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101.
102.
Colin I. Smith Benjamin T. Fuller Kyungcheol Choy Michael P. Richards 《Analytical biochemistry》2009,390(2):165-2988
We report a three-phase chromatographic method for the separation and analysis of δ13C values of underivatized amino acids from biological proteins (keratin, collagen, and casein) using liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). Both precision and accuracy of δ13C values for standard amino acid mixtures over the range of approximately 8 to 1320 ng of carbon per amino acid on the column were assessed. The precision of δ13C values of amino acids was found to be better at higher concentrations, whereas accuracy improved at lower concentrations. The optimal performance for this method was achieved with between 80 and 660 ng of carbon of each amino acid on the column. At amino acid amounts lower than 20 ng of carbon on the column, precision and accuracy may become compromised. The application of this new three-phase chromatographic technique will allow the analysis of δ13C of amino acids to be carried out as a routine method and benefit fields of research such as biomedicine, forensics, ecology, nutrition, and palaeodiet reconstruction in archaeology. 相似文献
103.
Background
Multidrug resistance (MDR) is a major factor which contributes to the failure of cancer chemotherapy, and numerous efforts have been attempted to overcome MDR. To date, none of these attempts have yielded a tolerable and effective therapy to reverse MDR; thus, identification of new agents would be useful both clinically and scientifically.Methodology/Principal Findings
To identify small molecule compounds that can reverse chemoresistance, we developed a 96-well plate high-throughput cell-based screening assay in a paclitaxel resistant ovarian cancer cell line. Coincubating cells with a sublethal concentration of paclitaxel in combination with each of 2,000 small molecule compounds from the National Cancer Institute Diversity Set Library, we identified a previously uncharacterized molecule, NSC23925, that inhibits Pgp1 and reverses MDR1 (Pgp1) but does not inhibit MRP or BCRP-mediated MDR. The cytotoxic activity of NSC23925 was further evaluated using a panel of cancer cell lines expressing Pgp1, MRP, and BCRP. We found that at a concentration of >10 µM NSC23925 moderately inhibits the proliferation of both sensitive and resistant cell lines with almost equal activity, but its inhibitory effect was not altered by co-incubation with the Pgp1 inhibitor, verapamil, suggesting that NSC23925 itself is not a substrate of Pgp1. Additionally, NSC23925 increases the intracellular accumulation of Pgp1 substrates: calcein AM, Rhodamine-123, paclitaxel, mitoxantrone, and doxorubicin. Interestingly, we further observed that, although NSC23925 directly inhibits the function of Pgp1 in a dose-dependent manner without altering the total expression level of Pgp1, NSC23925 actually stimulates ATPase activity of Pgp, a phenomenon seen in other Pgp inhibitors.Conclusions/Significance
The ability of NSC23925 to restore sensitivity to the cytotoxic effects of chemotherapy or to prevent resistance could significantly benefit cancer patients. 相似文献104.
Neudecker P Zarrine-Afsar A Choy WY Muhandiram DR Davidson AR Kay LE 《Journal of molecular biology》2006,363(5):958-976
Recent 15N and 13C spin-relaxation dispersion studies of fast-folding mutants of the Fyn SH3 domain have established that folding proceeds through a low-populated on-pathway intermediate (I) where the central beta-sheet is at least partially formed, but without interactions between the NH2- and COOH-terminal beta-strands that exist in the folded state (F). Initial studies focused on mutants where Gly48 is replaced; in an effort to establish whether this intermediate is a general feature of Fyn SH3 folding a series of 15N relaxation experiments monitoring the folding of Fyn SH3 mutants N53P/V55L and A39V/N53P/V55L are reported here. For these mutants as well, folding proceeds through an on-pathway intermediate with similar features to those observed for G48M and G48V Fyn SH3 domains. However, the 15N chemical shifts extracted for the intermediate indicate pronounced non-native contacts between the NH2 and COOH-terminal regions not observed previously. The kinetic parameters extracted for the folding of A39V/N53P/V55L Fyn SH3 from the three-state folding model F<-->I<-->U are in good agreement with folding and unfolding rates extrapolated to zero denaturant obtained from stopped-flow experiments analyzed in terms of a simplified two-state folding reaction. The folding of the triple mutant was studied over a wide range of temperatures, establishing that there is no difference in heat capacities between F and I states. This confirms a compact folding intermediate structure, which is supported by the 15N chemical shifts of the I state extracted from the dispersion data. The temperature-dependent relaxation data simplifies data analysis because at low temperatures (< 25 degrees C) the unfolded state (U) is negligibly populated relative to I and F. A comparison between parameters extracted at low temperatures where the F<-->I exchange model is appropriate with those from the more complex, three-state model at higher temperatures has been used to validate the protocol for analysis of three-site exchange relaxation data. 相似文献
105.
Tran BN Chen L Liu Y Wu J Velázquez-Campoy A Sivaraman J Hew CL 《Journal of virology》2011,85(17):9159-9166
Singapore grouper iridovirus (SGIV), a major pathogen of concern for grouper aquaculture, has a double-stranded DNA (dsDNA) genome with 162 predicted open reading frames, for which a total of 62 SGIV proteins have been identified. One of these, ORF158L, bears no sequence homology to any other known protein. Knockdown of orf158L using antisense morpholino oligonucleotides resulted in a significant decrease in virus yield in grouper embryonic cells. ORF158L was observed in nuclei and virus assembly centers of virus-infected cells. This observation led us to study the structure and function of ORF158L. The crystal structure determined at 2.2-Å resolution reveals that ORF158L partially exhibits a structural resemblance to the histone binding region of antisilencing factor 1 (Asf1), a histone H3/H4 chaperon, despite the fact that there is no significant sequence identity between the two proteins. Interactions of ORF158L with the histone H3/H4 complex and H3 were demonstrated by isothermal titration calorimetry (ITC) experiments. Subsequently, the results of ITC studies on structure-based mutants of ORF158L suggested Arg67 and Ala93 were key residues for histone H3 interactions. Moreover, a combination of approaches of ORF158L knockdown and isobaric tags/mass spectrometry for relative and absolute quantifications (iTRAQ) revealed that ORF158L may be involved in both the regulation and the expression of histone H3 and H3 methylation. Our present studies suggest that ORF158L may function as a histone H3 chaperon, enabling it to control host cellular gene expression and to facilitate viral replication. 相似文献
106.
107.
Xiao Lu Zhen-Du Zhang Xiaomei Guo Jenny Susana Choy Junrong Yang Mark Svendsen Ghassan Kassab 《PloS one》2014,9(8)
Although hemodynamics changes occur in heart failure (HF) and generally influence vascular function, it is not clear whether various HF models will affect the conduit vessels differentially or whether local hemodynamic forces or systemic factors are more important determinants of vascular response in HF. Here, we studied the hemodynamic changes in tachycardia or volume-overload HF swine model (created by either high rate pacing or distal abdominal aortic-vena cava fistula, respectively) on carotid, femoral, and renal arteries function and molecular expression. The ejection fraction was reduced by 50% or 30% in tachycardia or volume-overload model in four weeks, respectively. The LV end diastolic volume was increased from 65±22 to 115±78 ml in tachycardia and 67±19 to 148±68 ml in volume-overload model. Flow reversal was observed in diastolic phase in carotid artery of both models and femoral artery in volume-overload model. The endothelial function was also significantly impaired in carotid and renal arteries of tachycardia and volume-overload animals. The endothelial dysfunction was observed in femoral artery of volume-overload animals but not tachycardia animals. The adrenergic receptor-dependent contractility decreased in carotid and femoral arteries of tachycardia animals. The protein expressions of NADPH oxidase subunits increased in the three arteries and both animal models while expression of MnSOD decreased in carotid artery of tachycardia and volume-overload model. In conclusion, different HF models lead to variable arterial hemodynamic changes but similar vascular and molecular expression changes that reflect the role of both local hemodynamics as well as systemic changes in HF. 相似文献
108.
CJ Cooksey 《Biotechnic & histochemistry》2014,89(5):384-387
The history, origin, identity, chemistry and uses of Congo red are described. Originally patented in 1884, Congo red soon found applications in dyeing cotton, as a pH indicator for chemists and as a biological stain. Unlike the majority of the 19th century synthetic dyes, it still is available commercially. 相似文献
109.
A facile procedure for the small scale chemical synthesis of l,2-dipalmitoyl-Sn -glycero-3-phosphocholine (DPL) is reported. Under optimal conditions, 36% of the sn-glycero-3-phosphocholine was converted to DPL. This procedure is suitable for small scale preparation of DPL with very high specific radioactivity from labeled palmitic acid. Furthermore, the labeled DPL obtained will serve as precursor for the chemical or enzymatic synthesis of other labeled phospholipids. 相似文献
110.
Structural requirements of tropomodulin for tropomyosin binding and actin filament capping 总被引:2,自引:0,他引:2
Kostyukova AS Rapp BA Choy A Greenfield NJ Hitchcock-DeGregori SE 《Biochemistry》2005,44(12):4905-4910
Regulation of actin filament dynamics underlies many cellular functions. Tropomodulin together with tropomyosin can cap the pointed, slowly polymerizing, filament end, inhibiting addition or loss of actin monomers. Tropomodulin has an unstructured N-terminal region that binds tropomyosin and a folded C-terminal domain with six leucine-rich repeats. Of tropomodulin 1's 359 amino acids, an N-terminal fragment (Tmod1(1)(-)(92)) suffices for in vitro function, even though the C-terminal domain can weakly cap filaments independent of tropomyosin. Except for one short alpha-helix with coiled coil propensity (residues 24-35), the Tmod1(1)(-)(92) solution structure shows that the fragment is disordered and highly flexible. On the basis of the solution structure and predicted secondary structure, we have introduced a series of mutations to determine the structural requirements for tropomyosin binding (using native gels and CD) and filament capping (by measuring actin polymerization using pyrene fluorescence). Tmod1(1)(-)(92) fragments with mutations of an interface hydrophobic residue, L27G and L27E, designed to destroy the alpha-helix or coiled coil propensity, lost binding ability to tropomyosin but retained partial capping function in the presence of tropomyosin. Replacement of a flexible region with alpha-helical residues (residues 59-61 mutated to Ala) had no effect on tropomyosin binding but inhibited the capping function. A mutation in a region predicted to be an amphipathic helix (residues 65-75), L71D, destroyed the capping function. The results suggest that molecular flexibility and binding to actin via an amphipathic helix are both required for tropomyosin-dependent capping of the pointed end of the actin filament. 相似文献