Search PubMed⌕ Search

Biomedical subjects

D L Scott

Publications and source records attributed to D L Scott.

At least 19 recordsLinked to original sources
PubMed1995

The 2.4 A crystal structure of cholera toxin B subunit pentamer: choleragenoid.

Cholera toxin, a heterohexameric AB5 enterotoxin released by Vibrio cholera, induces a profuse secretory diarrhea in susceptible hosts. Choleragenoid, the B subunit pentamer of cholera toxin, directs the enzymatic A subunit to its target by binding the GM1 gangliosides exposed on the luminal surface of intestinal epithelial cells. The crystal structure of choleragenoid has been independently solved and refined at 2.4 A resolution by combining single isomorphous replacement with non-crystallographic symmetry averaging. The structure of the B subunits, and their pentameric arrangement, closely resembles that reported for the intact holotoxin, choleragen, the heat-labile enterotoxin from Escherichia coli, and for a choleragenoid-GM1 pentasaccharide complex. In the absence of the A subunit the central cavity of the B pentamer is a highly solvated channel. The binding of choleragenoid to the A subunit or to its receptor pentasaccharide modestly affects the local stereochemistry without perceptibly altering the subunit interface.

Amino Acid Sequence↗
PubMed1995

The three-dimensional crystal structure of cholera toxin.

The clinical manifestations of cholera are largely attributable to the actions of a secreted hexameric AB5 enterotoxin (choleragen). We have independently solved and refined the three-dimensional structure of choleragen at 2.5 A resolution. The structure of the crystalline toxin closely resembles that described for the heat-labile enterotoxin from Escherichia coli (LT) with which it shares 80% sequence homology. In both cases, the wedge-shaped A subunit is loosely held high above the plane of the pentameric B subunits by the tethering A2 chain. The most striking difference between the two toxins occurs at the carboxyl terminus of the A2 chain. Whereas the last 14 residues of the A2 chain of LT threading through the central pore of the B5 assembly form an extended chain with a terminal loop, the A2 chain of choleragen remains a nearly continuous alpha-helix throughout its length. The four carboxyl-terminal residues of the A2 chain (KDEL sequence), disordered in the crystal structure of LT, are clearly visible in choleragen's electron-density map. In the accompanying article we describe the three-dimensional structure of the isolated B pentamer of cholera toxin (choleragenoid). Comparison of the crystalline coordinates of choleragen, choleragenoid, and LT provides a solid three-dimensional foundation for further experimental investigation. These structures, along with those of related toxins from Shigella dysenteria and Bordetella pertussis, offer a first step towards the rational design of new vaccines and anti-microbial agents.

Amino Acid Sequence↗
PubMed1995

Are slow-acting anti-rheumatic drugs monitored too often? An audit of current clinical practice.

Rheumatologists usually recommend monthly blood monitoring when patients with rheumatoid arthritis (RA) are treated with slow-acting anti-rheumatic drugs (SAARDs). Is monthly monitoring needed or could its frequency be reduced? We audited the opinions of UK rheumatologists and reviewed clinical experience at three centres. To ascertain the interval at which patients are monitored and the determinants of monitoring policy we sent a questionnaire to 193 consultant rheumatologists; 143 (74%) replied. The majority use monthly monitoring for most SAARDs except sulphasalazine, chloroquine and hydroxychloroquine. There is extensive variation, which is not related to the type of rheumatology unit or whether a shared scheme with general practitioners is used. Reviewing experience in 390 patients treated with SAARDs at three adjacent rheumatology units in London showed that haematological adverse reactions were infrequent. During 1560 patient-years of treatment involving 18,720 monthly monitoring visits there were 13 haematological adverse reactions (11 thrombocytopenias and two leucopenias). Five thrombocytopenias developed after 6 months of treatment; five occurred gradually over 5 months or more and one borderline low platelet count was seen once. The two leucopenias were borderline low white cell counts occurring gradually over 3-6 months. Such frequent monitoring is expensive. The total cost of monitoring 390 patients for 1560 patient-years was 420,000 pounds. The cost of detecting each adverse reaction was 32,000 pounds. Three-monthly monitoring when therapy is established after an initial stabilizing period would have identified seven out of eight late adverse reactions. Monitoring policies are mainly based on clinical consensus with few prospective studies of their value; they need re-evaluation.

Antirheumatic Agents↗
PubMed1995

Identification of the central regulatory segment of plasmid R6K complexed with the membranes of Escherichia coli.

Understanding the mechanisms which control replication of chromosomes with multiple origins of replication have been the subject of many investigations. The plasmid, R6K, has three origins of replication, alpha, beta, and gamma. In vivo, alpha and beta are utilized with equal frequencies while the gamma origin is rarely used in the parental plasmid, or remains silent in several miniplasmids. Hence, in the present study, the multiple origin plasmid, R6K, was utilized as a model system to investigate DNA replication. A 1.6 kb PCR (polymerase chain reaction) amplified region of R6K containing the central regulatory segment (CRS) was examined to determine if membrane complexing is required for maintenance. Crude membrane extracts from exponentially growing cultures were prepared and sedimented through 30-50% (w/v) linear sucrose gradients. Fractions collected were probed using quantitative PCR which revealed two fractions containing CRS DNA. These complexes were localized in a region between the inner and outer membranes of Escherichia coli.

Base Sequence↗
PubMed1994

Mouse fibroblasts defective in thrombin mitogenesis possess functional proteolytically activated receptor for thrombin: requirement for a second signaling pathway.

Thrombin mitogenesis in fibroblasts requires two distinguishable subsets of signals; one generated by proteolytic cleavage, the other by high-affinity cell surface binding. Characterizing two closely related mouse embryo (ME) cell lines with high numbers of thrombin binding sites, we found that one line, B11-A, responds mitogenically to thrombin, epidermal growth factor (EGF), and serum, whereas the B11-B cell line is responsive to EGF and serum, but not to thrombin. The B11-B defect responsible for loss of thrombin responsiveness is not due to differences in the number of high-affinity binding sites, the affinity of thrombin binding to these sites, or to differences in cell surface expression of proteolytically activated receptors for thrombin (PART). The defect is also not associated with an inability of thrombin to activate PART since thrombin stimulates the cleavage-dependent induction of the proto-oncogene c-fos in both B11-A and B11-B cells. Various combinations of thrombin, synthetic thrombin receptor peptide, TRP-14 (SFFLRNPGENTFEL), platelet-derived growth factor (PDGF), and phorbol 12-myristate 13-acetate (PMA) were used to better define the defect in thrombin-mediated mitogenesis in B11-B cells. Direct activation of protein kinase C with PMA in combination with thrombin did not overcome B11-B nonresponsiveness. However, mitogenic responsiveness was regained in B11-B cells by simultaneous addition of PDGF and either thrombin or TRP-14. Therefore, the B11-B defect may involve a set of signals initiated by nonproteolytic thrombin interactions distinct from those initiated by PART, but related to the downstream signals initiated by the tyrosine kinase-associated growth factors, EGF and PDGF.

Amino Acid Sequence↗
PubMed1994

The electrostatic basis for the interfacial binding of secretory phospholipases A2.

Biochemical and structural data suggest that electrostatic forces play a critical role in the binding of secretory phospholipases A2 to substrate aggregates (micelles, vesicles, monolayers, and membranes). This initial binding (adsorption) of the enzyme to the interface is kinetically distinct from the subsequent binding of substrate to the buried active site. Thus, in the absence of specific active-site interactions, electrostatic forces operating at the molecular surface may orient and hold the enzyme at the interface. We have calculated the electrostatic potentials for 10 species of secretory phospholipases A2 whose atomic coordinates have been determined by x-ray crystallography. Most of these enzymes show a marked electrostatic sidedness that is accentuated to a variable degree by the presence of the essential cofactor calcium ion. This asymmetry suggests a discrete interfacial binding region on the protein's surface, the location of which is in general agreement with proposals derived from the results of chemical modification, mutational, and crystallographic experiments.

Amino Acid Sequence↗
PubMed1994

DC-ART: preventing or significantly decreasing the rate of progression of structural joint damage.

Progressive joint damage, increasing deformity, and declining function characterize rheumatoid arthritis (RA). The evidence suggests structural joint damage is the predominant cause of functional impairment. Structural changes of joints are evaluated by imaging methods. Plain joint radiographs remain the best method for determining the extent and nature of structural changes at present. Newer technologies such as magnetic resonance imaging may eventually replace them. Clinical studies of antirheumatic drugs involving the assessment of prevention or significant decrease in the rate of progression of structural joint damage in RA should meet several standards. All patients who enter a study, whether they continue medication or not, must be assessed on its completion. Studies require sufficient power to determine realistic differences due to therapy. They should last long enough for a reliable analysis of the effects of joint damage; 1 year would be the minimal period for such a study, and 2 years would be preferable. Evaluation should concentrate on erosions and related structural changes in juxtaarticular bone; mapping osteoporotic areas in early disease may be a sensitive and objective measure. Assessments ought to use changes within the hands and wrists to indicate overall progression, with the feet included in evaluating early disease. There should be different therapeutic aims at various stages of RA, focussing on preventing new erosions developing in early disease (< 2 years from diagnosis), preventing new erosions occurring in established disease (2-5 years from diagnosis), reducing the rate erosions develop in established disease (< 5 years from diagnosis), and reducing the rate of joint destruction in late disease (> 5 years from diagnosis).

Antirheumatic Agents↗
PubMed1994

Guidelines for use of antirheumatic drugs.

The Vth WHO/ILAR Task Force Meeting on Rhemuatic Diseases ratified guidelines for the use of antirheumatic drugs. Indications for use, contraindications, dose, administration and other usage principles, toxicity, and advice to patients have been agreed for analgesics, nonsteroidal antiinflammatory drugs, slower acting antirheumatic drugs, corticosteroids, and hypouricemic agents.

Adrenal Cortex Hormones↗
PubMed1994

Sex differences in inflammation induced cartilage damage in rodents. The influence of sex steroids.

OBJECTIVE: To investigate sex differences in granulomatous inflammation and its effects upon articular cartilage and to assess the potential role of sex steroids in the process. METHODS: The cotton-pellet cartilage implant model was used with male and female mice in the presence and absence of gonadectomy and hormone replacement. The effects of granulomatous tissue upon articular cartilage was assessed and tissue content of interleukin 1 (IL-1) was determined. The expression of sex hormone receptors in inflammatory tissue was investigated by immunocytochemistry. RESULTS: Female mice showed a higher ability than males to degrade cartilage irrespective of the sex of the cartilage implanted. Gonadectomy resulted in a significant acceleration of cartilage damage in both sexes, which was reverted by estrogen replacement in females and androgen replacement in males. Female granulomata had significantly higher IL-1 content than those from males. Gonadectomy was associated with an increased IL-1 content in males but not in females, the effects being abolished by androgen replacement in males. Estrogen and androgen receptors were identified in inflammatory cells from the granulomatous tissue. CONCLUSION: Our data demonstrate that sex hormones affect inflammation induced cartilage degradation in male and female mice probably through the modulation of cytokine production and release in the granulomatous tissue. Further investigation on the effects of sex steroids in inflammation induced cartilage degradation may help elucidate their pathogenic role and therapeutic potential in human disease.

Animals↗
PubMed1994

Developmental analysis of bone tumors in polyomavirus transgenic mice.

BACKGROUND: Transgenic mice carrying the polyoma (Py) early region gene develop both vascular and bone tumors that express the transgene (R. Wang and V. L. Bautch, J. Virol. 65:5174-5183, 1991). To determine the correlation between bone tumor formation and transgene expression, mice of two lines that showed differences in tumor pattern were analyzed. EXPERIMENTAL DESIGN: Py DNA encoding the early region gene was inserted into B6D2F1 mouse embryos. Transgenic mice were sacrificed at appropriate times in development, and the histopathology of the skulls and genotype analyses were performed independently. Py transgene expression was assayed in individual skulls or small groups of skulls. RESULTS: In mice of the Py-4 line, Py oncogene expression was detectable in skulls from 14.5 days of development. These mice had normal skulls at birth, and they developed lesions histologically resembling osteosarcoma synchronously 1 to 2 weeks after birth. Mice of the Py-3 line did not express detectable levels of transgene at 3 weeks of age, and they did not develop detectable skull pathology at this age. Less than 20% of adult Py-3 mice developed calvarial osteosarcoma-like lesions, and a similar number of these mice expressed the transgene in the skull. CONCLUSIONS: These data show that Py transgene expression in bone of the skull is differentially regulated in mice of the two lines, and they indicate that epigenetic changes may be sufficient to complement expression of the Py oncogene in neoplastic bone formation during development.

Animals↗
PubMed1993

Inflammation-induced cartilage degradation in female rodents. Protective role of sex hormones.

OBJECTIVE: To investigate the effects of physiologic levels of sex steroids on inflammation and cytokine production and their consequential cartilage degradation. METHODS: We used an in vivo model of inflammation-induced cartilage degradation in female mice to study the effects of ovariectomy and hormone treatment, and in vitro culture systems to examine the influence of sex steroids on cartilage metabolism, interleukin-1 (IL-1) production by granulomatous tissue, and its effects on female mouse articular cartilage. RESULTS: Ovariectomy resulted in accelerated cartilage breakdown associated with increased production of IL-1 by granulomatous tissue. The effects of ovariectomy on cartilage were reversed by treatment with estradiol and androgen, but not by progesterone treatment. Estradiol and progesterone reduced both spontaneous and IL-1-induced cartilage degradation in vitro. Testosterone antagonized the effects of IL-1 on both proteoglycan loss and proteoglycan synthesis. CONCLUSION: These data suggest that sex steroids have an important influence on inflammation-induced cartilage breakdown in female animals, with protective effects of both estradiol and androgens. Multiple mechanisms may be involved, and they are likely to include direct immunomodulatory effects as well as interactions with the effects of cytokine and of the glucocorticoid response to inflammation.

Animals↗