Refinement of the main chain directed assignment strategy for the analysis of 1H NMR spectra of proteins.

The underlying basis of the main chain directed (MCD) resonance assignment strategy for the analysis of 1H NMR spectra of proteins is reexamined. The criteria used in the construction of the patterns used in the MCD method have been extended to increase the robustness of the approach to the presence of variable protein secondary structure and significant spectral degeneracy. These criteria have led to the development of several dozen patterns exclusively involving the short distance relationships between main chain amide NH-C alpha-H-C beta H (NAB) J-coupled subspin systems of the amino acid residues. The MCD patterns have been examined for fidelity and frequency of occurrence in a database composed of the high resolution crystal structures of 39 proteins. The analysis has identified several extremely robust patterns, suitable for initiating a hierarchical construction of units of secondary structure based upon a systematic analysis of two-dimensional nuclear Overhauser effect spectra. A formal procedure, suitable for the computer assisted application of the MCD strategy, is developed. This procedure, termed MCDPAT, has been applied to the analysis of the crystal structures of human ubiquitin, T4 lysozyme, and ribonuclease A. It has been found that the MCDPAT procedure is conservative producing no significant errors and is globally successful in correctly identifying the appropriate units of secondary structure contained in these three proteins.     

Two-dimensional 1H NMR study of human ubiquitin: a main chain directed assignment and structure analysis

The 1H resonances of human ubiquitin were studied by two-dimensional nuclear magnetic resonance techniques. A recently introduced assignment algorithm termed the main chain directed (MCD) assignment [Englander, S. W., & Wand, A. J. (1987) Biochemistry 26, 5953-5958] was applied. This approach relies on an ordered series of searches for prescribed patterns of connectivities in two-dimensional J-correlated and nuclear Overhauser effect spectra and centers on the dipolar interactions involving main-chain amide NH, alpha-CH, and beta-CH. Unlike the sequential assignment procedure, the MCD approach does not rest upon definition of side-chain J-coupled networks and is generally not sequential with the primary sequence of the protein. The various MCD patterns and the general algorithm are reiterated and applied to the analysis of human ubiquitin. With this algorithm, the vast majority of amino acid residue amide NH-C alpha H-C beta H J-coupled subspin systems could be associated with and aligned within units of secondary structure without any knowledge of the identity of the side chains. This greatly simplified recognition of side-chain spin systems by restricting their identity. Essentially complete resonance assignments are presented. The MCD method is compared with the sequential assignment method in some detail. The MCD method is highly amenable to automation. Human ubiquitin is found, at pH 5.8 and 30 degrees C, to be composed of an extensive beta-sheet structure involving five strands. Three of these strands form an antiparallel set sharing a common strand and have a parallel orientation to two antiparallel strands. Two helical segments were also observed. The largest, spanning 13 residues, shows dipolar interactions consistent with an alpha-helix while the smaller 4-residue helical segment appears, on the basis of observed nuclear Overhauser effects, to be a 3(10) helix. Five classical tight turns could be demonstrated.     

Computer assisted management of warfarin treatment.

Maintenance of anticoagulation is laborious and costly, and the results are often indifferent. An automatic system which adjusts the dose of warfarin has been designed using a formula devised after a survey of prescribing habits. Programs running on a microcomputer maintain a file of the necessary information and deal with the bulk of the weekly clinic. Dosage is advised, the date of the next visit determined, and the file updated. A doctor uses the console to advise some 10% of patients reserved for special reasons. The system produces clinic and ambulance lists, copies of the advice sent to patients, and, as a protection against machine failure, a weekly copy of the updated file. The results after 16 months are at least as good as those achieved manually. Medical and secretarial time is saved, and statistics about the clinic and its efficacy are made available.     

Computer assisted shared care in hypertension.

A computer assisted shared care scheme for the long term management and follow up of hypertensive patients has been developed in the Grampian Region. The scheme aims at facilitating the exchange of clinically important information between doctors and at achieving target levels of blood pressure with treatment in patients at highest risk of cardiovascular events. The shared care scheme has been well received by the local practitioners. Two hundred and fifty seven patients (18%) of 1426 patients under current long term follow up are assigned to follow up in the hospital aspect of the scheme. At the most recent visit 32% of patients in the hospital aspect and 10% of 1169 patients in the general practice aspect had blood pressure recordings above the target levels of 160/95 mm Hg. The stratification of patients formerly attending hospital clinics into grades of risk has rationalised our follow up procedures to allow the specialist resources to be freed and concentrated on those patients at highest risk and with the most complex problems. This computer assisted patient records system could be applied to other groups of high risk patients in whom long term follow up and surveillance are necessary--for example, patients with diabetes mellitus--and has implications for optimising and monitoring the delivery and outcome of care without overwhelming limited hospital resources.     

Computer assisted simulations and molecular graphics methods in molecular design. 1. Theory and applications to enzyme active-site directed drug design

A survey is presented of model building techniques, computer-assisted molecular dynamics simulations and a new theory of enzyme catalysis. Some aspects of the theoretical formalism are given. Enzyme active-site directed drug design is illustrated with examples taken from molecular modeling studies using FAD-containing disulphide oxidoreductases, proteinases and carbonic anhydrases.     

Computer assisted analysis of S-potentials

S-potentials from the channel catfish were analyzed using a high speed biological data processing system developed at this Institute. The analysis has shown that, when moderately dark adapted, the relation between the (maximal) amplitude of S-potential and log intensity of flash was the hyperbolic tangent throughout the whole visible spectrum. This is what one could expect if the S-potential in the fish was generated by signals from a single class of pigment and each signal had discrete action on the S-potential generating mechanism. The maximal absorption of the pigment involved was at around 625 nm.     

Computer assisted microdensitometric analysis of footprinting autoradiographic DATA.

A system comprised of a linear scanning microdensitometer interfaced to a personal computer has been developed to facilitate analysis of ligand-DNA footprinting autoradiograms. The system, which can be used to record density and sequence information from autoradiographic films, enables the user to relate the area under an autoradiographic band to the concentration of radiolabeled molecules present in the electrophoresis gel. This report describes the computer program which performs the calculations and discusses the ability of the system to accurately determine oligonucleotide concentration, as a function of band separation, photographic response, and the computational algorithm used to calculate band areas.     

A random graph approach to NMR sequential assignment.

Nuclear magnetic resonance (NMR) spectroscopy allows scientists to study protein structure, dynamics and interactions in solution. A necessary first step for such applications is determining the resonance assignment, mapping spectral data to atoms and residues in the primary sequence. Automated resonance assignment algorithms rely on information regarding connectivity (e.g., through-bond atomic interactions) and amino acid type, typically using the former to determine strings of connected residues and the latter to map those strings to positions in the primary sequence. Significant ambiguity exists in both connectivity and amino acid type information. This paper focuses on the information content available in connectivity alone and develops a novel random-graph theoretic framework and algorithm for connectivity-driven NMR sequential assignment. Our random graph model captures the structure of chemical shift degeneracy, a key source of connectivity ambiguity. We then give a simple and natural randomized algorithm for finding optimal assignments as sets of connected fragments in NMR graphs. The algorithm naturally and efficiently reuses substrings while exploring connectivity choices; it overcomes local ambiguity by enforcing global consistency of all choices. By analyzing our algorithm under our random graph model, we show that it can provably tolerate relatively large ambiguity while still giving expected optimal performance in polynomial time. We present results from practical applications of the algorithm to experimental datasets from a variety of proteins and experimental set-ups. We demonstrate that our approach is able to overcome significant noise and local ambiguity in identifying significant fragments of sequential assignments.     

Computer assisted screening: effect on the patient and his consultation.

The initial impact of computer assisted preventive screening in general practice consultations has been monitored. The technology has not been found stressful by patients, and the power of the consultation to alleviate low arousal has been increased by computer use. No appreciable increase in the durations of consultation was detected, despite an average computer initiated input of two minutes eight seconds. The computer has successfully prompted preventive screening and health education with a sixfold increase in the number of potentially relevant procedures being mentioned. The actual information presented by the computer has been shown to be crucial, with the terminal''s mere presence an ineffective reminder. The computer terminal was used in 65% of the consultations for which it was available, which, if sustained, represents an effective screening programme for attending patients.     

Automated protein backbone assignment using the projection-decomposition approach

Production of sufficient amounts of human proteins is a frequent bottleneck in structural biology. Here we describe an Escherichia coli-based cell-free system which yields mg-quantities of human proteins in N-terminal fusion constructs with the GB1 domain, which show significantly increased translation efficiency. A newly generated E. coli BL21 (DE3) RIPL-Star strain was used, which contains a variant RNase E with reduced activity and an excess of rare-codon tRNAs, and is devoid of lon and ompT protease activity. In the implementation of the expression system we used freshly in-house prepared cell extract. Batch-mode cell-free expression with this setup was up to twofold more economical than continuous-exchange expression, with yields of 0.2-0.9 mg of purified protein per mL of reaction mixture. Native folding of the proteins thus obtained is documented with 2D [(15)N,(1)H]-HSQC NMR.     

A coalescent approach to the polymerase chain reaction.

A versatile algorithm is developed to model PCR on a computer. The method is based on a modification of the coalescent process and provides a general framework to analyse data from PCR. It allows for incorporation of the dynamics of the replication process as described in terms of the number of starting template molecules and cycle-dependent PCR efficiency. The simulation method generates, as a first step, the genealogy of a set of sequences sampled from a final PCR product. In a second step a mutation process is superimposed and the resulting data set is analysed. The efficiency of our algorithm enables us to get reliable approximations of various sample distributions. We demonstrate the relevance of our method with two applications: maximum likelihood estimation of the error rate in PCR and a test of homogeneity of the template.     

A monoclonal antibody (ST-1) directed to the native heparin chain.

A mouse monoclonal antibody, ST-1, was raised against heparin complexed to Salmonella minnesota. Characterization of this antibody showed that it recognizes an epitope in the intact molecule of heparin that is present regardless of its source or anticoagulant activity. ST-1 is the first monoclonal antibody specific for the intact unmodified molecule of heparin to be described. 3H-labeled heparin in solution was immunoprecipitated by ST-1, and the formation of the 3H-labeled immunocomplex was selectively inhibited by unlabeled heparin. No cross-reactivity of ST-1 was observed with other glycosaminoglycans such as heparan sulfate, chondroitin sulfate, hyaluronic acid, dermatan sulfate, and keratan sulfate, or with polyanionic polymers such as dextran sulfate. Selective removal of the N-sulfate groups or N,O-desulfation of heparin strongly reduced the binding of ST-1. Inhibition of binding was also observed after carbodiimide reduction of the carboxyl groups of the uronic acid units of heparin. Competitive assays of ST-1 binding to heparin immobilized on poly-L-lysine-coated plates using oligosaccharides of different sizes that arose from HNO2 cleavage of heparin showed that the minimum fragment required for reactivity of ST-1 is a decasaccharide.     

Computer assisted karyotype analysis of Pyrrhopappus carolinianus

With the help of Computer Aided Karyotyping procedures, Ag-NOR staining and C-banding techniques, the karyotype of Pyrrhopappus carolinianus (Asteraceae, Lactuceae) has been studied. The species has 2n=12 chromosomes. Silver staining reveals that the two shortest pairs of chromosomes possess NOR's. On the basis of chromosome length and centromere position, only the longest chromosome pair and the satellite chromosomes can be identified. Two types of C-banding can be obtained, dependent on the temperature of the hydrochloric acid hydrolysis of the root tips. Hydrolysis at 60°C results exclusively in centromeric bands, whereas a treatment at room temperature reveals a pattern of intercalary bands. A computer assisted analysis of the intercalary banding pattern resulted in the construction of schematic representation of the average C-banding pattern. This banding pattern allows an easy identification of each of the chromosome pairs.     

Synthesis of oligonucleotide derivatives containing a bis-pyrene residue in the main chain.

The oligonucleotide having the bis-pyrene residue in the main chain was synthesized. The preparation of the bis-pyrene was started from the conversion of 2,2-bis-(bromomethyl)-1,3-propanediol into the protected bis-amino derivative. The reaction of the bis-amino derivative with 1-pyrenebutyric acid using DCC/HOBT afforded the desired bis-pyrene. This compound was then converted to the protected phosphormidite. The oligonucleotides possessing the bis-pyrene were synthesized by using the amidite. The oligonucleotides having the bis-pyrene residue can bind to DNA sequence in an aqueous solution to give the duplex with comparable thermal stability as that of the unmodified DNA/DNA duplex. The significantly enhanced pyrene-excimer fluorescence was observed upon hybridization of the bis-pyrene modified oligonucleotides with DNA.     

Computer assisted semen analyzers in andrology research and veterinary practice.

The evaluation of sperm cell motility and morphology is an essential parameter in the examination of sperm quality and in the establishment of correlations between sperm quality and fertility. Computer-assisted sperm analysis (CASA) allows an objective assessment of different cell characteristics: motion, velocity, and morphology. The development and problems related to this technology are raised in this review, paying particular attention to the biases and standardization requirements absolutely needed to obtain useful results. Although some interesting results, mainly in humans, have already been obtained, many questions remain, which have to be answered to allow for further development of this technology in veterinary medicine, clinical fertility settings, physiological, and toxicology research activities. The main problem is related to the standardization and optimization of the equipment and procedures. The different CASA instruments have all demonstrated high levels of precision and reliability using different sperm classification methodology. Their availability gives us a great tool to objectively compare sperm motility and morphology and to improve our knowledge and ability to manipulate spermatozoa.