Proteomic analysis of the nucleus accumbens in rhesus monkeys of morphine dependence and withdrawal intervention

It has been known that the reinforcing effects and long-term consequences of morphine are closely associated with nucleus accumbens (NAc) in the brain, a key region of the mesolimbic dopamine pathway. However, the proteins involved in neuroadaptive processes and withdrawal symptom in primates of morphine dependence have not been well explored. In the present study, we performed proteomes in the NAc of rhesus monkeys of morphine dependence and withdrawal intervention with clonidine or methadone. Two-dimensional electrophoresis was used to compare changes in cytosolic protein abundance in the NAc. We found a total of 46 proteins differentially expressed, which were further identified by mass spectrometry analysis. The identified proteins can be classified into 6 classes: metabolism and mitochondrial function, synaptic transmission, cytoskeletal proteins, oxidative stress, signal transduction and protein synthesis and degradation. Importantly, we discovered 14 proteins were significantly but similarly altered after withdrawal therapy with clonidine or methadone, revealing potential pharmacological strategies or targets for the treatment of morphine addiction. Our study provides a comprehensive understanding of the neuropathophysiology associated with morphine addiction and withdrawal therapy in primate, which is helpful for the development of opiate withdrawal pharmacotherapies.     

Refolding and two-step purification by hydrophobic interaction chromatography of recombinant human bone morphogenetic protein-2 from Escherichia coli

To renature the inactive rhBMP-2 which overexpressed in Escherichia coli, post-expression treatments including inclusion bodies solubilization and in vitro refolding were systematically investigated. An optimized refolding process was established from screening and successfully scaled up with yield greater than 70%. Then, hydrophobic interaction chromatography (HIC) was adopted as two consecutive stages to separate the active rhBMP-2 homodimer from refolding mixture. Aiding additive N,N-dimethylformamide (DMF) was found to enhance the resolution of rhBMP-2 homodimer most effectively. The rhBMP-2 homodimer was purified to homogeneity through two HIC separations at different salt contents, the purified rhBMP-2 homodimer was fully bioactive and had equivalent biological activity to rhBMP-2 produced from Chinese hamster ovary cell (CHO). Under the optimal refolding and purification conditions, 80 mg rhBMP-2 homodimer with high purity could be obtained from 1 g wet weight of inclusion bodies. Finally, this efficient refolding and purification procedure was successfully scaled up in the pilot pharmaceutical plant.     

Beam-induced motion of vitrified specimen on holey carbon film

The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3-0.5 μm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.     

Engineering of an N-acetylneuraminic acid synthetic pathway in Escherichia coli

N-acetylneuraminic acid (NeuAc) has recently drawn much attention owing to its wide applications in many aspects. Besides extraction from natural materials, production of NeuAc was recently focused on enzymatic synthesis and whole-cell biocatalysis. In this study, we designed an artificial NeuAc biosynthetic pathway through intermediate N-acetylglucosamine 6-phosphate in Escherichia coli. In this pathway, N-acetylglucosamine 2-epimerase (slr1975) and glucosamine-6-phosphate acetyltransferase (GNA1) were heterologously introduced into E. coli from Synechocystis sp. PCC6803 and Saccharomyces cerevisiae EBY100, respectively. By derepressing the feedback inhibition of glucosamine-6-phosphate synthase, increasing the accumulation of N-acetylglucosamine and pyruvate, and blocking the catabolism of NeuAc, we were able to produce 1.62 g l^?1 NeuAc in recombinant E. coli directly from glucose. The NeuAc yield reached 7.85 g l^?1 in fed-batch fermentation. This process offered an efficient fermentative method to produce NeuAc in microorganisms using glucose as carbon source and can be optimized for further improvement.     

MOCAT: A Metagenomics Assembly and Gene Prediction Toolkit

MOCAT is a highly configurable, modular pipeline for fast, standardized processing of single or paired-end sequencing data generated by the Illumina platform. The pipeline uses state-of-the-art programs to quality control, map, and assemble reads from metagenomic samples sequenced at a depth of several billion base pairs, and predict protein-coding genes on assembled metagenomes. Mapping against reference databases allows for read extraction or removal, as well as abundance calculations. Relevant statistics for each processing step can be summarized into multi-sheet Excel documents and queryable SQL databases. MOCAT runs on UNIX machines and integrates seamlessly with the SGE and PBS queuing systems, commonly used to process large datasets. The open source code and modular architecture allow users to modify or exchange the programs that are utilized in the various processing steps. Individual processing steps and parameters were benchmarked and tested on artificial, real, and simulated metagenomes resulting in an improvement of selected quality metrics. MOCAT can be freely downloaded at http://www.bork.embl.de/mocat/.     

Expression pattern of Wnt inhibitor factor 1(Wif1) during the development in mouse CNS

Wnt inhibitor factor-1 (WIF-1) is an extracellular antagonist of Wnts secreted proteins. Here we describe the expression pattern of Wif1 throughout the development of the mouse central nervous system (CNS). Wif1 mRNA can be detected as early as the developmental stage E11, and expression persists to adulthood. In embryonic stages, the level of Wif1 expression was very prominent in several areas including the cerebral cortex, the diencephalon and the midbrain, with the strongest level in the hippocampal plate and the diencephalon. However, after birth, the expression level of Wif1 decreased in the cortex and diencephalon. By adulthood, Wif1 is mainly expressed in the medial habenular nucleus (MHb) in the epithalamus, the mitral layer cells in the olfactory bulb and a few nuclei in the hypothalamus. Our data shows that the expression of Wif1 was very strong during embryonic development of the CNS and suggests that Wif1 may play an essential role in the spatial and temporal regulation of Wnt signals.     

Domain movement of iron sulfur protein in cytochrome bc1 complex is facilitated by the electron transfer from cytochrome b(L) to b(H)

The key step of the "protonmotive Q-cycle" mechanism for cytochrome bc1 complex is the bifurcated oxidation of ubiquinol at the Qp site. ISP is reduced when its head domain is at the b-position and subsequent move to the c1 position, to reduce cytochrome c1, upon protein conformational changes caused by the electron transfer from cytochrome b(L) to b(H). Results of analyses of the inhibitory efficacy and the binding affinity, determined by isothermal titration calorimetry, of Pm and Pf, on different redox states of cytochrome bc1 complexes, confirm this speculation. Pm inhibitor has a higher affinity and better efficacy with the cytochrome b(H) reduced complex and Pf binds better and has a higher efficacy with the ISP reduced complex.     

Domain conformational switch of the iron-sulfur protein in cytochrome bc1 complex is induced by the electron transfer from cytochrome bL to bH

Intensive biochemical, biophysical and structural studies of the cytochrome (cyt) bc(1) complex in the past have led to the formulation of the "protonmotive Q-cycle" mechanism for electron and proton transfer in this vitally important complex. The key step of this mechanism is the separation of electrons during the oxidation of a substrate quinol at the Q(P) site with both electrons transferred simultaneously to ISP and cyt b(L) when the extrinsic domain of ISP (ISP-ED) is located at the b-position. Pre-steady state fast kinetic analysis of bc(1) demonstrates that the reduced ISP-ED moves to the c(1)-position to reduce cyt c(1) only after the reduced cyt b(L) is oxidized by cyt b(H). However, the question of how the conformational switch of ISP-ED is initiated remains unanswered. The results obtained from analysis of inhibitory efficacy and binding affinity of two types of Q(P) site inhibitors, Pm and Pf, under various redox states of the bc(1) complex, suggest that the electron transfer from heme b(L) to b(H) is the driving force for the releasing of the reduced ISP-ED from the b-position to c(1)-position to reduce cyt c(1).     

Enhancement of 5-aminolevulinate production with recombinant Escherichia coli using batch and fed-batch culture system

5-Aminolevulinate (ALA) production with recombinant Escherichia coli Rosetta (DE3)/pET28a(+)-hemA was studied. In batch fermentation, the addition of glucose and glycine was effective to improve ALA production. Then the fed-batch fermentation was conducted with continuous feeding of precursors. When the concentrations of succinic acid and glycine were 7.0 g/l and 4.0 g/l, respectively, in the feeding, the ALA yield reached 4.1g/l. But the molar yield (ALA/glycine) was decreased in the fed-batch fermentation compared to batch fermentation. And it was found that the pH control during fed-batch cultivation was very important for the cell growth and ALA production. A two-stage pH value controlling strategy was suggested, in which, the pH value in the first 6h was regulated at pH 5.9, after then at pH 6.2, and the ALA yield was as high as 6.6g/l via fed-batch fermentation.