Conditional knockout of the mouse NADPH-cytochrome p450 reductase gene

NADPH-cytochrome P450 reductase (CPR or POR) is the obligatory electron donor for all microsomal cytochrome P450 (CYP or P450)-catalyzed monooxygenase reactions. Disruption of the mouse Cpr gene has been reported to cause prenatal developmental defects and embryonic lethality. In this study, we generated a mouse model with a floxed Cpr allele (termed Cpr(lox)). Homozygous Cpr(lox) mice are fertile and without any histological abnormality or any change in CPR expression. The floxed Cpr allele was subsequently deleted efficiently by crossing Cpr(lox) mice with transgenic mice having liver-specific Cre expression (Alb-Cre); the result was a decrease in the level of CPR protein in liver microsomes. The Cpr(lox) strain will be valuable for conditional Cpr gene deletion and subsequent determination of the impact of CPR loss on the metabolism of endogenous and xenobiotic compounds, as well as on postnatal development and other biological functions.     

A new computational approach for real protein folding prediction

An effective and fast minimization approach is proposed for the prediction of protein folding, in which the 'relative entropy' is used as a minimization function and the off-lattice model is used. In this approach, we only use the information of distances between the consecutive Calpha atoms along the peptide chain and a generalized form of the contact potential for 20 types of amino acids. Tests of the algorithm are performed on the real proteins. The root mean square deviations of the structures of eight folded target proteins versus the native structures are in a reasonable range. In principle, this method is an improvement on the energy minimization approach.     

Glutathione protects chemokine-scavenging and antioxidative defense functions in human RBCs

Oxidant stress, in vivo or in vitro, isknown to induce oxidative changes in human red blood cells (RBCs). Ourobjective was to examine the effect of augmenting RBC glutathione(GSH) synthesis on 1) degenerative protein loss and2) RBC chemokine- and free radical-scavenging functions inthe oxidatively stressed human RBCs by using banked RBCs as a model.Packed RBCs were stored up to 84 days at 1-6°C in Adsol or inthe experimental additive solution (Adsol fortified with glutamine,glycine, and N-acetyl-L-cysteine). Supplementingthe conventional additive with GSH precursor amino acids improved RBCGSH synthesis and maintenance. The rise in RBC -glutamylcysteineligase activity was directly proportional to the GSH content andinversely proportional to extracellular homocysteine concentration,methemoglobin formation, and losses of the RBC proteins band 3, band4.1, band 4.2, glyceraldehyde-3-phosphate dehydrogenase, and Duffyantigen (P < 0.01). Reduced loss of Duffy antigencorrelated well with a decrease in chemokine RANTES (regulated uponactivation, normal T-cell expressed, and secreted) concentration. Weconclude that the concomitant loss of GSH and proteins in oxidatively stressed RBCs can compromise RBC scavenging function. Upregulating GSHsynthesis can protect RBC scavenging (free radical and chemokine) function. These results have implications not only in a transfusion setting but also in conditions like diabetes and sickle cell anemia, inwhich RBCs are subjected to chronic/acute oxidant stresses.

     

Activity coefficients for NaCl-monosaccharide (D-glucose, D-galactose, D-xylose, D-arabinose)-water systems at 298.15 K

Electrochemical cells with a sodium ion selectivity electrode (Na-ISE) versus a chloride ion selectivity electrode (Cl-ISE) as a reference electrode were used to determine the activity coefficients for NaCl-monosaccharide (D-glucose, D-galactose, D-xylose, and D-arabinose) systems in water at 298.15 K. A comparison of the results thus obtained was made with those determined by another electromotive force (emf) method. It is shown that agreement is excellent. The Gibbs free energy parameters of the interactions between these sugars and NaCl in water were evaluated together with the parameter C1(CHOH, exo), indicating the interaction of the exocyclic CHOH group of saccharide molecules and NaCl. The results suggested that the interactions of these monosaccharides with NaCl are controlled mostly by the dominant conformer of their molecules in water.     

N-Terminal Acetylation of Two Major Latex Proteins from Arabidopsis thaliana Using Electrospray Ionization Tandem Mass Spectrometry

The primary structure of two proteins named major latex protein in Arabidopsis thaliana were characterized by matrix-assisted laser desorption/ionization time of flight mass spectrometer and Nano-electrospray ionization tandem mass spectrometry (nanoESI-MS/MS) after two-dimensional gel electrophoresis separation. We revealed that the two proteins with the same N termini and the N-terminal alanine were acetylated after methionine cleavage by fragmentation of three doubly charged peptides using a quadrupole-time of flight 2 tandem mass spectrometer. It was worth noting that one peptide with sodium addition and acetylation was sequenced. It is usually difficult to analyze the peptide sequence of sodium adduct due to the 22-Da increment. The two proteins are highly homologous, and both their N-terminal and C-terminal peptides were sequenced. Of the two proteins, gi|15236568 (spot A) appears only in the seeding stage and flower organ, but gi|15236566 (spot B) appears throughout the whole life of A. thaliana. The biological mechanism of the two proteins and the function of N-terminal acetylation remain to be elucidated. This study showed that ESI-MS/MS was a powerful tool for the characterization of N-terminal acetylation of proteins.     

Selectivity of modification when latent and activated forms of the chloroplast F1-ATPase are inactivated by 7-chloro-4-nitrobenzofurazan

The characteristics and specificity of inactivation of the chloroplast F1-ATPase (CF1) with 7-chloro-4-nitrobenzofurazan (Nbf-Cl) have been investigated. Inactivation of the octylglucoside-dependent Mg2+-ATPase activity of latent CF1 by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. Following inactivation of CF1 with [14C]Nbf-Cl, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the majority of the radioactive reagent incorporated is present in the beta subunit. Treatment of the enzyme with [14C]Nbf-Cl following dithiothreitol heat activation, led to similar labeling of the beta subunit and substantial incorporation of 14C into the gamma subunit. On complete inactivation, about 4 mol of Nbf-S-Cys is formed per mole of dithiothreitol-heat-activated CF1. Incorporation of 14C into the gamma subunit is prevented by prior treatment of the latent CF1 or of the dithiothreitol-heat-activated CF1 with iodoacetamide. Following incubation of the dithiothreitol-heat-activated CF1 with iodoacetamide, complete inactivation of the octylglucoside-dependent Mg2+-ATPase activity by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. After stabilization of the [14C]Nbf-O-Tyr derivative by treatment with sodium dithionite, a labeled peptide was purified. Automatic Edman degradation of this peptide revealed the sequence V-X-V-P-A-D-(D). The majority of the radioactivity was cleaved in the second cycle, the position occupied in CF1 by Tyr-beta-328, which is homologous to Tyr-beta-311, the residue reactive with Nbf-Cl in the beef heart mitochondrial F1-ATPase. When CF1, modified at Tyr-beta-328 with Nbf-Cl, is incubated at pH 9.0, the Nbf-O-Tyr adduct is hydrolyzed, leading to concomitant recovery of the ATPase activity. In double labeling experiments, two-dimensional isoelectric focusing in the presence of urea followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that 2-azido-ADP, covalently bound at the tight ADP binding site, and the tyrosine modified by [14C]Nbf-Cl are located in different beta subunits.     

肝脏疾病微RNA胞间传递研究的现状与思考

微RNA(microRNAs)是一类通过调控基因表达参与机体生理、病理过程的非编码RNA.近来,研究证实其可在肝脏不同类型细胞间进行传递,以调控靶细胞的功能,从而参与肝脏疾病的发生发展.但其在不同类型肝脏细胞间传递的直接实验证据--细胞共培养实验仍需考虑:不同类型细胞在共培养时的数量比例及miRNAs在不同细胞间传递的方向.miRNAs的胞间传递作为肝脏疾病病理机制的重要理论创新,在研究过程中仍需要考虑临床实践.本文对近期关于微RNA胞间传递与肝脏疾病的研究进行综述,以期促进对相关研究的思考.     

唇形科植物在害虫控制应用中的研究进展

唇形科植物精油、挥发物或提取物具有对人畜安全、环境友好、对害虫不易造成农药抗性等特点。当前,唇形科植物被广泛应用于害虫的农药控制、理化诱控和生态调控等方面。本文综述了唇形科植物精油、挥发物或提取物的有效成分及含量,及其对害虫的活性。并进一步综述单体化合物对害虫的生物活性及其作用机制。在此基础上,概述了部分唇形科植物化合物的衍生物及其生物活性,最后提出了唇形科植物在害虫控制方面的研究方向。     

A synaptic model for pain: long-term potentiation in the anterior cingulate cortex

Investigation of molecular and cellular mechanisms of synaptic plasticity is the major focus of many neuroscientists. There are two major reasons for searching new genes and molecules contributing to central plasticity: first, it provides basic neural mechanism for learning and memory, a key function of the brain; second, it provides new targets for treating brain-related disease. Long-term potentiation (LTP), mostly intensely studies in the hippocampus and amygdala, is proposed to be a cellular model for learning and memory. Although it remains difficult to understand the roles of LTP in hippocampus-related memory, a role of LTP in fear, a simplified form of memory, has been established. Here, I will review recent cellular studies of LTP in the anterior cingulate cortex (ACC) and then compare studies in vivo and in vitro LTP by genetic/ pharmacological approaches. I propose that ACC LTP may serve as a cellular model for studying central sensitization that related to chronic pain, as well as pain-related cognitive emotional disorders. Understanding signaling pathways related to ACC LTP may help us to identify novel drug target for various mental disorders.