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Biomedical subjects

L Sawyer

Publications and source records attributed to L Sawyer.

At least 37 records · Page 2Linked to original sources
PubMed1998

Distribution of HIV type 1 (HIV-1) in blood components: detection and significance of high levels of HIV-1 associated with platelets.

BACKGROUND: Although inactivation of enveloped viruses transmitted by plasma derivatives has been successful, no methods for virus inactivation or removal have been established for platelet concentrates or red cell (RBC) components. Relatively little is known regarding the extent or significance of virus interactions with the cellular constituents in these components. STUDY DESIGN AND METHODS: Units of whole blood were collected from six HIV type 1 (HIV-1)-positive, asymptomatic individuals and separated into peripheral blood mononuclear cells (PBMNCs), cell-free plasma, white cell-reduced platelet concentrate, and white cell-reduced RBCs. DNA and RNA polymerase chain reaction and virus culture methods were used to study the compartmentalization of HIV-1 immediately after component preparation and after storage. RESULTS: As expected, HIV DNA and infectious virus were detected in fresh blood and in PBMNCs, and virion-associated RNA was detected in fresh plasma from all six donors. The levels of viral nucleic acids in these preparations remained relatively stable with 4 degrees C storage, whereas infectivity of PBMNCs was rapidly lost. Washed RBCs tested negative for HIV in all assays at all time points. Platelets retained high levels of HIV RNA (but not infectivity) after extensive washing, as well as after storage at 4 and 22 degrees C. High-level platelet-associated HIV-1 was also demonstrated in samples collected during early seroconversion. Periseroconversion and postseroconversion levels of platelet-associated HIV-1 correlated with the level of plasma viremia and with the rate of progression to AIDS. Cell-free virus from donor plasma and tissue culture fluid rapidly and firmly attached to platelets from noninfected donors. Infectivity of tissue culture virus bound to platelets was demonstrated in vitro. CONCLUSION: Significant levels of HIV-1 are associated with platelets during all stages of infection. Platelet-associated HIV could either mediate virus clearance or facilitate virus dissemination and expanded tropism. Finally, virus inactivation research must address virus associations with platelets.

Blood Platelets↗
PubMed1997

Synthetic, structural and biological studies of the ubiquitin system: synthesis and crystal structure of an analogue containing unnatural amino acids.

Ubiquitin is a 76-amino acid protein involved in the targeting for destruction of proteins in the cell. The protein can readily be synthesized chemically affording an extra dimension to studies of protein stability. Ubiquitin with various modifications to the hydrophobic core has been synthesized. In particular, two core amino acids have been replaced by aminobutyric acid (Val-26) and norvaline (for Ile-30) and the product crystallized. The refined crystal structure shows an overall contraction of the molecule and the side chain of Nva-30 rotates relative to Ile-30. However, the side chain rotation is not sufficient to compensate for the effect of the loss of the methyl group and hence a small cavity is introduced into the structure, which decreases the stability of the protein. The biological behaviour of the modified protein is unaltered. The observed changes in stability are of the magnitude expected for the removal of methyl groups from the hydrophobic core of a protein. Interestingly, the effect appears to be independent of the position of the removed methyl group. The intact structure, but not its stability, is important for recognition by the biological conjugating system.

Aminobutyrates↗
PubMed1997

Bovine beta-lactoglobulin at 1.8 A resolution--still an enigmatic lipocalin.

BACKGROUND: beta-Lactoglobulin (beta-Lg) is the major whey protein in the milk of ruminants and many other mammals. Its function is not known, but it undergoes at least two pH-dependent conformational changes which may be important. Bovine beta-Lg crystallizes in several different lattices, and medium-resolution structures of orthorhombic lattice Y and trigonal lattice Z have been published. Triclinic lattice X and lattice Z crystals grow at pH values either side of the pH at which one of the pH-induced conformational changes occurs. A full understanding of the structure is needed to help explain both the conformational changes and the different denaturation behaviour of the genetic variants. RESULTS: We have redetermined the structure of beta-Lg lattice Z at 3.0 A resolution by multiple isomorphous replacement and have partially refined it (R factor = 24.8%). Using the dimer from this lattice Z structure as a search model, the triclinic crystal form grown at pH 6.5 (lattice X) has been solved by molecular replacement. Refinement of lattice X at 1.8 A resolution gave an R factor of 18.1%. The structure we have determined differs from previously published structures in several ways. CONCLUSIONS: Incorrect threading of the sequence in the published structures of beta-Lg affects four of the nine beta strands. The basic lipocalin fold of the polypeptide chain is unchanged, however. The relative orientation of the monomers in the beta-Lg dimer differs in the two lattices. On raising the pH, there is a rotation of approximately 5 degrees, which breaks a number of intersubunit hydrogen bonds. It is not yet clear, however, why the stability of the structure should depend so heavily upon the external loop around residue 64 or the beta strand with the free thiol, each of which shows genetic variation.

Amino Acid Sequence↗
PubMed1997

Effects of metal salts on poly(DL-lactide-co-glycolide) polymer hydrolysis.

The effects of encapsulated metal salts on poly(DL-lactide-co-glycolide) (PLGA) water uptake and degradation properties were investigated in this work. Salts of varying aqueous solubility characteristics were incorporated into PLGA films either as particles or by codissolution in polymer solutions. Polymer films were characterized with respect to the kinetics of water uptake, morphology changes, degradation, and weight loss after hydration. It was found that these properties are strongly influenced by the presence and nature of encapsulated salts. Effects range from minor changes in water uptake profile with no significant difference in degradation kinetics to major alterations in water uptake kinetics together with a several-fold decrease in the polymer degradation rate. Possible mechanistic explanations for the observed effects are discussed.

Biocompatible Materials↗
PubMed1997

High-level expression of bovine beta-lactoglobulin in Pichia pastoris and characterization of its physical properties.

Bovine beta-lactoglobulin (BLG) variant A has been expressed in the methylotropic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the alpha-mating factor prepro-leader peptide from Saccharomyces cerevisiae. P. pastoris Mut+ transformants were obtained by single cross-over integration of the BLG-containing vector into the AOX1 locus. In a fed-batch fermenter, a cell density of approximately 300 mg/ml was achieved by controlled glycerol feeding for a total of 24 h. After 72 h of methanol induction, the secreted BLG reached levels of > 1 g/l. The secreted protein could be purified to homogeneity by ion-exchange chromatography. Amino-terminal sequencing of the secreted BLG revealed that the Glu-Ala spacer repeats inserted between the mature protein and the alpha-factor prepro-leader were still present. The purified protein was characterized by a number of methods, including CD spectroscopy, guanidine-HCl unfolding, crystallization and two-dimensional 1H-NMR spectroscopy. By all of these measures, the physical characteristics of recombinant BLG were indistinguishable from those of the native purified bovine BLG, making it useful as a model for protein folding and other biophysical studies.

Amino Acid Sequence↗
PubMed1996

Expression and secretion of recombinant ovine beta-lactoglobulin in Saccharomyces cerevisiae and Kluyveromyces lactis.

High expression and secretion of recombinant ovine beta-lactoglobulin has been achieved in the yeast Kluyveromyces lactis. The yield of beta-lactoglobulin is 40-50 mg per litre of culture supernatant and accounts for approx. 72% of the total secreted protein. Constitutive expression is under the control of the Saccharomyces cerevisiae phosphoglycerate kinase promoter from an intronless version of the beta-lactoglobulin gene. Secretion is specified by the ovine protein's own signal sequence. this system, coupled to an efficient and novel recovery protocol, allows 30 mg of pure protein to be isolated from a typical 1 litre culture. The protein is virtually indistinguishable from beta-lactoglobulin conventionally purified from sheep milk by its behaviour in native PAGE and SDS/PAGE, reactivity to antibodies, CD, fluorescence spectroscopy and N-terminal sequencing. Attempts to achieve a similar expression and secretion system in the yeast S. cerevisiae met with only limited success, although it was found that heat-shock treatment modestly increased the yield up to approx. 3-4 mg per litre of culture supernatant. Site-directed mutagenesis showed that secretion in S. cerevisiae depended upon correct formation of the two disulphide bonds present in beta-lactoglobulin.

Amino Acid Sequence↗
PubMed1996

International population and development: the United Nations' Cairo Action Plan for women's health.

The report of the International Conference on Population and Development (Cairo Action Document) was sponsored by 180 national governments and 1,200 nongovernmental organizations during the United Nations' conference on women, population, and development in Cairo, Egypt 1994. Many international conferences preceded the one in Cairo but it was there that women participated at an unprecedented level in shaping the debate. The resulting document represents a quantum leap for women's health. Nurses can play a major role in using the principles in the Cairo Action Document by empowering women and enhancing their health care. To invite dialogue, the authors provide an analysis of the Cairo Plan from a nursing perspective and identify three paradoxes that may hinder progress: Universal values and local cultures, global policy and local implementation, and national development and women's empowerment. Clinicians, scholars, and members of professional organizations are asked to consider the assumptions that underlie the recommendations for action and to propose ways to resolve the problems that may evolve during implementation.

Egypt↗
PubMed1995

Substrate binding and carboxylation by dethiobiotin synthetase--a kinetic and X-ray study.

BACKGROUND: The vitamin biotin is a ubiquitous prosthetic group of carboxylase and transcarboxylase enzymes. Biotin biosynthesis occurs by similar pathways in microorganisms and plants. The penultimate step in biotin biosynthesis, catalyzed by dethiobiotin synthetase (DTBS), involves a unique ATP-dependent N-carboxylation, resulting in formation of the ureido ring function of dethiobiotin. The first two steps of dethiobiotin formation, which is a complex, multistep enzymatic reaction, have been elucidated by a combination of X-ray crystallography and kinetic methods. RESULTS: The first step in catalysis by DTBS is the formation of an enzyme-substrate complex and the second is the enzymatic carboxylation of the bound substrate. Both steps are Mg2+ dependent. The kinetic constants in the presence and absence of Mg2+ have been measured and a set of X-ray structures determined at different stages of the reaction. The conformational changes in the active site of the enzyme, induced by Mg2+, substrate binding and substrate carboxylation, have been monitored crystallographically and are discussed. Sulfate ions bound to DTBS may mimic the behaviour of the alpha- and gamma-phosphates of ATP in Mg2+ binding and in the subsequent steps of the reaction. CONCLUSIONS: Mg2+ is an essential cation for both substrate binding and carbamate formation by DTBS, when sulfate is present. The conformational changes induced at the active site in the DTBS-substrate complex, when Mg2+ is present, are small yet highly significant and serve to optimize the interactions between substrate and enzyme. DTBS is active as a homodimer and the substrate-binding site straddles both monomers in the dimer. The carboxylation site is unambiguously identified as the N-7 amino group of the substrate, rather than the N-8 amino group, as previously suggested. The elongated nucleotide-binding loop (the P loop) binds both ATP and substrate in a manner which suggests that this feature may be of wider importance.

Acylation↗
PubMed1995

Arachidonic acid binds to apolipoprotein D: implications for the protein's function.

The lipocalin apolipoprotein D (ApoD) is associated in human plasma with lecithin-cholesterol acyl transferase. It has also been found in high concentration in the fluid of gross cystic disease of the mammary gland. Using protein fluorescence quenching, it is shown that ApoD binds arachidonic acid (Ka of 1.6 x 10(8) M-1) and as previously known progesterone (Ka of 2.5 x 10(6) M-1), but neither cholesterol nor any of the other prostanoid molecules examined had measurable affinity. This specific binding of arachidonate, also observable directly, suggests a role for ApoD in the mobilisation of arachidonic acid, and hence prostaglandin synthesis.

Apolipoproteins↗
PubMed1995

Matching versus cultural competence in research: methodological considerations.

Researcher/participant matching has been used in nursing research as a strategy for the development of culturally competent knowledge. In this manuscript, the complexities inherent in matching in nursing research are discussed. In raising and discussing issues and questions related to matching, we argue that although matching may be one of the strategies used to generate culturally competent knowledge, it is frequently too complex for operationalization. Therefore, we propose that researchers reflect on questions related to their knowledge of culture, their cultural sensitivity, and the nature of collaboration during every phase of the research. Reflecting on and responding to these questions in every research project may facilitate the development of more culturally competent knowledge.

Cultural Diversity↗
PubMed1995

The compensation of patients injured in clinical trials.

The problem of 'no fault' compensation for patients who suffer adverse effects as a result of their participation in clinical trials is discussed in the light of the guidelines issued by the Association of the British Pharmaceutical Industry (ABPI) and our recent experiences in reviewing protocols submitted to the local ethics of surgical research sub-committee. We have found a variety of qualifications being applied by pharmaceutical firms which are not in the spirit of the guidelines, let alone the interests of the patient, and we suggest a means whereby the patients can be assured of fair treatment in the event of 'no fault' injury.

Clinical Trials as Topic↗
PubMed1994

Mechanistic implications and family relationships from the structure of dethiobiotin synthetase.

BACKGROUND: Biotin is the vitamin essential for many biological carboxylation reactions, such as the conversion of acetyl-coenzyme A (CoA) to malonyl-CoA in fatty acid biosynthesis. Dethiobiotin synthetase (DTBS) facilitates the penultimate, ureido ring closure in biotin synthesis, which is a non-biotin-dependent carboxylation. DTBS displays no sequence similarity to any other protein in the database. Structural studies provide a molecular insight into the reaction mechanism of DTBS. RESULTS: We present the structure of DTBS refined to 1.80 A resolution with an R-factor of 17.2% for all terms plus unrefined data on the binding of the substrate, 7,8-diaminopelargonic acid and the product, dethiobiotin. These studies confirm that the protein forms a homodimer with each subunit folded as a single globular alpha/beta domain. The presence of sulphate ions in the crystals and comparisons with the related Ha-ras-p21 oncogene product are used to infer the ATP-binding site, corroborated by the difference electron density for the ATP analogue AMP-PNP. CONCLUSIONS: This study establishes that the enzyme active site is situated at the dimer interface, with the substrate binding to one monomer and ATP to the other. The overall fold of DTBS closely resembles that of three other enzymes, adenylosuccinate synthetase (purA), Ha-ras-p21, and nitrogenase iron protein, that are unrelated by sequence or function, indicating that DTBS is a member of a diverse family of enzymes.

Adenosine Triphosphate↗
PubMed1994

Structural analysis and classification of lipocalins and related proteins using a profile-search method.

A three-dimensional profile method of detecting amino-acid sequences compatible with the tertiary structure of any protein has been applied to the lipocalin family of 8-stranded beta-barrels. Profiles derived from six well-resolved lipocalin crystal structures were used to search a comprehensive, non-redundant protein sequence database. Each profile identified a sub-group of lipocalin sequences although no single profile was sufficient to identify the whole family. The alpha-1-acid glycoprotein sub-family was not identified by any lipocalin profile, indicating that known sequence differences in otherwise well conserved regions of these proteins may be reflected in structural differences. The predicted similarity between the beta-lactoglobulin and alpha-2u-globulin structures was much more marked than the similarity between their sequences, and alpha-1-microglobulin sequences were found to be compatible with the structure of epididymal retinoic acid binding protein which has an additional long C-terminal helix. Proteins of unknown structure which were predicted to be compatible with the lipocalin fold include a human mucin. In cases where a large protein family of low overall sequence similarity contains a small number of known structures, this technique can be useful in determining or confirming subtle structural relationships between family members.

Amino Acid Sequence↗
PubMed1994

Inadequacies in Health Recommendations Provided for International Travelers by North American Travel Health Advisors.

The rise of international travel has increased the need for more, improved travel advice from physicians and public health facilities. The quality of the health information given has not been examined on a large-scale basis by many studies, however. Surveys in Canada, Switzerland, and the United States, for example, report that only 20% to 50% of practitioners could give accurate information regarding immunization and prophylaxis about travel-related disease. Anonymous surveys were sent to 1165 American and 96 Canadian public health units and travel clinics. Using five scenarios on travel to developing countries, each source was asked to complete a standardized form giving their recommendations for immunization, antimalarials, travelers' diarrhea, and other travel issues. Of the American respondents, 60% were physicians equally distributed among private practice, university, and corporate clinics; nurses comprised 75% of the Canadian respondents, primarily from public health clinics. The number of travelers counseled per year ranged from 3 to 40,000 (American mean, 448; Canadian mean, 2180). Depending on the scenario, 20 to 75% of the immunization groups recommended were inadequate or inappropriate: most frequently, lack of tetanus/polio boosters; indiscriminant use of yellow fever/cholera vaccines; haphazard advice about meningococcal, rabies, and typhoid vaccines; and a lack of consideration of measles in young adults. Of the antimalarial recommendations given, 20 to 60% were incorrect, including prescribing medication for nonrisk areas, failure to recognize chloroquine-resistant areas, and failure to understand the use of, or contraindications to, mefloquine. Frequently, acclimatization, altitude sickness, sunscreens, and safe-sex issues were omitted. The prevention and treatment of travelers' diarrhea were adequately covered, however. Pre-travel advice given by North American health advisors shows a considerable variability in the accuracy and extent necessary for effective travel disease prevention and treatment. Despite the growing efforts to further educate those responsible, higher quality of health advice needs to become a priority.

Journal Article↗
PubMed1994

In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity.

A method is described that allows for the improvement of antibody affinity. This method, termed complementary-determining region (CDR) walking, does not require structural information on either antibody or antigen. Complementary-determining regions are targeted for random mutagenesis followed by selection for fitness, in this case increased binding affinity, by the phage-display approach. The current study targets a human CD4-binding-site anti-gp120 antibody that is potently and broadly neutralizing. Evolution of affinity of this antibody demonstrates in this case that affinity can be increased while reactivity to variants of human immunodeficiency virus type 1 is broadened. The neutralizing ability of this antibody is improved, as assayed with laboratory and primary clinical isolates of human immunodeficiency virus type 1. The ability to produce human antibodies of exceptional affinity and broad neutralizing ability has implications for the therapeutic and prophylactic application of antibodies for human immunodeficiency virus type 1 infection.

Amino Acid Sequence↗