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101.
We recently reported the pharmacological screening of a natural products-inspired library of spiroepoxide probes, resulting in the discovery of an agent MJE3 that displayed anti-proliferative effects in human breast cancer cells. MJE3 was found to covalently inactivate phosphoglycerate mutase-1 (PGAM1), a glycolytic enzyme with postulated roles in cancer cell metabolism and proliferation. Considering that MJE3 is one of the first examples of a cell-permeable, small-molecule inhibitor for PGAM1, we pursued a detailed examination of its mechanism and structural requirements for covalent inactivation. MJE3 was found to label PGAM1 on lysine-100, a conserved active site residue implicated in substrate recognition. Structural features of MJE3 important for PGAM1 labeling included two key recognition elements (an indole ring and carboxylic acid), the stereochemical orientation of the spiroepoxide, and presentation of these various binding/reactive groups on a rigid cyclohexane scaffold. Modeling studies of the docked MJE3-PGAM1 complex provide a structural rationale for these stringent requirements. Overall, these studies indicate that a special combination of binding and reactive elements are united in the MJE3 structure to inactivate PGAM1. More generally, our findings provide further evidence that useful pharmacological tools can emerge from screening structurally diverse libraries of protein-reactive probes. 相似文献
102.
Dimitra Sakoula Garrett J. Smith Jeroen Frank Rob J. Mesman Linnea F. M. Kop Pieter Blom Mike S. M. Jetten Maartje A. H. J. van Kessel Sebastian Lücker 《The ISME journal》2022,16(4):958
The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.Subject terms: Environmental microbiology, Sequencing, Microbiology 相似文献
103.
Jeannie M. Klein-Gordon Sujan Timilsina Yanru Xing Peter Abrahamian Karen A. Garrett Jeffrey B. Jones Gary E. Vallad Erica M. Goss 《The ISME journal》2022,16(2):591
Modern agricultural practices increase the potential for plant pathogen spread, while the advent of affordable whole genome sequencing enables in-depth studies of pathogen movement. Population genomic studies may decipher pathogen movement and population structure as a result of complex agricultural production systems. We used whole genome sequences of 281 Xanthomonas perforans strains collected within one tomato production season across Florida and southern Georgia fields to test for population genetic structure associated with tomato production system variables. We identified six clusters of X. perforans from core gene SNPs that corresponded with phylogenetic lineages. Using whole genome SNPs, we found genetic structure among farms, transplant facilities, cultivars, seed producers, grower operations, regions, and counties. Overall, grower operations that produced their own transplants were associated with genetically distinct and less diverse populations of strains compared to grower operations that received transplants from multiple sources. The degree of genetic differentiation among components of Florida’s tomato production system varied between clusters, suggesting differential dispersal of the strains, such as through seed or contaminated transplants versus local movement within farms. Overall, we showed that the genetic variation of a bacterial plant pathogen is shaped by the structure of the plant production system.Subject terms: Applied microbiology, Population genetics, Microbial ecology, Microbial ecology 相似文献
104.
K M Garrett R S Duman N Saito A J Blume M P Vitek J F Tallman 《Biochemical and biophysical research communications》1988,156(2):1039-1045
A cDNA clone of an alpha subunit of the human GABA-A receptor has been isolated. The human clone (pCLL800) contains 1055 nucleotides in an open reading frame and 260 nucleotides in the 5' non-coding region. The 351 amino acid sequence of this human alpha subunit shows 97% homology with its bovine counterpart. Hybridization of pCLL800 to Northern blots shows a 3.9/4.3 Kb RNA doublet in human cortex, rat whole brain, cortex, hippocampus, midbrain, olfactory bulb and cerebellum. Developmental studies show that the levels of the rat alpha mRNA increase between one and three weeks of age in a manner similar to the development of the benzodiazepine binding sites. 相似文献
105.
Cell cycle and biochemical effects of PD 0183812. A potent inhibitor of the cyclin D-dependent kinases CDK4 and CDK6 总被引:5,自引:0,他引:5
Fry DW Bedford DC Harvey PH Fritsch A Keller PR Wu Z Dobrusin E Leopold WR Fattaey A Garrett MD 《The Journal of biological chemistry》2001,276(20):16617-16623
Progression through the G1 phase of the cell cycle requires phosphorylation of the retinoblastoma gene product (pRb) by the cyclin D-dependent kinases CDK4 and CDK6, whose activity can specifically be blocked by the CDK inhibitor p16(INK4A). Misregulation of the pRb/cyclin D/p16(INK4A) pathway is one of the most common events in human cancer and has lead to the suggestion that inhibition of cyclin D-dependent kinase activity may have therapeutic value as an anticancer treatment. Through screening of a chemical library, we initially identified the [2,3-d]pyridopyrimidines as inhibitors of CDK4. Chemical modification resulted in the identification of PD 0183812 as a potent and highly selective inhibitor of both CDK4 and CDK6 kinase activity, which is competitive with ATP. Flow cytometry experiments showed that of the cell lines tested, only those expressing pRb demonstrated a G1 arrest when treated with PD 0183812. This arrest correlated in terms of incubation time and potency with a loss of pRb phosphorylation and a block in proliferation, which was reversible. These results suggest a potential use of this chemical class of compounds as therapeutic agents in the treatment of tumors with functional pRb, possessing cell cycle aberrations in other members of the pRb/cyclin D/p16(INK4A) pathway. 相似文献
106.
We have used the ligand binding domain of the recombinant human estrogen receptor (hER) to develop a nonisotopic assay for detection of estrogenic compounds. The assay is based on competition of the estrogenic ligand with 17beta-estradiol for binding to the receptor, which leaves 17beta-estradiol free to bind to an anti-17beta-estradiol antibody. Unbound anti-17beta-estradiol antibody then binds to immobilized 17beta-estradiol-protein conjugate (to which hER is unable to bind for steric reasons), and is detected by an enzyme-labeled anti-rabbit IgG antibody. We used the assay to detect estrogenic compounds (mainly members of the flavonoid group of plant polyphenols) in a variety of commonly consumed plant foods. 相似文献
107.
Genomewide linkage searches for Mendelian disease loci can be efficiently conducted using high-density SNP genotyping arrays 总被引:5,自引:0,他引:5 下载免费PDF全文
Sellick GS Longman C Tolmie J Newbury-Ecob R Geenhalgh L Hughes S Whiteford M Garrett C Houlston RS 《Nucleic acids research》2004,32(20):e164
Genomewide linkage searches aimed at identifying disease susceptibility loci are generally conducted using 300–400 microsatellite markers. Genotyping bi-allelic single nucleotide polymorphisms (SNPs) provides an alternative strategy. The availability of dense SNP maps coupled with recent technological developments in highly paralleled SNP genotyping makes it practical to now consider the use of these markers for whole-genome genetic linkage analyses. Here, we report the findings from three successful genomewide linkage analyses of families segregating autosomal recessively inherited neonatal diabetes, craniosynostosis and dominantly inherited renal dysplasia using the Affymetrix 10K SNP array. A single locus was identified for each disease state, two of which are novel. The performance of the SNP array, both in terms of efficiency and precision, indicates that such platforms will become the dominant technology for performing genomewide linkage searches. 相似文献
108.
Módos K Galántai R Bárdos-Nagy I Wachsmuth M Tóth K Fidy J Langowski J 《European biophysics journal : EBJ》2004,33(1):59-67
Fluorescence correlation spectroscopy was used to measure the diffusion behavior of a mixture of DMPC or DMPC/DMPG liposomes with human serum albumin (HSA) and mesoporphyrin (MP), which was used as the fluorescent label for liposomes and HSA as well. For decomposing the fluorescence intensity autocorrelation function (ACF) into components corresponding to a liposome population, HSA and MP, we used a maximum entropy procedure that computes a distribution of diffusion times consistent with the ACF data. We found that a simple parametric non-linear fit with a discrete set of decay components did not converge to a stable parameter set. The distribution calculated with the maximum entropy method was stable and the average size of the particles calculated from the effective diffusion time was in good agreement with the data determined using the discrete-component fit. 相似文献
109.
Zheng W Brandish PE Kolodin DG Scolnick EM Strulovici B 《Journal of biomolecular screening》2004,9(2):132-140
Inositol monophosphatase is a potential drug target for developing lithium-mimetic agents for the treatment of bipolar disorder. Enzyme-based assays have been traditionally used in compound screening to identify inositol monophosphatase inhibitors. A cell-based screening assay in which the compound needs to cross the cell membrane before reaching the target enzyme offers a new approach for discovering novel structure leads of the inositol monophosphatase inhibitor. The authors have recently reported a high-throughput measurement of G-protein-coupled receptor activation by determining inositol phosphates in cell extracts using scintillation proximity assay. This cell-based assay has been modified to allow the determination of inositol monophosphatase activity instead of G-protein-coupled receptors. The enzyme is also assayed in its native form and physiological environment. The authors have applied this cell-based assay to the high-throughput screening of a large compound collection and identified several novel inositol monophosphatase inhibitors. 相似文献
110.