Induced pluripotent stem cells (iPSCs) were reprogrammed from somatic cells using specific transcription factors. Bypassing the ethical issue caused by embryonic stem cells (ESCs), iPSCs can be successfully induced from a variety of cells, which makes iPSCs a powerful research tool for developmental biology. iPSCs have also become indispensable to the research of life science due to their broad potential applications. However, it's a big challenge to obtain iPSCs with high quality and genetic stability. Here, we review the research progress of increasing the reprogramming mechanism and genetic stability of iPSCs in order to provide references of reprogramming efficiency of iPSCs, reducing the cost, and addressing key points of iPSCs quality control, further promoting clinical application of the iPSCs.
Chemosensory proteins (CSPs) play important roles in chemical communication by insects, as they recognize and transport environmental chemical signals to receptors within sensilla. In this study, we identified HoblCSP1 and HoblCSP2 from a cDNA library of Holotrichia oblita antennae, successfully expressed them in E. coli and purified them by Ni ion affinity chromatography. We then measured the ligand-binding specificities of HoblCSP1 and HoblCSP2 to 50 selected ligands in a competitive binding assay. These results demonstrated that HoblCSP1 and HoblCSP2 have similar ligand-binding spectra. Both proteins displayed the highest affinity for ?-ionone, ?-ionone and cinnamaldehyde, indicating that they prefer binding to odorants other than sex pheromones. Additionally, immuno-localization revealed that HoblCSP1 is highly concentrated in sensilla basiconica, while HoblCSP2 is specifically localized to sensilla placodea. In conclusion, HoblCSP1 and HoblCSP2 are responsible for binding to general odorants with slightly different specificities due to their different in vivo environments.
Anisotropic polymetallic hollow nanostructures are highly desired for many applications because of their unique morphology, large specific surface areas and attractive electronic effects. Here, a simple method using gold nanorods as a self-sacrificed template has been developed for the fabrication of hollow dumbbell-like nanorods of Au@PtAg. The formation of the hollow structures involves the growth of another metallic shell first, and then the etching of gold nanorods, which is induced by oxygen and ascorbic acid. The lattice mismatch and cohesive energy of the shell, along with its surface passivation, greatly affect the subsequent etching and the resulting products, as has been demonstrated by a positive control in the case of Rh and a negative control in the case of Pd. Hollow dumbbell-like nanorods of Au@PtAg show great enhancement for the dehydrogenation pathway in the oxidation of formic acid, as compared to solid Au@PtAg nanorods, PtAu nanotubes and commercial Pt/C.
By anchoring 1,2,4,5-tetrazine-containing biomolecules onto trans-cyclooctene (TCO)-functionalized slides, a site-specific microarray immobilization approach is described in this study. Compared with existing immobilization methods, our approach offers several distinctive features, including fast kinetics and high chemoselectivity.
Induced pluripotent stem cells (iPSCs) have potential applications in the restoration of fertility, regenerative medicine, and animal biotechnology. In this study, we present the induction of iPSCs from mouse Sertoli cells (SCs) by introducing four factors--Oct4, Sox2, Klf4, and c-Myc. As early as day 3 after induction, expression of these factors was detected and typical embryonic stem-like cells began to form. On day 18, these exogenous genes were silenced and colonies were selected according to morphological characteristics. The iPSCs induced from SCs, termed SCiPSCs, strongly expressed pluripotent markers, showed a normal karyotype, and had proliferation and differentiation characteristics similar to those of embryonic stem cells (ESCs), both in vitro and in vivo. Furthermore, exposure of SCiPSCs to nitric oxide (NO) allowed them to maintain pluripotency through the activation of the pluripotent genes Oct4 and Sox2 and upregulation of Nanog expression. Moreover, NO prevented SCiPSCs from undergoing apoptosis by activating the antiapoptotic genes Bcl2 and Bcl2lll, downregulating the proapoptotic genes Bak1 and Casp7, and blocking the activation of the proapoptotic gene Bac. These effects were reversed by exposure to l-NG-monomethylarginine (l-NMMA), a NO inhibitor. These data demonstrate that iPSCs can be generated from SCs and that the self-renewal and pluripotency of SCiPS cells can be maintained in the presence of NO.
Cadmium (Cd) is a severe detrimental environmental pollutant. To adapt to Cd-induced deleterious effects, plants have evolved sophisticated defence mechanisms. In this study, a genome-wide transcriptome analysis was performed to identify the mechanisms of Cd tolerance using two barley genotypes with distinct Cd tolerance.
A series of new fluorogenic probes for monoamine oxidases (MAOs) were reported based on an oxidation and ?-elimination mechanism. The limits of detection of the probes for MAO-A and -B were determined to be 3.5 and 6.0 ?g mL(-1) respectively. These probes displayed strong activity towards MAOs, especially MAO-B. Cellular imaging studies were also successfully conducted with MCF-7 cells.
A meta-analysis was conducted to evaluate the association of ICAM-1 K469E gene polymorphism with diabetic retinopathy susceptibility in Type 2 diabetes mellitus. Seven studies involving 1094 cases and 909 controls were included. Current studies suggest that K469E polymorphism in ICAM-1 gene might not affect individual susceptibility to DR.
One-dimensional porous metallic nanomaterials have attracted much attention due to their unique shape and hollow structure. Herein, the gold nanorods in a porous shell of an AuAg alloy are synthesized via a dealloying process of the core-shell Au@AuAg nanorods at room temperature. The formation of tiny pores in the shell results in the huge red-shift, sharp decrease and drastic broadening of longitudinal surface plasmon resonance absorption. The continuous removal of silver from the porous nanorods leads to the breakage of tiny pores and leaves a rough surface on the nanorods behind. The rough surface gradually becomes smooth in the subsequent dealloying process. The surface structures of these intermediates are correlated with their absorption spectra and catalytic activities for the catalytic reduction of p-nitrophenol. The porous nanorods show a higher catalytic efficiency than the gold nanorods, the core-shell nanorods and the rough nanorods. The results indicate that the dealloying of anisotropic bimetal nanomaterials not only provides an effective pathway to carve the structures on the nanoscale but also offers numerous opportunities to observe novel optical properties and enhanced catalysis performances.
Fast and efficient: A library of trans-cyclooctene (TCO)-modified small molecules were immobilized on tetrazine-functionalized glass slides by using the fastest bioorthogonal reaction known. The resulting small-molecule microarray was screened against a variety of human bromodomains to identify protein-protein interaction inhibitors.
We report herein a new site-specific microarray immobilization method based on a biocompatible reaction between terminal cysteine and 2-cyanobenzothiazole (CBT). This immobilization strategy has been successfully applied to anchor small molecules, peptides and proteins onto microarrays.
In this study, a microfluidic platform was developed to generate single layer, linear array of microbeads for multiplexed high-throughput analysis of biomolecules. The microfluidic device is comprised of eight microbead-trapping units, where microbeads were immobilized in a linear array format by the exertion of a negative pressure in the control channel connected to each sieving microstructure. Multiplexed assays were achieved by using a mixture of different spectrally-encoded microbeads functionalized with specific probes, followed by on-chip reaction and detection. The microfluidic-based microbeads array platform was employed for multiplexed analysis of DNA and proteins, as demonstrated by the simultaneous discrimination of four HPV genotypes and the parallel detection of six different proteins. Compared with the off-chip protocols, the on-chip analysis exhibited better reaction efficiency, higher sensitivity and wider linear detection range. Visual inspection and identification of functionalized microbeads were facilitated by the single layer arrangement of microbeads so that accurate data acquisition can be performed during the detection process.
Protein phosphorylation is one of the most important and well-studied posttranslational modifications. Aberrant phosphorylation causes a wide spectrum of diseases, including cancers. As a result, many of the proteins involved in these pathways are seen as vital drug targets and biomarkers in treatment and diagnosis. The availability of broad-based platforms that identify changes across cellular states is critical in understanding unique disease characteristics and changes at the proteomic level. To highlight how microarrays can be applied in this regard, we describe here a comparative proteomic profiling method using two-color sample labeling and application on phosphopeptide microarrays, followed by a pull-down strategy and MS-based protein identification. This strategy has been applied to uncover candidate biomarkers in breast cancer and colon cancer cell lines. Apart from the synthesis of the phosphopeptide libraries and growth/isolation of cellular lysates, the protocol takes approximately 15 days to complete, once key steps have been optimized, and can be readily extended to other similarly complex biological specimens/samples.
Organelle-specific cell-permeable fluorescent dyes are invaluable tools in cell biology as they reveal intracellular dynamics in living cells. Mitrotracker is a family of dyes that strongly label the mitochondrion, a key organelle associated with many crucial cellular functions. Despite the popularity of these dyes, little is known about the molecular mechanism behind their staining specificity. Here, we aimed to identify the protein targets of one member of this dye family, mitotracker red (MTR), by 2DE and MS. MTR bound to cellular proteins covalently, and its fluorescence persisted even after cell lysis, protein solubilization, denaturation, and electrophoresis. This enabled us to display MTR-labeled proteins by 2DE. The MTR-specific fluorescent signals on the gel revealed the spots that contained MTR-conjugated proteins. These spots were analyzed by MS, resulting into the identification of ten proteins. We discovered that one major target is the mitochondrial protein HSP60 and that MTR staining could induce production of HSP60, predisposing cells to heat shock-like responses. The identification of the molecular targets of biological dyes, or "stainomics," can help correlate their intracellular staining properties with biochemical affinities. We believe this approach can be applied to a wide range of fluorescent probes.
A preliminary study was conducted on the development of an intelligent dental handpiece with functionality to detect subtle changes in mechanical properties of tooth tissue during milling. Such equipment would be able to adopt changes in cutting parameters and make real-time measurements to avoid tooth tissue damage caused by overexertion and overextension of the cutting tool. A modified dental handpiece, instrumented with strain gauges, microphone, displacement sensor, and air pressure sensor, was mounted to a linear movement table and used to mill three to four cavities in >50 bovine teeth. Extracted sound frequency and density were analyzed along with force, air pressure, and displacement for correlations and trends. Experimental results showed a high correlation (coefficient close to 0.7) between the feed force, the rotational frequency, and the averaged gray scale. These results could form the basis of a feedback control system to improve the safety of dental cutting procedures. This article is written in memory of Dr Hongyan Sun, who passed away in 2011 at a young age of 37.
Vibrio alginolyticus has been confirmed as an important pathogen for aquatic animals. However, the pathogenic mechanism of V. alginolyticus is not completely understood. A total of 31 isolates of V. alginolyticus from sea water, fish and shrimp on the mariculture systems were fingerprinted by pulsed-field gel electrophoresis. The pathogenicity of these isolates was tested by challenge and the 21 genes associated with the virulence of Vibrio cholerae or Vibrio parahaemolyticus were examined in V. alginolyticus using PCR. The results showed that the 31 V. alginolyticus isolates belonged to 26 PFGE genotypes and the isolates from different source had different genotypes. Nine of the 31 isolates were confirmed as pathogenic strains by challenge. Moreover, 12 vibrio virulence genes were detected in this study. Of the detected genes, VCtoxR, VCtoxS, hlyA, VPtoxR and tlh were found in both pathogenic and non-pathogenic isolates. However, the other 7 virulence genes, ctxB, zot, tagA, stn, sto, tdh and trh, were only present in pathogenic isolates. Analysis of the relationship between virulence associated genes and pathogenicity of V. alginolyticus provides a possible explanation that the pathogenic mechanism of V. alginolyticus might be similar to that of V. parahaemolyticus instead of V. cholerae.
Proteins are fundamental components of all living systems and critical drivers of biological functions. The large-scale study of proteins, their structures and functions, is defined as proteomics. This systems-wide analysis leads to a more comprehensive view of the intricate signaling transduction pathways that proteins engage in and improves the overall understanding of the complex processes supporting the living systems. Over the last two decades, the development of high-throughput analytical tools, such as microarray technologies, capable of rapidly analyzing thousands of protein-functioning and protein-interacting events, has fueled the growth of this important field. Herein, we review the most recent advancements in microarray technologies, with a special focus on peptide microarray, small molecule microarray, and protein microarray. These technologies have become prominent players in proteomics and have made significant changes to the landscape of life science and biomedical research. We will elaborate on their performance, advantages, challenges, and future directions.
To reveal grain physio-chemical and proteomic differences between two barley genotypes, Zhenong8 and W6nk2 of high- and low- grain-Cd-accumulation, grain profiles of ultrastructure, amino acid and proteins were compared. Results showed that W6nk2 possesses significantly lower protein content, with hordein depicting the greatest genotypic difference, compared with Zhenong8, and lower amino acid contents with especially lower proportion of Glu, Tyr, Phe and Pro. Both scanning and transmission electron microscopy observation declared that the size of A-type starch molecule in W6nk2 was considerably larger than that of Zhenong8. Grains of Zhenong8 exhibited more protein-rich deposits around starch granules, with some A-type granules having surface pits. Seventeen proteins were identified in grains, using 2-DE coupled with mass spectrometry, with higher expression in Zhenong8 than that in W6nk2; including z-type serpin, serpin-Z7 and alpha-amylase/trypsin inhibitor CM, carbohydrate metabolism, protein synthesis and signal transduction related proteins. Twelve proteins were less expressed in Zhenong8 than that in W6nk2; including barley trypsin inhibitor chloroform/methanol-soluble protein (BTI-CMe2.1, BTI-CMe2.2), trypsin inhibitor, dehydroascorbate reductase (DHAR), pericentrin, dynein heavy chain and some antiviral related proteins. The data extend our understanding of mechanisms underlying Cd accumulation/tolerance and provides possible utilization of elite genetic resources in developing low-grain-Cd barley cultivars.
The rate coefficients of H-abstraction reactions of butene isomers by the OH radical were determined by both canonical variational transition-state theory and transition-state theory, with potential energy surfaces calculated at the CCSD(T)/6-311++G(d,p)//BH&HLYP/6-311G(d,p) level and CCSD(T)/6-311++G(d,p)//BH&HLYP/cc-pVTZ level and quantum mechanical tunneling effect corrected by either the small-curvature tunneling method or the Eckart method. While 1-butene contains allylic, vinylic, and alkyl hydrogens that can be abstracted to form different butene radicals, results reveal that s-allylic H-abstraction channels have low and broad energy barriers, and they are the most dominant channels which can occur via direct and indirect H-abstraction channels. For the indirect H-abstraction s-allylic channel, the reaction can proceed via forming two van der Waals prereactive complexes with energies that are 2.7-2.8 kcal mol(-1) lower than that of the entrance channel at 0 K. Assuming that neither mixing nor crossover occurs between different reaction pathways, the overall rate coefficient was calculated by summing the rate coefficients of the s-allyic, methyl, and vinyl H-abstraction paths and found to agree well with the experimentally measured OH disappearance rate. Furthermore, the rate coefficients of p-allylic H abstraction of cis-2-butene, trans-2-butene, and isobutene by the OH radical were also determined at 300-1500 K, with results analyzed and compared with available experimental data.
Protein tyrosine phosphatases (PTPs) constitute a large family of enzymes that play key roles in cell signaling. Malfunctions of PTP activity have been linked to major human diseases including cancer. One key aspect in PTP biology is the elucidation of roles of PTPs, as well as substrates they act on, in different cellular events. Herein, a library of 144 putative peptide substrates against different PTPs was synthesized and immobilized onto a glass slide to generate the corresponding phosphopeptide microarray. Subsequent screening of the microarray against various PTPs provided a distinctive and comparative substrate fingerprint against each PTP. Several new substrates were identified, which might aid in the future design of potent and selective PTPs inhibitors. The signal-decrease microarray assay used in our studies provided a facile and efficient way for high-throughput determination of kinetic constants for peptide/PTP interactions en masse. Finally, our microarray results were independently verified by traditional microplate-based enzymatic assays.
The present study investigated Typha angustifolia leaf proteome in response to Cr, Cd and Pb stress. T. angustifolia of 90 (D90) and 130d (D130) old plants were subjected to 1mM Cr, Cd and Pb and samples were collected 30d after treatment. 2-DE coupled with MS (mass spectrometry) was used to analyze and identify Cr, Cd and Pb-responsive proteins. More than 1600 protein spots were reproducibly detected on each gel, wherein 44, 46, 66 and 33, 26, 62 spots in D90 and D130 samples were differentially expressed by Cr, Cd, Pb over the control, respectively. Of these differentially expressed proteins, 3, 1, 8 overlapped in D90 and D130; while 5, 8, 5 with regulation factors above 3 in one of D90 or D130 samples. Total of 22 and 4 up- and down-regulated proteins were identified using MS and data bank analysis. Cr-induced expression of ATP synthase, RuBisCO small subunit and coproporphyrinogen III oxidase; Cd-induced RuBisCO large subunit; Pb up-regulated carbohydrate metabolic pathway enzymes of fructokinase, and improved RuBisCO activase and large subunit, Mg-protoporphyrin IX chelatase. Contrarily, elF4F was inhibited by Cr/Pb, chloroplast FtsZ-like protein and GF14 omega impeded by Cd and Pb, respectively.
This study investigates the modulation of antioxidant defence system of Typha angustifolia after 30 days exposure of 1 mM chromium (Cr), cadmium (Cd), or lead (Pb). T. angustifolia showed high tolerance to heavy metal toxicity with no visual toxic symptom when exposed to metal stress, and Cd/Pb addition also increased plant height and biomass especially in Pb treatment. Along with increased Cr, Cd, and Pb uptake in metal treatments, there was enhanced uptake of plant nutrients including Ca and Fe, and Zn in Pb treatment. A significant increase in malondialdehyde (MDA) content and superoxide dismutase (SOD) and peroxidase (POD) activities were recorded in plants subjected to Cr, Cd, or Pb stress. Furthermore, Pb stress also improved catalase (CAT), ascorbate peroxidase (APX), and glutathione peroxidase (GPX) activities; whereas Cr stress depressed APX and GPX. The results indicate that enzymatic antioxidants and Ca/Fe uptake were important for heavy metal detoxification in T. angustifolia, stimulated antioxidative enzymes, and Ca, Fe, and Zn uptake could partially explain its hyper-Pb tolerance.
We report herein a novel phosphopeptide microarray capable of noncovalently "trapping" catalytically inactive mutants of protein tyrosine phosphatases (PTPs), and its application in high-throughput determination of PTP substrate specificity.
A capillary zone electrophoresis (CZE) method modified by ?-cyclodextrin (?-CD) and acetonitrile (ACN) was developed for simultaneous determination of seven structurally similar flavonoids in Scutellariae Barbatae Herba (SBH) and its preparations. Molecular selectivity of the analytes by ?-CD was in the following order: apigenin, luteolin, quercetin, scutellarin, baicalein, rutin and wogonin, based mainly on the "molecular fit" interaction between some ligands in the C ring of the flavonoid and the cavity of ?-CD. Flavonoids with hydroxyl substituent(s) at the C-ring, especially the 4 monohydroxyl, were highly selected by ?-CD although hydrophobicity of the guest molecule is the primary factor affecting the complexation. The function of acetonitrile in this study was to improve the separation of the analytes in the real SBH. The developed method was validated and applied to real samples. The principle of separation based on this CZE condition is also explained.
Induced pluripotent stem cells (iPSCs) are flourishing in the investigation of cell reprogramming. However, we still know little about the sequential molecular mechanism during somatic cell reprogramming (SCR). Here, we first observed rapid generation of colonies whereas mouse embryonic fibroblasts (MEFs) were induced by OCT4, SOX2, KLF4 (OSK), and vitamin C for 7 days. The colonys global transcriptional profiles were analyzed using Affymetrix microarray. Microarray data confirmed that SCR was a process in which transcriptome got reversed and pluripotent genes expressed de novo. There were many changes, especially substantial growth expression of epigenetic factors, on transcriptome during the transition from Day 7 to iPSCs indicating that this period may provide flexibility genome structure, chromatin remodeling, and epigenetic modifications to rebind to the transcriptional factors. Several biological processes such as viral immune response, apoptosis, cell fate specification, and cell communication were mainly involved before Day 7 whereas cell cycle, DNA methylation, and histone modification were mainly involved after Day 7. Furthermore, it was suggested that p53 signaling contributed to the transition hyperdynamic plastic cell state and assembled cell niche for SCR, and small molecular compounds useful for chromatin remodeling can enhance iPSCs by exciting epigenetic modification rather than the exogenous expression of more TFs vectors.
We report herein a novel fluorescent probe based on ?,?-unsaturated acyl sulfonamide to detect thiols. The probe has good water solubility and reacts with thiols under aqueous conditions. It reacts selectively with cysteine but not with the other natural amino acids. The probe was subsequently applied to detect intracellular thiols.
The thermal decomposition of the CH(3)N(•)NH(2), cis-CH(3)NHN(•)H, trans-CH(3)NHN(•)H, and C(•)H(2)NNH(2) radicals, which are the four radical products from the H-abstraction reactions of monomethylhydrazine, were theoretically studied by using ab initio Rice-Ramsperger-Kassel-Marcus (RRKM) transition-state theory and master equation analysis. Various decomposition pathways were identified by using either the QCISD(T)/cc-pV?Z//CASPT2/aug-cc-pVTZ or the QCISD(T)/cc-pV?Z//B3LYP/6-311++G(d,p) quantum chemistry methods. The results reveal that the ?-scission of NH(2) to form methyleneimine is the predominant channel for the decomposition of the C(•)H(2)NNH(2) radical due to its small energy barrier of 13.8 kcal mol(-1). The high pressure limit rate coefficient for the reaction is fitted by 3.88 × 10(19)T(-1.672) exp(-9665.13/T) s(-1). In addition, the pressure dependent rate coefficients exhibit slight temperature dependence at temperatures of 1000-2500 K. The cis-CH(3)NHN(•)H and trans-CH(3)NHN(•)H radicals are the two distinct spatial isomers with an energy barrier of 26 kcal mol(-1) for their isomerization. The ?-scission of CH(3) from the cis-CH(3)NHN(•)H radical to form trans-diazene has an energy barrier of 35.2 kcal mol(-1), and the ?-scission of CH(3) from the trans-CH(3)NHN(•)H radical to form cis-diazene has an energy barrier of 39.8 kcal mol(-1). The CH(3)N(•)NH(2) radical undergoes the ?-scission of methyl hydrogen and amine hydrogen to form CH(2)?NNH(2), trans-CH(3)N?NH, and cis-CH(3)N?NH products, with the energy barriers of 42.8, 46.0, and 50.2 kcal mol(-1), respectively. The dissociation and isomerization rate coefficients for the reactions were calculated via the E/J resolved RRKM theory and multiple-well master equation analysis at temperatures of 300-2500 K and pressures of 0.01-100 atm. The calculated rate coefficients associated with updated thermochemical property data are essential components in the development of kinetic mechanisms for the pyrolysis and oxidation of MMH and its derivatives.
The gas-phase kinetics of H-abstraction reactions of monomethylhydrazine (MMH) by OH radical was investigated by second-order multireference perturbation theory and two-transition-state kinetic model. It was found that the abstractions of the central and terminal amine H atoms by the OH radical proceed through the formation of two hydrogen bonded preactivated complexes with energies of 6.16 and 5.90 kcal mol(-1) lower than that of the reactants, whereas the abstraction of methyl H atom is direct. Due to the multireference characters of the transition states, the geometries and ro-vibrational frequencies of the reactant, transition states, reactant complexes, and product complexes were optimized by the multireference CASPT2/aug-cc-pVTZ method, and the energies of the stationary points of the potential energy surface were refined at the QCISD(T)/CBS level via extrapolation of the QCISD(T)/cc-pVTZ and QCISD(T)/cc-pVQZ energies. It was found that the abstraction reactions of the central and two terminal amine H atoms of MMH have the submerged energy barriers with energies of 2.95, 2.12, and 1.24 kcal mol(-1) lower than that that of the reactants respectively, and the abstraction of methyl H atom has a real energy barrier of 3.09 kcal mol(-1). Furthermore, four MMH radical-H(2)O complexes were found to connect with product channels and the corresponding transition states. Consequently, the rate coefficients of MMH + OH for the H-abstraction of the amine H atoms were determined on the basis of a two-transition-state model, with the total energy E and angular momentum J conserved between the two transition-state regions. In units of cm(3) molecule(-1) s(-1), the rate coefficient was found to be k(1) = 3.37 × 10(-16)T(1.295) exp(1126.17/T) for the abstraction of the central amine H to form the CH(3)N(•)NH(2) radical, k(2) = 2.34 × 10(-17)T(1.907) exp(1052.26/T) for the abstraction of the terminal amine H to form the trans-CH(3)NHN(•)H radical, k(3) = 7.41 × 10(-20)T(2.428) exp(1343.20/T) for the abstraction of the terminal amine H to form the cis-CH(3)NHN(•)H radical, and k(4) = 9.13 × 10(-21)T(2.964) exp(-114.09/T) for the abstraction of the methyl H atom to form the C(•)H(2)NHNH(2) radical, respectively. Assuming that the rate coefficients are additive, the total rate coefficient of these theoretical predictions quantitatively agrees with the measured rate constant at temperatures of 200-650 K, with no adjustable parameters.
Odorant binding proteins play a crucial role in transporting semiochemicals across the sensillum lymph to olfactory receptors within the insect antennal sensilla. In this study, the general odorant binding protein 2 gene was cloned from the antennae of Loxostege sticticalis, using reverse transcription PCR and rapid amplification of cDNA ends. Recombinant LstiGOBP2 was expressed in Escherichia coli and purified by Ni ion affinity chromatography. Real-time PCR assays indicated that LstiGOBP2 mRNA is expressed mainly in adult antennae, with expression levels differing with developmental age. Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN) as a fluorescent probe demonstrated that the LstiGOBP2 protein has binding affinity to a broad range of odorants. Most importantly, trans-11-tetradecen-1-yl acetate, the pheromone component of Loxostege sticticalis, and trans-2-hexenal and cis-3-hexen-1-ol, the most abundant plant volatiles in essential oils extracted from host plants, had high binding affinities to LstiGOBP2 and elicited strong electrophysiological responses from the antennae of adults.
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