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The complete amino acid sequence of subunit d of rat liver mitochondrial H(+)-ATP synthase.

Subunit d of H(+)-ATP synthase from rat liver mitochondria was isolated from the purified enzyme by reverse-phase high performance liquid chromatography. The partial amino acid sequence of the subunit was determined by automated Edman degradation of the peptide fragments. The nucleotide sequence of subunit d of rat liver H(+)-ATP synthase was determined from a recombinant cDNA clone isolated by screening a rat hepatoma cell line H4TG cDNA library with a probe DNA. The sequence was composed of 581 nucleotides including a coding region for the import precursor of subunit d and noncoding regions on the 5'- and 3'- sides. The possible precursor of subunit d and its mature polypeptide deduced from the open reading frame consisted of 161 and 160 amino acid residues with molecular weights of 18,763 and 18,631, respectively. Subunit d is a hydrophilic protein with an isoelectric point of 6.19. The sequence of the rat subunit d is highly homologous with that of subunit d of bovine heart and slightly similar to that of the subunit d of the yeast mitochondria. However, it had no homology with the sequence of any of the subunits of bacterial or chloroplast H(+)-ATP synthase.

Amino Acid Sequence↗

High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays.

A method was developed to rapidly identify high-affinity human antibodies from phage display library selection outputs. It combines high-throughput Fab fragment expression and purification with surface plasmon resonance (SPR) microarrays to determine kinetic constants (kon and koff) for 96 different Fab fragments in a single experiment. Fabs against human tissue kallikrein 1 (hK1, KLK1 gene product) were discovered by phage display, expressed in Escherichia coli in batches of 96, and purified using protein A PhyTip columns. Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest affinity Fabs discovered had dissociation constants of less than 1 nM. The described SPR method was also used to categorize Fabs according to their ability to recognize an apparent active site epitope. The ability to rapidly determine the affinities of hundreds of antibodies significantly accelerates the discovery of high-affinity antibody leads.

Automation↗

A small interfering RNA screen for modulators of tumor cell motility identifies MAP4K4 as a promigratory kinase.

Cell motility is a complex biological process, involved in development, inflammation, homeostasis, and pathological processes such as the invasion and metastatic spread of cancer. Here, we describe a genomic screen designed to identify inhibitors of cell migration. A library of 10,996 small interfering RNAs (targeting 5,234 human genes) was screened for their ability to block the migration of a highly motile ovarian carcinoma cell line, SKOV-3, by using a 384-well wound-healing assay coupled with automated microscopy and wound quantification. Two or more small interfering RNAs against four genes, CDK7, DYRK1B, MAP4K4 (NIK/HGK) (MAP4K4, mitogen-activated protein 4 kinase 4), and SCCA-1 (SerpinB3), potently blocked the migration of SKOV-3 cells, concordant with reduced transcript levels. Further studies of the promigratory role of MAP4K4 showed that the knockdown of this transcript inhibited the migration of multiple carcinoma cell lines, indicating a broad role in cell motility and potently suppressed the invasion of SKOV-3 cells in vitro. The effect of MAP4K4 on cellular migration was found to be mediated through c-Jun N-terminal kinase, independent of AP1 activation and downstream transcription. Accordingly, small molecule inhibition of c-Jun N-terminal kinase suppressed SKOV-3 cell migration, underscoring the potential therapeutic utility of mitogen-activated protein kinase pathway inhibition in cancer progression.

Base Sequence↗

Method enabling fast partial sequencing of cDNA clones.

Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine the needed length for finding a unique DNA sequence, 100 sequence tags from human were retrieved from the database and different lengths from each sequence were randomly analyzed. An homology search based on 20 and 30 nucleotides produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was employed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences shared homology with one gene at different positions. Two of the 64 clones had unique sequences. The search results from pyrosequencing were in 100% agreement with conventional DNA sequencing methods. The possibility of using a fully automated pyrosequencer machine for future high-throughput tag sequencing is discussed.

Base Sequence↗

Peptide backbone reconstruction using dead-end elimination and a knowledge-based forcefield.

A novel, yet simple and automated, protocol for reconstruction of complete peptide backbones from C(alpha) coordinates only is described, validated, and benchmarked. The described method collates a set of possible backbone conformations for each set of residue triads from a structural library derived from the PDB. The optimal permutation of these three residue segments of backbone conformations is determined using the dead-end elimination (DEE) algorithm. Putative conformations are evaluated using a pairwise-additive knowledge-based forcefield term and a fragment overlap term. The protocol described in this report is able to restore the full backbone coordinates to within 0.2-0.6 A of the actual crystal structure from C(alpha) coordinates only. In addition, it is insensitive to errors in the input C(alpha) coordinates with RMSDs of 3.0 A, and this is illustrated through application to deliberately distorted C(alpha) traces. The entire process, as described, is rapid, requiring of the order of a few minutes for a typical protein on a typical desktop PC. Approximations enable this to be reduced to a few seconds, although this is at the expense of prediction accuracy. This compares very favorably to previously published methods, being sufficiently fast for general use and being one of the most accurate methods. Because the method is not restricted to the reconstruction from only C(alpha) coordinates, reconstruction based on C(beta) coordinates is also demonstrated.

Hydrogen Bonding↗

Characterization of bovine prothrombin mRNA and its translation product.

Prothrombin mRNA has been enriched 20-60-fold by using specific immunoadsorption of bovine liver polysomes. The enriched mRNA was translated in a cell-free protein synthesizing system derived from rabbit reticulocytes, and the radiolabeled translation product was isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radiolabeled prothrombin synthesized in the cell-free system was then subjected to automated Edman degradation and shown to contain a leader sequence of at least 30 residues that was rich in leucine, phenylalanine, and alanine. In order to fully characterize the leader sequence for prothrombin, a bovine liver cDNA library was constructed containing DNA inserts of over 1000 base pairs. Two cDNA clones coding for bovine prothrombin were isolated from the library and their nucleotide sequences determined. A leader sequence of 43 amino acids was predicted from the sequence of the cDNA, and the first 30 residues were in agreement with the partial sequence obtained by the cell-free protein synthesizing system. From the amino acid sequence of the leader sequence, it is proposed that bovine prothrombin is synthesized with a prepro leader sequence starting with a methionine residue at position -43. The amino acid sequence of the mature prothrombin molecule circulating in plasma was also predicted from the cDNA and shown to be in good agreement with that determined previously by conventional amino acid sequence analysis.

Amino Acid Sequence↗

Human glucose-6-phosphate dehydrogenase: primary structure and cDNA cloning.

The X-chromosome-linked glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ oxidoreductase, EC 1.1.1.49) of humans and other mammals consists of a subunit with a molecular weight of about 58,000. The enzyme plays a key role in the generation of NADPH, particularly in matured erythrocytes, and the genetic deficiency of the enzyme is associated with chronic and drug- or food-induced hemolytic anemia in humans. The enzyme was purified to homogeneity from human erythrocytes. The complete amino acid sequence of the subunit, consisting of 531 amino acid residues, was determined by automated and manual Edman degradation of tryptic, chymotryptic, thermolytic, and cyanogen bromide peptides obtained from the enzyme. Based on the amino acid sequence data thus obtained, a 41-mer oligonucleotide with unique sequence was prepared. Two cDNA libraries constructed in phage lambda gt11--i.e., a human liver cDNA library and a human hepatoma Li-7 cDNA library--were screened with the synthetic nucleotide probe. Two positive clones, lambda G6PD-19 and lambda G6PD-25, were obtained from the hepatoma library. lambda G6PD-19 contained an insertion of 2.0 kilobase pairs (kbp), and encoded 204 amino acid residues that were completely compatible with the COOH-terminal portion of the enzyme. The insertion of the clone had a 3' noncoding region of 1.36 kbp. The other clone, lambda G6PD-25, had an insertion of 1.8 kbp and encoded 362 amino acid residues of G6PD. Southern blot analysis of DNA samples obtained from cells with and without the human X chromosome indicated that the cDNA hybridizes with a sequence in the X chromosome.

Amino Acid Sequence↗

Volume rendering of visible human data for an anatomical virtual environment.

In this work, we utilize the axial anatomical human male sections from the National Library of Medicine's Visible Human Project to generate three-dimensional (3-D) volume representations of the human male subject. The two-dimensional (2-D) projection images were produced by combining ray tracing techniques with automated image segmentation routines. The resultant images provide accurate and realistic volumetric representations of the Visible Human data set which is ultimately needed in medical virtual environment simulation. Ray tracing techniques provide methods by which 2-D volume views of a 3-D voxel array can be produced. The cross-sectional images can be scanned at different angles to produce rotated views of the voxel array. By combining volume views at incremental angles over 360 degrees a full volumetric representation of the voxel array, in this case the human male data set, can be computer generated and displayed without the speed and memory limitations of trying to display the entire data array. Additional texture and feature information can be obtained from the data by applying optical property equations to the ray scans. The imaging effects that can be added to volume renderings using these equations include shading, shadowing, and transparency. The automated segmentation routines provide a means to distinguish between various anatomical structures of the body. These routines can be used to differentiate between skin, fat, muscle, cartilage, blood vessels, and bone. By combining automated segmentation routines with the ray-tracing techniques, 2-D volume views of various anatomical structures and features can be isolated from the full data set. Examples of these segmentation abilities are demonstrated for the human male data set which include volume views of the skeletal systems, the musculoskeletal system, and part of the vascular system. The methods described above allow us to generate lifelike images, NURBS surface models, and realistic texture maps of specific anatomical structures. We have the capability to generate images that are both accurate and lifelike, much like photographic anatomical atlases. We can also generate images, models, and textures that have the clarity of medical artwork/illustrations, by highlighting the coloring of the ray traced structures with conventional colors instead of the natural color of the specimen. We are currently in the process of generating a comprehensive reference atlas of volume rendered images of the human body, soon to be published by Mosby-Year Book. The segmentation techniques needed to create this atlas also offer the accuracy and realism needed to create surface models and texture maps for a virtual environment for surgery simulation.

Anatomy, Cross-Sectional↗

High-throughput techniques for compound characterization and purification.

A new paradigm in drug discovery is the synthesis of structurally diverse collections of compounds, so-called libraries, followed by high-throughput biological screening. High-throughput characterization and purification techniques are required to provide high-quality compounds and reliable biological data, which has led to the development of faster methods, system automation and parallel approaches. This review summarizes recent advances in support of analytical characterization and preparative purification technologies. Notably, mass spectrometry (MS) and supercritical fluid chromatography (SFC) are among the areas where new developments have had a major impact on defining these high-throughput applications.

Chromatography↗

Applying Hybrid-Asbru clinical guidelines using the Spock system.

Clinical Guidelines are a major tool for improving the quality of medical care. Currently, a major research direction is automating the application of guidelines at the point of care. To support that automation, several requirements must be fulfilled, such as specification in a machine-interpretable format, and connection to an electronic patent record. We propose an innovative approach to guideline application, which capitalizes on our Digital electronic Guidelines Library (DeGeL). The DeGeL framework includes a new hybrid model for incremental specification of free-text guidelines, using several intermediate representations. The new approach was implemented, in the case of the Asbru guideline ontology, as the Spock system. Spock's hybrid application engine supports application of guideline represented at an intermediate format. Spock was evaluated in a preliminary fashion by applying several guidelines to sample patient data.

Decision Making, Computer-Assisted↗

Support vector machine applications in bioinformatics.

The support vector machine (SVM) approach represents a data-driven method for solving classification tasks. It has been shown to produce lower prediction error compared to classifiers based on other methods like artificial neural networks, especially when large numbers of features are considered for sample description. In this review, the theory and main principles of the SVM approach are outlined, and successful applications in traditional areas of bioinformatics research are described. Current developments in techniques related to the SVM approach are reviewed which might become relevant for future functional genomics and chemogenomics projects. In a comparative study, we developed neural network and SVM models to identify small organic molecules that potentially modulate the function of G-protein coupled receptors. The SVM system was able to correctly classify approximately 90% of the compounds in a cross-validation study yielding a Matthews correlation coefficient of 0.78. This classifier can be used for fast filtering of compound libraries in virtual screening applications.

Algorithms↗

Identification of genes encoding zinc finger motifs in the cardiovascular system.

The Zn2+-finger DNA-binding domain has been identified in several developmental control proteins, transcription factors and gene products associated with diseases, as well as in several RNA-binding proteins. We applied library screening, expressed sequence tagging (EST sequencing), Zn2+-binding assays and Northern blot hybridization, in order to characterize novel cDNA clones of the human cardiovascular system which contain Zn2+-finger motifs. An embryonic (8-10 weeks gestation) heart lambda ZAP Express cDNA library was screened with an oligonucleotide probe deduced from a consensus amino acid sequence which is highly conserved for Zn2+-finger proteins, and approximately 350 positive clones were isolated from 1 x 10(4) plaque-forming units (pfu) initially plated. The isolated clones were classified as known and novel following single pass automated DNA sequencing. Analysis of Northern blot hybridization delineated the tissue specificity of these clones, as well as their association with cardiac growth and development. Existence of Zn2+-finger motifs in the novel clones was confirmed by Zn2+-binding assay. In this report, we present the characterization of eight novel clones, including the complete cDNA sequences of one of these clones (HHZ-123).

Amino Acid Sequence↗

Identification of human liver mitochondrial aldehyde dehydrogenase as a potential target for microcystin-LR.

Microcystins (MCs) are hepatotoxins produced by a variety of freshwater cyanobacteria. The toxicity of these hepatotoxins is a severe health issue for both humans and livestock; MCs have been implicated in the development of liver cancer, necrosis, and even deadly intrahepatic bleeding. Microcystin-LR (MC-LR) is the MC variant most commonly encountered in a contaminated aquatic system. Thus far, MC-LR has only been shown to target the serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A) and it is still unknown whether MC-LR can bind and inhibit any other protein targets inside the cell. To find potential MC-LR targets, we screened a phage display library for peptide ligands that specifically recognize MC-LR. Using these peptide sequences as guides, we performed a series of bioinformatics analyses revealing that MC-LR binds human liver aldehyde dehydrogenase 2 (ALDH2) at residues 447-451. We confirmed MC-LR binding of ALDH2 via automated docking computation, which yielded results matching our experimental and bioinformatics analyses. ALDH2 dysfunction may lead to aldehyde-induced reactive oxygen species (ROS) generation and, in turn, apoptosis. Therefore, ALDH2 could potentially be a target of MC-LR associated with the process of ROS-induced apoptosis. Our current study presents a new approach to the study of interactions of biological molecules by combining phage display technology with computational methods.

Aldehyde Dehydrogenase↗

A structure-based strategy for discovery of small ligands binding to functionally unknown proteins: combination of in silico screening and surface plasmon resonance measurements.

In the postgenomic era, many researchers and organizations have been engaged in structural and functional analyses of proteins. As a part of these efforts, searching for small organic compounds that bind specifically to target proteins is quite important. In this study, we have developed a rational strategy for ligand discovery based on the three-dimensional structures of target proteins, which were elucidated by X-ray crystallography and nuclear magnetic resonance spectroscopy. The strategy has three features: (i) rapid selection of candidate compounds by in silico screening, (ii) automated preparation of sample solutions with robotics, and (iii) reliable evaluation of the candidates with surface plasmon resonance. Applying the strategy to a protein, At2g24940 from Arabidopsis thaliana, we discovered four small ligands out of a commercially available library of about 150 000 compounds. Although these compounds had only weak affinities to the target protein, with dissociation constants ranging from 68 to 120 microM, they apparently possess common structural features. They would be leads for the development of specific inhibitors/drugs for At2g24940, and provide important clues toward elucidation of the protein function.

Arabidopsis Proteins↗

Notechis 11'2, a non-toxic phospholipase A2 from the venom of Notechis scutatus scutatus.

Previously, we deduced the amino acid sequence of a novel phospholipase-A2-like protein (PLA2) from the nucleotide sequence of a cDNA isolated from a library prepared from the venom gland of the Australian elapid Notechis scutatus scutatus. The corresponding protein has now been identified, purified from the venom and named Notechis 11'2. Its complete amino acid sequence has been determined by automated Edman degradation of both the whole protein and peptides generated by Staphylococcus aureus protease digestion and chemical cleavage at a tryptophan residue. As predicted from its sequence which contains all the residues putatively required for PLA2 activity, Notechis 11'2 exhibits an esterase activity, preferentially against neutral phospholipids. However, despite its sequence homology with other highly toxic PLA2 present in the venom of Notechis scutatus scutatus, notechis 11'2 has no lethal activity. This observation further supports the view that the lethal activity of PLA2 from Notechis scutatus scutatus is not due to the esterasic activity only.

Amino Acid Sequence↗

An algorithm for automated bacterial identification using matrix-assisted laser desorption/ionization mass spectrometry.

An algorithm for bacterial identification using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is being developed. This mass spectral fingerprint comparison algorithm is fully automated and statistically based, providing objective analysis of samples to be identified. Based on extraction of reference fingerprint ions from test spectra, this approach should lend itself well to real-world applications where samples are likely to be impure. This algorithm is illustrated using a blind study. In the study, MALDI-MS fingerprints for Bacillus atrophaeus ATCC 49337, Bacillus cereus ATCC 14579T, Escherichia coli ATCC 33694, Pantoea agglomerans ATCC 33243, and Pseudomonas putida F1 are collected and form a reference library. The identification of test samples containing one or more reference bacteria, potentially mixed with one species not in the library (Shewanella alga BrY), is performed by comparison to the reference library with a calculated degree of association. Out of 60 samples, no false positives are present, and the correct identification rate is 75%. Missed identifications are largely due to a weak B. cereus signal in the bacterial mixtures. Potential modifications to the algorithm are presented and result in a higher than 90% correct identification rate for the blind study data, suggesting that this approach has the potential for reliable and accurate automated data analysis of MALDI-MS.

Algorithms↗

An efficient rapid system for profiling the cellular activities of molecular libraries.

Rapid quantitative methods for characterizing small molecules, peptides, proteins, or RNAs in a broad array of cellular assays would allow one to discover new biological activities associated with these molecules and also provide a more comprehensive profile of drug candidates early in the drug development process. Here we describe a robotic system, termed the automated compound profiler, capable of both propagating a large number of cell lines in parallel and assaying large collections of molecules simultaneously against a matrix of cellular assays in a highly reproducible manner. To illustrate its utility, we have characterized a set of 1,400 kinase inhibitors in a panel of 35 activated tyrosine-kinase-dependent cellular assays in dose-response format in a single experiment. Analysis of the resulting multidimensional dataset revealed subclusters of both inhibitors and kinases with closely correlated activities. The approach also identified activities for the p38 inhibitor BIRB796 and the dual src/abl inhibitor BMS-354825 and exposed the expected side activities for Glivec/STI571, including cellular inhibition of c-kit and platelet-derived growth factor receptor. This methodology provides a powerful tool for unraveling the cellular biology and molecular pharmacology of both naturally occurring and synthetic chemical diversity.

Animals↗

The computer as a tool for learning.

Experimenters from the beginning recognized the advantages computers might offer in medical education. Several medical schools have gained experience in such programs in automated instruction. Television images and graphic display combined with computer control and user interaction are effective for teaching problem solving. The National Board of Medical Examiners has developed patient-case simulation for examining clinical skills, and the National Library of Medicine has experimented with combining media. Advances from the field of artificial intelligence and the availability of increasingly powerful microcomputers at lower cost will aid further development. Computers will likely affect existing educational methods, adding new capabilities to laboratory exercises, to self-assessment and to continuing education.

Computer-Assisted Instruction↗