Genome analysis with gene-indexing databases.

The recent release of the draft sequence and the eventual completion of the human genome present the scientific community with a rich source of data to mine. Yet, these data are content poor in the absence of additional correlative information. Expressed sequence tag (EST) datasets and their associated gene indices have existed for many years, and represent the first attempt at understanding the complexity of the genome. These datasets remain extremely important as information sources and, in particular, as tools for analyzing the completed genomes. Here, we discuss the nature of ESTs and their associated tools and gene-indexing databases. In particular, we will compare three EST gene indices (UNIGENE, Merck Gene Index Version 2.0 and Doubletwist CAT), discuss how these gene indices are applied for both genome analysis and drug discovery, and demonstrate their importance as a complementary dataset to the annotated human genome.

The human inward rectifier K(+) channel subunit kir5.1 (KCNJ16) maps to chromosome 17q25 and is expressed in kidney and pancreas.

A novel human Kir5.1 (inward rectifier K+ channel subunit, gene name KCNJ16) was identified through database searches. This human KCNJ16 was mapped to chromosome 17q25. The full-length cDNA was identified and its genomic structure was determined. Tissue distribution studies showed that human KCNJ16 is significantly expressed in human kidney, pancreas and thyroid gland. In situ hybridization revealed expression in convoluted tubule cells of kidney and in the acinar and ductal cells of pancreas. These suggest that human Kir5.1 may be involved in the regulation of fluid and pH balance, thus making it a potential therapeutic target for hypertension, renal failure, or pancreatic disease.

Storing biological sequence databases in relational form.

SUMMARY: We have created a set of applications using Perl and Java in combination with XML technology to install biological sequence databases into an Oracle RDBMS. An easy-to-use interface using Java has been created for database query and other tools developed to integrate with our in-house bioinformatics applications. AVAILIBILITY: The database schema, DTD file, and source codes are available from the authors via email. CONTACT: guochun_ xie@merck. com

Adenosine as an adjunct to thrombolytic therapy for acute myocardial infarction: results of a multicenter, randomized, placebo-controlled trial: the Acute Myocardial Infarction STudy of ADenosine (AMISTAD) trial.

OBJECTIVES: The Acute Myocardial Infarction STudy of ADenosine (AMISTAD) trial was designed to test the hypothesis that adenosine as an adjunct to thrombolysis would reduce myocardial infarct size. BACKGROUND: Reperfusion therapy for acute myocardial infarction (MI) has been shown to reduce mortality, but reperfusion itself also may have deleterious effects. METHODS: The AMISTAD trial was a prospective, open-label trial of thrombolysis with randomization to adenosine or placebo in 236 patients within 6 h of infarction onset. The primary end point was infarct size as determined by Tc-99 m sestamibi single-photon emission computed tomography (SPECT) imaging 6+/-1 days after enrollment based on multivariable regression modeling to adjust for covariates. Secondary end points were myocardial salvage index and a composite of in-hospital clinical outcomes (death, reinfarction, shock, congestive heart failure or stroke). RESULTS: In all, 236 patients were enrolled. Final infarct size was assessed in 197 (83%) patients. There was a 33% relative reduction in infarct size (p = 0.03) with adenosine. There was a 67% relative reduction in infarct size in patients with anterior infarction (15% in the adenosine group vs. 45.5% in the placebo group) but no reduction in patients with infarcts located elsewhere (11.5% for both groups). Patients randomized to adenosine tended to reach the composite clinical end point more often than those assigned to placebo (22% vs. 16%; odds ratio, 1.43; 95% confidence interval, 0.71 to 2.89). CONCLUSIONS: Many agents thought to attenuate reperfusion injury have been unsuccessful in clinical investigation. In this study, adenosine resulted in a significant reduction in infarct size. These data support the need for a large clinical outcome trial.

MULTICLUSTAL: a systematic method for surveying Clustal W alignment parameters.

MULTICLUSTAL is a Perl script designed to automate the process of alignment parameter choice for Clustal W with the goal of generating high quality multiple sequence alignments.

The Merck Gene Index browser: an extensible data integration system for gene finding, gene characterization and EST data mining.

MOTIVATION: To make effective use of the vast amounts of expressed sequence tag (EST) sequence data generated by the Merck-sponsored EST project and other similar efforts, sequences must be organized into gene classes, and scientists must be able to 'mine' the gene class data in the context of related genomic data. RESULTS: This paper presents the Merck Gene Index browser, an easily extensible, World Wide Web-based system for mining the Merck Gene Index (MGI) and related genomic data. The MGI is a non-redundant set of clones and sequences, each representing a distinct gene, constructed from all high-quality 3' EST sequences generated by the Merck-sponsored EST project. The MGI browser integrates data from a variety of sources and storage formats, both local and remote, using an eclectic integration strategy, including a federation of relational databases, a local data warehouse and simple hypertext links. Data currently integrated include: LENS cDNA clone and EST data, dbEST protein and non-EST nucleic acid similarity data, WashU sequence chromatograms. Entrez sequence and Medline entries, and UniGene gene clusters. Flatfile sequence data are accessed using the Bioapps server, an internally developed client-server system that supports generic sequence analysis applications. Browser data are retrieved and formatted by means of the Bioinformatics Data Integration Toolkit (B-DIT), a new suite of Perl routines.

Toward the development of a gene index to the human genome: an assessment of the nature of high-throughput EST sequence data.

A rigorous analysis of the Merck-sponsored EST data with respect to known gene sequences increases the utility of the data set and helps refine methods for building a gene index. A highly curated human transcript data base was used as a reference data set of known genes. A detailed analysis of EST sequences derived from known genes was performed to assess the accuracy of EST sequence annotation. The EST data was screened to remove low-quality and low-complexity sequences. A set of high-quality ESTs similar to the transcript data base was identified using BLAST; this subset of ESTs was compared with the set of known genes using the Smith-Waterman algorithm. Error rates of several types were assessed based on a flexible match criterion defining sequence identity. The rate of lane-tracking errors is very low, approximately 0.5%. Insert size data is accurate within approximately 20%. Reversed clone and internal priming error rates are approximately 5% and 2.5%, respectively, contributing to the incorrect identification of reads as 3' ends of genes. Follow-up investigation reveals that a significant number of clones, miscategorized as reversed, represent overlapping genes on the opposite strand of entries in the transcript data base. Relevance of these results to the creation of a high-quality index to the human genome capable of supporting diverse genomic investigations is discussed.

PROFILER: a tool for automatic searching of internally maintained databases.

A new application program, the BIOINFORMATICS PROFILER, is described which simplifies the analysis of new genome sequence information appearing on a daily basis. Control tasks are defined through an intuitive graphical user interface and are executed at user defined nightly intervals. Electronic mail is sent to indicate that search results have been found. All task output is presented to users in the form of hypertext (HTML), allowing easy browsing. Currently supported tasks include BLAST and FastA sequence searching, keyword based searching of network news articles and WAIS databases, examination of GenBank sequence entries using regular expressions and boolean operations and protein sequence motif searching.

Mutagenicity and antimutagenicity testing of six chemicals associated with the pungent properties of specific spices as revealed by the Ames Salmonella/microsomal assay.

Three compounds, capsaicin, thymol and borneol, were initially screened for mutagenic activity using Salmonella typhimurium strains TA97, TA98 and TA100, with and without S9 metabolic activation, and 20 min standard preincubation time. Three other compounds, allyl isothiocyanate, eugenol and cinnamaldehyde, were screened for mutagenic activity as above, but with a prolonged, nonstandard preincubation time of up to 120 minutes. All six test compounds used in the assays are associated with the pungent properties of some specific spices in which the test compounds can be found to exist naturally. The first objective of this study was to observe if mutagenic activity can be correlated to the pungent properties of these six test compounds. However, due to toxicity and the observation that only capsaicin was mutagenic, using strain TA100 in the presence of S9 metabolic activation, it was not possible to deduce any relationship between mutagenicity and the test chemicals' pungent properties. Naturally occurring capsaicin, found in the spice Capsicum annum, was detected and quantified using thin layer and gas chromatographic techniques. The final objective was to detect the presence of antimutagenic factor(s) in C. annum that would suppress the mutagenicity of capsaicin. When the mutagenic capsaicin and 2-aminoanthracene were assayed in the presence of C. annum acetone extract, using strain TA100 with S9 metabolic activation, the mutagenic response of both the mutagens were reduced by approximately 50%. Assaying capsaicin and 2-aminoanthracene in the presence of chlorophyll, the mutagenic response of the two mutagens was reduced by less than 40%. From this observation it was inferred that chlorophyll can successfully suppress the mutagenicity activities of capsaicin and 2-aminoanthracene, together with other antimutagenic factors that were present in the acetone extract of C. annum.

Direct database searching with MALDI-PSD spectra of peptides.

The analysis of matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) mass spectra of peptides by using the cross-correlation method for database searching is illustrated. MALDI-PSD mass spectra are shown to contain sufficient fragmentation information to uniquely identify the correct amino acid sequence from large protein databases (approximately 160,000 entries). A search employing the MALDI-PSD mass spectrum of a phosphorylated peptide that correctly identifies the amino acid sequence and the site of phosphorylation is also illustrated.

NMR View: A computer program for the visualization and analysis of NMR data.

NMR View is a computer program designed for the visualization and analysis of NMR data. It allows the user to interact with a practically unlimited number of 2D, 3D and 4D NMR data files. Any number of spectral windows can be displayed on the screen in any size and location. Automatic peak picking and facilitated peak analysis features are included to aid in the assignment of complex NMR spectra. NMR View provides structure analysis features and data transfer to and from structure generation programs, allowing for a tight coupling between spectral analysis and structure generation. Visual correlation between structures and spectra can be done with the Molecular Data Viewer, a molecular graphics program with bidirectional communication to NMR View. The user interface can be customized and a command language is provided to allow for the automation of various tasks.

Chemically-induced histone modification as a predictor of carcinogenicity.

The interaction between carcinogens and DNA is believed to initiate neoplastic transformation, but evidence suggests that epigenetic mechanisms may also be of importance. Because the histone proteins have important roles in chromatin structure and cellular function, they provide a reasonably well understood epigenetically-based system for the detection of carcinogens. In this study, human foreskin fibroblastic cells were exposed to one of several mutagens and/or carcinogens for 3, 12, or 24 h to determine if induced histone modification may be a means of predicting chemical carcinogenicity. Butyric acid (5 mM), known to result in acetylation of histones H3 and H4, and 12-O-tetradecanoylphorbol-13-acetate (3 microM), known to result in phosphorylated histone H1, were tested initially. Electrophoresis of the histone fractions was capable of resolving multiple forms of histones H1, H3, and H4. Propane sultone (0.1 mM) induced a broadening of the H2A and H2B bands after a 24 h exposure and carbon tetrachloride (1 mM) induced the formation of new histone forms in the H1 fraction after 24 h and in the H3 fraction after 3 h. Experimental variability limited the statistically significant modifications to carbon tetrachloride and propane sultone, two known carcinogens, where new forms of modified histone were detected. Therefore, the histone modification assay, with further experimentation, may be an alternate method of detecting carcinogens, especially when conventional genotoxic tests prove unreliable.

The effect of pravastatin on plasma lipoprotein and apolipoprotein levels in primary hypercholesterolemia. The Southeastern Michigan Collaborative Group.

Pravastatin is a metabolic product of mevastatin and a potent inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. It was investigated for its cholesterol-lowering properties in a double-blind, placebo-controlled, multicenter study of 82 patients with primary hypercholesterolemia. Following a 6- to 8-week dietary lead-in period, patients were randomized to twice-daily placebo or active drug for 16 weeks. Patients receiving 10 mg of pravastatin twice a day for 8 weeks experienced mean total cholesterol and low-density lipoprotein cholesterol (LDL-C) level reductions of 20% (6.85 vs 5.48 mmol/L [265 vs 212 mg/dL]) and 28% (5.17 vs 3.75 mmol/L [200 vs 145 mg/dL]), respectively. At 20 mg twice a day for an additional 8 weeks, pravastatin reduced plasma total cholesterol, LDL-C, and apolipoprotein B-100 levels by 23% (6.85 vs 5.30 mmol/L [265 vs 205 mg/dL]), 31% (5.17 vs 3.59 mmol/L [200 vs 139 mg/dL]), and 23% (118 vs 91 mg/dL), respectively. High-density lipoprotein cholesterol (HDL-C), HDLb-C, HDLb-C, and apolipoprotein A-I plasma concentrations increased by 11%, 60%, 7%, and 10%. Plasma triglyceride concentrations decreased in both the pravastatin- and placebo-treated patients. Pravastatin was generally well tolerated and an effective agent for the treatment of primary hypercholesterolemia.

2,3,7,8 Tetrachlorodibenzodioxin in fish from the Pigeon River of eastern Tennessee, USA: its toxicity and mutagenicity as revealed by the Ames Salmonella assay.

Levels of 2,3,7,8 tetrachlorodibenzodioxin (TCDD) were determined in both striated muscle (fillets) and whole body extracts of fish specimens harvested during a two-year period (1987-1989) from the Pigeon River (between Hartford and Newport) of Eastern Tennessee (USA). Whole body (wet weight) fish extract levels as high as 117 micrograms/kg body weight and composite fish fillet (wet weight) extract levels as high as 87 micrograms/kg fillet weight were observed. Pure TCDD was found to be highly toxic to the Salmonella typhimurium strains TA97, TA98, and TA100 at dosages which exceeded 825 ng TCDD/ml in the top agar of the Ames Salmonella assay. An 825 ng/ml TCDD dosage was not mutagenic to any of the tested Salmonella strains, either with or without metabolic activation (S9 mix). However, when acidic fish extracts from the Pigeon River were tested for mutagenicity, most of the fish extracts were mutagenic to Salmonella strains TA97, TA98, and TA100. These mutagenic extracts also demonstrated mutagenic dose-response curves. Other chemicals within the extracts as well as synergistic effects may account for the mutagenicity.

Fate of mutagenic activity during conventional treatment of municipal wastewater sludge.

The mutagenic activity of wastewater was followed during conventional activated sludge treatment at a municipal plant. Raw wastewater was initially screened for mutagenic potential, using the AmesSalmonella/mammalian microsomal test and employer tester strains. The combined raw wastewater produced dose-related mutagenic responses in the presence of S9 metabolic activation. Raw wastewater from domestic sources alone was not mutagenic. Mutagenic activity was observed throughout the treatment process. Activated sludge prior to chlorination contained the highest specific mutagenic activity. Chlorination decreased the specific mutagenic activity at pH 11. Mutagenic activity in municipal wastewater containing industrial discharges is not removed by conventional treatment processes and can be enhanced by activated sludge treatment.

The relationship between apolipoprotein B-100 and low-density lipoprotein cholesterol after treatment with an HMG CoA reductase inhibitor.

Measurement of apolipoproteins AI and B-100 has been shown to provide at least equivalent information to measurement of lipoprotein cholesterol levels for evaluating risk of cardiovascular disease. In the present study, the authors examined the relationship between APO B-100 and low-density lipoprotein (LDL) cholesterol in a hypercholesterolemic population that was treated with 16 weeks of hydroxymethylglutaryl CoA reductase inhibitor therapy to lower plasma cholesterol levels. The average decrease in APO B-100 was -0.27 g/L (23% of baseline), which was similar to the decrease in LDL cholesterol, -1.6 mmol/L (-0.61 g/L) (30% of baseline). Correlation data (r = 0.902) indicated that the information provided by the two parameters corresponded in individual cases. Apolipoproteins assayed on automated equipment by kit methods are simpler, more straightforward, and provide more reproducible results than measurement of lipoprotein cholesterols. The authors conclude that, in addition to being more reliable for appraising risk of coronary artery disease, measurement of apolipoproteins may be equally useful for monitoring lipoprotein-lowering therapy.

Dopamine and dobutamine induce hypokalemia in anesthetized dogs.

Epinephrine-induced hypokalemia appears to be mediated by beta 2-agonist activation of Na+/K+ ATPase. To determine whether dopamine and dobutamine induce hypokalemia, eight adult mongrel dogs were anesthetized and studied in random crossover fashion. Potassium [K+] was measured with an ion-selective microelectrode, and central hemodynamics were measured continuously. After stabilization, dopamine and dobutamine were infused at doses of 2, 4, 8, and 20 micrograms/kg/min (15-min increments/dose), and 0.9% NaCl was infused at equivalent volumes, with a 1-h washout between treatments. The mean change in [K+] at each infusion rate was compared between treatments among dogs with an adequate hemodynamic response. Among dopamine responders (n = 5), [K+] decreased from 3.74 +/- 0.42 mEq/L at baseline to 3.63 +/- 0.51 at 2 micrograms/kg/min (p less than 0.02) and was not significantly different at higher doses. Among dobutamine responders (n = 7), [K+] decreased from 3.52 +/- 0.74 at baseline to 3.31 +/- 0.87 at 8 micrograms/kg/min (p less than 0.02) and 3.25 +/- 0.86 at 20 micrograms/kg/min (p less than 0.02), and was not significantly different at lower doses. We conclude that dopamine and dobutamine induce significant hypokalemia, consistent with their adrenergic agonist activity, and this may be related to the known arrhythmogenicity of these agents.

The incidence, magnitude, and time course of the amiodarone-warfarin interaction.

Eight patients concurrently treated with amiodarone and warfarin sodium were studied to characterize the interaction between these drugs. All fulfilled the following criteria: (1) stable and therapeutic prothrombin time (PT) at baseline, defined as at least two consecutive PTs obtained within two weeks before beginning amiodarone therapy that varied by less than or equal to 15%; (2) no warfarin dosage adjustment in the two weeks prior to amiodarone therapy; (3) no other drugs given that alter coagulation study results; and (4) follow-up PTs obtained 1, 2, 4, and 8 weeks after initiation of amiodarone treatment. A clinically significant change in PT was defined as greater than 15%. Mean baseline PT was 19.8 s for patients receiving 5.99 mg/d of warfarin sodium. Patients had a mean maximum increase in PT of 44% (range, 22% to 108%), which occurred during the first two weeks. In six patients, the PT returned to within 15% of baseline by week 4 or 8, and the daily warfarin requirement had decreased by 35% (range, 25% to 50%). Two patients had PTs varying by greater than 15% from baseline at week 8 despite a 33% reduction in warfarin dosage in each case. No patient in this series encountered complications of anticoagulant therapy, perhaps due to early recognition and dosage reduction. Although the mechanism remains unclear, our study indicates that amiodarone potentiation of warfarin effects occurs in all patients, occurs in the first two weeks of amiodarone therapy, variably increases PT by 22% to 108%, and lowers the warfarin requirement by 25% to 50%. We recommend a 25% prophylactic reduction of warfarin dosage and weekly measurements of PT for one month when amiodarone therapy is initiated.