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81.
We developed a gas chromatography-mass spectrometry (GC-MS) assay to measure the activity of malonyl-coenzyme A (CoA) decarboxylase (MCD) in crude tissue homogenates. Liver extracts are incubated with [U-(13)C(3)]malonyl-CoA to form [U-(13)C(2)]acetyl-CoA by the action of MCD. The reaction mixture contains 2 mM ADP to prevent the hydrolysis of [1,2-(13)C(2)]acetyl-CoA by acetyl-CoA hydrolase present in the extracts. Newly formed [U-(13)C(2)]acetyl-CoA and internal standard of [(2)H(3),1-(13)C]acetyl-CoA are analyzed as thiophenol derivatives by GC-MS. This assay was applied to a study of the kinetics of MCD in rat liver. Using the Lineweaver-Burke plot of MCD kinetics, K(m) of 202microM and V(max) of 3.3micromol min(-1) (g liver)(-1) were calculated. The liver MCD activities (micromol min(-1) g(-1)+/-SD) in three groups of rats with different nutritional statuses-fed, 1-day fasted, and 2-day fasted-were 1.80+/-0.41, 2.59+/-0.37 (P<0.05), and 3.07+/-0.70 (P<0.05), respectively. We report a practical, nonradioactive, sensitive assay of MCD in crude tissue extract. 相似文献
82.
Stiff CM Zhong M Sarver RW Gao H Ho AM Sweeney MT Zurenko GE Romero DL 《Bioorganic & medicinal chemistry letters》2007,17(19):5479-5482
Previously we reported the discovery and initial optimization of a novel anthranilic acid derived class of antibacterial agents which suffered from extensive protein binding. This report describes efforts directed toward understanding the relationship of the acidity of the carboxylic acid with the extent of protein binding. The pK(a) of the acid was modified via the synthesis of a number of anthranilic acid analogs which vary the aromatic ring substituent at the 4-position. The pK(a) and HSA binding constants have been determined for each of the analogs. Our results indicate a correlation between pK(a) and HSA K(d). The physical properties and antibacterial activities will be discussed as well as how these results help address the protein binding issue with this series of compounds. 相似文献
83.
Reconstructing the evolutionary history of protein sequences will provide a better understanding of divergence mechanisms of protein superfamilies and their functions. Long-term protein evolution often includes dynamic changes such as insertion, deletion, and domain shuffling. Such dynamic changes make reconstructing protein sequence evolution difficult and affect the accuracy of molecular evolutionary methods, such as multiple alignments and phylogenetic methods. Unfortunately, currently available simulation methods are not sufficiently flexible and do not allow biologically realistic dynamic protein sequence evolution. We introduce a new method, indel-Seq-Gen (iSG), that can simulate realistic evolutionary processes of protein sequences with insertions and deletions (indels). Unlike other simulation methods, iSG allows the user to simulate multiple subsequences according to different evolutionary parameters, which is necessary for generating realistic protein families with multiple domains. iSG tracks all evolutionary events including indels and outputs the "true" multiple alignment of the simulated sequences. iSG can also generate a larger sequence space by allowing the use of multiple related root sequences. With all these functions, iSG can be used to test the accuracy of, for example, multiple alignment methods, phylogenetic methods, evolutionary hypotheses, ancestral protein reconstruction methods, and protein family classification methods. We empirically evaluated the performance of iSG against currently available methods by simulating the evolution of the G protein-coupled receptor and lipocalin protein families. We examined their true multiple alignments, reconstruction of the transmembrane regions and beta-strands, and the results of similarity search against a protein database using the simulated sequences. We also presented an example of using iSG for examining how phylogenetic reconstruction is affected by high indel rates. 相似文献
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86.
Evans DW Müller-Loennies S Brooks CL Brade L Kosma P Brade H Evans SV 《Glycobiology》2011,21(8):1049-1059
The structure of the antigen-binding fragment from the monoclonal antibody S64-4 in complex with a pentasaccharide bisphosphate fragment from chlamydial lipopolysaccharide has been determined by x-ray diffraction to 2.6 ? resolution. Like the well-characterized antibody S25-2, S64-4 displays a pocket formed by the residues of germline sequence corresponding to the heavy and light chain V gene segments that binds the terminal Kdo residue of the antigen; however, although S64-4 shares the same heavy chain V gene segment as S25-2, it has a different light chain V gene segment. The new light chain V gene segment codes for a combining site that displays greater affinity, different specificity, and allows a novel antigen conformation that brings a greater number of antigen residues into the combining site than possible in S25-2. Further, while antibodies in the S25-2 family use complementarity determining region (CDR) H3 to discriminate among antigens, S64-4 achieves its specificity via the new light chain V gene segment and resulting change in antigen conformation. These structures reveal an intriguing parallel strategy where two different combinations of germline-coded V gene segments can act as starting points for the generation of germline antibodies against chlamydial antigens and show how anti-carbohydrate antibodies can exploit the conformational flexibility of this class of antigens to achieve high affinity and specificity independently of CDR H3. 相似文献
87.
Labroli M Paruch K Dwyer MP Alvarez C Keertikar K Poker C Rossman R Duca JS Fischmann TO Madison V Parry D Davis N Seghezzi W Wiswell D Guzi TJ 《Bioorganic & medicinal chemistry letters》2011,21(1):471-474
Previous efforts by our group have established pyrazolo[1,5-a]pyrimidine as a viable core for the development of potent and selective CDK inhibitors. As part of an effort to utilize the pyrazolo[1,5-a]pyrimidine core as a template for the design and synthesis of potent and selective kinase inhibitors, we focused on a key regulator in the cell cycle progression, CHK1. Continued SAR development of the pyrazolo[1,5-a]pyrimidine core at the C5 and C6 positions, in conjunction with previously disclosed SAR at the C3 and C7 positions, led to the discovery of potent and selective CHK1 inhibitors. 相似文献
88.
Repifermin, a truncated form of fibroblast growth factor-10 (FGF-10) also known as keratinocyte growth factor-2 (KGF-2), is a heparin-binding protein with potent regenerative properties. The protein unfolds and aggregates at relatively low temperature (~37 °C). Electrostatic interactions between polyanions and several FGFs have been reported to enhance the thermal stability of these proteins. Polyethylene glycol (PEG) was grafted to the polyanions pentosan polysulfate (PPS) and dextran sulfate (DS) as an alternative means to stabilize and noncovalently PEGylate KGF-2. Physical characteristics of KGF-2:polyanion-PEG complexes were examined using a variety of methods including circular dichroism (CD), intrinsic tryptophan fluorescence, differential scanning calorimetry, and dynamic light scattering. When compared to KGF-2 alone, subtle changes in CD spectra and fluorescence emission maxima were found when KGF-2 was formulated with the synthetic PEG-polyanions. Highly PEGylated polyanions (DS-PEG5) did not bind KGF-2 as well as conjugates with fewer PEG chains. The molecular weight of PEG did not have a noticeable effect on KGF-2 binding to the various PEG-polyanion conjugates. At optimal molar ratios, PPS-PEG and DS-PEG conjugates were able to stabilize KGF-2 by increasing the melting temperature by approximately 9-17 °C. Thus, polyanion-PEG conjugates improved the stability of KGF-2 and also offered a new electrostatic PEGylation scheme that may be extrapolated to other heparin-binding proteins. 相似文献
89.
Cory Pelletier William C Speed Trupti Paranjape Katie Keane Rachel Blitzblau Antoinette Hollestelle Kyan Safavi Ans van den Ouweland Daniel Zelterman Frank J Slack Kenneth K Kidd Joanne B Weidhaas 《Cell cycle (Georgetown, Tex.)》2011,10(1):90-99
Genetic markers identifying women at an increased risk of developing breast cancer exist, yet the majority of inherited risk remains elusive. While numerous BRCA1 coding sequence mutations are associated with breast cancer risk, BRCA1 mutations account for less then 5% of breast cancer risk. Since 3′ untranslated region (3′UTR) polymorphisms disrupting microRNA (miRNA) binding can be functional and can act as genetic markers of cancer risk, we tested the hypothesis that such polymorphisms in the 3′UTR of BRCA1 and haplotypes containing these functional polymorphisms may be associated with breast cancer risk. We sequenced the BRCA1 3′UTR from breast cancer patients to identify miRNA disrupting polymorphisms. We further evaluated haplotypes of this region including the identified 3′UTR variants in a large population of controls and breast cancer patients (n = 221) with known breast cancer subtypes and ethnicities. We identified three 3′UTR variants in BRCA1 that are polymorphic in breast cancer populations, and haplotype analysis including these variants revealed that breast cancer patients harbor five rare haplotypes not generally found among controls (9.50% for breast cancer chromosomes, 0.11% for control chromosomes, p = 0.0001). Three of these rare haplotypes contain the rs8176318 BRCA1 3′UTR functional variant. These haplotypes are not biomarkers for BRCA1 coding mutations, as they are found rarely in BRCA1 mutant breast cancer patients (1/129 patients = 0.78%). These rare BRCA1 haplotypes and 3′UTR SNPs may represent new genetic markers of breast cancer risk.Key words: BRCA1, haplotype, microRNA, SNP, 3′UTR, breast cancer, triple negative breast cancer 相似文献
90.
Park KS Martelotto LG Peifer M Sos ML Karnezis AN Mahjoub MR Bernard K Conklin JF Szczepny A Yuan J Guo R Ospina B Falzon J Bennett S Brown TJ Markovic A Devereux WL Ocasio CA Chen JK Stearns T Thomas RK Dorsch M Buonamici S Watkins DN Peacock CD Sage J 《Nature medicine》2011,17(11):1504-1508
Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment. Using a mouse model in which deletion of Rb1 and Trp53 in the lung epithelium of adult mice induces SCLC, we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rb1 and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC. 相似文献