Creatine kinase (EC 2.7.3.2) (CK) isoenzymes are crucial to energy metabolism, particularly in tissues with high energy requirements.
Nuclear genes encode four known CK subunits: cytoplasmic muscle, ...cytoplasmic brain, ubiquitous mitochondrial (uMtCK), and
sarcomeric mitochondrial (sMtCK). Herein, we report the isolation and complete structural characterization of the human sMtCK
gene. It contains 11 exons and encompasses more than 37 kilobase pairs (kb). The sites of exon localization in the sMtCK-coding
region and their precise sizes are identical with the human uMtCK gene. The translation start codon is in the third exon and
lies 17 kb from the transcription start site. The human sMtCK gene is located on chromosome 5. Sequence analysis of the sMtCK
genomic upstream sequences reveals a typical TATAA box within the 80 base pairs (bp) that, by transfection experiments, are
sufficient to promote expression of chimeric plasmids with the chloramphenicol acetyltransferase reporter. Cis-acting sequences
in a fragment containing 3360 bp of upstream sequence, the first exon, and 750 bp of the first intron are sufficient to mediate
tissue-specific expression. However, these sequences only partially regulate induction of sMtCK expression in differentiating
mouse myoblasts. MEF1/MYOD and MEF2 sequence motifs present in the sMtCK gene are not sufficient to regulate differentiation-specific
expression. The sMtCK gene contains sequences homologous to several motifs that are shared among some nuclear genes encoding
mitochondrial proteins and that may be essential for the coordinated activation of these genes during mitochondrial biogenesis.
V(D)J rearrangement is the molecular mechanism by which an almost infinite array of specific immune receptors are generated. Defects in this process result in profound immunodeficiency as is the case ...in the C.B-17 SCID mouse or in RAG-1 (recombination-activating gene 1) or RAG-2 deficient mice. It has recently become clear that the V(D)J recombinase most likely consists of both lymphoid-specific factors and ubiquitously expressed components of the DNA double-strand break repair pathway. The deficit in SCID mice is in a factor that is required for both of these pathways. In this report, we show that the factor defective in the autosomal recessive severe combined immunodeficiency of Arabian foals is required for (i) V(D)J recombination, (ii) resistance to ionizing radiation, and (iii) DNA-dependent protein kinase activity.
Fragment-Based Screen against HIV Protease Perryman, Alexander L; Zhang, Qing; Soutter, Holly H ...
Chemical biology & drug design,
March 2010, Letnik:
75, Številka:
3
Journal Article
Recenzirano
Odprti dostop
We have employed a fragment-based screen against wild-type (NL4-3) HIV protease (PR) using the Active Sight fragment library and X-ray crystallography. The experiments reveal two new binding sites ...for small molecules. PR was co-crystallized with fragments, or crystals were soaked in fragment solutions, using five crystal forms, and 378 data sets were collected to 2.3-1.3 Å resolution. Fragment binding induces a distinct conformation and specific crystal form of TL-3 inhibited PR during co-crystallization. One fragment, 2-methylcyclohexanol, binds in the 'exo site' adjacent to the Gly¹⁶Gly¹⁷Gln¹⁸loop where the amide of Gly¹⁷is a specific hydrogen bond donor, and hydrophobic contacts occur with the side chains of Lys¹⁴and Leu⁶³. Another fragment, indole-6-carboxylic acid, binds on the 'outside/top of the flap' via hydrophobic contacts with Trp⁴², Pro⁴⁴, Met⁴⁶, and Lys⁵⁵, a hydrogen bond with Val⁵⁶, and a salt-bridge with Arg⁵⁷. 2-acetyl-benzothiophene also binds at this site. This study is the first fragment-based crystallographic screen against HIV PR, and the first time that fragments were screened against an inhibitor-bound drug target to search for compounds that both bind to novel sites and stabilize the inhibited conformation of the target.
The Organ Procurement and Transplantation Network (OPTN) Deceased Donor Potential Study, funded by the Health Resources and Services Administration, characterized the current pool of potential ...deceased donors and estimated changes through 2020. The goal was to inform policy development and suggest practice changes designed to increase the number of donors and organ transplants. Donor estimates used filtering methodologies applied to datasets from the OPTN, the National Center for Health Statistics, and the Agency for Healthcare Research and Quality and used these estimates with the number of actual donors to estimate the potential donor pool through 2020. Projected growth of the donor pool was 0.5% per year through 2020. Potential donor estimates suggested unrealized donor potential across all demographic groups, with the most significant unrealized potential (70%) in the 50–75‐year‐old age group and potential Donation after Circulatory Death (DCD) donors. Actual transplants that may be realized from potential donors in these categories are constrained by confounding medical comorbidities not identified in administrative databases and by limiting utilization practices for organs from DCD donors. Policy, regulatory, and practice changes encouraging organ procurement and transplantation of a broader population of potential donors may be required to increase transplant numbers in the United States.
Findings from the OPTN Deceased Donor Potential Study suggest possible transplant center and organ procurement organization practice changes, as well as potential transplant system regulatory changes, which may expand the donor pool.
Since the onset of fisheries science, monitoring programs have been implemented to support stock assessments and fisheries management. Here, we take inventory of the monitoring programs of the U.S. ...Gulf of Mexico (GOM) surveying fish and invertebrates and conduct a gap analysis of these programs. We also compile a large monitoring database encompassing much of the monitoring data collected in the U.S. GOM using random sampling schemes and employ this database to fit statistical models to then map the spatial distributions of 61 fish and invertebrate functional groups, species and life stages of the U.S. GOM. Finally, we provide recommendations for improving current monitoring programs and designing new programs, and guidance for more comprehensive use and sharing of monitoring data, with the ultimate goal of enhancing the inputs provided to stock assessments and ecosystem-based fisheries management (EBFM) projects in the U.S. GOM. Our inventory revealed that 73 fisheries-independent and fisheries-dependent programs have been conducted in the U.S. GOM, most of which (85%) are still active. One distinctive feature of monitoring programs of the U.S. GOM is that they include many fisheries-independent surveys conducted almost year-round, contrasting with most other marine regions. A major sampling recommendation is the development of a coordinated strategy for collecting diet information by existing U.S. GOM monitoring programs for advancing EBFM.
Creatine kinase (CK; EC 2.7.3.2) isoenzymes play prominent roles in energy metabolism. Nuclear genes encode three known CK subunits: cytoplasmic muscle (MCK), cytoplasmic brain (BCK), and ...mitochondrial (MtCK). We have isolated the gene and cDNA encoding human placental MtCK. By using a dog heart MCK cDNA-derived probe, the 7.0-kb EcoRI fragment from one cross-hybridizing genomic clone was isolated and its complete nucleotide sequence determined. A region of this clone encoded predicted amino acid sequence identical to residues 15–26 of the human heart MtCK NH2-terminal protein sequence. The human placental MtCK cDNA was isolated by hybridization to a genomic fragment encoding this region. The human placental MtCK gene contains 9 exons encoding 416 amino acids, including a 38-amino acid transit peptide, presumably essential for mitochondrial import. Residues 1–14 of human placental MtCK cDNA-derived NH2-terminal sequence differ from the human heart MtCK protein sequence, suggesting that tissue-specific MtCK mRNAs are derived from multiple MtCK genes. RNA blot analysis demonstrated abundant MtCK mRNA in adult human ventricle and skeletal muscle, low amounts in placenta and small intestine, and a dramatic increase during in vitro differentiation induced by serum-deprivation in the non-fusing mouse smooth muscle cell line, BC3H1. These findings demonstrate coordinate regulation of MtCK and cytosolic CK gene expression and support the phosphocreatine shuttle hypothesis.
The export of nitrogen from urban catchments is a global problem, and denitrifying bacteria in stream ecosystems are critical for reducing in-stream N. However, the environmental factors that control ...the composition of denitrifying communities in streams are not well understood. We determined whether denitrifying community composition in sediments of nine streams on the eastern fringe of Melbourne, Australia was correlated with two measures of catchment urban impact: effective imperviousness (El, the proportion of a catchment covered by impervious surfaces with direct connection to streams) or septic tank density (which affects stream water chemistry, particularly stream N concentrations). Denitrifying community structure was examined by comparing terminal restriction fragment length polymorphisms of nosZ genes in the sediments, as the nosZ gene codes for nitrous oxide reductase, the last step in the denitrification pathway. We also determined the chemical and physical characteristics of the streams that were best correlated with denitrifying community composition. El was strongly correlated with community composition and sediment physical and chemical properties, while septic tank density was not. Sites with high El were sandier, with less fine sediment and lower organic carbon content, higher sediment cations (calcium, sodium and magnesium) and water filterable reactive phosphorus concentrations. These were also the best small-scale environmental variables that explained denitrifying community composition. Among our study streams, which differed in the degree of urban stormwater impact, sediment grain size and carbon content are the most likely drivers of change in community composition. Denitrifying community composition is another in a long list of ecological indicators that suggest the profound degradation of streams is caused by urban stormwater runoff. While the relationships between denitrifying community composition and denitrification rates are yet to be unequivocally established, landscape-scale indices of environmental impact such as El may prove to be useful indicators of change in microbial communities.
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