Impact of gender on basal and insulin-like growth factor I-regulated nitric oxide synthase activity in adult rat left ventricular myocytes

Cardiovascular morbidity and mortality are far less in pre-menopausal women compared to age-matched men. Ovarian hormones are believed to be mainly responsible for this "female advantage" in cardiovascular function although the underlying mechanism has not been fully elucidated. A gender difference exists in vascular nitric oxide (NO) synthesis, which may play a key role in ventricular function and cardiac remodeling. This study was designed to compare NO production, basal NO synthase (NOS) expression and activity, as well as insulin-like growth factor I (IGF-1)-induced response on NOS activity in left ventricular myocytes from age-matched adult male and female Sprague-Dawley rats. NO production and protein expression of NOS, IGF-1 receptor (IGF-1R) and IGF binding protein-3 (IGFBP-3) were measured by Griess assay and Western blot analysis, respectively. NOS activity was evaluated by conversion of (3)H-arginine to (3)H-citrulline. Basal NO production, endothelial NOS expression and NOS activity were both significantly higher in female left ventricular myocytes than their male counterparts. However, protein expression of inducible and neuronal NOS as well as IGFBP-3 was comparable between the two genders. IGF-1R expression was less in female than male group. IGF-1 (10(-10)-10(-6) m) induced a concentration-dependent inhibition of NOS activity in male myocytes with a maximal inhibition of 22.2%. However, the IGF-1-induced inhibition in NOS activity was not present in left ventricular myocytes from female rats. These data revealed a gender difference in myocardial basal NO levels, endothelial NOS expression, basal NOS activity and IGF-1-induced inhibition on NOS activity, which may contribute to the gender-related difference of cardiac function.     

P-glycoprotein-ATPase modulation: the molecular mechanisms

P-glycoprotein-ATPase is an efflux transporter of broad specificity that counteracts passive allocrit influx. Understanding the rate of allocrit transport therefore matters. Generally, the rates of allocrit transport and ATP hydrolysis decrease exponentially with increasing allocrit affinity to the transporter. Here we report unexpectedly strong down-modulation of the P-glycoprotein-ATPase by certain detergents. To elucidate the underlying mechanism, we chose 34 electrically neutral and cationic detergents with different hydrophobic and hydrophilic characteristics. Measurement of the P-glycoprotein-ATPase activity as a function of concentration showed that seven detergents activated the ATPase as expected, whereas 27 closely related detergents reduced it significantly. Assessment of the free energy of detergent partitioning into the lipid membrane and the free energy of detergent binding from the membrane to the transporter revealed that the ratio, q, of the two free energies of binding determined the rate of ATP hydrolysis. Neutral (cationic) detergents with a ratio of q = 2.7 ± 0.2 (q > 3) followed the aforementioned exponential dependence. Small deviations from the optimal ratio strongly reduced the rates of ATP hydrolysis and flopping, respectively, whereas larger deviations led to an absence of interaction with the transporter. P-glycoprotein-ATPase inhibition due to membrane disordering by detergents could be fully excluded using ²H-NMR-spectroscopy. Similar principles apply to modulating drugs.     

Enhanced uridine 5′-monophosphate production by whole cell of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> through rational redistribution of metabolic flux

A whole-cell biocatalytic process for uridine 5′-monophosphate (UMP) production from orotic acid by Saccharomyces cerevisiae was developed. To rationally redistribute the metabolic flux between glycolysis and pentose phosphate pathway, statistical methods were employed first to find out the critical factors in the process. NaH₂PO₄, MgCl₂ and pH were found to be the important factors affecting UMP production significantly. The levels of these three factors required for the maximum production of UMP were determined: NaH₂PO₄ 22.1 g/L; MgCl₂ 2.55 g/L; pH 8.15. An enhancement of UMP production from 6.12 to 8.13 g/L was achieved. A significant redistribution of metabolic fluxes was observed and the underlying mechanism was discussed.     

Folin-ciocalteu比色法测桑叶中多酚含量

采用以没食子酸为标准,研究了Folin-ciocalteu比色法测定桑叶中多酚含量的适宜条件。结果表明,桑叶提取液在Folin-ciocalteu试剂2.0 mL、20%Na2CO3溶液5.0 mL、反应温度30℃、反应时间2 h的条件下,测定其760 nm处的吸光值,多酚浓度在10.0～100.0 mg/L范围内与吸光值呈良好的线性关系;稳定性、精密度、重现性和回收率实验的相对标准偏差为0.2969%～2.502%。该法是一种简便、快速、准确测定桑叶多酚含量的可靠方法。     

Proteomic analysis of early-responsive redox-sensitive proteins in Arabidopsis

Regulation of protein function through oxidative modification has emerged as an important molecular mechanism modulating various biological processes. Here, we report a proteomic study of redox-sensitive proteins in Arabidopsis cells subjected to H(2)O(2) treatment. Four gel-based approaches were employed, leading to the identification of four partially overlapping sets of proteins whose thiols underwent oxidative modification in the H(2)O(2)-treated cells. Using a method based on differential labeling of thiols followed by immunoprecipitation and Western blotting, five of the six selected putative redox-sensitive proteins were confirmed to undergo oxidative modification following the oxidant treatment in Arabidopsis leaves. Another method, which is based on differential labeling of thiols coupled with protein electrophoretic mobility shift assay, was adopted to reveal that one of the H(2)O(2)-sensitive proteins, a homologue of cytokine-induced apoptosis inhibitor 1 (AtCIAPIN1), also underwent oxidative modification in Arabidopsis leaves after treatments with salicylic acid or the peptide elicitor flg22, two inducers of defense signaling. The redox-sensitive proteins identified from the proteomic study are involved in various biological processes such as metabolism, the antioxidant system, protein biosynthesis and processing, and cytoskeleton organization. The identification of novel redox-sensitive proteins will be helpful toward understanding of cellular components or pathways previously unknown to be redox-regulated.     

Adaptive Evolution and the Birth of CTCF Binding Sites in the Drosophila Genome

Changes in the physical interaction between cis-regulatory DNA sequences and proteins drive the evolution of gene expression. However, it has proven difficult to accurately quantify evolutionary rates of such binding change or to estimate the relative effects of selection and drift in shaping the binding evolution. Here we examine the genome-wide binding of CTCF in four species of Drosophila separated by between ∼2.5 and 25 million years. CTCF is a highly conserved protein known to be associated with insulator sequences in the genomes of human and Drosophila. Although the binding preference for CTCF is highly conserved, we find that CTCF binding itself is highly evolutionarily dynamic and has adaptively evolved. Between species, binding divergence increased linearly with evolutionary distance, and CTCF binding profiles are diverging rapidly at the rate of 2.22% per million years (Myr). At least 89 new CTCF binding sites have originated in the Drosophila melanogaster genome since the most recent common ancestor with Drosophila simulans. Comparing these data to genome sequence data from 37 different strains of Drosophila melanogaster, we detected signatures of selection in both newly gained and evolutionarily conserved binding sites. Newly evolved CTCF binding sites show a significantly stronger signature for positive selection than older sites. Comparative gene expression profiling revealed that expression divergence of genes adjacent to CTCF binding site is significantly associated with the gain and loss of CTCF binding. Further, the birth of new genes is associated with the birth of new CTCF binding sites. Our data indicate that binding of Drosophila CTCF protein has evolved under natural selection, and CTCF binding evolution has shaped both the evolution of gene expression and genome evolution during the birth of new genes.     

Loss of BRCA1-A complex function in RAP80 null tumor cells

Receptor Associated Protein 80 (RAP80) is a subunit of the BRCA1-A complex and targets BRCA1 to DNA damage sites in response to DNA double strand breaks. Since mutations of BRCA1 are associated with familial ovarian cancers, we screened 26 ovarian cancer-derived cell lines for RAP80 mutations and found that TOV-21G cells harbor a RAP80 mutation (c.1107G >A). This mutation generates a stop codon at Trp369, which deletes the partial AIR region and the C-terminal zinc fingers of RAP80. Interestingly, both the mutant and wild type alleles of RAP80 lose their expression due to promoter hypermethylation, suggesting that TOV-21G is a RAP80-null cell line. In these cells, not only is the BRCA1-A complex disrupted, but the relocation of the remaining subunits in the BRCA1-A complex including BRCA1, CCDC98, NBA1, BRCC36 and BRE is significantly suppressed. Moreover, TOV-21G cells are hypersensitive to ionizing radiation, which is due to the compromised DNA damage repair capacity in these cells. Reconstitution of TOV-21G cells with wild type RAP80 rescues these cellular defects in response to DNA damage. Thus, our results demonstrate that RAP80 is a scaffold protein in the BRCA1-A complex. Identification of TOV-21G as a RAP80 null tumor cell line will be very useful for the study of the molecular mechanism in DNA damage response.     

Frequent recent origination of brain genes shaped the evolution of foraging behavior in Drosophila

The evolution of the brain and behavior are coupled puzzles. The genetic bases for brain evolution are widely debated, yet whether newly evolved genes impact the evolution of the brain and behavior is vaguely understood. Here, we show that during recent evolution in Drosophila, new genes have frequently acquired neuronal expression, particularly in the mushroom bodies. Evolutionary signatures combined with expression profiling showed that natural selection influenced the evolution of young genes expressed in the brain, notably in mushroom bodies. Case analyses showed that two young retrogenes are expressed in the olfactory circuit and facilitate foraging behavior. Comparative behavioral analysis revealed divergence in foraging behavior between species. Our data suggest that during adaptive evolution, new genes gain expression in specific brain structures and evolve new functions in neural circuits, which might contribute to the phenotypic evolution of animal behavior.     

Selective separation of biobutanol from acetone-butanol-ethanol fermentation broth by means of sorption methodology based on a novel macroporous resin

The traditional distillation method for recovery of butanol from fermentation broth is an energy-intensive process. Separation of butanol based on adsorption methodology has advantages in terms of biocompatibility and stability, as well as economy, and therefore gains much attention. However, the application of the commercial adsorbents in the integrated acetone-butanol-ethanol (ABE) fermentation process is restricted due to the low recovery (less than 85%) and the weak capability of enrichment in the eluent (3-4 times). In this study, we investigated the sorption properties of butanol onto three kinds of adsorbents with different polarities developed in our laboratory, that is, XD-41, H-511, and KA-I resin. The sorption behaviors of single component and ABE ternary mixtures presented in the fermentation broths on KA-I resin were investigated. KA-I resin had higher affinity for butanol than for acetone, ethanol, glucose, acetic acid, and butyric acid. Multicomponent ABE sorption on KA-I resin was modeled using a single site extended Langmuir isotherm model. In a desorption study, all the adsorbed components were desorbed in one bed volume of methanol, and the recovery of butanol from KA-I resin was 99.7%. The concentration of butanol in the eluent was increased by a factor of 6.13. In addition, KA-I resin was successfully regenerated by two bed volumes of water. Because of its quick sorption, high sorption capacity, low cost, and ease of desorption and regeneration, KA-I resin exhibits good potential for compatibility with future ABE fermentation coupled with in situ recovery product removal techniques.     

Nonparametric Method for Genomics-Based Prediction of Performance of Quantitative Traits Involving Epistasis in Plant Breeding

Genomic selection (GS) procedures have proven useful in estimating breeding value and predicting phenotype with genome-wide molecular marker information. However, issues of high dimensionality, multicollinearity, and the inability to deal effectively with epistasis can jeopardize accuracy and predictive ability. We, therefore, propose a new nonparametric method, pRKHS, which combines the features of supervised principal component analysis (SPCA) and reproducing kernel Hilbert spaces (RKHS) regression, with versions for traits with no/low epistasis, pRKHS-NE, to high epistasis, pRKHS-E. Instead of assigning a specific relationship to represent the underlying epistasis, the method maps genotype to phenotype in a nonparametric way, thus requiring fewer genetic assumptions. SPCA decreases the number of markers needed for prediction by filtering out low-signal markers with the optimal marker set determined by cross-validation. Principal components are computed from reduced marker matrix (called supervised principal components, SPC) and included in the smoothing spline ANOVA model as independent variables to fit the data. The new method was evaluated in comparison with current popular methods for practicing GS, specifically RR-BLUP, BayesA, BayesB, as well as a newer method by Crossa et al., RKHS-M, using both simulated and real data. Results demonstrate that pRKHS generally delivers greater predictive ability, particularly when epistasis impacts trait expression. Beyond prediction, the new method also facilitates inferences about the extent to which epistasis influences trait expression.