DNA crosslinking and biological activity of a hairpin polyamide–chlorambucil conjugate

A prototype of a novel class of DNA alkylating agents, which combines the DNA crosslinking moiety chlorambucil (Chl) with a sequence-selective hairpin pyrrole-imidazole polyamide ImPy-beta-ImPy-gamma-ImPy-beta-Dp (polyamide 1), was evaluated for its ability to damage DNA and induce biological responses. Polyamide 1-Chl conjugate (1-Chl) alkylates and interstrand crosslinks DNA in cell-free systems. The alkylation occurs predominantly at 5'-AGCTGCA-3' sequence, which represents the polyamide binding site. Conjugate-induced lesions were first detected on DNA treated for 1 h with 0.1 muM 1-Chl, indicating that the conjugate is at least 100-fold more potent than Chl. Prolonged incubation allowed for DNA damage detection even at 0.01 muM concentration. Treatment with 1-Chl decreased DNA template activity in simian virus 40 (SV40) in vitro replication assays. 1-Chl inhibited mammalian cell growth, genomic DNA replication and cell cycle progression, and arrested cells in the G(2)/M phase. Moreover, cellular effects were observed at 1-Chl concentrations similar to those needed for DNA damage in cell-free systems. Neither of the parent compounds, unconjugated Chl or polyamide 1, demonstrated any cellular activity in the same concentration range. The conjugate molecule 1-Chl possesses the sequence-selectivity of a polyamide and the enhanced DNA reactivity of Chl

Fmoc solid phase synthesis of polyamides containing pyrrole and imidazole amino acids

Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids are synthetic ligands that have an affinity and specificity for DNA comparable to those of many naturally occurring DNA binding proteins. A machine-assisted Fmoc solid phase synthesis of polyamides has been optimized to afford high stepwise coupling yields (>99%). Two monomer building blocks, Fmoc-Py acid and Fmoc-Im acid, were prepared in multigram scale. Cleavage by aminolysis followed by HPLC purification affords up to 200 mg quantities of polyamide with purities and yields greater than or equal to those reported using Boc chemistry. A broader set of reaction conditions will increase the number and complexity of minor groove binding polyamides which may be prepared and help ensure compatibility with many commercially available peptide synthesizers

Archeological and Bioarcheological Investigations at Campbell’s Bayou Cemetery Galveston County, Texas

This report documents the removal of individuals buried within Campbell’s Bayou Cemetery (41GV171) to avoid potential impact to the remains during implementation of remediation activities at the Malone Service Company Superfund Site (Site) in Texas City, TX. An oil recovery and waste processing facility had operated at the Site for more than 30 years, ending in the mid1990s. The facility had stored, processed, and disposed of industrial solid wastes and hazardous wastes. In July 2012, a group of companies known as the Malone Cooperating Parties (MCP) entered into a Consent Decree with the U.S. Government, the U.S. Environmental Protection Agency, and the State of Texas to implement a remedial design and remedial action at the Site. It was determined that if the remains in the cemetery were not relocated, there was the potential that remediation activities could impact the cemetery. Archival research, review of historic maps and aerial photographs, and reconnaissance survey revealed the extent of potential remains at the cemetery, and, given the location of the cemetery and the scope of the planned remediation activities, it was deemed impractical for the environmental remediation contractors to work around the cemetery. (41GV171). The MCP consulted with EPA, Campbell family descendants, the Galveston County Historical Commission, and the Texas Historical Commission and developed a plan to relocate the remains to a perpetual care cemetery in accordance with Texas state law and associated rules and procedures. In accordance with Texas Health and Safety Code §711.004, the landowner Land Navigator, Ltd., on behalf of the MCP, petitioned the Galveston County Judicial District Court for removal of the dedication of the cemetery and the transfer of the human remains to the perpetual care cemetery operated by Forest Park East Funeral Home and Cemetery (FPE), 21620 Gulf Freeway, Webster, TX 77598. On February 11, 2014, Land Navigator was granted a Summary Judgment allowing Land Navigator to disinter and relocate the remains to FPE. Versar, Inc. (formerly Geo-Marine, Inc.), on behalf of the MCP, provided all archeological and human osteological expertise for the disinterment and analysis of the human remains. Disinterment permits from the State Registrar of the Vital Statistics Unit of the Department of State Health Services, as required by Texas Administrative Code, Title 13, Chapter 22 (Texas Historical Commission, Cemeteries), were obtained for each burial. The disinterment excavations at Campbell’s Bayou Cemetery revealed 34 burials from which 35 individuals were excavated. No graves were marked by headstones. It is the professional judgment of Versar that, of the 35 individual sets of remains identified, 11 were determined to be adults (5 male and 3 female; 3 of indeterminate sex), and 24 were determined to be children. The majority of children at Campbell’s Bayou Cemetery (n=18) are under 5 years of age and six are premature infants aged 30–40 weeks. Burials could not be associated conclusively with any individuals identified by the descendants; however, the combination of bioarcheological analysis, coffin hardware analysis, census data, and descendant identifications resulted in a list of individuals that may have been interred in certain graves. Some of the interments include James and Mary Campbell, Charlie Meyers, Benjamin Ninnie Dick, Phoebe Rutlage, and Shelby McNeil, Jr. Children were difficult to identify; however, there is good potential the graves of Frank Campbell, Mary Jane Campbell, Charles Munson, and Grace Dick were identified. Data are conclusive that the children Levi and Joseph (Joe) Parr were both interred together in Burial 6, the concrete crypt with brick covering. Grace Dick was the last individual interred at the cemetery in 1904

Improved nuclear localization of DNA-binding polyamides

Regulation of endogenous genes by DNA-binding polyamides requires effective nuclear localization. Previous work employing confocal microscopy to study uptake of fluorophore-labeled polyamides has demonstrated the difficulty of predicting a priori the nuclear uptake of a given polyamide. The data suggest that dye identity influences uptake sufficiently such that a dye-conjugate cannot be used as a proxy for unlabeled analogs. Polyamides capable of nuclear localization unaided by fluorescent dyes are desirable due to size and other limitations of fluorophores. Recently, a polyamide-fluorescein conjugate targeted to the hypoxia response element (HRE) was found to inhibit vascular endothelial growth factor (VEGF) expression in cultured HeLa cells. The current study uses inhibition of VEGF expression as a biological read-out for effective nuclear localization of HRE-targeted polyamides. We synthesized a focused library of non-fluorescent, HRE-targeted polyamides in which the C-terminus ‘tail’ has been systematically varied. Members of this library bind the HRE with affinities comparable or superior to that of the fluorescein-labeled analog. Although most library members demonstrate modest or no biological activity, two non-fluorescent polyamides are reported with activity rivaling that of the previously reported fluorescein-labeled polyamide. We also show evidence that promoter occupancy by HIF-1, the transcription factor that binds the HRE, is inhibited by HRE-targeted polyamides

Metabolic Biomarker Discovery for Risk of Peripheral Artery Disease Compared With Coronary Artery Disease : Lipoprotein and Metabolite Profiling of 31 657 Individuals From 5 Prospective Cohorts

Background Peripheral artery disease (PAD) and coronary artery disease (CAD) represent atherosclerosis in different vascular beds. We used detailed metabolic biomarker profiling to identify common and discordant biomarkers and clarify pathophysiological differences for these vascular diseases. Methods and Results We used 5 prospective cohorts from Finnish population (FINRISK 1997, 2002, 2007, and 2012, and Health 2000; n=31 657; median follow-up time of 14 years) to estimate associations between >200 metabolic biomarkers and incident PAD and CAD. Metabolic biomarkers were measured with nuclear magnetic resonance, and disease events were obtained from nationwide hospital records. During the follow-up, 498 incident PAD and 2073 incident CAD events occurred. In age- and sex-adjusted Cox models, apolipoproteins and cholesterol measures were robustly associated with incident CAD (eg, hazard ratio [HR] per SD for higher apolipoprotein B/A-1 ratio, 1.30; 95% CI, 1.25-1.36), but not with incident PAD (HR per SD for higher apolipoprotein B/A-1 ratio, 1.04; 95% CI, 0.95-1.14; P-heterogeneity0.05). Lower proportion of polyunsaturated fatty acids relative to total fatty acids, and higher concentrations of monounsaturated fatty acids, glycolysis-related metabolites, and inflammatory protein markers were strongly associated with incident PAD, and many of these associations were stronger for PAD than for CAD (P-heterogeneityPeer reviewe

Metabolic Biomarker Discovery for Risk of Peripheral Artery Disease Compared With Coronary Artery Disease : Lipoprotein and Metabolite Profiling of 31 657 Individuals From 5 Prospective Cohorts

Background Peripheral artery disease (PAD) and coronary artery disease (CAD) represent atherosclerosis in different vascular beds. We used detailed metabolic biomarker profiling to identify common and discordant biomarkers and clarify pathophysiological differences for these vascular diseases. Methods and Results We used 5 prospective cohorts from Finnish population (FINRISK 1997, 2002, 2007, and 2012, and Health 2000; n=31 657; median follow-up time of 14 years) to estimate associations between >200 metabolic biomarkers and incident PAD and CAD. Metabolic biomarkers were measured with nuclear magnetic resonance, and disease events were obtained from nationwide hospital records. During the follow-up, 498 incident PAD and 2073 incident CAD events occurred. In age- and sex-adjusted Cox models, apolipoproteins and cholesterol measures were robustly associated with incident CAD (eg, hazard ratio [HR] per SD for higher apolipoprotein B/A-1 ratio, 1.30; 95% CI, 1.25-1.36), but not with incident PAD (HR per SD for higher apolipoprotein B/A-1 ratio, 1.04; 95% CI, 0.95-1.14; P-heterogeneity0.05). Lower proportion of polyunsaturated fatty acids relative to total fatty acids, and higher concentrations of monounsaturated fatty acids, glycolysis-related metabolites, and inflammatory protein markers were strongly associated with incident PAD, and many of these associations were stronger for PAD than for CAD (P-heterogeneityPeer reviewe

PENGARUH KUALITAS PRODUK DAN HARGA TERHADAP KEPUASAN KONSUMEN PADA PT. MARGA TIRTA KENCANA (Survei Pada Penghuni Perumahan Permata Buah Batu 1)

ABSTRAK Penelitian ini bertujuan untuk mengetahui pengaruh Kualitas Produk dan Harga terhadap Kepuasan Konsumen pada PT. Marga Tirta Kencana (Survei Pada Perumahan Permata Buah Batu 1 Bandung). Rumusan masalah dalam penelitian ini adalah bagaimana tanggapan konsumen mengenai kualitas produk yang ditawarkan, bagaimana tanggapan konsumen mengenai harga yang ditawarkan, bagaimana kepuasan konsumen, dan seberapa besar pengaruh kualitas produk dan harga terhadap kepuasan konsumen di PT. Marga Tirta Kencana Bandung secara simultan dan parsial. Metode yang digunakan penulis dalam penelitian ini adalah penelitian deskriptif dan verifikatif dengan tehnik pengumpulan data dengan interview (wawancara), kuesioner (angket) dan observasi (pengamatan). Adapun ukuran populasinya 619 orang dengan sampel 87 orang. Sedangkan tehnik sampling yang digunakan untuk menghitung besarnya ukuran sampel dalam non-probability sampling. Sesuai dengan perhitungan statistik, Kualitas Produk berada dalam kategori baik dan Harga berada dalam kategori baik terhadap Kepuasan Konsumen pada PT. Marga Tirta Kencana yang berada dalam kategori puas. Kata Kunci : Kualitas Produk, Harga, Kepuasan Konsume

Fmoc solid phase synthesis of polyamides containing pyrrole and imidazole amino acids

Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids are synthetic ligands that have an affinity and specificity for DNA comparable to those of many naturally occurring DNA binding proteins. A machine-assisted Fmoc solid phase synthesis of polyamides has been optimized to afford high stepwise coupling yields (>99%). Two monomer building blocks, Fmoc-Py acid and Fmoc-Im acid, were prepared in multigram scale. Cleavage by aminolysis followed by HPLC purification affords up to 200 mg quantities of polyamide with purities and yields greater than or equal to those reported using Boc chemistry. A broader set of reaction conditions will increase the number and complexity of minor groove binding polyamides which may be prepared and help ensure compatibility with many commercially available peptide synthesizers