P2X7 receptors mediate NADH transport across the plasma membranes of astrocytes

NADH plays critical roles in mitochondrial functions and energy metabolism. There has been no study demonstrating that NADH can be transported across the plasma membranes of cells. In this study we tested our hypothesis that NADH can be transported across the plasma membranes of astrocytes by a P2X7 receptor (P2X7R)-mediated mechanism. We found that treatment of astrocytes with NADH led to increases in both intracellular NADH and NAD+. Three lines of studies suggest that P2X7R mediates the NADH transport into astrocytes: the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) blocked the NADH transport; RNAi knockdown of P2X7R led to decreased NADH transport; and transfection of HEK293 cells with mouse P2X7R cDNA led to increased NADH transport. Collectively, our study provides the first direct evidence demonstrating that NADH can be transported across the plasma membranes of astrocytes by a P2X7R-mediated mechanism. Our study also suggests a novel approach for manipulating intracellular NADH and NAD+ levels.     

Construction of mathematical model for high-level expression of foreign genes in pPIC9 vector and its verification

In this report, we introduced a mathematical model for high-level expression of foreign genes in pPIC9 vector. At first, we collected 40 heterologous genes expressed in pPIC9 vector, and these 40 genes were classified into high-level expression group (expression level >100mg/L, 12 genes) and low-level expression group (expression level <100mg/L, 28 genes). Then, the Naive Bayes method was used to construct the model with RNA secondary structure profile of 3'-end of foreign genes as features. The classification accuracy from leave-one-out cross-validation was 100%. Finally, another five genes collected from literatures were used to test the ability of the model. The results indicated that there were four genes correctly predicted. In addition, the model was also verified by expressing human neutrophil gelatinase-associated lipocalin (NGAL) gene with expression level more than 100mg/L. Therefore, we propose that the model can be used to predict the expression level of heterologous genes before experiments and optimize the experiment designs to obtain the high-level expression. Furthermore, we have developed a web server for evaluation and design for high-level expression of foreign genes, which is accessible at http://ppic9.med.stu.edu.cn/ppic9.     

Joint analysis of longitudinal data with informative right censoring

Longitudinal data arise when subjects are followed over a period of time. A commonly encountered complication in the analysis of such data is the variable length of follow-up due to right censorship. This can be further exacerbated by the possible dependency between the censoring time and the longitudinal measurements. This article proposes a combination of a semiparametric transformation model for the censoring time and a linear mixed effects model for the longitudinal measurements. The dependency is handled via latent variables which are naturally incorporated. We show that the likelihood function has an explicit form and develops a two-stage estimation procedure to avoid direct maximization over a high-dimensional parameter space. The resulting estimators are shown to be consistent and asymptotically normal, with a closed form for the variance-covariance matrix that can be used to obtain a plug-in estimator. Finite sample performance of the proposed approach is assessed through extensive simulations. The method is applied to renal disease data.     

A Novel Ferric Reductase Purified from <Emphasis Type="Italic">Magnetospirillum gryphiswaldense</Emphasis> MSR-1

A ferric reductase was purified into an electrophoretically homologous state from Magnetospirillum gryphiswaldense MSR-1 strain. The enzyme was found within the cytoplasm and associated with the cytoplasmic membrane. The molecular weight of the purified enzyme was calculated as 16.1 kDa using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and was almost identical to that calibrated using agarose gel filtration chromatography. It was NADH-dependent and required flavin mononucleotide as a cofactor. The optimal reaction temperature and pH values were 30°C and 6.5, respectively. The K _m and Vmax values for ferric citrate were 45.1 μM and 1.216 μM min⁻¹, respectively. Though ferric reductase activity could be inhibited by Co²⁺, Cu²⁺, Mn²⁺, and Zn²⁺, even high concentrations of Mg²⁺ ions have failed to accomplish such enzyme inhibition. Furthermore, the molecular weight, the N-terminal sequence, and the activity of ferric reductase from MSR-1 are not matching with the enzyme preparation obtained from an analogous strain M. magnetotacticum (MS-1). Therefore, it is concluded that the ferric reductase of M. grysphiwaldense and M. magnetotacticum strains are two different enzymes.     

Purified and sterilized magnetosomes from Magnetospirillum gryphiswaldense MSR-1 were not toxic to mouse fibroblasts in vitro

AIMS: To establish a criterion for measuring the purity of purified and sterilized magnetosomes from Magnetospirillum gryphiswaldense and to evaluate their toxicity for mouse fibroblasts in vitro. METHODS AND RESULTS: The purification of magnetosomes involves disrupting bacterial cells with a French Press, washing directly with PBS buffer accompanied by treatment with low power ultrasonication, and using a magnet to collect the magnetosomes. Five characteristic peaks were displayed by Fourier-transform infrared spectroscopy (FT-IR), which was used to detect the quality of the purified magnetosomes, at 3273, 2921, 1735, 1645 and 1531 cm(-1). The purified magnetosomes showed no evidence of impurities when observed by transmission electron microscopy and energy disperse spectroscopy. The particles could be stored at -20 degrees C after lyophilization and treatment by gamma-rays. Purified and sterilized magnetosomes had no obvious negative effects on the viability of mouse fibroblasts by 3-(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide assay. CONCLUSIONS: Purified and sterilized magnetosomes were not toxic to mouse fibroblasts in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides methods for evaluating the purity and safety of magnetosomes from M. gryphiswaldense. The magnetosomes have the potential to be used as novel drug or gene carriers for tumour therapy.     

Temporal and spatial distribution of biogenic silica and its significance in the Wuliangsuhai Lake and Daihai Lake

In this work, the concentrations, temporal and spatial distributions and the relationship between biogenic silica (BSi) and primary productivity are discussed on the basis of the geochemistry character of BSi in the water–sediment system of the Wuliangsuhai Lake and Daihai Lake. The results show that the average concentrations of SiO₃^2- and BSi are 3.0 mg/L and 3.5 mg/g in the overlying water and sediments from the Wuliangsuhai Lake, respectively, while they are 1.0 mg/L and 7.5 mg/g, respectively, in the Daihai Lake. It is the uptake and assimilation of diatom phytoplankton which results in the significant difference of the SiO₃^2- concentrations between the two lakes, and the inputs of surface runoff is one of the important factors in impacting the spatial distributions of SiO₃^2- in the overlying water. The spatial distributions of BSi suggest the Si source of the two lakes and indicate the differences of eutrophication types and ancient primary producer between the two lakes. The eutrophication precesses and ancient primary productivity of diatom phytoplankton are reconstructed by applying the geochemistry information of BSi archived in the vertical concentration profiles in the two lake sediments. The geochemistry information of BSi well responds to the paleoenvironment and paleoclimate of the Daihai drainage basin indicating silicate limitation of primary productivity by diatoms phytoplankton in the Daihai Lake.     

Co-translational binding of GroEL to nascent polypeptides is followed by post-translational encapsulation by GroES to mediate protein folding

The eubacterial chaperonins GroEL and GroES are essential chaperones and primarily assist protein folding in the cell. Although the molecular mechanism of the GroEL system has been examined previously, the mechanism by which GroEL and GroES assist folding of nascent polypeptides during translation is still poorly understood. We previously demonstrated a co-translational involvement of the Escherichia coli GroEL in folding of newly synthesized polypeptides using a reconstituted cell-free translation system (Ying, B. W., Taguchi, H., Kondo, M., and Ueda, T. (2005) J. Biol. Chem. 280, 12035-12040). Employing the same system here, we further characterized the mechanism by which GroEL assists folding of translated proteins via encapsulation into the GroEL-GroES cavity. The stable co-translational association between GroEL and the newly synthesized polypeptide is dependent on the length of the nascent chain. Furthermore, GroES is capable of interacting with the GroEL-nascent peptide-ribosome complex, and experiments using a single-ring variant of GroEL clearly indicate that GroES association occurs only at the trans-ring, not the cis-ring, of GroEL. GroEL holds the nascent chain on the ribosome in a polypeptide length-dependent manner and post-translationally encapsulates the polypeptide using the GroES cap to accomplish the chaperonin-mediated folding process.