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81.
Among the key issues determining success of a study employing molecular genetics tools in wildlife monitoring or research is a large enough set of highly informative genetic markers and a reliable, cost effective method for their analysis. While optimized commercial genotyping kits have been developed for humans and domestic animals, such protocols are rare in wildlife research. We developed a highly optimized multiplex PCR that genotypes 12 microsatellite loci and a sex determination locus in brown bear (Ursus arctos) faecal samples in a single multiplex PCR and a single sequencer run. We used this protocol to genotype 1053 faecal samples of bears from the Dinaric population, and obtained useful genotypes for 88% of the samples, a very high success rate. The new protocol outperformed the multiplex pre-amplification strategy used in a previous study of 473 faecal samples with a 78.4% success rate. On a subset of 182 samples we directly compared the performance of both approaches, and found no advantage of the multiplex pre-amplification. While pre-amplification protocols might still improve PCR success and reliability on a small fraction of low-quality samples, the higher costs and workload do not justify their use when analysing reasonably fresh non-invasive material. Moreover, the high number of multiplexed loci in the new protocol makes it comparable to commercially developed genotyping kits developed for domestic animals and humans. 相似文献
82.
Tina Kamčeva Maja Radisavljević Iva Vukićević Jürgen Arnhold Marijana Petković 《化学与生物多样性》2013,10(11):1972-1986
Phospholipase A2 is involved in propagation of inflammatory processes and carcinogenesis through its role in phospholipid metabolism, and release of arachidonic acid and lysophospholipids. Recent findings on correlation between elevated PLA2 activity and metastatic cancer render this enzyme an attractive target for cancer therapy. On the other hand, due to a broad range of oxidation states under physiological conditions and a high affinity for protein binding, platinum and ruthenium coordination complexes are promising candidates for PLA2 inhibitors. In this article, we discuss the interactions of Pt and Ru coordination complexes with PLA2 and phospholipids, as well as the application of MALDI‐TOF mass spectrometry for screening PLA2 inhibitors. Owing to the ability of this technique to simultaneously detect and monitor changes in substrate and product concentrations, the inhibitor mechanisms of both Pt and Ru complexes with various ligands were determined. 相似文献
83.
Victoria L. Wyckelsma Tomas Venckunas Peter J. Houweling Maja Schlittler Volker M. Lauschke Chrystal F. Tiong Harrison D. Wood Niklas Ivarsson Henrikas Paulauskas Nerijus Eimantas Daniel C. Andersson Kathryn N. North Marius Brazaitis Håkan Westerblad 《American journal of human genetics》2021,108(3):446-457
84.
Protein synthesis elongation factor 2 (EF-2) from eukaryotes contains a conserved post-translationally modified histidine residue known as diphthamide. Diphthamide is a unique site of ADP-ribosylation by diphtheria toxin (DT), which is responsible for cell killing. In this report, we describe the construction of DT-resistant HeLa cell lines by engineering the toxin-resistant form of its specific substrate, protein elongation factor-2. Using site-specific mutagenesis of the histidine precursor of diphthamide, the histidine residue of codon 715 in human EF-2 cDNA was substituted with one of four amino acid residue codons: leucine, methionine, asparagine or glutamine. Mutant EF-2s were subcloned into a pCMVexSVneo expression vector, transfected into HeLa cells, and DT-resistant cell clones were isolated. The protective effect of mutant EF-2s against cell killing by DT, after exposing all four mutant strains derived from HeLa cells to different concentrations of the toxin (5-20 ng/mL) was demonstrated by: (1) the normal morphological appearance of the cells; (2) their unaffected or slightly slower growth rates; (3) their undisturbed electrophoretic DNA profiles whose integrity was virtually preserved. Mutant cell strains showed also considerable levels of resistance to very high concentrations of DT, in that they maintained slower but consistent rates of cell growth. It was hence concluded that despite its strict conservation and unique modification, the diphthamide histidine appears not to be essential to the function of human EF-2 in protein synthesis. In addition, DT-resistant HeLa cell clones should prove valuable hosts for various DT gene-containing vectors that express the toxin intracellularly. 相似文献
85.
Francesco Marabita Malin Almgren Maléne E. Lindholm Sabrina Ruhrmann Fredrik Fagerstr?m-Billai Maja Jagodic Carl J. Sundberg Tomas J. Ekstr?m Andrew E. Teschendorff Jesper Tegnér David Gomez-Cabrero 《Epigenetics》2013,8(3):333-346
The proper identification of differentially methylated CpGs is central in most epigenetic studies. The Illumina HumanMethylation450 BeadChip is widely used to quantify DNA methylation; nevertheless, the design of an appropriate analysis pipeline faces severe challenges due to the convolution of biological and technical variability and the presence of a signal bias between Infinium I and II probe design types. Despite recent attempts to investigate how to analyze DNA methylation data with such an array design, it has not been possible to perform a comprehensive comparison between different bioinformatics pipelines due to the lack of appropriate data sets having both large sample size and sufficient number of technical replicates. Here we perform such a comparative analysis, targeting the problems of reducing the technical variability, eliminating the probe design bias and reducing the batch effect by exploiting two unpublished data sets, which included technical replicates and were profiled for DNA methylation either on peripheral blood, monocytes or muscle biopsies. We evaluated the performance of different analysis pipelines and demonstrated that: (1) it is critical to correct for the probe design type, since the amplitude of the measured methylation change depends on the underlying chemistry; (2) the effect of different normalization schemes is mixed, and the most effective method in our hands were quantile normalization and Beta Mixture Quantile dilation (BMIQ); (3) it is beneficial to correct for batch effects. In conclusion, our comparative analysis using a comprehensive data set suggests an efficient pipeline for proper identification of differentially methylated CpGs using the Illumina 450K arrays. 相似文献
86.
Asparagine‐linked glycosylation is a common post‐translational modification of proteins catalyzed by oligosaccharyltransferase that is important in regulating many aspects of protein function. Analysis of protein glycosylation, including glycoproteomic measurement of the site‐specific extent of glycosylation, remains challenging. Here, we developed methods combining enzymatic deglycosylation and protease digestion with SWATH‐MS to enable automated measurement of site‐specific occupancy at many glycosylation sites. Deglycosylation with peptide‐endoglycosidase H, leaving a remnant N‐acetylglucosamine on asparagines previously carrying high‐mannose glycans, followed by trypsin digestion allowed robust automated measurement of occupancy at many sites. Combining deglycosylation with the more general peptide‐N‐glycosidase F enzyme with AspN protease digest allowed robust automated differentiation of nonglycosylated and deglycosylated forms of a given glycosylation site. Ratiometric analysis of deglycosylated peptides and the total intensities of all peptides from the corresponding proteins allowed relative quantification of site‐specific glycosylation occupancy between yeast strains with various isoforms of oligosaccharyltransferase. This approach also allowed robust measurement of glycosylation sites in human salivary glycoproteins. This method for automated relative quantification of site‐specific glycosylation occupancy will be a useful tool for research with model systems and clinical samples. 相似文献
87.
Novak-Stroligo M Alpeza-Dunato Z Kovacević D Caljkusić-Mance T 《Collegium antropologicum》2010,34(Z2):209-210
The endothelial cells are one of the most important structures in a donor cornea. Morphology and concentration of endothelial cells must be carefully evaluated with a specular microscope before transplantation. The aim of this study is to evaluate the status of corneal endothelium in glaucoma patients. Prospective study included 50 patients suffering from glaucoma and 50 patients in control group. Patients had no corneal disease, ocular inflammation, previous trauma or ocular surgery. Patients were not contact lens wearers. They were also analyzed in groups according to type of glaucoma. Specular microscopy was performed on central corneas. This study showed that patients with glaucoma have lower central corneal endothelial cell density than those without glaucoma of the same age group. Also, patients with pseudoexfoliative glaucoma had lower values of central endothelial cell density comparing to patients with open angle or angle closure glaucoma. 相似文献
88.
Limitation of the Ellman method: cholinesterase activity measurement in the presence of oximes 总被引:2,自引:0,他引:2
The Ellman method for assaying thiols is widely used for cholinesterase activity measurement. Cholinesterase activity is measured indirectly by quantifying the concentration of 5-thio-2-nitrobenzoic acid (TNB) ion formed in the reaction between the thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and thiocholine, a product of substrate (i.e., acetylthiocholine [ATCh]) hydrolysis by the cholinesterase. Oximes, reactivators of inhibited cholinesterase, are nucleophiles that also react with ATCh (oximolysis), producing thiocholine and (indirectly) TNB ion. The aim of this study was to characterize ATCh oximolysis. Therefore, we measured the oximolysis between oximes (K027 and HI-6) and ATCh in the presence of DTNB at different pH values, taking into account the final concentration of a product that is thiocholine. To confirm oximate ion involvement in the nucleophilic attack, we also determined the reaction rate between the oximes and ATCh, without DTNB, at different pH values by measuring the decrease in oximate ion absorption over time. The oximate ion of K027 reacted 14 times faster with ATCh (306M(-1)min(-1)) than the oximate ion of HI-6 (22M(-1)min(-1)). However, the rate constants obtained with the Ellman method were 84M(-1)min(-1) for K027 and 22M(-1)min(-1) for HI-6. Our results confirmed that the rate obtained with K027 using the Ellman method is actually the rate of the Ellman reaction itself. This suggests that the Ellman method cannot be used uncritically to evaluate oxime reaction with choline esters, in particular when oximolysis is faster than the Ellman reaction itself at a given pH. 相似文献
89.
Chloupková M Pickert A Lee JY Souza S Trinh YT Connelly SM Dumont ME Dean M Urbatsch IL 《Biochemistry》2007,46(27):7992-8003
Human ATP-binding cassette (ABC) transporters comprise a family of 48 membrane-spanning transport proteins, many of which are associated with genetic diseases or multidrug resistance of cancers. In this study, we present a comprehensive approach for the cloning, expression, and purification of human ABC transporters in the yeast Pichia pastoris. We analyzed the expression of 25 proteins and demonstrate that 11 transporters, including ABCC3, ABCB6, ABCD1, ABCG1, ABCG4, ABCG5, ABCG8, ABCE1, ABCF1, ABCF2, and ABCF3, were expressed at high levels comparable to that of ABCB1 (P-glycoprotein). As an example of the purification strategy via tandem affinity chromatography, we purified ABCC3 (MRP3) whose role in the transport of anticancer drugs, bile acids, and glucuronides has been controversial. The yield of ABCC3 was 3.5 mg/100 g of cells in six independent purifications. Purified ABCC3, activated with PC lipids, exhibited significant ATPase activity with a Vmax of 82 +/- 32 nmol min-1 mg-1. The ATPase activity was stimulated by bile acids and glucuronide conjugates, reaching 170 +/- 28 nmol min-1 mg-1, but was not stimulated by a variety of anticancer drugs. The glucuronide conjugates ethinylestradiol-3-glucuronide and 17beta-estradiol-17-glucuronide stimulated the ATPase with relatively high affinities (apparent Km values of 2 and 3 microM, respectively) in contrast to bile acids (apparent Km values of >130 microM), suggesting that glucuronides are the preferred substrates for this transporter. Overall, the availability of a purification system for the production of large quantities of active transporters presents a major step not only toward understanding the role of ABCC3 but also toward future structure-function analysis of other human ABC transporters. 相似文献
90.
Melanoma patients are subject to different degrees of psychosocial distress. The emotional impact of malignant melanoma can be long lasting and profound, with the most common reactions to melanoma being depression, anxiety and deterioration in quality of life. Coping styles have been shown to have a significant influence on patients' quality of life and their emotional reaction to the illness. The aim of this paper was to investigate the quality of life, emotional status and coping styles in patients with melanoma. 31 patients suffering from malignant melanoma were included in the study. Results of this study show that melanoma has a medium influence on patients' psychological status and quality of life. The most "constructive" coping style--problem focused coping is the mostly used style by the patients, which might be one of the reasons why the illness didn't have a more severe influence on patients' psychological status. 相似文献