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Sedimentation velocity analytical ultracentrifugation (SV) is a powerful first-principle technique for the study of protein interactions, and allows a rigorous characterization of binding stoichiometry and affinities. A recently introduced commercial fluorescence optical detection system (FDS) permits analysis of high-affinity interactions by SV. However, for most proteins the attachment of an extrinsic fluorophore is an essential prerequisite for analysis by FDS-SV. Using the glutamate receptor GluA2 amino terminal domain as a model system for high-affinity homo-dimerization, we demonstrate how the experimental design and choice of fluorescent label can impact both the observed binding constants as well as the derived hydrodynamic parameter estimates for the monomer and dimer species. Specifically, FAM (5,6-carboxyfluorescein) was found to create different populations of artificially high-affinity and low-affinity dimers, as indicated by both FDS-SV and the kinetics of dimer dissociation studied using a bench-top fluorescence spectrometer and Förster Resonance Energy Transfer. By contrast, Dylight488 labeled GluA2, as well as GluA2 expressed as an EGFP fusion protein, yielded results consistent with estimates for unlabeled GluA2. Our study suggests considerations for the choice of labeling strategies, and highlights experimental designs that exploit specific opportunities of FDS-SV for improving the reliability of the binding isotherm analysis of interacting systems. 相似文献
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Hong Sung Hyun Singh Sudhir Tripathi Bhumi Nath Mondal Suvendu Lee Sangmin Jung Hyun Suk Cho Chuloh Kaur Shubhpreet Kim Jin-Hong Lee Sungbeom Bai Hyoung-Woo Bae Hyeun-Jong Lee Sang Yeol Lee Seung Sik Chung Byung Yeoup 《Protoplasma》2020,257(3):807-817
Protoplasma - Alkyl hydroperoxide reductase subunit F (AhpF) is a well-known flavoprotein that transfers electrons from pyridine nucleotides to the peroxidase protein AhpC via redox-active... 相似文献
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Gautam A Gupta A Lomash V Pant SC Vijayaraghavan R 《Indian journal of experimental biology》2010,48(7):752-761
Sulphur mustard, [bis (2-chloroethyl)] sulphide (SM), is a bifunctional alkylating agent. SM forms sulphonium ion in the body which alkylates DNA and several other macromolecules, and induces oxidative stress. Although several antidotes have been screened for the treatment of systemic toxicity of SM in experimental animals none of them are recommended so far. In the search for more effective and less toxic antidotes, various combinations were tried against SM induced toxicity and skin lesions. SM exposed through percutaneous route was used to evaluate the prophylactic efficacy of various combinations. Low dose of DRDE-07 (S-2(2-aminoethylamino) ethyl phenyl sulphide), DRDE-30 [S-2(2-aminoethyl amino) ethyl propyl sulphide], DRDE-35 [S-2(2-aminoethyl amino) ethyl butyl sulphide] with amifostine combinations, were given orally 30 min prior to SM exposure. Significant depletion was observed in body weight, organ body weight index and hepatic GSH and GSSG content in mice after SM exposure. Pretreatment with low dose of different combinations of DRDE-07, DRDE-30 and DRDE-35 with amifostine could recover biochemical alterations and histopathological changes caused by SM exposures. 相似文献
4.
Sethi KK Sahoo SK Pichikala JN Suresh P 《Journal of enzyme inhibition and medicinal chemistry》2012,27(1):97-100
Novel chemotypes with carbonic anhydrase (CA; EC 4.2.1.1) inhibitory action, in addition to the sulphonamide and sulphamate were discovered, many of which are based on natural products. Caffeine and piperine were extracted and tested for inhibition of the human (h) cytosolic isoforms hCA I and II. The IC(50) values of caffeine against hCA I was of 55?mM, whereas that of piperine of 60?mM. The IC(50) values of caffeine and piperine against hCA II were of 2?mM. Although these are quite weak inhibitors they may constitute leads for developing tighter binding compounds. 相似文献
5.
Suvendu Mondal A. M. Badigannavar S. F. D’Souza 《Molecular breeding : new strategies in plant improvement》2012,29(2):467-476
Rust is a serious and the most prevalent groundnut disease in tropical and subtropical growing regions of the world. A total
of 164 recombinant inbred lines derived from resistant (VG 9514) and susceptible (TAG 24) cultivated groundnut parents were
screened for rust resistance in five environments. Subsequent genotyping of these lines with 109 simple sequence repeat (SSR)
markers generated a genetic linkage map with 24 linkage groups. The total length of the linkage map was 882.9 cM with an average
of 9.0 cM between neighbouring markers. The markers pPGPseq4A05 and gi56931710 flanked the rust resistance gene at map distances
of 4.7 cM and 4.3 cM, respectively, in linkage group 2. The significant association of these two markers with the rust reaction
was also confirmed by discriminant analysis. The informative SSR markers classified rust-resistant and susceptible groups
with 99.97% correctness. The SSR markers pPGPseq4A05 and gi56931710 were able to identify all the susceptible genotypes from
a set of 20 cultivated genotypes differing in rust reaction. Tagging of the rust resistance locus with linked SSR markers
will be useful in selecting the rust resistant genotypes from segregating populations and in introgressing the rust resistance
genes from diploid wild species. 相似文献
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Cultivated groundnut (Arachis hypogaea L.) is considered as one of the primary oilseed crops and a major fodder for cattle industry in most of the developing countries, owing to its rich source of protein. It is due to its geocarpic nature of growth that the overall yield performance of groundnut is hindered by several biotic and abiotic stress factors. Multidimensional attempts were undertaken to combat these factors by developing superior groundnut varieties, modified with integral mechanism of tolerance/resistance; however this approach proved to be futile, owing to inferior pod and kernel quality. As a superior alternative, biotechnological intervention like transformation of foreign genes, either directly (biolistic) or via Agrobacterium, significantly aided in the development of advanced groundnut genotypes equipped with integral resistance against stresses and enhanced yield attributing traits. Several genes triggered by biotic and abiotic stresses, were detected and some of them were cloned and transformed as major parts of transgenic programmes. Application of modern molecular biological techniques, in designing biotic and abiotic stress tolerant/resistant groundnut varieties that exhibited mechanisms of resistance, relied on the expression of specific genes associated to particular stress. The genetically transformed stress tolerant groundnut varieties possess the potential to be employed as donor parents in traditional breeding programmes for developing varieties that are resilient to fungal, bacterial, and viral diseases, as well as to draught and salinity. The present review emphasizes on the retrospect and prospect of genetic transformation tools, implemented for the enhancement of groundnut varieties against key biotic and abiotic stress factors. 相似文献
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An understanding of the genetic affinity and the past history of the tribal populations of India requires the untangling of the confounding influences of language, ethnicity, and geography on the extant diverse tribes. The present study examines the genetic relationship of linguistically (Dravidian, Austro‐Asiatic, and Tibeto‐Burman) and ethnically (Australian and East Asian) diverse tribal populations (46) inhabiting different regions of the Indian subcontinent. For the purpose, we have utilized the published data on allele frequency of 15 autosomal STR loci of our study on six Adi sub‐tribes of Arunachal Pradesh and compared the same with the reported allele frequency data, for nine common autosomal STR loci, of 40 other tribes. Phylogenetic and principal component analyses exhibit geography based clustering of Tibeto‐Burman speakers and separation of the Mundari and Mon‐Khmer speaking Austro‐Asiatic populations. The combined analyses of all 46 populations show clustering of the groups belonging to same ethnicity and inhabiting contiguous geographic regions, irrespective of their different languages. These results help us to reconstruct and understand three plausible scenarios of the antiquity of Indian tribal populations: the Dravidian and Austro‐Asiatic (Mundari) tribes were possibly derived from common early settlers; the Tibeto‐Burman tribes possibly belonged to a different ancestry and the Mon‐Khmer speaking Austro‐Asiatic populations share a common ancestry with some of the Tibeto‐Burman speakers. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc. 相似文献
10.
Indian subcontinent harbours both the human mtDNA macrohaplogroups M and N, of which M is the most prevalent. In this study,
we discuss the overall distribution of the various haplogroups and sub-haplogroups of M among the different castes and tribes
to understand their diverse pattern with respect to geographical location and linguistic affiliation of the populations. An
overview of about 170 studied populations, belonging to four distinct linguistic families and inhabiting different geographic
zones, revealed wide diversity of about 22 major haplogroups of M. The tribal populations belonging to the same linguistic
family but inhabiting different geographical regions (Dravidian and Austro-Asiatic speakers) exhibited differences in their
haplogroup diversity. The northern and southern region castes showed greater diversity than the castes of other regions. 相似文献