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1.
Background
[FeFe] hydrogenases are metalloenzymes involved in the anaerobic metabolism of H2. These proteins are distinguished by an active site cofactor known as the H-cluster. This unique [6Fe–6S] complex contains multiple non-protein moieties and requires several maturation enzymes for its assembly. The pathways and biochemical precursors for H-cluster biosynthesis have yet to be elucidated.Principal Findings
We report an in vitro maturation system in which, for the first time, chemical additives enhance [FeFe] hydrogenase activation, thus signifying in situ H-cluster biosynthesis. The maturation system is comprised of purified hydrogenase apoprotein; a dialyzed Escherichia coli cell lysate containing heterologous HydE, HydF, and HydG maturases; and exogenous small molecules. Following anaerobic incubation of the Chlamydomonas reinhardtii HydA1 apohydrogenase with S-adenosyl methionine (SAM), cysteine, tyrosine, iron, sulfide, and the non-purified maturases, hydrogenase activity increased 5-fold relative to incubations without the exogenous substrates. No conditions were identified in which addition of guanosine triphosphate (GTP) improved hydrogenase maturation.Significance
The in vitro system allows for direct investigation of [FeFe] hydrogenase activation. This work also provides a foundation for studying the biosynthetic mechanisms of H-cluster biosynthesis using solely purified enzymes and chemical additives. 相似文献2.
Kuchenreuther JM Grady-Smith CS Bingham AS George SJ Cramer SP Swartz JR 《PloS one》2010,5(11):e15491
Background
The realization of hydrogenase-based technologies for renewable H2 production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases.Principal Findings
In this report, we describe an improved Escherichia coli-based expression system capable of producing 8–30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H2 evolution with rates comparable to those of enzymes isolated from their respective native organisms.Significance
The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H2-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments. 相似文献3.
Weisenberg SA Schuetz AN Alexander EA Eiss B Behta M Saiman L Larone DH Jenkins SG Rhee KY 《PloS one》2011,6(12):e28566
Background
Acinetobacter baumannii is an increasingly multidrug-resistant (MDR) cause of hospital-acquired infections, often associated with limited therapeutic options. We investigated A. baumannii isolates at a New York hospital to characterize genetic relatedness.Methods
Thirty A. baumannii isolates from geographically-dispersed nursing units within the hospital were studied. Isolate relatedness was assessed by repetitive sequence polymerase chain reaction (rep-PCR). The presence and characteristics of integrons were assessed by PCR. Metabolomic profiles of a subset of a prevalent strain isolates and sporadic isolates were characterized and compared.Results
We detected a hospital-wide group of closely related carbapenem resistant MDR A. baumannii isolates. Compared with sporadic isolates, the prevalent strain isolates were more likely to be MDR (p = 0.001). Isolates from the prevalent strain carried a novel Class I integron sequence. Metabolomic profiles of selected prevalent strain isolates and sporadic isolates were similar.Conclusion
The A. baumannii population at our hospital represents a prevalent strain of related MDR isolates that contain a novel integron cassette. Prevalent strain and sporadic isolates did not segregate by metabolomic profiles. Further study of environmental, host, and bacterial factors associated with the persistence of prevalent endemic A. baumannii strains is needed to develop effective prevention strategies. 相似文献4.
5.
Haiyan Ren Zhuwen Xu Wenhao Zhang Lin Jiang Jianhui Huang Shiping Chen Lixin Wang Xingguo Han 《Annals of botany》2013,112(9):1879-1885
Background and Aims
Leaf longevity is an important plant functional trait that often varies with soil nitrogen supply. Ethylene is a classical plant hormone involved in the control of senescence and abscission, but its role in nitrogen-dependent leaf longevity is largely unknown.Methods
Pot and field experiments were performed to examine the effects of nitrogen addition on leaf longevity and ethylene production in two dominant plant species, Agropyron cristatum and Stipa krylovii, in a temperate steppe in northern China.Key Results
Nitrogen addition increased leaf ethylene production and nitrogen concentration but shortened leaf longevity; the addition of cobalt chloride, an ethylene biosynthesis inhibitor, reduced leaf nitrogen concentration and increased leaf longevity. Path analysis indicated that nitrogen addition reduced leaf longevity mainly through altering leaf ethylene production.Conclusions
These findings provide the first experimental evidence in support of the involvement of ethylene in nitrogen-induced decrease in leaf longevity. 相似文献6.
Background
[FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of throughputs high enough to adequately sample heavily mutated DNA libraries. In vitro compartmentalization (IVC) is a powerful method capable of screening for multiple-turnover enzymatic activity at very high throughputs. Recent advances have allowed [FeFe] hydrogenases to be expressed and activated in the cell-free protein synthesis reactions on which IVC is based; however, IVC is a demanding technique with which many enzymes have proven incompatible.Methodology/Principal Findings
Here we describe an extremely high-throughput IVC screen for oxygen-tolerant [FeFe] hydrogenases. We demonstrate that the [FeFe] hydrogenase CpI can be expressed and activated within emulsion droplets, and identify a fluorogenic substrate that links activity after oxygen exposure to the generation of a fluorescent signal. We present a screening protocol in which attachment of mutant genes and the proteins they encode to the surfaces of microbeads is followed by three separate emulsion steps for amplification, expression, and evaluation of hydrogenase mutants. We show that beads displaying active hydrogenase can be isolated by fluorescence-activated cell-sorting, and we use the method to enrich such beads from a mock library.Conclusions/Significance
[FeFe] hydrogenases are the most complex enzymes to be produced by cell-free protein synthesis, and the most challenging targets to which IVC has yet been applied. The technique described here is an enabling step towards the development of biocatalysts for a biological hydrogen economy. 相似文献7.
Background and Aims
The herbicide quinclorac has been reported to inhibit incorporation of glucose both into cellulose and other cell wall polysaccharides. However, further work has failed to detect any apparent effect of this herbicide on the synthesis of the wall. In order to elucidate whether quinclorac elicits the inhibition of cellulose biosynthesis directly, in this study bean cell calli were habituated to grow on lethal concentrations of the herbicide and the modifications in cell wall composition due to the habituation process were analysed.Methods
Fourier transform infrared spectroscopy associated with multivariate analysis, cell wall fractionation techniques, biochemical analyses and the immunolocation of different cell wall components with specific monoclonal antibodies were used to characterize the cell walls of quinclorac-habituated cells.Key Results
Quinclorac-habituated cells were more irregularly shaped than non-habituated cells and they accumulated an extracellular material, which was more abundant as the level of habituation rose. Habituated cells did not show any decrease in cellulose content, but cell wall fractionation revealed that changes occurred in the distribution and post-depositional modifications of homogalacturonan and rhamnogalacturonan I during the habituation process. Therefore, since the action of quinclorac on the cell wall does not seem to be due to a direct inhibition of any cell wall component, it is suggested that the effect of quinclorac on the cell wall could be due to a side-effect of the herbicide.Conclusions
Long-term modifications of the cell wall caused by the habituation of bean cell cultures to quinclorac did not resemble those of bean cells habituated to the well-known cellulose biosynthesis inhibitors dichlobenil or isoxaben. Quinclorac does not seem to act primarily as an inhibitor of cellulose biosynthesis.Key words: Quinclorac, herbicide, Phaseolus vulgaris, cell culture habituation, primary cell wall, cellulose, FTIR spectroscopy 相似文献8.
9.
10.
Tianhu Wang Zhenhua Yang Fang Yang Mingye Zhang Jinlong Zhao Jinwu Chen Yongming Li 《PloS one》2014,9(4)
Objective
The study was performed to compare the 3D pharyngeal airway dimensions in adult skeletal Class II patients with different vertical growth patterns (low, normal, and high angle) and to investigate whether the upper airway dimensions of untreated skeletal Class II adults were affected by vertical skeletal variables.Methods
Cone-beam computed tomography (CBCT) records of 64 untreated adult skeletal Class II patients (34 male and 30 female) were collected to evaluate the pharyngeal airway dimensions. Subjects were divided into three subgroups according to the GoGn-SN angle (low angle, normal angle or high angle). All subgroups were matched for sex. ANOVA and SNK - q tests were used to identify differences within and among groups (p<0.05). Coefficient of product-moment correlation (Pearson correlation coefficient) was used to analyze the association between pharyngeal airway dimensions and vertical growth patterns.Results
The results showed that pharyngeal airway measurements were statistically significantly less (p<0.05) in high angle group as compared to normal angle or low angle group.Conclusions
Adult skeletal Class II subjects with vertical growth patterns have significantly narrower pharyngeal airways than those with normal or horizontal growth patterns, confirming an association between pharyngeal airway measurements and a vertical skeletal pattern. 相似文献11.
12.
Mohammad Salmani Izadi Abbas Ali Naserian Mohammad Reza Nasiri Reza Majidzadeh Heravi 《Reports of Biochemistry & Molecular Biology》2014,3(1):1-6
Background:
Stearoyl-CoA desaturase (SCD) is a key enzyme that converts saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs) in fat biosynthesis. Despite being crucial for interpreting SCDs’ roles across species, the evolutionary relationship of SCD proteins across species has yet to be elucidated. This study aims to present this evolutionary relationship based on amino acid sequences.Methods:
Using Multiple Sequence Alignment (MSA) and phylogenetic construction methods, a hypothetical evolutionary relationship was generated between the stearoyl-CoA desaturase (SCD) protein sequences between 18 different species.Results:
SCD protein sequences from Homo sapiens, Pan troglodytes (chimpanzee), and Pongo abelii (orangutan) have the lowest genetic distances of 0.006 of the 18 species studied. Capra hircus (goat) and Ovis aries (Sheep) had the next lowest genetic distance of 0.023. These farm animals are 99.987% identical at the amino acid level.Conclusions:
The SCD proteins are conserved in these 18 species, and their evolutionary relationships are similar. Key Words: Phylogenetic analysis, Stearoyl-CoA desaturase (SCD) proteins, Multiple sequence alignment 相似文献13.
Jisheng Li Yimei Cai Lupeng Ye Shaohua Wang Jiaqian Che Zhengying You Jun Yu Boxiong Zhong 《BMC genomics》2014,15(1)
Background
The growth and development of the posterior silk gland and the biosynthesis of the silk core protein at the fifth larval instar stage of Bombyx mori are of paramount importance for silk production.Results
Here, aided by next-generation sequencing and microarry assay, we profile 1,229 microRNAs (miRNAs), including 728 novel miRNAs and 110 miRNA/miRNA* duplexes, of the posterior silk gland at the fifth larval instar. Target gene prediction yields 14,222 unique target genes from 1,195 miRNAs. Functional categorization classifies the targets into complex pathways that include both cellular and metabolic processes, especially protein synthesis and processing.Conclusion
The enrichment of target genes in the ribosome-related pathway indicates that miRNAs may directly regulate translation. Our findings pave a way for further functional elucidation of these miRNAs and their targets in silk production.Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-410) contains supplementary material, which is available to authorized users. 相似文献14.
Zhao JY Sun JW Gu ZY Wang J Wang EL Yang XY Qiao B Duan WY Huang GY Wang HY 《PloS one》2012,7(2):e31644
Background
Clinical research indicates that periconceptional administration of folic acid can reduce the occurrence of congenital cardiac septal defects (CCSDs). The vital roles of folate exhibits in three ways: the unique methyl donor for DNA expression regulation, the de novo biosynthesis of purine and pyrimidine for DNA construction, and the serum homocysteine removal. Thymidylate synthase (TYMS) is the solo catalysis enzyme for the de novo synthesis of dTMP, which is the essential precursor of DNA biosynthesis and repair process. To examine the role of TYMS in Congenital Cardiac Septal Defects (CCSDs) risk, we investigated whether genetic polymorphisms in the TYMS gene associated with the CCSDs in a Han Chinese population.Method
Polymorphisms in the noncoding region of TYMS were identified via direct sequencing in 32 unrelated individuals composed of half CCSDs and half control subjects. Nine SNPs and two insertion/deletion polymorphisms were genotyped from two independent case-control studies involving a total of 529 CCSDs patients and 876 healthy control participants. The associations were examined by both single polymorphism and haplotype tests using logistic regression.Result
We found that TYMS polymorphisms were not related to the altered CCSDs risk, and even to the changed risk of VSDs subgroup, when tested in both studied groups separately or in combination. In the haplotype analysis, there were no haplotypes significantly associated with risks for CCSDs either.Conclusion
Our results show no association between common genetic polymorphisms of the regulatory region of the TYMS gene and CCSDs in the Han Chinese population. 相似文献15.
Introduction
Stroma cells and extracellular matrix (ECM) components provide the pivotal microenvironment for tumor development. The study aimed to evaluate the importance of the pancreatic stroma for tumor development.Methods
Pancreatic tumor cells were implanted subcutaneously into green fluorescent protein transgenic mice, and stroma cells invading the tumors were identified through immunohistochemistry. Inhibition of tumor invasion by stroma cells was achieved with halofuginone, an inhibitor of TGFβ/Smad3 signaling, alone or in combination with chemotherapy. The origin of tumor ECM was evaluated with species-specific collagen I antibodies and in situ hybridization of collagen α1(I) gene. Pancreatic fibrosis was induced by cerulean injection and tumors by spleen injection of pancreatic tumor cells.Results
Inhibition of stroma cell infiltration and reduction of tumor ECM levels by halofuginone inhibited development of tumors derived from mouse and human pancreatic cancer cells. Halofuginone reduced the number only of stroma myofibroblasts expressing both contractile and collagen biosynthesis markers. Both stroma myofibroblasts and tumor cells generated ECM that contributes to tumor growth. Combination of treatments that inhibit stroma cell infiltration, cause apoptosis of myofibroblasts and inhibit Smad3 phosphorylation, with chemotherapy that increases tumor-cell apoptosis without affecting Smad3 phosphorylation was more efficacious than either treatment alone. More tumors developed in fibrotic than in normal pancreas, and prevention of tissue fibrosis greatly reduced tumor development.Conclusions
The utmost importance of tissue fibrosis and of stroma cells for tumor development presents potential new therapy targets, suggesting combination therapy against stroma and neoplastic cells as a treatment of choice. 相似文献16.
Christopher O. Ciccolella Nathan A. Raynard John H-M. Mei Derek C. Church Robert A. Ludwig 《PloS one》2010,5(8)
Background
In symbiotic legume nodules, endosymbiotic rhizobia (bacteroids) fix atmospheric N2, an ATP-dependent catalytic process yielding stoichiometric ammonium and hydrogen gas (H2). While in most legume nodules this H2 is quantitatively evolved, which loss drains metabolic energy, certain bacteroid strains employ uptake hydrogenase activity and thus evolve little or no H2. Rather, endogenous H2 is efficiently respired at the expense of O2, driving oxidative phosphorylation, recouping ATP used for H2 production, and increasing the efficiency of symbiotic nodule N2 fixation. In many ensuing investigations since its discovery as a physiological process, bacteroid uptake hydrogenase activity has been presumed a single entity.Methodology/Principal Findings
Azorhizobium caulinodans, the nodule endosymbiont of Sesbania rostrata stems and roots, possesses both orthodox respiratory (exo-)hydrogenase and novel (endo-)hydrogenase activities. These two respiratory hydrogenases are structurally quite distinct and encoded by disparate, unlinked gene-sets. As shown here, in S. rostrata symbiotic nodules, haploid A. caulinodans bacteroids carrying single knockout alleles in either exo- or-endo-hydrogenase structural genes, like the wild-type parent, evolve no detectable H2 and thus are fully competent for endogenous H2 recycling. Whereas, nodules formed with A. caulinodans exo-, endo-hydrogenase double-mutants evolve endogenous H2 quantitatively and thus suffer complete loss of H2 recycling capability. More generally, from bioinformatic analyses, diazotrophic microaerophiles, including rhizobia, which respire H2 may carry both exo- and endo-hydrogenase gene-sets.Conclusions/Significance
In symbiotic S. rostrata nodules, A. caulinodans bacteroids can use either respiratory hydrogenase to recycle endogenous H2 produced by N2 fixation. Thus, H2 recycling by symbiotic legume nodules may involve multiple respiratory hydrogenases. 相似文献17.
Mironova VV Omelyanchuk NA Novoselova ES Doroshkov AV Kazantsev FV Kochetov AV Kolchanov NA Mjolsness E Likhoshvai VA 《Annals of botany》2012,110(2):349-360
Background and Aims
The root apical meristem (RAM) is the plant stem cell niche which provides for the formation and continuous development of the root. Auxin is the main regulator of RAM functioning, and auxin maxima coincide with the sites of RAM initiation and maintenance. Auxin gradients are formed due to local auxin biosynthesis and polar auxin transport. The PIN family of auxin transporters plays a critical role in polar auxin transport, and two mechanisms of auxin maximum formation in the RAM based on PIN-mediated auxin transport have been proposed to date: the reverse fountain and the reflected flow mechanisms.Methods
The two mechanisms are combined here in in silico studies of auxin distribution in intact roots and roots cut into two pieces in the proximal meristem region. In parallel, corresponding experiments were performed in vivo using DR5::GFP Arabidopsis plants.Key Results
The reverse fountain and the reflected flow mechanism naturally cooperate for RAM patterning and maintenance in intact root. Regeneration of the RAM in decapitated roots is provided by the reflected flow mechanism. In the excised root tips local auxin biosynthesis either alone or in cooperation with the reverse fountain enables RAM maintenance.Conclusions
The efficiency of a dual-mechanism model in guiding biological experiments on RAM regeneration and maintenance is demonstrated. The model also allows estimation of the concentrations of auxin and PINs in root cells during development and under various treatments. The dual-mechanism model proposed here can be a powerful tool for the study of several different aspects of auxin function in root. 相似文献18.
Hui-Yeng Y Yap Yit-Heng Chooi Mohd Firdaus-Raih Shin-Yee Fung Szu-Ting Ng Chon-Seng Tan Nget-Hong Tan 《BMC genomics》2014,15(1)