结核分枝杆菌耐吡嗪酰胺分子机制研究

吡嗪酰胺(PZA)是结核病短程化疗中的一线抗结核药物,由吡嗪酰胺酶转换成为活性形式吡嗪酸而生效。吡嗪酰胺酶由pncA基因编码,pncA基因突变会导致该酶活性丧失,与PZA耐药性产生有关。为了进一步明确PZA耐药性产生的基因学基础和PZA耐药株的pncA基因突变率,对中国100株结核分枝杆菌临床分离株进行了DNA序列测定,其中85株为PZA耐药株,15株为PZA敏感株。PZA耐药株有27%(23/85)发生了pncA基因突变,从而导致吡嗪酰胺酶基本氨基酸序列的改变,突变分布在pncA基因开读框架17-546位的核苷酸。其中有一株突变位于pncA基因的调节区域-11位处。同时发现20%(3/15)pncA敏感株也发生了pncA基因突变。敏感株发生突变可能是由于PZA敏感性实验不准确或存在其它耐药机制。实验表明,pncA基因突变是PZA耐药的主要机制之一,中国PZA耐药临床分离株尚存在其它耐药分子机制。     

Role of Glu312 in binding and positioning of the substrate for the hydride transfer reaction in choline oxidase

Choline oxidase catalyzes the oxidation of choline to glycine betaine, a compatible solute that accumulates in pathogenic bacteria and plants so they can withstand osmotic and temperature stresses. The crystal structure of choline oxidase was determined and refined to a resolution of 1.86 A with data collected at 100 K using synchrotron X-ray radiation. The structure reveals a covalent linkage between His99 Nepsilon2 and FAD C8M atoms, and a 123 A3 solvent-excluded cavity adjacent to the re face of the flavin. A hypothetical model for choline docked into the cavity suggests that several aromatic residues and Glu312 may orient the cationic substrate for efficient catalysis. The role of the negative charge on Glu312 was investigated by engineering variant enzymes in which Glu312 was replaced with alanine, glutamine, or aspartate. The Glu312Ala enzyme was inactive. The Glu312Gln enzyme exhibited a Kd value for choline at least 500 times larger than that of the wild-type enzyme. The Glu312Asp enzyme had a kcat/KO2 value similar to that of the wild-type enzyme but kcat and kcat/Km values that were 230 and 35 times lower, respectively, than in the wild-type enzyme. These data are consistent with the spatial location of the negative charge on residue 312 being important for the oxidation of the alcohol substrate. Solvent viscosity and substrate kinetic isotope effects suggest the presence of an internal equilibrium in the Glu312Asp enzyme prior to the hydride transfer reaction. Altogether, the crystallographic and mechanistic data suggest that Glu312 is important for binding and positioning of the substrate in the active site of choline oxidase.     

Molecular phylogeny of Pneumocystis based on 5.8S rRNA gene and the internal transcribed spacers of rRNA gene sequences

To clarify the phylogenetic relationships and species status of Pneumocystis, the 5.8S rRNA gene and the internal transcribed spacers (ITS, 1 and 2) of Pneumocystis rRNA derived from rat, gerbil and human were amplified, cloned and sequenced. The genetic distance matrix of six Pneumocystis species compared with other fungi like Taphrina and Saccharomyces indicated that the Pneumocystis genus contained multiple species including Pneumocystis from gerbil. The phylogenetic tree also showed that Pneumocystis from human and monkey formed one group and four rodent Pneumocystis formed another group. Among the four members, Pneumocystis wakefieldiae was most closely related to Pneumocystis murina and Pneumocystis carinii, and was least related to gerbil Pneumocystis.     

Efficient suspension bioreactor expansion of murine embryonic stem cells on microcarriers in serum-free medium

Large numbers of cells will be required for successful embryonic stem cell (ESC)-based cellular therapies or drug discovery, thus raising the need to develop scaled-up bioprocesses for production of ESCs and their derived progeny. Traditionally, ESCs have been propagated in adherent cultures in static flasks on fibroblasts layers in serum-containing medium. Direct translation of two-dimensional flatbed cultures to large-scale production of the quantities of cells required for therapy simply by increasing the number of dishes or flasks is not practical or economical. Here, we describe successful scaled-up production of ESCs on microcarriers in a stirred culture system in a serum-free medium. Cells expanded on CultiSpher S, Cytodex 3, and Collagen microcarriers showed superior cell-fold expansions of 439, 193, and 68, respectively, without excessive agglomeration, compared with 27 in static culture. In addition, the ESCs maintained their pluripotency after long-term culture (28 days) in serum-free medium. This is the first time mESCs have been cultured on microcarriers without prior exposure to serum and/or fibroblasts, while also eliminating the excessive agglomeration plaguing earlier studies. These protocols provide an economical, practical, serum-free means for expanding ESCs in a stirred suspension bioprocess.     

Changes in proteomics profile during maturation of marrow-derived dendritic cells treated with oxidized low-density lipoprotein

Increasing evidence suggests that dendritic cells (DCs) and oxidized low-density lipoprotein (ox-LDL) participate in atherosclerosis. However, few data on the molecular mechanisms of this process are available. To address this question, we used iTRAQ labeling followed by LC-MS/MS analysis to identify many proteins that changed markedly during the maturation of dendritic cells stimulated with ox-LDL. Among a total of 781 identified proteins, 93 were upregulated and 100 were downregulated. The major and significant changes in upregulated proteins were that ox-LDL not only affected the levels of intracellular cathepsins G, Z, D and S, but also significantly enhanced cathepsin S secretion by the treated cells. Our results may provide clues for a more comprehensive understanding the pathogenesis of atherosclerosis.     

Genetic model of selective COX2 inhibition reveals novel heterodimer signaling

Selective inhibitors of cyclooxygenase-2 (COX2) have attracted widespread media attention because of evidence of an elevated risk of cardiovascular complications in placebo-controlled trials, resulting in the market withdrawal of some members of this class. These drugs block the cyclooxygenase activity of prostaglandin H synthase-2 (PGHS2), but do not affect the associated peroxidase function. They were developed with the rationale of conserving the anti-inflammatory and analgesic actions of traditional nonsteroidal anti-inflammatory drugs (tNSAIDs) while sparing the ability of PGHS1-derived prostaglandins to afford gastric cytoprotection. PGHS1 and PGHS2 coexist in the vasculature and in macrophages, and are upregulated together in inflammatory tissues such as rheumatoid synovia and atherosclerotic plaque. They are each believed to function as homodimers. Here, we developed a new genetic mouse model of selective COX2 inhibition using a gene-targeted point mutation, resulting in a Y385F substitution. Structural modeling and biochemical assays showed the ability of PGHS1 and PGHS2 to heterodimerize and form prostaglandins. The heterodimerization of PGHS1-PGHS2 may explain how the ductus arteriosus closes normally at birth in mice expressing PGHS2 Y385F, but not in PGHS2-null mice.     

Calcium carbonate-methylene blue nanohybrids for photodynamic therapy and ultrasound imaging

Photodynamic therapy plays an important role in cancer treatment. In this work, methylene blue (MB)-embedded calcium carbonate nanorods (CaCO₃-MB NRs) have been synthesized for pH-responsive photodynamic therapy and ultrasound imaging. The morphology of CaCO₃-MB NRs can be controlled by modulating the concentration of Na₂CO₃ aqueous solution. The generation of effective reactive oxygen species (ROS) were confirmed by 1,3-diphenylisobenzofuran (DPBF) probe. Both photodynamic therapy performance and echogenic performance of CaCO₃-MB NRs were investigated to confirm the feasibility of CaCO₃-MB nanohybrids for ultrasound image-guided photodynamic therapy.     

Microbial Reduction of Cr (VI)-loaded Schwertmannite by Shewanella oneidensis MR-1

Microbial transformation of sulfate minerals plays an important role in controlling the behavior of heavy metals in mining areas. Here, the anaerobic reduction of Cr (VI)-loaded schwertmannite by Shewanella oneidensis MR-1 (S. oneidensis MR-1) was investigated. The release of ferrous iron (Fe(II)) to the solution demonstrated the microbial reduction of structural Fe(III) from the schwertmannite to Fe(II). The concentration of Cr in solution decreased in all treatments, indicating that no Cr was released to the solution during this bio-reduction process of schwertmannite. The incorporation of chromate into the mineral structure of schwertmannite increased the microbial stability of the mineral, retarding the formation of secondary phases during bio-reduction process. Analysis of the XRD, SEM and fourier transform infrared spectroscopy (FT-IR) results further showed that goethite formed after 3 or 7 days with a lower content (0.22% or 0.37%) of Cr in schwertmannite, while no secondary mineral was observed with a higher concentration of Cr (0.6 wt%) incorporated in schwertmannite until 22 days. These results imply that microbial reduction of Cr(VI)-loaded schwertmannite does not lead to the release of Cr to the solution, and the microbial stability of schwertmannite will be increased by the incorporation of chromate.