Data mining the protein data bank: residue interactions

The protein databank contains a vast wealth of structural and functional information. The analysis of this macromolecular information has been the subject of considerable work in order to advance knowledge beyond the collection of molecular coordinates. This article presents a method that determines local structural information within proteins using mathematical data mining techniques. The mine program described returns many known configurations of residues such as the catalytic triad, metal binding sites and the N-linked glycosylation site; as well as many other multiple residue interactions not previously categorized. Because mathematical constructs are used as targets, this method can identify new information not previously known, and also provide unbiased results of typical structure and their expected deviations. Because the results are defined mathematically, they cannot indicate the biological implications of the results. Therefore two support programs are described that provide insight into the biological context for the mine results. The first allows a weighted RMSD search between a template set of coordinates and a list of PDB files, and the second allows the labeling of a protein with the template results from mining to aid in the classification of this protein.     

Two simple programs for the analysis of data from enzyme-linked immunosorbent (ELISA) assays on a programmable desk-top calculator

We have designed two programs for use with an inexpensive programmable calculator which rapidly and accurately convert raw data generated from enzyme-linked immunosorbent assays directly into antigen concentration. The first program computes and compares effective doses (ED₅₀)'s between a standard and each unknown sample assayed. The ED₅₀ from the unknown sample is then multiplied by a concentration factor which yields the unknown concentration. The second program linearizes the sigmoidal enzyme-linked immunosorbent assay titration curve using a logit-log transformation of the data in order to compute unknown concentration values. Both programs employ stringent limit conditions to decrease “nonsense” calculations. Data are then processed by a least-squares best-fit linear regression analysis.     

Methylation blockage and other improvements to a comprehensive DNA analysis program.

A comprehensive DNA analysis computer program was described in the second special issue of Nucleic Acids Research on the applications of computers to research on nucleic acids by Stone and Potter (1). Criteria used in designing the program were user friendliness, ability to handle large DNA sequences, low storage requirement, migratability to other computers and comprehensive analysis capability. The program has been used extensively in an industrial-research environment. This paper talks about improvements to that program. These improvements include testing for methylation blockage of restriction enzyme recognition sites, homology analysis, RNA folding analysis, integration of a large DNA database (GenBank), a site specific mutagenesis analysis, a protein database and protein searching programs. The original design of the DNA analysis program using a command executive from which any analytical programs can be called, has proven to be extremely versatile in integrating both developed and outside programs to the file management system employed.     

Chemicals from alternative feedstocks in the United States

Abstract: Growing industrial interest in products from renewable alternative feedstocks has resulted in the creation of more industry-led federal research and development programs. The basis of this interest is introduced, followed by an overview of the various federal programs that support basic through applied research relevant to chemical products from renewable resources, and evidence of the increased coordination efforts between programs. The majority of the paper outlines a new program within the U.S. Department of Energy, the Alternative Feedstocks Program, which specifically targets chemicals from renewables which have the potential to become part of the next generation of high-volume chemical feedstocks for the manufacturing industries. The first product of the program, an iterative process technology decision analysis tool, is broadly presented using the first process development project: succinic acid from fermentable sugars.     

Preparing faculty to teach in a problem-based learning curriculum: the Sherbrooke experience.

Over the last 6 years Sherbrooke Medical School has undertaken a major reform of its undergraduate curriculum. A new student-centred, community-oriented curriculum was implemented in September 1987. Problem-based learning (PBL) is now the main educational method. To adequately prepare teachers for the curriculum a series of faculty development programs in pedagogy were offered: first, a 2-day introductory workshop to initiate teachers into educational principles and their application in the new program; second, a 1-year basic training program in medical pedagogy; third, a 1-day workshop on PBL; and fourth, a comprehensive 3-day training program in PBL tutoring. Over 60% of all full-time teachers attended the introductory program and 80% the tutor training program. The 1-year basic training program was completed by 33% of the faculty members. The implementation of these programs, coupled with a high participation rate, resulted in a more student-centred educational philosophy and a greater interest in medical education. This had a significant impact when the new curriculum was instituted. Lessons learned from the experience are discussed.     

Trends and values of ‘Land for Wildlife’ programs for private land conservation

The Land for Wildlife program started in Victoria in 1981 as a voluntary program with the broad aim of supporting landholders in providing habitat for wildlife on their property. The program has since spread across Australia and is implemented in a range of guises, through a variety of governance approaches. This research collected qualitative and quantitative data on Land for Wildlife programs across Australia to conduct the first national review. Data were gathered on changes in program membership to assess different participation trends. In addition, phone interviews with Land for Wildlife coordinators throughout Australia were conducted to explore how the programs are positioned in delivering biodiversity outcomes in a range of different regions. Over 14,000 properties covering 2.3 million ha are currently registered under Land for Wildlife programs. with at least 500,000 ha of habitat managed for conservation. Limited resources present a large challenge faced by a number of programs, with generally low funding and staffing resulting in restricted biodiversity focus and conservation outcomes. We suggest options to enhance the programs and propose future research directions.     

Prediction of RNA secondary structure, including pseudoknotting, by computer simulation.

A computer program is presented which determines the secondary structure of linear RNA molecules by simulating a hypothetical process of folding. This process implies the concept of 'nucleation centres', regions in RNA which locally trigger the folding. During the simulation, the RNA is allowed to fold into pseudoknotted structures, unlike all other programs predicting RNA secondary structure. The simulation uses published, experimentally determined free energy values for nearest neighbour base pair stackings and loop regions, except for new extrapolated values for loops larger than seven nucleotides. The free energy value for a loop arising from pseudoknot formation is set to a single, estimated value of 4.2 kcal/mole. Especially in the case of long RNA sequences, our program appears superior to other secondary structure predicting programs described so far, as tests on tRNAs, the LSU intron of Tetrahymena thermophila and a number of plant viral RNAs show. In addition, pseudoknotted structures are often predicted successfully. The program is written in mainframe APL and is adapted to run on IBM compatible PCs, Atari ST and Macintosh personal computers. On an 8 MHz 8088 standard PC without coprocessor, using STSC APL, it folds a sequence of 700 nucleotides in one and a half hour.     

Thermal denaturation of double-stranded nucleic acids: prediction of temperatures critical for gradient gel electrophoresis and polymerase chain reaction.

A program is described which calculates the thermal stability and the denaturation behaviour of double-stranded DNAs and RNAs up to a length of 1000 base pairs. The algorithm is based on recursive generation of conditional and a priori probabilities for base stacking. Output of the program may be compared directly to experimental results; thus the program may be used to optimize the nucleic acid fragments, the primers and the experimental conditions prior to experiments like polymerase chain reactions, temperature-gradient gel electrophoresis, denaturing-gradient gel electrophoresis and hybridizations. The program is available in three versions; the first version runs interactively on VAXstations producing graphics output directly, the second is implemented as part of the HUSAR package at GENIUSnet, the third runs on any computer producing text output which serves as input to available graphics programs.     

Retriever and CompareTable, two informatics tools for data analysis of high-density oligonucleotide arrays.

High-density oligonucleotide arrays are widely employed for detecting global changes in gene expression profiles of cells or tissues exposed to specific stimuli. Presented with large amounts of data, investigators can spend significant amounts of time analyzing and interpreting this array data. In our application of GeneChip arrays to analyze changes in gene expression in viral-infected epithelium, we have needed to develop additional computational tools that may be of utility to other investigators using this methodology. Here, I describe two executable programs to facilitate data extraction and multiple data point analysis. These programs run in a virtual DOS environment on Microsoft Windows 95/98/2K operating systems on a desktop PC. Both programs can be freely downloaded from the BioTechniques Software Library (www.BioTechniques.com). The first program, Retriever, extracts primary data from an array experiment contained in an Affymetrix textfile using user-inputted individual identification strings (e.g., the probe set identification numbers). With specific data retrieved for individual genes, hybridization profiles can be examined and data normalized. The second program, CompareTable, is used to facilitate comparison analysis of two experimental replicates. CompareTable compares two lists of genes, identifies common entries, extracts their data, and writes an output text file containing only those genes present in both of the experiments. The output files generated by these two programs can be opened and manipulated by any software application recognizing tab-delimited text files (e.g., Microsoft NotePad or Excel).     

Antigraviceptive neck muscle responses to "moving up and moving down" in human

The responses of neck muscle to sudden transit from one 'g' to hyper 'g', work to support the head and remain the relative position of head on trunk as common observed: i.e. in sudden acceleration or deceleration by car or ejection of pilot from aircraft. Accordingly it is highly possible that the neck muscle responses to moving up may be important to prevent the neck injury due to sudden linear acceleration such as moving up against gravity. However little is known about the evaluation of mechanism of this reflex. Therefore the present study was conducted with two aims. The first aim was to investigate the neck muscle responses to vertical linear acceleration bv 0.4 g produced with an electro-hydraulic servo-system. We chose the vertical linear acceleration because it activates mainly sacculus, from which afferents have been demonstrated to be connected directly to sternocleidomastoid muscle in animals and human. The second aim was to determine whether there is a difference of neck muscle response to moving down and moving up.     

Computer-controlled discontinuous rotating gel electrophoresis for separation of very large DNA molecules

The newly designed equipment for alternating field gel electrophoresis which permits the separation of very large DNA molecules and the simultaneous analysis of up to 35 samples is described. The field alternation is effected by intermittently rotating the submerged agarose gel by optitional angles. The time intervals between changes of position are controlled by a computer program driving a simple switching device which was designed to suit any technique using periodic switching or inversion of the electrical field. Because the electrophoresis unit provides an absolutely homogeneous electrical field, no distortion of migration lanes occurs and the resolution is very good. Moreover, by using a switching time interval gradient an almost perfect linear relationship between migration distances and molecule sizes in the range of about 100-1250 kilobase pairs is observed. In two-dimensional separations, different switching time programs for the first and second dimension allow maximum resolution of selected size ranges. Field inversion gel electrophoresis is possible as well. The performance of the method is demonstrated by comparing the chromosome sizes of different yeast strains.     

MitoAnalyzer, a computer program and interactive web site to determine the effects of single nucleotide polymorphisms and mutations in human mitochondrial DNA

MitoAnalyzer provides information about the effects of single nucleotide polymorphisms (SNPs) and mutations in human mitochondrial DNA (mtDNA). This program determines if a single base pair (bp) change is in the non-coding or coding region, in the first, second or third bp of the codon, in a rRNA, tRNA or a protein, causes an amino acid (aa) change, the nature of that change, the position of the aa change in the protein, and the new aa sequence of the changed protein. Mutations associated with published mitochondrial diseases are noted. This program, thus, facilitates rapid analysis and evaluation of SNPs and mutations in human mtDNA.     

Comparison of two different approaches in the detection of intermittent cardiorespiratory coordination during night sleep

Background

The objective was to evaluate and to compare two completely different detection algorithms of intermittent (short-term) cardiorespiratory coordination during night sleep. The first method is based on a combination of respiratory flow and electrocardiogram recordings and determines the relative phases of R waves between successive onsets of inspiration. Intermittent phase coordination is defined as phase recurrence with accuracy α over at least k heartbeats. The second, recently introduced method utilizes only binary coded variations of heart rate (acceleration = 1, deceleration = 0) and identifies binary pattern classes which can be assigned to respiratory sinus arrhythmia (RSA). It is hypothesized that RSA pattern class recurrence over at least k heartbeats is strongly related with the intermittent phase coordination defined above.

Results

Both methods were applied to night time recordings of 20 healthy subjects. In subjects <45 yrs and setting k = 3 and α = 0.03, the phase and RSA pattern recurrence were highly correlated. Furthermore, in most subjects the pattern predominance (PP) showed a pronounced oscillation which is most likely linked with the dynamics of sleep stages. However, the analysis of bivariate variation and the use of surrogate data suggest that short-term phase coordination mainly resulted from central adjustment of heart rate and respiratory rate rather than from real phase synchronization due to physiological interaction.

Conclusion

Binary pattern analysis provides essential information on short-term phase recurrence and reflects nighttime sleep architecture, but is only weakly linked with true phase synchronization which is rare in physiological processes of man.     

Efficient computation of three-dimensional protein structures in solution from nuclear magnetic resonance data using the program DIANA and the supporting programs CALIBA, HABAS and GLOMSA.

A novel procedure for efficient computation of three-dimensional protein structures from nuclear magnetic resonance (n.m.r.) data in solution is described, which is based on using the program DIANA in combination with the supporting programs CALIBA, HABAS and GLOMSA. The first part of this paper describes the new programs DIANA. CALIBA and GLOMSA. DIANA is a new, fully vectorized implementation of the variable target function algorithm for the computation of protein structures from n.m.r. data. Its main advantages, when compared to previously available programs using the variable target function algorithm, are a significant reduction of the computation time, and a novel treatment of experimental distance constraints involving diastereotopic groups of hydrogen atoms that were not individually assigned. CALIBA converts the measured nuclear Overhauser effects into upper distance limits and thus prepares the input for the previously described program HABAS and for DIANA. GLOMSA is used for obtaining individual assignments for pairs of diastereotopic substituents by comparison of the experimental constraints with preliminary results of the structure calculations. With its general outlay, the presently used combination of the four programs is particularly user-friendly. In the second part of the paper, initial results are presented on the influence of the novel DIANA treatment of diastereotopic protons on the quality of the structures obtained, and a systematic study of the central processing unit times needed for the same protein structure calculation on a range of different, commonly available computers is described.     

Complexes of vitamin B6XX: equilibrium and mechanistic studies of the reaction of pyridoxal-5'-phosphate with pyridoxamine-5'-phosphate in the presence of copper(II)

The interaction of Cu(II) with pyridoxamine-5'-phosphate (PMP) and pyridoxal-5'-phosphate (PLP) was studied potentiometrically. The titration data were assessed by MINIQUAD program. Several protonated and nonprotonated complexes have been found to exist in solution. The reaction of PLP with Cu(II)-PMP has been studied kinetically, using the stopped-flow technique. Two rate steps have been observed. The first step has been attributed to the formation of a Schiff's base metal complex. The second step may be due to the formation of a ternary complex formation. A mechanism was suggested.     

Respiratory effects of transient axial acceleration.

Whereas gravity has an inspiratory effect in upright subjects, transient upward acceleration is reported to have an expiratory effect. To explore the respiratory effects of transient axial accelerations, we measured axial acceleration at the head and transrespiratory pressure or airflow in five subjects as they were dropped or lifted on a platform. For the first 100 ms, upward acceleration caused a decrease in mouth pressure and inspiratory flow, and downward acceleration caused the opposite. We also simulated these experimental observations by using a computational model of a passive respiratory system based on anatomical data and normal respiratory characteristics. After 100 ms, respiratory airflow in our subjects became highly variable, no longer varying with acceleration. Electromyograms of thoracic and abdominal respiratory muscles showed bursts of activity beginning 40-125 ms after acceleration, suggesting reflex responses responsible for subsequent flow variability. We conclude that, in relaxed subjects, transient upward axial acceleration causes inspiratory airflow and downward acceleration causes expiratory airflow, but that after ~100 ms, reflex activation of respiratory musculature largely determines airflow.     

Analytical platform for metabolome analysis of microbial cells using methyl chloroformate derivatization followed by gas chromatography-mass spectrometry

This protocol describes an analytical platform for the analysis of intra- and extracellular metabolites of microbial cells (yeast, filamentous fungi and bacteria) using gas chromatography-mass spectrometry (GC-MS). The protocol is subdivided into sampling, sample preparation, chemical derivatization of metabolites, GC-MS analysis and data processing and analysis. This protocol uses two robust quenching methods for microbial cultures, the first of which, cold glycerol-saline quenching, causes reduced leakage of intracellular metabolites, thus allowing a more reliable separation of intra- and extracellular metabolites with simultaneous stopping of cell metabolism. The second, fast filtration, is specifically designed for quenching filamentous micro-organisms. These sampling techniques are combined with an easy sample-preparation procedure and a fast chemical derivatization reaction using methyl chloroformate. This reaction takes place at room temperature, in aqueous medium, and is less prone to matrix effect compared with other derivatizations. This protocol takes an average of 10 d to complete and enables the simultaneous analysis of hundreds of metabolites from the central carbon metabolism (amino and nonamino organic acids, phosphorylated organic acids and fatty acid intermediates) using an in-house MS library and a data analysis pipeline consisting of two free software programs (Automated Mass Deconvolution and Identification System (AMDIS) and R).