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Biomedical subjects

L Hooker

Publications and source records attributed to L Hooker.

7 recordsLinked to original sources
PubMed2001

A sensitive, single-tube assay to measure the enzymatic activities of influenza RNA polymerase and other poly(A) polymerases: application to kinetic and inhibitor analysis.

We describe a fast and robust new assay format to measure poly(A) polymerase (PAP) activity in a microtiter plate format. The new assay principle uses only natural nucleotide triphosphates and avoids a labour-intensive filtration step. A coupled enzymatic system combining PAP and reverse transcriptase forms the basis of the assay. The PAP generates a poly(A) tail on a RNA substrate and the reverse transcriptase is used to quantify the polyadenylated RNA by extension of a biotinylated oligo-dT primer. We demonstrate the principle of the assay using influenza virus RNA polymerase and yeast PAP as examples. A specific increase in the K(m) value for ATP and the observation of burst kinetics in the polyadenylation dependent, but not in the polyadenylation independent, assay suggest that a rate limiting step of influenza polymerase activity occurs after transcription elongation. Yeast PAP was used to validate the assay as an example of a template independent PAP. The new yeast PAP assay was approximately 100-fold more sensitive than the conventional TCA precipitation assay for yeast PAP, but the kinetic analysis of the PAP reaction gave similar results in both assays. The two enzymes show important differences with respect to inhibition by 3'-deoxy-ATP. Whereas the K(i) value for 3'-deoxy-ATP (105-117 microM) is similar to the K(m) value for ATP (186 microM) in the case of influenza RNA polymerase, the K(i) value for 3'-deoxy-ATP (0.4-0.6 microM) is approximately 100-fold lower than the K(m) value for ATP (50 microM) in the case of yeast PAP.

Adenosine Triphosphate↗
PubMed2001

Influenza virus endoribonuclease.

The influenza virus polymerase complex contains two associated enzymatic activities, an endoribonuclease and a RNA-dependent RNA polymerase activity. Both activities have so far been observed only with the complete polymerase complex consisting of three subunits, PB1, PB2, and PA. This chapter describes a robust and optimized procedure for the purification of active influenza virus polymerase in complex with genomic RNA and the single-stranded RNA-binding protein nucleoprotein from influenza virus particles. It also explains the synthesis of capped RNA molecules as substrates of the influenza virus endonuclease. The enzymatic properties of influenza virus-derived endoribonuclease activity have been characterized with a model RNA substrate of 20-nucleotide length, termed G20 RNA. The rate of RNA cleavage under steady state conditions appears to be limited by product dissociation. Therefore conditions have been optimized to study the chemical step of RNA cleavage under single turnover conditions. The enzyme requires divalent metal ions for activity and can use Mn(II), Co(II), and Fe(II) efficiently at pH 7, Mg(II) with intermediate efficiency, and Ni(II) and Zn(II) with lower efficiency. The reaction progress curves show slow binding of Zn(II) and Ni(II) to the protein, suggesting a conformational change of the active site as a prerequisite for endonuclease activity in the presence of these two metal ions. Low concentrations of the detergent DOC inhibit the activity and also disrupt the trimeric polymerase complex, whereas other detergents do not have a significant effect on the activity.

Base Sequence↗
PubMed2000

Taking practice forward in paediatric oncology: the impact of a newly developed education programme for nurses working in shared-care hospitals.

Fundamental to any change or development in health care is the quality of the service received by patients and their families. Practitioner-led initiatives that are designed to improve the quality of care are more likely to succeed if they are underpinned with appropriate educational input. A structured programme in paediatric oncology nursing was developed to meet the needs of nurses working in District General Hospitals. Nurses' perceptions of the consequences of undertaking the programme were explored using a focus group approach. Findings have shown that there have been significant changes to nurses' roles and this in turn has had an impact on the individual and the organization. It was perceived that there are positive outcomes for both the service and quality of care for children with cancer and their families. The shared-care nurses believe that this has been the result of improved knowledge, skill and expertise, better communication between care providers, and the collaborative frameworks which have evolved.

Journal Article↗
PubMed1999

Safety, efficacy, and acceptability of home intravenous therapy administered by parents of pediatric oncology patients.

BACKGROUND: A prospective study of patients receiving intravenous therapy administered at home by parents was conducted over 1 year at a United Kingdom regional pediatric oncology center. PROCEDURE: The study defined the patient groups for whom this approach was used and evaluated the safety and efficacy of home treatment. Parents' evaluation of home therapy was sought to determine the acceptability of the scheme from their perspective. Eight-nine courses of antibiotics, chemotherapy or antiemetics were given, comprising a total of 469 days of home treatment. RESULTS: Few clinical problems were encountered. Parents felt that home treatment helped them to cope (72%); they felt more in control (75%) and learned more about their child's illness and treatment (82%). CONCLUSIONS: Home treatment was perceived as less stressful than hospital treatment (79%), and all reported benefits to family life. There is a need for parents to be taught vigilance in observing for and reporting signs of deterioration.

Adolescent↗
PubMedDate not supplied

Parent-held shared care records: bridging the communication gaps.

Specialist centres provide excellent treatment and expert care, but the best interests of children may not be served if they must always attend a hospital far from home. Systems of shared care have been developed to offer care nearer the family home whenever possible. Parent-held shared care records were introduced at a paediatric haematology/oncology centre in response to issues raised by parents and professionals concerning the management of shared care. These were set up to facilitate communication, teamwork and family involvement. Consultation took place within the multidisciplinary team to ensure that the design of the records met the needs of each group. During a multidisciplinary study day the records were introduced to professionals from shared care hospitals. Before formally incorporating these records into regular clinical practice they were piloted with a group of families. The records have become an accepted part of the service provided for family and professional partners in shared care. Formal evaluation of this innovation is currently taking place.

Child↗