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81.
The lion Panthera leo is one of the world's most charismatic carnivores and is one of Africa's key predators. Here, we used a large dataset from 357 lions comprehending 1.13 megabases of sequence data and genotypes from 22 microsatellite loci to characterize its recent evolutionary history. Patterns of molecular genetic variation in multiple maternal (mtDNA), paternal (Y-chromosome), and biparental nuclear (nDNA) genetic markers were compared with patterns of sequence and subtype variation of the lion feline immunodeficiency virus (FIV(Ple)), a lentivirus analogous to human immunodeficiency virus (HIV). In spite of the ability of lions to disperse long distances, patterns of lion genetic diversity suggest substantial population subdivision (mtDNA Phi(ST) = 0.92; nDNA F(ST) = 0.18), and reduced gene flow, which, along with large differences in sero-prevalence of six distinct FIV(Ple) subtypes among lion populations, refute the hypothesis that African lions consist of a single panmictic population. Our results suggest that extant lion populations derive from several Pleistocene refugia in East and Southern Africa ( approximately 324,000-169,000 years ago), which expanded during the Late Pleistocene ( approximately 100,000 years ago) into Central and North Africa and into Asia. During the Pleistocene/Holocene transition ( approximately 14,000-7,000 years), another expansion occurred from southern refugia northwards towards East Africa, causing population interbreeding. In particular, lion and FIV(Ple) variation affirms that the large, well-studied lion population occupying the greater Serengeti Ecosystem is derived from three distinct populations that admixed recently.  相似文献   
82.
Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re‐quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.  相似文献   
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Alpha(1)-proteinase inhibitor (alpha(1)-PI) of the domestic cat (Felis catus) was purified from serum and a radioimmunoassay (RIA) for the measurement of feline alpha(1)-PI concentration in serum was developed and validated. Feline alpha(1)-PI (falpha(1)-PI) was isolated using ammonium sulfate precipitation, anion-exchange, size-exclusion, ceramic hydroxyapatite, and hydrophobic interaction chromatography. The molecular weight of falpha(1)-PI was estimated at 57,000 and the relative molecular mass (M(r)) was determined to be approximately 54.5 kDa. Isoelectric focusing revealed four bands with isoelectric points (pI) between 4.3 and 4.5. The N-terminal amino acid sequence of the first 19 residues was Glu-Gly-Leu-Gln-Gly-Ala-Ala-Val-Gln-Glu-Thr-Val-Ala-Ser-Gln-His-Asp-Gln-Glu. Antiserum against feline alpha(1)-PI was raised in rabbits. Tracer was produced by iodination ((125)I) of feline alpha(1)-PI using the chloramine T method. A radioimmunoassay was established and validated by determination of sensitivity, dilutional parallelism, spiking recovery, intra-assay variability, and inter-assay variability. A control range for serum feline alpha(1)-PI concentration was established from 50 healthy cats using the central 95th percentile. The sensitivity of the assay was 0.042 mg/ml. Observed to expected ratios for serial dilutions ranged from 105% to 141.18% for four different serum samples at dilutions of 1 in 35,000, 1 in 70,000, 1 in 140,000 and 1 in 280,000. Observed to expected ratios for spiking recovery ranged from 88.14% to 152.17% for four different serum samples and five different spiking concentrations. Coefficients of variation for four different serum samples were 4.57%, 6.45%, 8.52%, and 4.27% for intra-assay variability and 6.88%, 9.57%, 7.44%, and 9.94% for inter-assay variability. The reference range was established as 0.25-0.6 mg/ml. In summary, feline alpha(1)-PI was successfully purified from serum using a rapid and efficient method. The radioimmunoassay described here is sensitive, linear, accurate, precise, and reproducible and will facilitate further studies of the physiological or potential pathological role of alpha(1)-PI in cats.  相似文献   
85.
A series of squaramide-based hydroxamic acids were designed, synthesized and evaluated against human HDAC enzyme. Squaramides were found to be potent in the Hut78 cell line, but initially suffered from low solubility. Leads with improved solubility and metabolic profiles were shown to be class I, IIB and IV selective.  相似文献   
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A general field bioassay for detecting biologically active compounds in plants and insects has been developed and tested for efficacy and sensitivity. Methanolic extracts, in sucrose solution, of 20 plant and six caterpillar species were offered to the ponerine ant Paraponera clavata and the feeding preferences observed. The bioassay resulted in the detection of nine plant and three caterpillar species with ant-deterrent extracts, and 11 plant and three caterpillar species with neutral or attractant extracts. All of the plants showing ant-deterrent characteristics which had been chemically investigated in our laboratory, or for which chemical literature was available, contained secondary metabolites of known deterrence. Both naturally occurring and artificial differences in chemical concentrations could be detected using the bioassay. The method provides a means of screening plants and insects for compounds that are insect anti-feedants or that can modify insect behaviour.  相似文献   
89.
Although the relevance of three-dimensional (3-D) culture has been recognized for years and exploited at an academic level, its translation to industrial applications has been slow. The development of reliable high-throughput technologies is clearly a prerequisite for the industrial implementation of 3-D models. In this study the robustness of spherical microtissue production and drug testing in a 96-well hanging-drop multiwell plate format was assessed on a standard 96-well channel robotic platform. Microtissue models derived from six different cell lines were produced and characterized according to their growth profile and morphology displaying high-density tissue-like reformation and growth over at least 15 days. The colon cancer cell line HCT116 was chosen as a model to assess microtissue-based assay reproducibility. Within three individual production batches the size variations of the produced microtissues were below 5%. Reliability of the microtissue-based assay was tested using two reference compounds, staurosporine and chlorambucil. In four independent drug testings the calculated IC(50) values were benchmarked against 2-D multiwell testings displaying similar consistency. The technology presented here for the automated production of a variety of microtissues for efficacy testing in a standard 96-well format will aid the implementation of more organotypic models at an early time point in the drug discovery process.  相似文献   
90.
Drosophila Smaug is a sequence-specific RNA-binding protein that can repress the translation and induce the degradation of target mRNAs in the early Drosophila embryo. Our recent work has uncovered a new mechanism of Smaug-mediated translational repression whereby it interacts with and recruits the Argonaute 1 (Ago1) protein to an mRNA. Argonaute proteins are typically recruited to mRNAs through an associated small RNA, such as a microRNA (miRNA). Surprisingly, we found that Smaug is able to recruit Ago1 to an mRNA in a miRNA-independent manner. This work suggests that other RNA-binding proteins are likely to employ a similar mechanism of miRNA-independent Ago recruitment to control mRNA expression. Our work also adds yet another mechanism to the list that Smaug can use to regulate its targets and here we discuss some of the issues that are raised by Smaug’s multi-functional nature.  相似文献   
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