Structure-Function Analysis of Porcine Cytochrome P450 3A29 in the Hydroxylation of T-2 Toxin as Revealed by Docking and Mutagenesis Studies

T-2 toxin, one of the type A trichothecenes, presents a potential hazard to human and animal health. Our previous work demonstrated that porcine cytochrome P450 3A29 (CYP3A29) played an important role in the hydroxylation of T-2 toxin. To identify amino acids involved in this metabolic process, T-2 toxin was docked into a homology model of CYP3A29 based on a crystal structure of CYP3A4 using AutoDock 4.0. Nine residues of CYP3A29, Arg105, Arg106, Phe108, Ser119, Lys212, Phe213, Phe215, Arg372 and Glu374, which were found within 5 Å around T-2 toxin were subjected to site-directed mutagenesis. In the oxidation of nifedipine, the CL _int value of R106A was increased by nearly two-folds compared with the wild-type CYP3A29, while the substrate affinities and CL _int values of S119A and K212A were significantly reduced. In the hydroxylation of T-2 toxin, the generation of 3′-OH-T-2 by R105A, S119A and K212A was significantly less than that by the wild-type, whereas R106A slightly increased the generation of 3′-OH-T-2. These results were further confirmed by isothermal titration calorimetry analysis, suggesting that these four residues are important in the hydroxylation of T-2 toxin and Arg105 may be a specific recognition site for the toxin. Our study suggests a possible structure-function relationship of CYP3A29 in the hydroxylation of T-2 toxin, providing with new insights into the mechanism of CYP3A enzymes in the biotransformation of T-2 toxin.     

Dissecting the sequential assembly and localization of intraflagellar transport particle complex B in chlamydomonas

Intraflagellar transport (IFT), the key mechanism for ciliogenesis, involves large protein particles moving bi-directionally along the entire ciliary length. IFT particles contain two large protein complexes, A and B, which are constructed with proteins in a core and several peripheral proteins. Prior studies have shown that in Chlamydomonas reinhardtii, IFT46, IFT52, and IFT88 directly interact with each other and are in a subcomplex of the IFT B core. However, ift46, bld1, and ift88 mutants differ in phenotype as ift46 mutants are able to form short flagella, while the other two lack flagella completely. In this study, we investigated the functional differences of these individual IFT proteins contributing to complex B assembly, stability, and basal body localization. We found that complex B is completely disrupted in bld1 mutant, indicating an essential role of IFT52 for complex B core assembly. Ift46 mutant cells are capable of assembling a relatively intact complex B, but such complex is highly unstable and prone to degradation. In contrast, in ift88 mutant cells the complex B core still assembles and remains stable, but the peripheral proteins no longer attach to the B core. Moreover, in ift88 mutant cells, while complex A and the anterograde IFT motor FLA10 are localized normally to the transition fibers, complex B proteins instead are accumulated at the proximal ends of the basal bodies. In addition, in bld2 mutant, the IFT complex B proteins still localize to the proximal ends of defective centrioles which completely lack transition fibers. Taken together, these results revealed a step-wise assembly process for complex B, and showed that the complex first localizes to the proximal end of the centrioles and then translocates onto the transition fibers via an IFT88-dependent mechanism.     

Prevalence and correlations with depression, anxiety, and other features in outpatients with chronic obstructive pulmonary disease in China: a cross-sectional case control study

Background

While it is suggested that the prevalence of asthma in developed countries may have stabilized, this is not clear in currently developing countries. Current available information for both adults and children simultaneously on the burden and impact of allergic conditions in Colombia and in many Latin American countries is limited. The objectives of this study were to estimate the prevalence for asthma, allergic rhinitis (AR), atopic eczema (AE), and atopy in six colombian cities; to quantify costs to the patient and her/his family; and to determine levels of Immunoglobulin E (IgE) in asthmatic and healthy subjects.

Methods

We conducted a cross-sectional, population-based study in six cities during the academic year 2009?C2010. We used a school-based design for subjects between 5?C17?years old. We carried out a community-based strategy for subjects between 1?C4?years old and adults between 18?C59?years old. Serum samples for total and antigen-specific (IgE) levels were collected using a population-based, nested, case?Ccontrol design.

Results

We obtained information on 5978 subjects. The largest sample of subjects was collected in Bogotá (2392). The current prevalence of asthma symptoms was 12% (95% CI, 10.5-13.7), with 43% (95% CI, 36.3-49.2) reporting having required an emergency department visit or hospitalization in the past 12?months. Physician diagnosed asthma was 7% (95% CI, 6.1-8.0). The current prevalence of AR symptoms was 32% (95% CI, 29.5-33.9), and of AE symptoms was 14% (95% CI, 12.5-15.3). We collected blood samples from 855 subjects; 60.2% of asthmatics and 40.6% of controls could be classified as atopic.

Conclusions

In Colombia, symptom prevalence for asthma, AR and AE, as well as levels of atopy, are substantial. Specifically for asthma, symptom severity and absence from work or study due to symptoms are important. These primary care sensitive conditions remain an unmet public health burden in developing countries such as Colombia.     

Arresten在烟草中的表达及其生物学活性分析

采用5'端引入His-tag的引物从携带有Arresten基因的质粒pCA中扩增血管生成抑制因子Arresten编码基因,构建其植物表达载体pCAMBIAarr并通过冻融法转化根癌农杆菌LBA4404,获得携带目的基因的重组农杆菌.采用叶盘法以重组农杆菌转化烟草,在50 μg/mL潮霉素B为选择压力下获得再生烟草植株,经过Southern杂交、RT-PCR和Western blotting检测,获得稳定整合有Arresten编码基因的烟草转基因植株.牛血管内皮细胞BCE增殖抑制实验表明,采用镍离子螯合次氨基三乙酸亲和层析法从转基因烟草叶片中分离纯化的重组Arresten蛋白具有明显的抑制牛血管内皮细胞增殖的生物活性.     

A novel fluorescent indicator-based fluorene derivative for rapid and visual trace water sensing

Fluorene nucleus derivatives show great potential for building outstanding fluorescence probes. In this paper, a novel fluorescent probe was developed by reacting with fluorene core with azacyclobutane, which exhibits typical solvation chromogenic effect in solvent. The fluorescence of the probe quenched in highly polar solvent. Based on this phenomenon, a novel fluorescence system for trace water was constructed. The response of this probe was fast (30 s) and sensitive for the detection of trace water in organic solvents, and the detection limit of water content in DMSO reached 0.13%. In addition, the probe can also be made as a test strip combined with homemade portable device and a smartphone for rapid detection of trace water. The luminescence mechanism of the probe is theoretically calculated based on time-contained density functional theory (TDDFT). To showcase its practicality, it has been applied for the detection of trace water in honey and alcohol by dipstick. This method provides a new idea for designing efficient fluorescent probes based on dipstick and mobile phone rapid detection.     

Flagellar length control system: testing a simple model based on intraflagellar transport and turnover

Flagellar length regulation provides a simple model system for addressing the general problem of organelle size control. Based on a systems-level analysis of flagellar dynamics, we have proposed a mechanism for flagellar length control in which length is set by the balance of continuous flagellar assembly and disassembly. The model proposes that the assembly rate is length dependent due to the inherent length dependence of intraflagellar transport, whereas disassembly is length independent, such that the two rates can only reach a balance point at a single length. In this report, we test this theoretical model by using three different measurements: 1) the quantity of intraflagellar transport machinery as a function of length, 2) the variation of flagellar length as a function of flagellar number, and 3) the rate of flagellar growth as a function of length. We find that the quantity of intraflagellar transport machinery is independent of length, that flagellar length is a decreasing function of flagellar number, and that flagellar growth rate in regenerating flagella depends on length and not on the time since regeneration began. These results are consistent with the balance-point model for length control. The three strategies used here are not limited to flagella and can in principle be adapted to probe size control systems for any organelle.     

Intraflagellar transport is required for the vectorial movement of TRPV channels in the ciliary membrane

The membranes of all eukaryotic motile (9 + 2) and immotile primary (9 + 0) cilia harbor channels and receptors involved in sensory transduction (reviewed by). These membrane proteins are transported from the cytoplasm onto the ciliary membrane by vesicles targeted for exocytosis at a point adjacent to the ciliary basal body. Here, we use time-lapse fluorescence microscopy to demonstrate that select GFP-tagged sensory receptors undergo rapid vectorial transport along the entire length of the cilia of Caenorhabditis elegans sensory neurons. Transient receptor potential vanilloid (TRPV) channels OSM-9 and OCR-2 move in ciliary membranes at rates comparable to the intraflagellar transport (IFT) machinery located between the membrane and the underlying axonemal microtubules. OSM-9 motility is disrupted in certain IFT mutant backgrounds. Surprisingly, motility of transient receptor potential polycystin (TRPP) channel PKD-2 (polycystic kidney disease-2), a mechano-receptor, was not detected. Our study demonstrates that IFT, previously shown to be necessary for transport of axonemal components, is also involved in the motility of TRPV membrane protein movement along cilia of C. elegans sensory cells.     

Crystal structures of T cell receptor (beta) chains related to rheumatoid arthritis

The crystal structures of the Vbeta17+ beta chains of two human T cell receptors (TCRs), originally derived from the synovial fluid (SF4) and tissue (C5-1) of a patient with rheumatoid arthritis (RA), have been determined in native (SF4) and mutant (C5-1(F104-->Y/C187-->S)) forms, respectively. These TCR beta chains form homo-dimers in solution and in crystals. Structural comparison reveals that the main-chain conformations in the CDR regions of the C5-1 and SF4 Vbeta17 closely resemble those of a Vbeta17 JM22 in a bound form; however, the CDR3 region shows different conformations among these three Vbeta17 structures. At the side-chain level, conformational differences were observed at the CDR2 regions between our two ligand-free forms and the bound JM22 form. Other significant differences were observed at the Vbeta regions 8-12, 40-44, and 82-88 between C5-1/SF4 and JM22 Vbeta17, implying that there is considerable variability in the structures of very similar beta chains. Structural alignments also reveal a considerable variation in the Vbeta-Cbeta associations, and this may affect ligand recognition. The crystal structures also provide insights into the structure basis of T cell recognition of Mycoplasma arthritidis mitogen (MAM), a superantigen that may be implicated in the development of human RA. Structural comparisons of the Vbeta domains of known TCR structures indicate that there are significant similarities among Vbeta regions that are MAM-reactive, whereas there appear to be significant structural differences among those Vbeta regions that lack MAM-reactivity. It further reveals that CDR2 and framework region (FR) 3 are likely to account for the binding of TCR to MAM.