise a class of biodegradable polymers which offer an environmentally sustainable alternative to petroleum-based plastics. Production of PHAs in plants is attractive since current fermentation technology is prohibitively expensive. The PHA homopolymer poly(β-hydroxybutyrate) (PHB) has previously been produced in leaves of Arabidopsis thaliana (Nawrath et al., 1994, Proc Natl Acad Sci USA 91: 12760–12764). However, Brassica napus oilseed may provide a better system for PHB production because acetyl-CoA, the substrate required in the first step of PHB biosynthesis, is prevalent during fatty acid biosynthesis. Three enzymatic activities are needed to synthesize PHB: a β-ketothiolase, an acetoacetyl-CoA reductase and a PHB synthase. Genes from the bacterium Ralstonia eutropha encoding these enzymes were independently engineered behind the seed-specific Lesquerella fendleri oleate 12-hydroxylase promoter in a modular fashion. The gene cassettes were sequentially transferred into a single, multi-gene vector which was used to transform B. napus. Poly(β-hydroxybutyrate) accumulated in leukoplasts to levels as high as 7.7% fresh seed weight of mature seeds. Electron-microscopy analyses indicated that leukoplasts from these plants were distorted, yet intact, and appeared to expand in response to polymer accumulation. Received: 26 May 1999 / Accepted: 16 June 1999     

Transformation of nuclear and plastomic plant genomes by biolistic particle bombardment

Microprojectile bombardment is a powerful method for the transformation of various organisms and tissues. For plants, the biolistic approach is primarily used for transformation of cereals and other monocotyledons, as well as for dicotyledonous plants shown to be recalcitrant to Agrobacterium-based transformation of organellar genomes, and transformation of plant and algal chloroplasts has recently been reported. In this protocol paper we provide methods for nuclear and plastomic transformation of plants using the biolistic technique.     

Progesterone neuroprotection in spinal cord trauma involves up-regulation of brain-derived neurotrophic factor in motoneurons

Progesterone (PROG) provides neuroprotection to the injured central and peripheral nervous system. These effects may be due to regulation of myelin synthesis in glial cells and also to direct actions on neuronal function. Both types of cells express classical intracellular PROG receptors (PR), while neurons additionally express the PROG membrane-binding site called 25-Dx. In motoneurons from rats with spinal cord injury (SCI), PROG restores to normal the deficient levels of choline acetyl-transferase and of alpha3 subunit Na,K-ATPase mRNA, while levels of the growth associated protein GAP-43 mRNA are further stimulated. Recent studies suggest that neurotrophins are possible mediators of hormone action, and in agreement with this assumption, PROG treatment of rats with SCI increases the expression of brain-derived neurotrophic factor (BDNF) at both the mRNA and protein levels in ventral horn motoneurons. In situ hybridization (ISH) has shown that SCI reduces BDNF mRNA levels by 50% in spinal motoneurons, while PROG administration to injured rats (4mg/kg/day during 3 days, s.c.) elicits a three-fold increase in grain density. In addition to enhancement of mRNA levels, PROG increases BDNF immunoreactivity in perikaryon and cell processes of motoneurons of the lesioned spinal cord, and also prevents the lesion-induced chromatolytic degeneration of spinal cord motoneurons as determined by Nissl staining. Our findings strongly indicate that motoneurons of the spinal cord are targets of PROG, as confirmed by the expression of PR and the regulation of molecular parameters. PROG enhancement of endogenous neuronal BDNF could provide a trophic environment within the lesioned spinal cord and might be part of the PROG activated-pathways to provide neuroprotection. Thus, PROG treatment constitutes a new approach to sustain neuronal function after injury.     

A codon window in mRNA downstream of the initiation codon where NGG codons give strongly reduced gene expression in Escherichia coli

The influences on gene expression by codons at positions +2, +3, +5 and +7 downstream of the initiation codon have been compared. Most of the +2 codons that are known to give low gene expression are associated with a higher expression if placed at the later positions. The NGG codons AGG, CGG, UGG and GGG, but not GGN or GNG (where N is non-G), are unique since they are associated with a very low gene expression also if located at positions +2, +3 and +5. All codons, including NGG, give a normal gene expression if placed at positions +7. The negative effect by the NGG codons is true for both the lacZ and 3A′ model genes. The low expression is suggested to originate at the translational level, although it is not the result of mRNA secondary structure or a lowered intracellular mRNA pool.     

Label-free quantification and shotgun analysis of complex proteomes by one-dimensional SDS-PAGE/NanoLC-MS: evaluation for the large scale analysis of inflammatory human endothelial cells

To perform differential studies of complex protein mixtures, strategies for reproducible and accurate quantification are needed. Here, we evaluated a quantitative proteomic workflow based on nanoLC-MS/MS analysis on an LTQ-Orbitrap-VELOS mass spectrometer and label-free quantification using the MFPaQ software. In such label-free quantitative studies, a compromise has to be found between two requirements: repeatability of sample processing and MS measurements, allowing an accurate quantification, and high proteomic coverage of the sample, allowing quantification of minor species. The latter is generally achieved through sample fractionation, which may induce experimental bias during the label-free comparison of samples processed, and analyzed independently. In this work, we wanted to evaluate the performances of MS intensity-based label-free quantification when a complex protein sample is fractionated by one-dimensional SDS-PAGE. We first tested the efficiency of the analysis without protein fractionation and could achieve quite good quantitative repeatability in single-run analysis (median coefficient of variation of 5%, 99% proteins with coefficient of variation <48%). We show that sample fractionation by one-dimensional SDS-PAGE is associated with a moderate decrease of quantitative measurement repeatability while largely improving the depth of proteomic coverage. We then applied the method for a large scale proteomic study of the human endothelial cell response to inflammatory cytokines, such as TNFα, interferon γ, and IL1β, which allowed us to finely decipher at the proteomic level the biological pathways involved in endothelial cell response to proinflammatory cytokines.     

Canalization and developmental instability of the fetal skull in a mouse model of maternal nutritional stress

Nutritional imbalance is one of the main sources of stress in both extant and extinct human populations. Restricted availability of nutrients is thought to disrupt the buffering mechanisms that contribute to developmental stability and canalization, resulting in increased levels of fluctuating asymmetry (FA) and phenotypic variance among individuals. However, the literature is contradictory in this regard. This study assesses the effect of prenatal nutritional stress on FA and among‐individual variance in cranial shape and size using a mouse model of maternal protein restriction. Two sets of landmark coordinates were digitized in three dimensions from skulls of control and protein restricted specimens at E17.5 and E18.5. We found that, by the end of gestation, maternal protein restriction resulted in a significant reduction of skull size. Fluctuating asymmetry in size and shape exceeded the amount of measurement error in all groups, but no significant differences in the magnitude of FA were found between treatments. Conversely, the pattern of shape asymmetry was affected by the environmental perturbation since the angles between the first eigenvectors extracted from the covariance matrix of shape asymmetric component of protein restricted and control groups were not significantly different from the expected for random vectors. In addition, among‐individual variance in cranial shape was significantly higher in the protein restricted than the control group at E18.5. Overall, the results obtained from a controlled experiment do not support the view of fluctuating asymmetry of cranial structures as a reliable index for inferring nutritional stress in human populations. Am J Phys Anthropol 154:544–553, 2014. © 2014 Wiley Periodicals, Inc.     

Targeted elimination of peroxisome proliferator-activated receptor gamma in beta cells leads to abnormalities in islet mass without compromising glucose homeostasis

The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) is an important regulator of lipid and glucose homeostasis and cellular differentiation. Studies of many cell types in vitro and in vivo have demonstrated that activation of PPAR gamma can reduce cellular proliferation. We show here that activation of PPAR gamma is sufficient to reduce the proliferation of cultured insulinoma cell lines. We created a model with mice in which the expression of the PPARG gene in beta cells was eliminated (beta gamma KO mice), and these mice were found to have significant islet hyperplasia on a chow diet. Interestingly, the normal expansion of beta-cell mass that occurs in control mice in response to high-fat feeding is markedly blunted in these animals. Despite this alteration in beta-cell mass, no effect on glucose homeostasis in beta gamma KO mice was noted. Additionally, while thiazolidinediones enhanced insulin secretion from cultured wild-type islets, administration of rosiglitazone to insulin-resistant control and beta gamma KO mice revealed that PPAR gamma in beta cells is not required for the antidiabetic actions of these compounds. These data demonstrate a critical physiological role for PPAR gamma function in beta-cell proliferation and also indicate that the mechanisms controlling beta-cell hyperplasia in obesity are different from those that regulate baseline cell mass in the islet.