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Biomedical subjects

A Boyd

Publications and source records attributed to A Boyd.

At least 19 recordsLinked to original sources
PubMed1997

Expression of bcl-2 in lichen planus, acute graft-versus-host disease, and erythema multiforme.

The bcl-2 gene family plays a significant role in the propagation of cell survival and tissue modeling. Bcl-2 was originally described in follicular lymphomas and is associated with the suppresion of cellular apoptosis. Evaluation for this protein has been performed for a variety of benign and malignant cutaneous tumors but not to any significant extent on inflammatory disorders. Therefore, we stained biopsy specimens from diseases with interface inflammation (lichen planus, acute graft-versus-host disease, and erythema multiforme) for Bcl-2. Epidermal expression of this protein was minimal for all three diseases; however, lymphocytes stained prominently in lichen planus. The data suggest that Bcl-2 is not prominently involved in the epidermal changes in these diseases. The role of other members of this oncogene family in interface dermatitis still needs to be elucidated.

Erythema Multiforme↗
PubMed1996

Expression of active, human lysyl oxidase in Escherichia coli.

Lysyl oxidase (LO) is a copper amine oxidase of the extracellular matrix which initiates covalent cross-linking in collagens and elastin. Human LO was expressed in Escherichia coli. At 37 degrees C, large amounts of protein were obtained, but in the form of insoluble aggregates. Lowering the growth temperature, and reducing the amount of inducer, resulted in the production of soluble LO, which was active on a degrees [3H]lysine-labeled elastin substrate. LO was also targeted to the periplasm as a fusion protein with the pelb signal peptide. The periplasmic enzyme was soluble, active and inhibited by beta-aminopropionitrile. Production of the carbonyl co-factor is therefore not a limitation in the expression of active LO in bacteria.

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

Chiral kinetics and dynamics of ketorolac.

It has been shown that the analgesic and cyclooxygenase inhibitor activity of ketorolac tromethamine (KT), which is marketed as the racemic mixture of (-)S and (+)R enantiomers, resides primarily with (-)S ketorolac and that the ulcerogenic activity of this agent also resides in (-)S ketorolac. Resolution of individual enantiomers for analysis in plasma samples has been accomplished by two methods: derivatization to form diastereomers that are separated by HPLC, or direct HPLC using a chiral phase column. When mice and rats were given oral solutions of (-)S and (+) KT, it was found that the kinetics and interconversion of the enantiomers were species and dose dependent. Interconversion was higher in mice than in rats; when (-)S KT was administered, 71% of the area under the concentration-time curve (AUC) was due to (+)R ketorolac in mice, compared with 12% in rats. More interconversion was observed at higher doses; the percent of AUC due to (-)S ketorolac when (+)R KT was administered increased from 12% to 25% in mice and from 2% to 8% in rats. In general, more interconversion occurred from (-)S to (+)R ketorolac in the animal studies. Human subjects were given single oral solution doses of racemic KT (30 mg), (-)S KT (15 mg), and (+)R KT (15 mg). The plasma concentrations of (-)S ketorolac were lower than (+)R ketorolac at all sample times after racemic KT (22% of the AUC was due to (-)S ketorolac). When (+)R KT was administered, (-)S ketorolac was not detectable and interconversion was essentially 0%. When (-)S KT was administered, significant levels of (+)R ketorolac were detectable and interconversion was 6.5%. After all doses, plasma half-life was shorter and clearance greater for (-)S ketorolac than for (+)R ketorolac. Thus, in humans very little or no interconversion of (+)R to (-)S was observed, and interconversion of (-)S to (+)R was minimal (6.5%). These data demonstrate that the kinetics and interconversion of the enantiomers of ketorolac is different in animals and humans as well as from most other NSAIDs. This may be due to more rapid excretion or metabolism of (-)S ketorolac and a different mechanism of interconversion.

Animals↗
PubMed1996

An indirect (derivatization) and a direct HPLC method for the determination of the enantiomers of ketorolac in plasma.

An indirect and a direct HPLC method for the quantification of the (R) and (S) enantiomers of ketorolac are described here. The indirect method employs the chiral amine (+)-R-1-(1-naphthyl)ethylamine to form disastereomeric amides; separation of the disastereomeric derivates is achieved by normal-phase HPLC with a mobile phase of ethyl acetate-hexane. The direct method uses a C18 solid-phase extraction column to extract ketorolac enantiomers from plasma; the reconstituted extract is then injected onto an alpha 1-acid glycoprotein chiral column using a mobile phase of isopropanol-phosphate buffer (0.05 M; pH 5.5). Both methods are reproducible, accurate, and stereospecific, and both have equivalent quantification limits (0.02 microgram ml-1 of plasma for each enantiomer), ranges (0.02-2.0 micrograms per aliquot of plasma), precision (% relative standard deviations of < or = 10.5% and < or = 10.8% for (R)- and (S)-ketorolac respectively), and accuracy (mean recoveries of 88.4-110% and 90.1-110% for (R)- and (S)-ketorolac respectively). Results of analyses of clinical samples by the two methods showed excellent agreement (slope near 1.0 and coefficients of correlation between 0.9740 and 0.9864 for both enantiomers).

Analgesics, Non-Narcotic↗
PubMed1996

Public perceptions of brain tumours in Scotland: the need for access to appropriate information.

The aim of the study was to evaluate knowledge and perceptions of brain tumours in a heterogeneous, highly educated group of adults living in Edinburgh. Two hundred and forty four respondents answered a questionnaire about the causes, effects and treatments of brain tumours. There were misconceptions about several clinical and biological aspects of intracranial tumours although most respondents were aware of the bleak prognosis associated with many neoplasms. These results, from the intellectual elite of Scotland's capital, suggest that access to information about brain tumours should be readily available. Five leaflets, covering various aspects of brain tumours, that are available through the recently established Scottish charity Brain Tumour Action seem well formulated to address areas of concern and misconception.

Adult↗
PubMed1995

Localization of a protein A-tagged Kex2 protein to the vacuole of Saccharomyces cerevisiae allows rapid purification of vacuolar membranes.

We have previously reported an immunoisolation procedure which allows purification of Kex2p-containing Golgi membranes from lysed yeast cells. In order to evaluate the use of tagging procedures in organelle isolation we set out to isolate the same Golgi membrane fraction using a version of the Kex2 protease that had been affinity-tagged at its C-terminus. This protein is found to be localized in the vacuole, providing the basis of a method for the affinity-purification of vacuolar membranes.

Base Sequence↗
PubMed1995

Pseudomonas aeruginosa biofilms: role of the alginate exopolysaccharide.

Pseudomonas aeruginosa synthesizes an exopolysaccharide called alginate in response to environmental conditions. Alginate serves to protect the bacteria from adversity in its surroundings and also enhances adhesion to solid surfaces. Transcription of the alginate biosynthetic genes is induced upon attachment to the substratum and this leads to increased alginate production. As a result, biofilms develop which are advantageous to the survival and growth of the bacteria. In certain circumstances, P. aeruginosa produces an alginate lyase enzyme which cleaves the polymer into short oligosaccharides. This negates the anchoring properties of the alginate and results in increased detachment of the bacteria away from the surface, allowing them to spread and colonize new sites. Thus, both alginate biosynthetic and degradative enzymes are important for the development, maintenance and spread of P. aeruginosa biofilms.

Alginates↗
PubMed1995

Bromocriptine and psychosis: a literature review.

Bromocriptine is an ergot-derived dopamine agonist. Its current uses include the treatment of Parkinson's disease, postpartum ablaction, prolactinomas, acromegaly, and amenorrhea and galactorrhea secondary to neuroleptic use. It is often reported to produce psychiatric side effects such as confusion, hallucinations, and delusions. The literature is reviewed and supports a strong anecdotal relationship between bromocriptine use and psychosis.

Amenorrhea↗
PubMed1995

High sun protection factor sunscreens in the suppression of actinic neoplasia.

BACKGROUND AND DESIGN: A controlled trial was undertaken from December 1987 to December 1990 to test the hypothesis that a strong sunscreen can reduce the number of cancerous and precancerous skin lesions. Candidates were selected from a high-risk population attending either a university- or Veterans Affairs-based dermatology practice in Lubbock, Tex, for a prospective, double-blind, controlled trial of daily application of sunscreen vs placebo over a 2-year period. Participants were asked to volunteer if they had demonstrated premalignant changes (actinic keratoses) or nonmelanoma skin cancer (basal cell carcinoma or squamous cell carcinoma), had continuing sun exposure, and were not using sunscreen on a regular basis. Fifty-three volunteers were initially enrolled in the study, and 37 came for the final 24-month visit. RESULTS: The rate of appearance of new precancerous skin lesions was less for the treatment group than for control subjects. People with darker skin had fewer actinic keratoses, women had fewer lesions than men, and people with fewer lesions at enrollment had fewer lesions during the study. The numbers of new nonmelanoma skin cancers appearing during the study period were too small for statistical analysis. CONCLUSIONS: The regular use of sunscreens can significantly reduce cutaneous neoplasia, as indicated by its suppression of precancerous lesions. A longer and/or larger study would be necessary to demonstrate an effect on malignant lesions.

Adult↗
PubMed1994

Identification of amino acid residues involved in the activity of phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa.

Phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase (PMI-GMP), which is encoded by the algA gene, catalyzes two noncontiguous steps in the alginate biosynthetic pathway of Pseudomonas aeruginosa; the isomerization of D-fructose 6-phosphate to D-mannose 6-phosphate and the synthesis of GDP-D-mannose and PPi from GTP and D-mannose 1-phosphate. Amino acids that are required for the GMP enzyme activity were identified through site-directed mutagenesis of the algA gene. Mutation of Lys-175 to arginine, glutamine, or glutamate produced an enzyme whose Km for D-mannose 1-phosphate was 470-3,200-fold greater than that measured for the wild type enzyme. In addition, these mutant enzymes had a lower Vmax for the GMP activity as compared with the wild type PMI-GMP. These results indicate that Lys-175 is primarily involved in the binding of the substrate D-mannose 1-phosphate, although it is likely that other residues are required for the specificity of binding. Mutation of Arg-19 to glutamine, histidine, or leucine resulted in a 2-fold lower Vmax for the GMP enzyme activity and a 4-7-fold increase in the Km for GTP as compared with the wild type enzyme. Thus, it appears that Arg-19 functions in the binding of GTP. In addition, chymotryptic digestion of PMI-GMP showed that the carboxyl terminus is critical for PMI activity but not for GMP activity. Taken together, these results support the hypothesis that the bifunctional PMI-GMP protein is composed of two independent enzymatic domains.

Alginates↗
PubMed1994

Role of alginate lyase in cell detachment of Pseudomonas aeruginosa.

The exopolysaccharide alginate of Pseudomonas aeruginosa was shown to be important in determining the degree of cell detachment from an agar surface. Nonmucoid strain 8822 gave rise to 50-fold more sloughed cells than mucoid strains 8821 and 8830. Alginate anchors the bacteria to the agar surface, thereby influencing the extent of detachment. The role of the P. aeruginosa alginate lyase in the process of cell sloughing was investigated. Increased expression of the alginate lyase in mucoid strain 8830 led to alginate degradation and increased cell detachment. Similar effects were seen both when the alginate lyase was induced at the initial stage of cell inoculation and when it was induced at a later stage of growth. It appears that high-molecular-weight alginate polymers are required to efficiently retain the bacteria within the growth film. When expressed from a regulated promoter, the alginate lyase can induce enhanced sloughing of cells because of degradation of the alginate. This suggests a possible role for the lyase in the development of bacterial growth films.

Agar↗
PubMed1994

Venous embolization after deflation of lower extremity tourniquets.

Massive pulmonary embolism has been reported to occur with the use of lower extremity tourniquets. We used transesophageal echocardiography to determine the incidence of venous embolism during lower extremity orthopedic surgery performed with a pneumatic tourniquet. The hemodynamic and respiratory consequences of all embolic events were assessed. Venous emboli were detected after tourniquet deflation in 8 of 30 procedures. The incidence of embolism was unrelated to the type of surgical procedure performed or the duration of tourniquet inflation. There were no significant differences in preoperative characteristics or postdeflation hemodynamic and respiratory responses between patients with and without emboli. Venous embolization is a relatively common event after tourniquet deflation. The clinical significance of these events remains to be determined.

Adult↗
PubMed1993

Sequence of the algL gene of Pseudomonas aeruginosa and purification of its alginate lyase product.

The alginate lyase-encoding gene (algL) of Pseudomonas aeruginosa was localized to a 1.7-kb EcoRI-XbaI fragment within the alginate biosynthetic gene cluster at 34 minutes on the chromosome. The nucleotide sequence of this DNA fragment revealed an ORF encoding a protein of M(r) 40,885 which is transcribed in the same orientation as the other alg genes within the biosynthetic gene cluster. The predicted protein has a potential N-terminal signal peptide which is consistent with its proposed periplasmic location. The AlgL protein was overproduced in Escherichia coli and purified. The purified protein was shown to have alginate lyase activity. In addition, an algL insertion mutant of the mucoid P. aeruginosa 8830 was constructed. This mutant (alm1) had a nonmucoid phenotype due to a polar effect on the transcription of an essential alg gene, algA. Thus, the algL gene is located within a region of the alginate biosynthetic gene cluster that appears to be non-essential for alginate production.

Alginates↗
PubMed1993

Molecular characterization of the SEC1 gene of Saccharomyces cerevisiae: subcellular distribution of a protein required for yeast protein secretion.

Strains of Saccharomyces cerevisiae harbouring temperature-sensitive mutations in the SEC1 and SEC5 genes exhibit an accumulation of post-Golgi secretory vesicles at 37 degrees C. We have cloned a fragment of yeast DNA which carries two distinct genes, one of which complements a sec1 mutation, and the other a sec5 mutation. Genetic test confirm that the sec1-complementing gene is indeed SEC1, and is essential for cell growth. Nucleotide sequence analysis reveals that the cloned SEC1 gene is the same as a previously sequenced sec1-complementing gene. The SEC1 sequence encodes a protein of 724 amino acids with a predicted molecular mass of 83 kDa. Antibodies purified from a polyclonal antiserum raised against the protein product of the cloned gene recognize a yeast protein of apparent molecular mass 78 kDa which is found in a detergent-resistant association with a rapidly sedimenting yeast subcellular fraction, behaviour which is suggestive of an interaction with a component of the yeast cytoskeleton.

Cloning, Molecular↗
PubMed1993

Surface antigens of human thymocyte populations defined by CD3, CD4 and CD8 expression: CD1a is expressed by mature thymocytes but not peripheral T cells.

Three colour flow cytometric analysis has been used to analyze the expression of a series of surface antigens on human thymic and peripheral T-cell populations. CD4, CD8 and CD3 were used to divide the populations into the conventional major categories, and the distribution of CD1a, CD2, CD7, CD34, CD44, class I MHC and class II MHC was then determined. Some characteristics of 'single positive' (CD4+8- and CD4-8+) T-lineage cells were unexpected. Amongst thymocytes, some 'immature single positives' were delineated as larger sized cells lacking cell surface CD3 and expressing low levels of class I MHC; however, in contrast with murine thymocytes, these were all CD4+8-, rather than being predominantly CD4-8+. Amongst peripheral T cells, a small proportion of CD7- cells were detected, within both the CD4+8-3+ and the CD4-8+3+ categories. Finally, in contrast to previous conclusions, CD1a was expressed at high levels on mature (CD4+8-3+ and CD4-8+3+) human thymocytes, although in agreement with previous reports it was absent from peripheral T cells. CD1a is therefore a useful marker of post-selection, post-thymic T-cell maturation.

Adolescent↗