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Biomedical subjects

J B Cooper

Publications and source records attributed to J B Cooper.

At least 37 records · Page 2Linked to original sources
PubMed2003

Heritability of intramammary infections at first parturition and relationships with sire transmitting abilities for somatic cell score, udder type traits, productive life, and protein yield.

The objective of this study was to determine the relationships among daughter intramammary infections at first parturition and sire transmitting abilities for somatic cell score, udder type traits, productive life, and protein yield. Quarter milk samples from 958 daughters (in eight Pennsylvania herds and one Nebraska herd) of 182 Holstein sires were collected within a few days of first calving and cultured to determine intramammary infection status. A total of 446 cows had intramammary infections in 835 quarters at first parturition. Incidence of intramammary infections at first parturition and the proportion of quarters infected per cow were regressed on age at first calving, days in milk at sample collection, herd-season of calving (a classification variable), and sire transmitting abilities taken one at a time. Linear effects, non-linear effects, and odds ratios were estimated for sire transmitting abilities. Separate, preplanned analyses were conducted on data from one herd that gave all heifers an intramammary antibiotic infusion in each quarter 30 d prior to the expected calving date. Separate analyses were also conducted on dependent variables that considered intramammary infections at first parturition from: all organisms, coagulase-negative staphylococci, coliform species, streptococci other than Streptococcus agalactiae, and the most common environmental organisms (coliform species and streptococci other than Streptococcus agalactiae). Daughters of sires that transmit the lowest somatic cell score had the fewest intramammary infections at first parturition. Daughters of sires that transmit longer productive life, shorter teats, and closely spaced front teats had fewer intramammary infections at first parturition. Selection for lower somatic cell score, longer productive life, shorter teats, or closely spaced front teats may reduce the incidence of intramammary infections at first parturition.

Age Factors↗
PubMed2002

Five atomic resolution structures of endothiapepsin inhibitor complexes: implications for the aspartic proteinase mechanism.

Endothiapepsin is derived from the fungus Endothia parasitica and is a member of the aspartic proteinase class of enzymes. This class of enzyme is comprised of two structurally similar lobes, each lobe contributing an aspartic acid residue to form a catalytic dyad that acts to cleave the substrate peptide bond. The three-dimensional structures of endothiapepsin bound to five transition state analogue inhibitors (H189, H256, CP-80,794, PD-129,541 and PD-130,328) have been solved at atomic resolution allowing full anisotropic modelling of each complex. The active sites of the five structures have been studied with a view to studying the catalytic mechanism of the aspartic proteinases by locating the active site protons by carboxyl bond length differences and electron density analysis. In the CP-80,794 structure there is excellent electron density for the hydrogen on the inhibitory statine hydroxyl group which forms a hydrogen bond with the inner oxygen of Asp32. The location of this proton has implications for the catalytic mechanism of the aspartic proteinases as it is consistent with the proposed mechanism in which Asp32 is the negatively charged aspartate. A number of short hydrogen bonds (approximately 2.6 A) with ESD values of around 0.01 A that may have a role in catalysis have been identified within the active site of each structure; the lengths of these bonds have been confirmed using NMR techniques. The possibility and implications of low barrier hydrogen bonds in the active site are considered.

Aspartic Acid Endopeptidases↗
PubMed2002

5-Aminolaevulinic acid dehydratase: metals, mutants and mechanism.

5-Aminolaevulinic acid dehydratase catalyses the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid. The studies described highlight the importance of a bivalent metal ion and two active-site lysine residues for the functioning of 5-aminolaevulinic acid dehydratase. Dehydratases fall into two main categories: zinc-dependent enzymes and magnesium-dependent enzymes. Mutations that introduced zinc-binding ligands into a magnesium-dependent enzyme conferred an absolute requirement for zinc. Mutagenesis of lysine residues 247 and 195 in the Escherichia coli enzyme lead to dramatic effects on enzyme activity, with lysine 247 being absolutely essential. Mutation of either lysine 247 or 195 to cysteine, and treatment of the mutant enzyme with 2-bromethylamine, resulted in the recovery of substantial enzyme activity. The effects of the site-directed alkylating inhibitor, 5-chlorolaevulinic acid, and 4,7-dioxosebacic acid, a putative intermediate analogue, were investigated by X-ray crystallography. These inhibitors reacted with both active-site lysine residues. The role of these two lysine residues in the enzyme mechanism is discussed.

Amino Acid Sequence↗
PubMed2002

Aspartic proteinases in disease: a structural perspective.

The aspartic proteinases are a family of enzymes involved in a number of important biological processes. In animals the enzyme renin has a hypertensive action through its role in the renin-angiotensin system. The retroviral aspartic proteinases, such as the HIV proteinase, are essential for maturation of the virus particle and inhibitors have a proven therapeutic record in the treatment of AIDS. The lysosomal aspartic proteinase cathepsin D has been implicated in tumorigenesis and the stomach enzyme pepsin, which plays a major physiological role in hydrolysis of acid-denatured proteins, is responsible for much of the tissue damage in peptic ulcer disease. Since aspartic proteinases also play major roles in amyloid disease, malaria and common fungal infections such as candidiasis, inhibitors to these enzymes are much sought after as potential therapeutic agents. In all aspartic proteinases, the catalytic aspartate residues are involved in an intricate arrangement of hydrogen bonds involving a solvent molecule which is presumed to be water. The catalytic mechanism is thought to involve nucleophilic attack of the active site water molecule on the scissile bond carbonyl generating a tetrahedral gem-diol intermediate. The design of inhibitors generally involves the use of short oligopeptides containing a transition state analogue which mimic this tetrahedral intermediate. The application of structure-based drug design to members of the aspartic proteinase family is the main subject of this review.

Amyloidosis↗
PubMed2002

Relationships among severity and duration of clinical mastitis and sire transmitting abilities for somatic cell score, udder type traits, productive life, and protein yield.

The objective of this study was to determine the relationships among severity and duration of clinical mastitis during first and second lactation and sire transmitting abilities for somatic cell score, udder type traits, productive life, and protein yield. Recording of clinical episodes began at first parturition for 1704 Holstein cows (in six Pennsylvania herds and one Nebraska herd) and continued into second lactation for 1055 of these cows. A total of 456 cows (sired by 168 bulls) had at least one clinical episode during first lactation, and 230 cows (sired by 100 bulls) had at least one clinical episode during second lactation. A severity code from 1 (normal milk) to 5 (acute systemic mastitis) was assigned daily (for up to 30 d after detection) to all quarters that had clinical mastitis. Only the severity codes for the first clinical episode to occur during first and second lactation are considered here. The initial and maximum severity codes, as well as the natural logarithms of both the sum of severity codes that were above normal (> 1) and the total days severity codes were above normal were regressed on herd (a classification variable), age at first calving, days in milk at clinical detection, and sire transmitting abilities taken one at a time. Linear and nonlinear effects were estimated for sire transmitting abilities. Separate analyses were conducted on dependent variables that considered severity and duration of clinical mastitis from: all organisms, coagulase-negative staphylococci, coliform species, streptococci other than Streptococcus agalactiae, and the most common environmental organisms (coliform species and streptococci other than Streptococcus agalactiae). Daughters of sires that transmit the lowest somatic cell score had the least severe and shortest clinical episodes from environmental organisms during first lactation. Selection for lower somatic cell score may reduce the severity and duration of clinical episodes from environmental organisms during first lactation.

Animals↗
PubMed2001

A neutron Laue diffraction study of endothiapepsin: implications for the aspartic proteinase mechanism.

Current proposals for the catalytic mechanism of aspartic proteinases are largely based on X-ray structures of bound oligopeptide inhibitors possessing nonhydrolyzable analogues of the scissile peptide bond. However, the positions of protons on the catalytic aspartates and the ligand in these complexes have not been determined with certainty. Thus, our objective was to locate crucial protons at the active site of an inhibitor complex since this will have major implications for a detailed understanding of the mechanism of action. We have demonstrated that high-resolution neutron diffraction data can be collected from crystals of the fungal aspartic proteinase endothiapepsin bound to a transition state analogue (H261). The neutron structure of the complex has been refined at a resolution of 2.1 A to an R-factor of 23.5% and an R(free) of 27.4%. This work represents the largest protein structure studied to date by neutron crystallography at high resolution. The neutron data demonstrate that 49% of the main chain nitrogens have exchanged their hydrogen atoms with D2O in the mother liquor. The majority of residues resisting exchange are buried within core beta-sheet regions of the molecule. The neutron maps confirm that the protein has a number of buried ionized carboxylate groups which are likely to give the molecule a net negative charge even at very low pH, thereby accounting for its low pI. The functional groups at the catalytic center have clearly undergone H-D exchange despite being buried by the inhibitor occupying the active site cleft. Most importantly, the data provide convincing evidence that Asp 215 is protonated and that Asp 32 is the negatively charged residue in the transition state complex. This has an important bearing on mechanistic proposals for this class of proteinase.

Aspartic Acid↗
PubMed2001

The x-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with substrate and three inhibitors.

The structures of 5-aminolaevulinic acid dehydratase (ALAD) complexed with substrate (5-aminolaevulinic acid) and three inhibitors: laevulinic acid, succinylacetone and 4-keto-5-aminolaevulinic acid, have been solved at high resolution. The ligands all bind by forming a covalent link with Lys263 at the active site. The structures define the interactions made by one of the two substrate moieties that bind to the enzyme during catalysis. All of the inhibitors induce a significant ordering of the flap covering the active site. Succinylacetone appears to be unique by inducing a number of conformational changes in loops covering the active site, which may be important for understanding the co-operative properties of ALAD enzymes. Succinylacetone is produced in large amounts by patients suffering from the hereditary disease type I tyrosinaemia and its potent inhibition of ALAD also has implications for the pathology of this disease. The most intriguing result is that obtained with 4-keto-5-amino-hexanoic acid, which seems to form a stable carbinolamine intermediate with Lys263. It appears that we have defined the structure of an intermediate of Schiff base formation that the substrate forms upon binding to the P-site of the enzyme.

Aminolevulinic Acid↗
PubMed2001

Site-directed mutagenesis and X-ray crystallography of the PQQ-containing quinoprotein methanol dehydrogenase and its electron acceptor, cytochrome c(L).

Two proteins specifically involved in methanol oxidation in the methylotrophic bacterium Methylobacterium extorquens have been modified by site-directed mutagenesis. Mutation of the proposed active site base (Asp303) to glutamate in methanol dehydrogenase (MDH) gave an active enzyme (D303E-MDH) with a greatly reduced affinity for substrate and with a lower activation energy. Results of kinetic and deuterium isotope studies showed that the essential mechanism in the mutant protein was unchanged, and that the step requiring activation by ammonia remained rate limiting. No spectrally detectable intermediates could be observed during the reaction. The X-ray structure, determined to 3 A resolution, of D303E-MDH showed that the position and coordination geometry of the Ca2+ ion in the active site was altered; the larger Glu303 side chain was coordinated to the Ca2+ ion and also hydrogen bonded to the O5 atom of pyrroloquinoline quinone (PQQ). The properties and structure of the D303E-MDH are consistent with the previous proposal that the reaction in MDH is initiated by proton abstraction involving Asp303, and that the mechanism involves a direct hydride transfer reaction. Mutation of the two adjacent cysteine residues that make up the novel disulfide ring in the active site of MDH led to an inactive enzyme, confirming the essential role of this remarkable ring structure. Mutations of cytochrome c(L), which is the electron acceptor from MDH was used to identify Met109 as the sixth ligand to the heme.

Alcohol Oxidoreductases↗
PubMed2001

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors.

The structures of 5-aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4-oxosebacic acid and 4,7-dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P-side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A- and P-side substrates). The most intriguing result is the novel finding that 4,7-dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P-side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A-side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A- and P-sites is proposed.

Catalytic Domain↗
PubMed2001

Pilot study of electrical stimulation on median nerve in comatose severe brain injured patients: 3-month outcome.

PRIMARY OBJECTIVE: To determine if electrical stimulation (ES) benefits (waking time, 3-month outcomes) treated coma patients. RESEARCH DESIGN: Double blind randomized-controlled study. METHODS AND PROCEDURES: Ten coma patients; six treatment and four controls, using the 'Respond Select' by EMPI. EXPERIMENTAL INTERVENTIONS: Treatment group received radial nerve ES applied in 300 ms intermittent pulses at 40 Hz, 15-20m A 8 hours a day up to 14 days of coma; control group received sham stimulation. MAIN OUTCOMES AND RESULTS: ES group emerged from coma mean 2 days earlier than controls, although this result was not statistically significant. At 3 months post-injury, there was no group difference in Glasgow Outcome Scale, although the ES group had improved function over controls as measured by the FIM/FAM (mean of 114 and 64.5, respectively, n.s.). CONCLUSIONS: These data show an interesting trend, although statistical power was limited in this small pilot study, suggesting the need for a larger trial.

Adult↗
PubMed2001

Accidents and mishaps in anesthesia: how they occur; how to prevent them.

The problem of safety and accidents prevention is a primary issue in modern anesthesia. Throughout the last twenty years, much effort has been made to assess the causes of mishaps, introducing new technology and safer drugs. The author presents a well known model to describe factors affecting accidents causality, proposing old and new strategies to ensure safety during the everyday practice.

Anesthesia↗
PubMed2000

X-ray structures of five renin inhibitors bound to saccharopepsin: exploration of active-site specificity.

Saccharopepsin is a vacuolar aspartic proteinase involved in activation of a number of hydrolases. The enzyme has great structural homology to mammalian aspartic proteinases including human renin and we have used it as a model system to study the binding of renin inhibitors by X-ray crystallography. Five medium-to-high resolution structures of saccharopepsin complexed with transition-state analogue renin inhibitors were determined. The structure of a cyclic peptide inhibitor (PD-129,541) complexed with the proteinase was solved to 2.5 A resolution. This inhibitor has low affinity for human renin yet binds very tightly to the yeast proteinase (K(i)=4 nM). The high affinity of this inhibitor can be attributed to its bulky cyclic moiety spanning P(2)-P(3)' and other residues that appear to optimally fit the binding sub-sites of the enzyme. Superposition of the saccharopepsin structure on that of renin showed that a movement of the loop 286-301 relative to renin facilitates tighter binding of this inhibitor to saccharopepsin. Our 2.8 A resolution structure of the complex with CP-108,420 shows that its benzimidazole P(3 )replacement retains one of the standard hydrogen bonds that normally involve the inhibitor's main-chain. This suggests a non-peptide lead in overcoming the problem of susceptible peptide bonds in the design of aspartic proteinase inhibitors. CP-72,647 which possesses a basic histidine residue at P(2), has a high affinity for renin (K(i)=5 nM) but proves to be a poor inhibitor for saccharopepsin (K(i)=3.7 microM). This may stem from the fact that the histidine residue would not bind favourably with the predominantly hydrophobic S(2) sub-site of saccharopepsin.

Aspartic Acid Endopeptidases↗
PubMed2000

Video teleconferencing with realistic simulation for medical education.

This report describes how realistic patient simulation can be used with video teleconferencing to conduct long-distance clinical case discussions with realistic re-enactments of critical events. By observing what appears to be a real procedure unfolding in real time, it is intended that audience members will better learn and appreciate the lessons from conferences. A commercially available mannequin simulator and video teleconferencing technology were used in nine sessions between a free-standing simulation center and different conference sites throughout the U.S. Transmission was via high-speed telephone lines. In each conference, a clinical scenario was simulated on a screen. Audience members asked questions of a live simulated "patient" and family and later advised the care team on routine treatments and management of urgent clinical problems that arose during management of the mannequin simulator in a highly realistic clinical setting. Ninety-eight percent of respondents from one audience of 150 (response rate 60%) judged the quality of the presentation as "very good or excellent." In response to the statement that "the educational value of the presentation was much greater than that of a standard case conference," 95% scored 4 or 5 on a five-point Likert scale (where 5 is highest agreement). While all conferences were conducted successfully, there were instances of technical challenge in using teleconferencing technology. Technical information about the teleconferencing system and scenario preparation, contingency planning for failures, and other details of using this new teaching modality are described. Although audiences were enthusiastic in their response to this approach to clinical case conferences, further study is needed to assess the added value of interactive simulation for education compared to standard conference formats.

Education, Medical↗
PubMed2000

MAD analyses of yeast 5-aminolaevulinate dehydratase: their use in structure determination and in defining the metal-binding sites.

MAD experiments attempting to solve the structure of 5--aminolaevulinic acid dehydratase using Zn and Pb edges are described. The data obtained proved insufficient for a complete structure solution but were invaluable in subsequent identification of metal-binding sites using anomalous difference Fourier analyses once the structure of the enzyme had been solved. These sites include the highly inhibitory substitution of an enzymic cofactor Zn(2+) ion by Pb(2+) ions, which represents a major contribution towards understanding the molecular basis of lead poisoning. The MAD data collected at the Pb edge were also used with isomorphous replacement data from the same Pb co-crystal and a Hg co-crystal to provide the first delineation of the enzyme's quaternary structure. In this MADIR analysis, the Hg co-crystal data were treated as native data. Anomalous difference Fouriers were again used, revealing that Hg(2+) had substituted for the same Zn(2+) cofactor ion as had Pb(2+), a finding of fundamental importance for the understanding of mercury poisoning. In addition, Pt(2+) ions were found to bind at the same place in the structure. The refined structures of the Pb- and the Hg-complexed enzymes are presented at 2.5 and 3.0 A resolution, respectively.

Absorptiometry, Photon↗
PubMed2000

A preliminary neutron Laue diffraction study of the aspartic proteinase endothiapepsin.

Until now, no aspartic proteinase has been subjected to a successful neutron diffraction analysis, owing to the limited size of the crystals. However, the recent development of the neutron Laue technique at ILL and EMBL (Grenoble) has allowed the collection of data to 2.2 A on a complex of endothiapepsin with a transition-state analogue. The objective is to define the positions of the protons at the active site by refinement using the neutron data. In line with work on serine proteinases, where neutron diffraction has provided some of the most definitive data on the catalytic mechanism, it is expected that this work will have a major significance for studies of the aspartic proteinase enzymes.

Aspartic Acid Endopeptidases↗
PubMed2000

Purification, co-crystallization and preliminary X--ray analysis of the natural aspartic proteinase inhibitor IA3 complexed with saccharopepsin from Saccharomyces cerevisiae.

The vacuolar aspartic proteinase from baker's yeast, saccharopepsin, has been co-crystallized with its natural inhibitor I(A)3, found in the cytosol. The I(A)3-saccharopepsin complex crystals belong to the space group P6(2)22, with unit-cell parameters a = b = 192.1, c = 59. 80 A and one molecule per asymmetric unit. The initial X-ray analysis of the complex indicates that the crystals diffract to 5.0 A, similar to native saccharopepsin crystals. This is probably a consequence in part of glycosylation of the native saccharopepsin. Full structural analysis of the complex crystal is in progress.

Aspartic Acid Endopeptidases↗
PubMed2000

A computerized mastitis decision aid using farm-based records: an artificial neural network approach.

A computer module was developed and tested that used field survey and Dairy Herd Improvement Association (DHIA) data to broadly classify bacterial causes of mastitis in dairy herds. Further development of the computer model could aid interpretation of DHIA data by dairy record processing centers and herd consultants. This diagnostic module was developed with an artificial neural network, a technology that processes complex data in a manner similar to human brain function. Information describing herd management practices, quarter milk samples, and monthly DHIA data was collected from Pennsylvania dairy herds with moderate to high somatic cell counts. This information was used to develop or train an artificial neural network model that discriminated among four categories of bacterial organisms (contagious, environmental, no significant growth, and other) associated with clinical and subclinical mastitis. After training the model, new DHIA and management data were presented to the model to assess its ability to classify bacteriological etiology. When the artificial neural network was used, the probabilities of diagnosing the bacteriologic status from three randomly selected cow groups and from new untested herds ranged from 57 to 71%. Performance of the artificial neural network model was best in herds with higher frequency of minor and contagious pathogens. Prediction results for the same test data with linear discriminant analysis were less successful, ranging from 42 to 57%.

Animals↗