Search PubMed⌕ Search

Biomedical subjects

P Ward

Publications and source records attributed to P Ward.

At least 91 records · Page 5Linked to original sources
PubMed1995

Asymmetric replication in vitro from a human sequence element is dependent on adeno-associated virus Rep protein.

The DNA of human parvovirus adeno-associated virus type 2 (AAV) integrates preferentially into a defined region of human chromosome 19. Southern blots of genomic DNA from latently infected cell lines revealed that the provirus was not simply inserted into the cellular DNA. Both the proviral and adjoining cellular DNA organization indicated that integration occurred by a complex, coordinated process involving limited DNA replication and rearrangements. However, the mechanism for targeted integration has remained obscure. The two larger nonstructural proteins (Rep68 and Rep78) of AAV bind to a sequence element that is present in both the integration locus (P1) and the AAV inverted terminal repeat. This binding may be important for targeted integration. To investigate the mechanism of targeted integration, we tested the cloned integration site subfragment in a cell-free replication assay in the presence or absence of recombinant Rep proteins. Extensive, asymmetric replication of linear or open-circular template DNA was dependent on the presence of P1 sequence and Rep protein. The activities of Rep on the cloned P1 element are analogous to activities on the AAV inverted terminal repeat. Replication apparently initiates from a 3'-OH generated by the sequence-specific nicking activity of Rep. This results in a covalent attachment between Rep and the 5'-thymidine of the nick. The complexity of proviral structures can be explained by the participation of limited DNA replication facilitated by Rep during integration.

Base Sequence↗
PubMed1995

Neuronal ceroid-lipofuscinosis in a cat.

Neuronal ceroid-lipofuscinosis was diagnosed in a young adult domestic short-haired cat euthanatized because of severe progressive neurologic disease. Clinical signs included blindness, seizures, and decreased mentation. An autofluorescent pigment, identified as ceroid-lipofuscin by electron microscopy and staining properties, was found within neurons of the central and peripheral nervous systems. A diffuse reactive astrocytosis accompanied by multifocal microgliosis was visible in all areas of the brain. Retinal atrophy with intraneuronal lipopigment accumulation was also identified. Contrary to the human neuronal ceroid-lipofuscinoses, pigment deposition appeared to be restricted to neural tissues.

Animals↗
PubMed1995

Assessment of the completeness and accuracy of computer medical records in four practices committed to recording data on computer.

BACKGROUND: General practice computer databases are being increasingly seen as a source of data for public health monitoring and commissioning. Such ambitions depend on routine clinical data being recorded with acceptable completeness and accuracy. AIM: The aim of this study was to assess the completeness and accuracy of the computer medical records in four high-recording general practices. METHOD: Four general practices in the Trent Region that use the EMIS computer system, and were known to be high recorders of clinical data on their computer databases, were selected. A retrospective analysis of the computer records, a prospective comparison of a sample of computer records with manual records, and a prospective comparison between videorecorded consultations and their manual and computer records were undertaken. RESULTS: Checks for completeness in computer recording of diabetes mellitus and glaucoma showed high levels of accurate recording, 97% and 92% respectively. Prevalence rates between practices were reasonably comparable. No practice consistently, across 10 diagnoses, recorded prevalences higher or lower than the other practices; those diagnoses with recognized objective diagnostic criteria were recorded with a more consistent prevalence than those without. Lifestyle data recording was low; overall, smoking habits and alcohol consumption were recorded for 52% and 38% of patients aged over 16 years, respectively. Comparison of the manual records with the computer records showed that the computer records were sufficiently complete with regard to diagnoses (82% of all items recorded), prescriptions (100%) and referrals (67%), but missed most of the remaining data that a manual record captured. The videorecorded validation study showed that there were no important lapses in the recording of diagnoses, prescriptions or referrals when the computer recording was compared to the actual process of the consultations. CONCLUSION: In these four high-recording practices the data in computer records were of sufficient completeness and accuracy to allow meaningful data aggregation for some diagnoses, prescriptions and referrals. Standardized protocols for defining which patients are included and excluded from major disease groups are required.

England↗
PubMed1994

Inhibition of human platelet aggregation by GR91669, a prototype fibrinogen receptor antagonist.

In order to produce more potent and specific fibrinogen receptor (GpIIb/IIIa) antagonists, the Arg-Gly of a chemical series based upon Arg-Gly-Asp was replaced by alkyl chains of varying lengths. The most potent in this series, GR91669, inhibited aggregation of human gel-filtered platelets (GFP) in vitro induced by ADP or the thromboxane A2 mimetic, U46619, with IC50 values of 200nM and 500nM respectively and was selected for further studies. Its inhibitory effects on GFP were reversed by addition of excess fibrinogen. The compound also inhibited ADP- or U46619-induced platelet aggregation in human whole blood (IC50 values of 700nM in both cases). 125I-Fibrinogen binding to ADP-stimulated platelets was inhibited by GR91669 with an IC50 (65nM) similar to that against platelet aggregation. GR91669 (1mM) did not inhibit U46619-induced platelet shape change or 14C-5HT secretion from platelets stimulated by collagen, U46619 or thrombin. Therefore GR91669 inhibits aggregation but has no significant effect on stimulus-response events, a profile consistent with fibrinogen receptor blockade. In addition, GR91669 (1mM), unlike echistatin or Gly-Arg-Gly-Asp-Ser, did not disrupt vitronectin recptor-dependent attachment of cultured HUVECS in vitro and similarly did not inhibit Mac-1 dependent adhesion of human granulocytes. Thus, of the integrins tested, GR91669 appears to be specific for GpIIb/IIIa. Following intravenous administration to marmosets of 1 or 10 mg/kg GR91669, ADP (10 microM)-induced platelet aggregation ex vivo was abolished for 15 and 60 minutes respectively. Greater than 50% inhibition was maintained for 30 minutes and 2 hours respectively. GR91669, therefore appears to be a potent, specific fibrinogen receptor antagonist in vitro and which is also active in vivo.

Amino Acid Sequence↗
PubMed1994

GR138676, a novel peptidic tachykinin antagonist which is potent at NK3 receptors.

GR138676, a conformationally constrained analogue of neurokinin B, is a novel, potent NK3 receptor antagonist. GR138676 was a competitive antagonist of neurokinin B-dependent arachidonic acid mobilization from prelabelled Chinese hamster ovary cells stably transfected with a human NK3 receptor gene (pKB 8.3) and of contractions induced by senktide in rat portal vein (pKB 8.2). However, GR138676 was also a competitive antagonist of the increase in intracellular calcium evoked by the selective NK1 agonist, GR73632, in the human astrocytoma U373MG cell-line (pKB 8.3). GR138676 had little activity at NK2 receptors, inhibiting binding of the NK2 antagonist radioligand [3H]-GR100679 to Chinese hamster ovary cells transfected with the human ileum NK2 receptor with a pKi of 6.0. In summary, despite its activity at NK1 receptors, GR138676 will be a useful tool for characterizing NK3 receptors as well as defining the physiological and pathophysiological function of this receptor subtype.

Amino Acid Sequence↗
PubMed1994

Generating social class data in primary care.

The objective of this study was to compare three methods of collecting social class data in general practice. The setting was a rural dispensing practice on the Nottinghamshire/Lincolnshire border. The methods examined were: (a) a self-administered questionnaire to 200 patients to determine their social class based on the occupation of the head of household; (b) members of the practice staff were asked to assign a social class to these households based on their local knowledge; and (c) use of small area statistics from the 1991 census data using modal and weighted methods. It was found that the practice staff were unable reliably to assign a social class to the households. The modal method of using small area statistics to assign social class to households through their postcode and its link to the census data was also inaccurate. While a personal questionnaire will remain the only method for assigning a social class to individual patients for clinical care or most research, the weighted method of small area statistics is shown to be a cost-effective and sufficiently accurate method for health needs assessment in general practice.

Data Collection↗
PubMed1994

Effect of age and gender on tirilazad pharmacokinetics in humans.

Tirilazad mesylate pharmacokinetics were assessed in 12 young and 12 elderly volunteers (six men and six women per age group). Subjects received single 10-minute intravenous infusions of 1.5 mg/kg and 3.0 mg/kg tirilazad mesylate. Plasma tirilazad mesylate concentrations were determined by HPLC. There were no significant dose effects on clearance, but half-life increased with dose because of assay insensitivity at the lower dose. Mean half-lives were 16.3 +/- 15.5 and 21.4 +/- 12.6 hours for young and elderly subjects, respectively, at the 3.0 mg/kg dose. At the same dose, mean tirilazad mesylate systemic clearance was 0.630 +/- 0.254 and 0.428 +/- 0.090 L/hr/kg, respectively. The decreased clearance in elderly volunteers was primarily attributable to a lower clearance in elderly women relative to young women. The small effect of age on tirilazad clearance is likely to have minimum clinical impact. Tirilazad clearance was approximately 40% higher in young women than in young men. The clinical importance of this observation is unknown.

Adult↗
PubMed1994

Intermediates of adeno-associated virus DNA replication in vitro.

Intermediates of adeno-associated virus type 2 (AAV) DNA replication in an in vitro assay have been characterized. The assay involves rescue and replication of an AAV insert in pBR322. Intermediates were shown to be duplex molecules in which at least one terminus was in the extended configuration, in contrast to the hairpinned ends seen after rescue in the absence of AAV DNA replication. Also present were linear double-stranded dimers, which were characterized as either head-to-head or tail-to-tail tandems; no head-to-tail dimers were detected. The results are in accord with the current model of AAV DNA replication.

DNA Replication↗
PubMed1994

Adeno-associated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein.

The adeno-associated virus (AAV) nonstructural protein Rep 68 is required for viral DNA replication. An in vitro assay has been developed in which addition of Rep 68 to an extract from uninfected HeLa cells supports AAV DNA replication. In this paper, we report characterization of the replication process when a fusion of the maltose binding protein and Rep 68, expressed in Escherichia coli, was used in the assay. Replication was observed when the template was either linear double-stranded AAV DNA or a plasmid construct containing intact AAV DNA. When the recombinant plasmid construct was used as the template, there was replication of pBR322 DNA as well as the AAV DNA; however, linear pBR322 DNA was not replicated. When the plasmid construct was the template, replication appeared to initiate on the intact plasmid and led to separation of the AAV sequences from those of the vector, a process which has been termed rescue. There was no evidence that replication could initiate on the products of rescue. Rep 68 can make a site-specific nick 124 nucleotides from the 3' end of AAV DNA; the site of the nick has been called the terminal resolution site. Our data are most consistent with initiation occurring at the terminal resolution site and proceeding toward the 3' terminus. When the template was the plasmid construct, either elongation continued past the junction into pBR322 sequences or the newly synthesized sequence hairpinned, switched template strands, and replicated the AAV DNA. Replication was linear for 4 h, during which time 70% of the maximal synthesis took place. An additional finding was that the Rep fusion could resolve AAV dimer length duplex intermediates into monomer duplexes without DNA synthesis.

ATP-Binding Cassette Transporters↗
PubMed1993

Anti-emetic profile of a non-peptide neurokinin NK1 receptor antagonist, CP-99,994, in ferrets.

In the ferret, 5-HT3 receptor antagonists are effective in controlling emesis produced by cytotoxic agents or radiation. To investigate the possibility that substance P has a role, as well as 5-HT, in the emetic reflex pathway, we have examined the anti-emetic effects of a NK1 receptor antagonist (racemic CP-99,994) in the ferret. Racemic CP-99,994 was effective against a range of emetogens, comprising cytotoxic drugs, radiation, morphine, ipecacuanha and copper sulphate.

Animals↗
PubMed1993

Improved potency and specificity of Arg-Gly-Asp (RGD) containing peptides as fibrinogen receptor blocking drugs.

A range of cyclic RGD based peptides have been developed to mimic the conformation of RGD within fibrinogen. These peptides, as well as echistatin (IC50 = 0.05 microM) and GRGDS (IC50 = 25 microM) fully inhibited adenosine diphosphate (ADP) (10 microM)-induced platelet aggregation of human gel-filtered platelets (GFP). RGDF was the most potent linear peptide in inhibiting ADP-induced aggregation (IC50 = 8 microM) but cyclisation, using a 6,5 bicyclic coupling group to produce GR83895, led to an approximately 10-fold increase in potency (IC50 = 0.9 microM). In GFP, ADP-induced 125I-fibrinogen binding was inhibited (> 80%) by echistatin, GRGDS or GR83895 at concentrations (IC50 values 0.05 microM, 25 microM and 1.4 microM respectively) similar to those needed to inhibit aggregation. All three compounds also completely inhibited ADP- and U46619-induced aggregation in both platelet rich plasma (PRP) and whole blood. In contrast to platelet aggregation, U-46619-induced 14C-5HT secretion in PRP was not inhibited by GR83895 or echistatin, indicating that agonist-induced signal transduction is not affected by either agent, a profile consistent with that predicted for a specific fibrinogen receptor blocking drug. To test specificity of action, echistatin, GR83895 and GRGDS were also examined for their ability to detach cultured human umbilical vein endothelial cells attached to plastic through a vitronectin receptor dependent process. GR83895 only caused detachment at concentrations 100-fold greater than those required to inhibit platelet aggregation, in contrast to GRGDS and echistatin which caused cell detachment at concentrations similar to those inhibiting aggregation. In summary, cyclisation of RGD-containing peptides has led to both improved potency and specificity of action. Such specificity of action may prove to be an important consideration for the successful development of a fibrinogen receptor blocking drug as an anti-thrombotic drug.

Amino Acid Sequence↗
PubMed1993

Care management and mental health.

The community care reforms will produce a new kind of key worker who will organise and budget for packages of care: the care manager. Care management goes live in April 1993 but is still poorly rehearsed and its performance may yet disappoint. This overview sets out the origins of case management, its transformation into care management, and the principles guiding its practice. To spell out how the concept works, plans for care management in Southwark's mental health services are described.

Budgets↗
PubMed1993

Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac-1).

Full-length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac-1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu-CAMs). The biosynthesis of recombinant Mac-1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac-1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac-1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC50) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac-1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac-1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet-Leu-Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF-alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac-1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.

Carbohydrates↗
PubMed1993

Accuracy of a computer-based anaesthetic audit system.

The accuracy of an anaesthetic computer-based audit system was assessed. In a retrospective survey of operations performed 4 months previously only 50% of the patients' notes could be found, and 40% of these notes did not contain an anaesthetic chart. A prospective survey was also performed. The accuracy with which the computer output reflected the anaesthetic technique was found to be 52% in the retrospective survey and 33% in the prospective survey. The authors conclude that audit systems should be audited before their generated data are used for decision-making.

Anesthesia↗