Evaluating the roles of the heme a side chains in cytochrome c oxidase using designed heme proteins

Heme a is a redox cofactor unique to cytochrome c oxidases and vital to aerobic respiration. Heme a differs from the more common heme b by two chemical modifications, the C-8 formyl group and the C-2 hydroxyethylfarnesyl group. The effects of these porphyrin substituents on ferric and ferrous heme binding and electrochemistry were evaluated in a designed heme protein maquette. The maquette scaffold chosen, [Delta7-H3m](2), is a four-alpha-helix bundle that contains two bis(3-methyl-l-histidine) heme binding sites with known absolute ferric and ferrous heme b affinities. Hemes b, o, o+16, and heme a, those involved in the biosynthesis of heme a, were incorporated into the bis(3-methyl-l-histidine) heme binding sites in [Delta7-H3m](2). Spectroscopic analyses indicate that 2 equiv of each heme binds to [Delta7-H3m](2), as designed. Equilibrium binding studies of the hemes with the maquette demonstrate the tight affinity for hemes containing the C-2 hydroxyethylfarnesyl group in both the ferric and ferrous forms. Coupled with the measured equilibrium midpoint potentials, the data indicate that the hydroxyethylfarnesyl group stabilizes the binding of both ferrous and ferric heme by at least 6.3 kcal/mol via hydrophobic interactions. The data also demonstrate that the incorporation of the C-8 formyl substituent in heme a results in a 179 mV, or 4.1 kcal/mol, positive shift in the heme reduction potential relative to heme o due to the destabilization of ferric heme binding relative to ferrous heme binding. The two substituents appear to counterbalance each other to provide for tighter heme a affinity relative to heme b in both the ferrous and ferric forms by at least 6.3 and 2.1 kcal/mol, respectively. These results also provide a rationale for the reaction sequence observed in the biosynthesis of heme a.     

Purification and electron microscopic characterization of the membrane subunit (IICB(Glc)) of the Escherichia coli glucose transporter

The glucose transporter of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It consists of a cytoplasmic subunit IIA(Glc) and the transmembrane subunit IICB(Glc). IICB(Glc) was purified to homogeneity by urea/alkali washing of membranes and nickel-chelate affinity chromatography. About 1.5 mg highly pure IICB(Glc) representing 77% of the total activity present in the membranes was obtained from 8g (wet weight) of cells. IICB(Glc) was reconstituted into lipid bilayers by temperature-controlled dialysis to yield small 2D crystals and by a rapid detergent-dilution procedure to yield densely packed vesicles. Electron microscopy and digital image processing of the negatively stained 2D crystals revealed a trigonal lattice with a unit cell size of a = b = 14.5 nm. The unit cell morphology exhibited three dimers of IICB(Glc) surrounding the threefold symmetry center. Single particle analysis of IICB(Glc) in proteoliposomes obtained by detergent dialysis also showed predominantly dimeric structures.     

Genomic mapping of chromosomal region 2p15-p21 (D2S378-D2S391): integration of Genemap'98 within a framework of yeast and bacterial artificial chromosomes

The region of chromosome 2 encompassed by the polymorphic markers D2S378 (centromeric) and D2S391 (telomeric) spans an approximately 10-cM distance in cytogenetic bands 2p15-p21. This area is frequently involved in cytogenetic alterations in human cancers. It also harbors the genes for several genetic disorders, including Type I hereditary nonpolyposis colorectal cancer (HNPCC), familial male precocious puberty (FMPP), Carney complex (CNC), Doyne's honeycomb retinal dystrophy (DHRD), and one form of familial dyslexia (DYX-3). Only a handful of known genes have been mapped to 2p16. These include MSH2, which is responsible for HNPCC, FSHR, the gene responsible for FMPP, EFEMP-1, the gene mutated in DHRD, GTBP, a DNA repair gene, and SPTBN1, nonerythryocytic beta-spectrin. The genes for CNC and DYX-3 remain unknown, due to lack of a contig of this region and its underrepresentation in the existing maps. This report presents a yeast- and bacterial-artificial chromosome (YAC and BAC, respectively) resource for the construction of a sequence-ready map of 2p15-p21 between the markers D2S378 and D2S391 at the centromeric and telomeric ends, respectively. The recently published Genemap'98 lists 146 expressed sequence tags (ESTs) in this region; we have used our YAC-BAC map to place each of these ESTs within a framework of 40 known and 3 newly cloned polymorphic markers and 37 new sequence-tagged sites. This map provides an integration of genetic, radiation hybrid, and physical mapping information for the region corresponding to cytogenetic bands 2p15-p21 and is expected to facilitate the identification of disease genes from the area.     

Uncovering the pathogenesis and identifying novel targets of pancreatic cancer using bioinformatics approach

Pancreatic cancer is a uniformly lethal disease that can be difficult to diagnose at its early stage. Thus, our present study aimed to explore the underlying mechanism and identify new targets for this disease. The data GSE16515, including 36 tumor and 16 normal samples were available from Gene Expression Omnibus. Differentially expressed genes (DEGs) were screened out using Robust Multichip Averaging and LIMMA package. Moreover, gene ontology and pathway enrichment analyses were performed to DEGs. Followed with protein–protein interaction (PPI) network construction by STRING and Cytoscape, module analysis was conducted using ClusterONE. Finally, based on PubMed, text mining about these DEGs was carried out. Total 274 up-regulated and 93 down-regulated genes were identified as the common DEGs and these genes were discovered significantly enriched in cell adhesion and extracellular region terms, as well as ECM-receptor interaction pathway. In addition, five modules were screened out from the up-regulated PPI network with none in down-regulated network. Finally, the up-regulated genes, including MIA, MET and CEACAMS, and down-regulated genes, such as FGF, INS and LAPP, had the most references in text mining analysis. Our findings demonstrate that the up- and down-regulated genes play important roles in pancreatic cancer development and might be new targets for the therapy.     

Analysis of fungal community structure in the soil of Zoige Alpine Wetland

To understand the soil fungal community diversity in different zones of the Zoige Alpine Wetland, BIOLOG analysis and traditional culture method were employed in our research. Three sample sites namely the Conservatory Station up-hill slope (CSUS), the Flower Lake side (FLS) and the Conservatory Station down slope (CSDS) with increasing by water content were investigated. The results of BIOLOG showed that fungal catabolic richness index (S) and Shannon diversity index (H) increasingly rose with water content augmented from CSUS to CSDS, while different from the former tendency, the fungal catabolic activity was highest at CSDS and lowest at FLS. Principal component analysis (PCA) results demonstrated the functional diversity of fungal community varied among the three sample sites, showing us more similarity between CSDS and FLS, and considerable difference between CSUS and the former two sites. The outcome of traditional culture method illustrated the number of soil fungi increased from CSUS to CSDS, while the sort of fungal species that could be cultured did not show much difference among the three sample sites.     

Ursolic acid benzaldehyde chalcone,leads to inhibition of cell proliferation and arrests cycle in G1/G0 phase in colon cancer

The present study investigates the effect of matrine on colon cancer cell viability and apoptosis and tumor growth in mice xenograft model. The results from MTT assay revealed a concentration and time dependent reduction in viability of HCT8 and HT29 colon cancer cells by matrine. The viability of HCT8 and HT29 cells was reduced to 24.67 and 29.32% on treatment with 4 µM/ml concentration of matrine after 48 h (P < 0.05). The results from flow cytometry revealed increase in population of HCT8 and HT29 cells to 77.6 ± 0.3 and 54.0 ± 5.4%, respectively compared to 1.4 ± 0.3 and 2.4 ± 0.7% in control on exposure to 1 µM/ml concentration of matrine. Histone H2AX phosphorylation and expression of Myt1, cyclin A2, cyclin B1 and p53 were increased in HCT8 and HT29 cells on treatment with matrine for 48 h. Matrine treatment also increased the phosphorylation of cdc2 significantly compared to control cells at 48 h (P < 0.05). Results from Annexin-V/FITC-staining showed increase in proportion of apoptotic cells in HCT8 and HT29 cells 67.52 and 68.56 on treatment with 1 µM/ml of matrine. Matrine treatment caused a marked reduction in the growth of HCT8 cell xenograft after 21 days. Thus matrine inhibits cell viability, induces apoptosis and inhibits tumor growth in colon cancer.     

Effect of lignocellulosic inhibitory compounds on growth and ethanol fermentation of newly-isolated thermotolerant Issatchenkia orientalis

A newly isolated thermotolerant ethanologenic yeast strain, Issatchenkia orientalis IPE 100, was able to produce ethanol with a theoretical yield of 85% per g of glucose at 42 °C. Ethanol production was inhibited by furfural, hydroxymethylfurfural and vanillin concentrations above 5.56 g L⁻¹, 7.81 g L⁻¹, and 3.17 g L⁻¹, respectively, but the strain was able to produce ethanol from enzymatically hydrolyzed steam-exploded cornstalk with 93.8% of theoretical yield and 0.91 g L⁻¹ h⁻¹ of productivity at 42 °C. Therefore, I. orientalis IPE 100 is a potential candidate for commercial lignocelluloses-to-ethanol production.     

基于稳定同位素技术的塔里木河下游不同林龄胡杨的水分利用来源

水分是制约很多陆地生态系统植物生长和繁殖的重要因素, 在干旱地区尤为明显。利用稳定同位素技术探究塔里木河下游不同林龄胡杨(Populus euphratica)的水分来源情况, 了解生态输水背景下荒漠河岸林的水分利用循环与利用策略, 可为生态输水提供科学依据, 同时也可对同类地区的生态恢复提供借鉴。本研究通过测定塔里木河下游胡杨茎干水和各潜在水源(土壤水、地下水)的稳定氢氧同位素值(δD、δ¹⁸O), 应用多源线性混合模型(IsoSource)分析了各潜在水源对不同林龄胡杨的贡献比例, 并结合3种林龄胡杨不同土壤深度含水量的变化, 分析了胡杨的主要吸水层位。结果表明: (1)不同林龄胡杨样地的不同深度区间上的土壤水δ¹⁸O值存在显著差异(P < 0.05): 胡杨幼龄木、成熟木、过熟木木质部δ¹⁸O分别为-7.83 ± 0.07‰、-8.53 ± 0.11‰、-9.36 ± 0.21‰; 而δD值不存在显著差异(P > 0.05)。可据此来推断胡杨的主要吸水层位。(2)总体上, 三种林龄胡杨土壤水δ¹⁸O值随土壤深度增加而减小, 并趋于接近地下水的δ¹⁸O值。其中, 0-60 cm土壤水受蒸发影响比较大, 其同位素组成经历了强烈的蒸发分馏过程, 土壤含水量极少, 土壤水δ¹⁸O值偏正。(3)不同林龄胡杨所利用的水分来源不同: 胡杨幼龄木对于地表80 cm以下的土壤水以及地下水均有一定程度的利用, 对80-140 cm、140-220 cm和220-340 cm的土壤水平均利用比率依次为16.2%、21.4%和24.6%, 对地下水平均利用比率为24.5%; 成熟木主要利用220-340 cm的土壤水及地下水, 平均利用比率分别为36.9%和42.3%; 过熟木主要利用140-340 cm的土壤水及地下水, 平均利用比率分别为32.8%和49.3%。