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Butterfield DA Howard B Yatin S Koppal T Drake J Hensley K Aksenov M Aksenova M Subramaniam R Varadarajan S Harris-White ME Pedigo NW Carney JM 《Life sciences》1999,65(18-19):1883-1892
Age-associated neurodegenerative disorders are becoming more prevalent as the mean age of the population increases in the United States over the next few decades. Both normal brain aging and Alzheimer's disease (AD) are associated with oxidative stress. Our laboratory has used a wide variety of physical and biochemical methods to investigate free radical oxidative stress in several models of aging and AD. Beta-amyloid (A beta), the peptide that constitutes the central core of senile plaques in AD brain, is associated with free radical oxidative stress and is toxic to neurons. This review summarizes some of our studies in aging and A beta-associated free radical oxidative stress and on the modulating effects of free radical scavengers on neocortical synaptosomal membrane damage found in aging and A beta-treated systems. 相似文献
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In an effort to advance the use of whole-embryo culture (WEC) techniques in the rabbit, we recently developed a gestational-age-based quantitative morphologic evaluation system for rabbit embryos. In the current study, we applied this new morphological scoring system to assess the development of rabbit gestational day (gd) 9 embryos exposed for 48 hr in WEC to the teratogens ethanol (EtOH, 154 mM), 6-aminonicotinamide (6AN, 0.15 mM), and methoxyacetic acid (MAA, 5.0 mM), and the nonteratogen penicillin G (PG, 2.0 mM). Each teratogen at the concentration tested markedly inhibited morphological development, as indicated by significantly lower morphologic scores (10.1+/-0.05, EtOH; 10.2+/-0.05, 6AN; and 9.8, MAA) relative to controls (10.6+/-0.04), and resulted in an increased percentage of malformed embryos (53%, EtOH; 57%, 6AN; 90%, MAA; and 3%, control). Embryonic growth, as measured by head length, somite number, and total embryonic protein, was significantly decreased by each teratogen. The abnormalities produced by teratogen exposure, which included brain, somite, and facial defects, were often similar to those produced following in vivo exposure in rabbits and rodents, and/or in vitro exposure in rodents. In contrast to the teratogen exposure groups, PG had no effect on embryo growth parameters, or on malformation rate (6%), although a slight but statistically significant decrease in morphology score (10.5+/-0.03) was noted. Our preliminary studies demonstrate the usefulness of the morphology evaluation system by quantifying graded differences in development, and indicate that rabbit WEC may be a useful adjunct to rodent WEC in gaining insights regarding differential interspecies sensitivity. 相似文献
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The molecular basis of Sjögren-Larsson syndrome: mutation analysis of the fatty aldehyde dehydrogenase gene 下载免费PDF全文
Sj?gren-Larsson syndrome (SLS) is an autosomal recessive disorder characterized by ichthyosis, mental retardation, spasticity, and deficient activity of fatty aldehyde dehydrogenase (FALDH). To define the molecular defects causing SLS, we performed mutation analysis of the FALDH gene in probands from 63 kindreds with SLS. Among these patients, 49 different mutations-including 10 deletions, 2 insertions, 22 amino acid substitutions, 3 nonsense mutations, 9 splice-site defects, and 3 complex mutations-were found. All of the patients with SLS were found to carry mutations. Nineteen of the missense mutations resulted in a severe reduction of FALDH enzyme catalytic activity when expressed in mammalian cells, but one mutation (798G-->C [K266N]) seemed to have a greater effect on mRNA stability. The splice-site mutations led to exon skipping or utilization of cryptic acceptor-splice sites. Thirty-seven mutations were private, and 12 mutations were seen in two or more probands of European or Middle Eastern descent. Four single-nucleotide polymorphisms (SNPs) were found in the FALDH gene. At least four of the common mutations (551C-->T, 682C-->T, 733G-->A, and 798+1delG) were associated with multiple SNP haplotypes, suggesting that these mutations originated independently on more than one occasion or were ancient SLS genes that had undergone intragenic recombination. Our results demonstrate that SLS is caused by a strikingly heterogeneous group of mutations in the FALDH gene and provide a framework for understanding the genetic basis of SLS and the development of DNA-based diagnostic tests. 相似文献
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A model of structure and catalysis for ketoreductase domains in modular polyketide synthases 总被引:7,自引:0,他引:7
Reid R Piagentini M Rodriguez E Ashley G Viswanathan N Carney J Santi DV Hutchinson CR McDaniel R 《Biochemistry》2003,42(1):72-79
A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr --> Phe, Ser --> Ala, and Lys --> Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivo analysis. In this case, the Tyr --> Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser --> Ala and Lys --> Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides. The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains. Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin. Immobilized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution. 相似文献
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fRFLP and fAFLP: medium-throughput genotyping by fluorescently post-labeling restriction digestion 总被引:1,自引:0,他引:1
Genome-scale studies of population structure and high-resolution mapping of genetically complex traits both require techniques for accurately and efficiently genotyping large numbers of polymorphic sites in multiple individuals. Many high-throughput genotyping technologies require the purchase of expensive equipment or consumables and are therefore out of reach of some individual research laboratories. Conversely, less expensive technologies are often labor intensive so that the effort involved in typing large numbers of samples or polymorphic sites is prohibitive. Here we present a method of fluorescently post-labeling restriction digestion using standard dye-terminator sequencing chemistry so that RFLP and AFLP products can be visualized on an automated sequencer This labeling method is efficient, inexpensive, easily multiplexed, and requires no unusual equipment or reagents, thus striking a balance between cost and throughput that should be appropriate for many research groups and core facilities. 相似文献
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Molecular analysis of the cyclic AMP-dependent protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene in patients with Carney complex and primary pigmented nodular adrenocortical disease (PPNAD) reveals novel mutations and clues for pathophysiology: augmented PKA signaling is associated with adrenal tumorigenesis in PPNAD 下载免费PDF全文
Groussin L Kirschner LS Vincent-Dejean C Perlemoine K Jullian E Delemer B Zacharieva S Pignatelli D Carney JA Luton JP Bertagna X Stratakis CA Bertherat J 《American journal of human genetics》2002,71(6):1433-1442
We studied 11 new kindreds with primary pigmented nodular adrenocortical disease (PPNAD) or Carney complex (CNC) and found that 82% of the kindreds had PRKAR1A gene defects (including seven novel inactivating mutations), most of which led to nonsense mRNA and, thus, were not expressed in patients' cells. However, a previously undescribed base substitution in intron 6 (exon 6 IVS +1G-->T) led to exon 6 skipping and an expressed shorter PRKAR1A protein. The mutant protein was present in patients' leukocytes and tumors, and in vitro studies indicated that the mutant PRKAR1A activated cAMP-dependent protein kinase A (PKA) signaling at the nuclear level. This is the first demonstration of an inactivating PRKAR1A mutation being expressed at the protein level and leading to stimulation of the PKA pathway in CNC patients. Along with the lack of allelic loss at the PRKAR1A locus in most of the tumors from this kindred, these data suggest that alteration of PRKAR1A function (not only its complete loss) is sufficient for augmenting PKA activity leading to tumorigenesis in tissues affected by CNC. 相似文献