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D Goode

Publications and source records attributed to D Goode.

At least 19 recordsLinked to original sources
PubMed2003

Reduction of experimental Salmonella and Campylobacter contamination of chicken skin by application of lytic bacteriophages.

Lytic bacteriophages, applied to chicken skin that had been experimentally contaminated with Salmonella enterica serovar Enteritidis or Campylobacter jejuni at a multiplicity of infection (MOI) of 1, increased in titer and reduced the pathogen numbers by less than 1 log(10) unit. Phages applied at a MOI of 100 to 1,000 rapidly reduced the recoverable bacterial numbers by up to 2 log(10) units over 48 h. When the level of Salmonella contamination was low (< log(10) 2 per unit area of skin) and the MOI was 10(5), no organisms were recovered. By increasing the number of phage particles applied (i.e., MOI of 10(7)), it was also possible to eliminate other Salmonella strains that showed high levels of resistance because of restriction but to which the phages were able to attach.

Animals↗
PubMed2001

Perceptions and experiences of primary nursing in an ICU: a combined methods approach.

With the introduction of the Patient's Charter, greater emphasis has been placed on 'named nursing' (Department of Health 1991). While there is much literature extolling the benefits of this method of delivering care to patients (Reed 1988; Manley 1989; Macguire 1991); there is a dearth of empirical studies exploring primary nursing in an adult intensive care unit (ICU). In addition, little is known about how nurses feel about being a 'primary nurse'. The aim of this study was to determine qualified nurses' perceptions and experiences of the effect of primary nursing on patient care in an intensive care environment (ICU) and to explore nurses' experiences of being a primary nurse. This study was carried out in an ICU in Northern Ireland. Primary team nursing was the system of organizing care within this unit. This system consisted of teams of 'primary' and 'associate' nurses. A total of 65 qualified nurses were surveyed. Sixteen nurses took part in focus group interviews. A combined methods approach comprising a questionnaire and focus group interviews was employed for the study. Part one of the questionnaire provided data on professional and demographic details. Part two facilitated nurses to elaborate on their feelings and perceptions of the concept of primary nursing. The focus groups facilitated in-depth exploration of the respondents' personal feelings and experiences about their role as a primary nurse. The data obtained from the two-part questionnaire were analysed using descriptive statistic and content analysis. The data obtained from the focus groups were analysed using content analysis and the development of emerging themes. Analysis of the data revealed that the nurses' views were similar to those highlighted in the existing literature. Nurses perceived primary nursing to relate to the responsibility for the care of one patient, from admission to discharge with the primary nurse assessing, planning, implementing and evaluating care and the associate nurse assisting in the delivery of this care. Thus, continuity of care was seen as the major advantage of primary nursing. The findings, however, suggested that further teaching on the concept of primary nursing was required. In addition, many of the nurses admitted they experienced considerable stress, particularly in relation to their close proximity over a long period of time, with patients and their relatives. This is an important finding and highlights the need for primary nurses in ICU to have the opportunity (in some instances), to be relieved of their responsibility for a named patient, thereby reducing some of the potential for stress. It is acknowledged that the findings of this study may not be generalized beyond the identified sample. Further empirical studies exploring nurses' perceptions and experiences of primary nursing in an ICU are therefore needed.

Adult↗
PubMed1999

Genetic differentiation and natural hybridization between two morphological forms of the common woodlouse, oniscus asellus linnaeus 1758

The common woodlouse Oniscus asellus can be divided into two forms on the basis of morphology, particularly male accessory genitalia. Where these taxa meet, morphological intermediates are found, and the forms were therefore described as subspecies; O. a. asellus and O. a. occidentalis. In this study allozyme loci are used to test the hypothesis that intermediate forms result from hybridization, and to study the nature of hybridization. Thirteen enzyme loci were scored across five English sites representative of each subspecies and intermediates. Ten loci showed strong frequency differences between asellus and occidentalis populations, although no loci showed completely fixed differences. These data confirm that asellus and occidentalis represent genetically distinct taxa, and that intermediate populations are of hybrid origin. There is apparently substantial population substructuring in the contact zone, as indicated by deficits of heterozygotes (FIS) and sporadic gametic (i. e. linkage) disequilibria. Population structure in the Oniscus hybrid zone appears to be analogous to that seen in plant hybrid swarms rather than the narrow hybrid zones observed in many animal taxa. Values of Nei's genetic distance between the subspecies range from 0.65 to 0.70; these are much higher than between typical conspecific taxa and are indicative of ancient genetic divergence. However, because asellus and occidentalis do not remain distinct in areas of overlap, it is simplest to regard these taxa as members of the same species.

Journal Article↗
PubMed1998

Effect of mutations of murine lens alphaB crystallin on transfected neural cell viability and cellular translocation in response to stress.

We examined the influence of over-expressed native and mutant murine lens alphaB crystallin on the response of a murine neural cell line to heat and ionic strength shock. Native and mutant (F27R and KK174/175LL) murine alphaB crystallin amplicons were subcloned into a Lac-Switch IPTG-inducible RSV promoter eukaryotic vector, and transfected into N1E-115 cells using lipofectin. Expression was induced maximally 8 h after addition of IPTG (optimal final concentration 1 mM) to the medium. Cells grew normally after transfection with native and mutant murine alphaB crystallin. We demonstrated expression of the protein using specific anti-alpha crystallin antibodies. Viability under severe heat and ionic strength stress increased when cells had been transfected with native alphaB crystallin, but not with mutants F27R or KK174/175LL. Heat shock caused translocation of both native and mutant alphaB crystallins into the central region of the cells. These results show that mutations in alphaB crystallin that effect its chaperone-like activity may also influence viability of N1E-115 neural cells under stress, while not influencing the distribution of the protein within the cell.

Amino Acid Sequence↗
PubMed1998

Studies on the binding of alpha-crystallin to recombinant prochymosins and chymosin.

PURPOSE: To further investigate the binding of alpha-crystallin to other proteins as part of its chaperone-like activity, we studied interactions of alpha-crystallin with recombinant calf prochymosins and chymosin. METHODS: Recombinant calf prochymosin B and one C-terminal mutant (PC+2, with two additional residues, Histidine-Glycine) were expressed as inclusion bodies in E. coli. Native and mutant proteins were denatured in 8 M urea before being refolded by dilution slowly in phosphate buffer, pH 10.7, in the presence and absence of alpha-crystallin at different concentration ratios. After dialysis, the folded proteins were converted to the active chymosin by acidification. The resulting enzyme activities at standard protein concentrations were determined by a microtitre milk-clotting assay. RESULTS: Refolding of 1.0 mg/ml of protein inclusion bodies diluted in phosphate buffer at 0.32 M urea in the presence of alpha-crystallin resulted in enhanced chymosin activity relative to the control without alpha-crystallin. When lower inclusion body concentrations were used, enzyme activity was not enhanced relative to the control. The mutant enzyme (PC+2) showed no conversion to the active form in the presence of alpha-crystallin. alpha-Crystallin formed a complex with refolded prochymosin, as well as with prochymosin during refolding, but not with active chymosin. Removal of the 43-residue propeptide resulted in loss of alpha-crystallin binding. The addition of two residues (Histidine-Glycine) to the prochymosin C-terminus resulted in precipitation of the mutant prochymosin-alpha-crystallin complex and loss of enzyme activity. CONCLUSIONS: Our experiments show that even under stringent refolding conditions, alpha-crystallin, which retains its gross oligomeric integrity, can bind to unfolded proteins in inclusion bodies and enhance the apparent yield of chymosin activity from high concentrations of inclusion bodies. alpha-Crystallin shows some specificity for binding to its target protein; this specificity may be based on steric considerations as well as residue-specific interactions.

Animals↗
PubMed1998

Effects of controlled mutations on the N- and C-terminal extensions of chick lens beta B1 crystallin.

BACKGROUND: While gamma-crystallin exists as a monomer, beta-crystallin, which unlike gamma-crystallin contains N- and C-terminal arms, associates to form homodimers. METHODS: In order to answer the question of whether the extensions are involved in dimerisation of chick lens beta B1 crystallin, we have developed a heterologous expression system for chicken beta B1 crystallin in Escherichia coli, and produced three mutations by site-directed mutagenesis. We have substituted residues in the PAPA segment of the N-terminal extension, curtailed the N-terminal extension by five residues, and deleted 16 residues from the C-terminal extension. RESULTS: High-resolution gel filtration chromatography and non-denaturing gel electrophoresis show that the mutations did not influence dimerisation of the beta B1 crystallin, while circular dichroism and tryptophan fluorescence indicated that the mutations did not have a major influence on beta B1 crystallin structure or its heat stability. CONCLUSIONS: Our experiments show that as with rat lens beta B2 crystallin, dimerisation of beta B1 crystallin is not affected by alterations to the conserved PAPA region and that the peptide linker region rather than the N- and C-terminal extensions must be important in dimerisation.

Amino Acid Sequence↗
PubMed1998

Aldose reductase: a window to the treatment of diabetic complications?

Kinetic studies on the aldose reductase protein (AR2) have shown that it does not behave as a classical enzyme in relation to ring aldose sugars. These results have been confirmed by X-ray crystallography studies, which have pinpointed binding sites for pharmacological "aklose reductase inhibitors" (ARIs). As with non-enzymic glycation reactions, there is probably a free-radical element involved derived from monosaccharide autoxidation. In the case of AR2, there is free radical oxidation of NADPH by autoxidising monosaccharides, enhanced in the presence of the NADPH-binding protein. Whatever the behaviour of AR2, many studies have showed that sorbitol production is not an initiating aetiological factor in the development of diabetic complications in humans. Vitamin E (alpha-tocopherol), other antioxidants and high fat diets can delay or prevent cataract in diabetic animals even though sorbitol and fructose levels are not modified; vitamin C acts as an AR1 in humans. Protein post-translational modification by glyc-oxidation or other events is probably the key factor in the aetiology of diabetic complications. There is now no need to invoke AR2 in xylitol biosynthesis. Xylitol can be produced in the lens from glucose, via a pathway involving the enzymes myo-inositol-oxygen oxidoreductase, D-glucuronate reductase. L-gulonate NAD(+)-3-oxidoreductase and L-iditol-NAD(+)-5-oxidoreductase, all of which have recently been found in bovine and rat lens. This chapter investigates the molecular events underlying AR2 and its binding and kinetics. Induction of the protein by osmotic response elements is discussed, with detailed analysis of recent in vitro and in vivo experiments on numerous ARIs. These have a number of actions in the cell which are not specific, and which do not involve them binding to AR2. These include peroxy-radical scavenging and recently discovered effects of metal ion chelation. In controlled experiments, it has been found that incubation of rat lens homogenate with glucose and the copper chelator o-phenanthroline abolishes production of sorbitol. Taken together, these results suggest AR2 is a vestigial NADPH-binding protein, perhaps similar in function to a number of non-mammalian crystallins which have been recruited into the lens. There is mounting evidence for the binding of reactive aldehyde moieties to the protein, and the involvement of AR2 either as a 'housekeeping' protein, or in a free-radial-mediated 'catalytic' role. Interfering with the NADPH binding and flux levels--possibly involving free radicals and metal ions--has a deleterious effect. We have yet to determine whether aldose reductase is the black sheep of the aldehyde reductase family, or whether it is a skeleton in the cupboard, waiting to be clothed in the flesh of new revelations in the interactions between proteins, metal ions and redox metabolites.

Aldehyde Reductase↗
PubMed1997

Ibuprofen protects alpha-crystallin against posttranslational modification by preventing protein cross-linking.

Posttranslational modification of bovine alpha-crystallin by D-erythrose-4-phosphate, fructose-6-phosphate, D-ribose-5-phosphate and carbamylation using potassium cyanate induced the loss of chaperone-like activity, as assessed by gamma-crystallin aggregation. The presence of high-molecular-weight aggregates indicated that erythrosylated, fructosylated and carbamylated alpha-crystallins were modified by non-reducible cross-linking. In contrast, ribosylation of alpha-crystallin induced the formation of reducible cross-links. Analysis of ribosylated, erythrosylated and carbamylated alpha-crystallin using non-denaturing acrylamide gels showed that the cross-linking did not sterically inhibit the normal aggregate formation or alter the oligomerisation of the aggregate. Co-incubation of ibuprofen in the presence of alpha-crystallin and the modifying agents protected the chaperone-like activity of alpha-crystallin, enabling the inhibition of gamma-crystallin aggregation. In addition, ibuprofen inhibited the formation of both reducible and non-reducible cross-linked high-molecular-weight alpha-crystallin aggregates. We show in this paper that ibuprofen can inhibit in vitro cross-linking events responsible for the loss of chaperone-like activity of alpha-crystallin and suggest that the protective effect of ibuprofen may be exerted by the binding of ibuprofen breakdown products to alpha-crystallin lysine groups, preventing posttranslational modification responsible for the loss of chaperone-like activity.

Animals↗
PubMed1996

Effects of site-directed mutations on the chaperone-like activity of alphaB-crystallin.

Recombinant alphaB-crystallin has been shown to exhibit chaperone-like activity, suppressing the thermal aggregation of gamma-crystallin and aggregation of the reduced insulin B chain conferring thermotolerance to Escherichia coli BL21(DE3) cells. Mutations were made in three specific areas of the alphaB-crystallin, the N terminus D2G, the conserved phenylalanine-rich region, F24R, F27R, F27A, and the two C-terminal lysines K174L/K175L, K174G/K175G. Biophysical characterization of the mutant alphaB-crystallins using far-UV CD revealed no change in secondary structural elements. Tryptophan fluorescence demonstrated global structural changes. Heat stability of the mutant alphaB-crystallins was not significantly affected as indicated by tryptophan fluorescence of heat-treated proteins. Mutations within the phenylalanine-rich region abolish the chaperone-like activity as measured by both in vivo and in vitro assays. Proteins with mutations at the C terminus demonstrated no significant chaperone-like activity, failing to confer thermotolerance on E. coli and demonstrating no significant inhibition of protein aggregation in either gamma-crystallin or reduced insulin B chain assays. The N-terminal mutation D2G demonstrated a significant reduction in efficiency of the chaperone-like activity although some thermotolerance was conferred in the E. coli assay. In vitro assays showed that complete inhibition of aggregation was only achieved at 10-fold higher concentrations of D2G than that required by the native alphaB-crystallin. Consistent changes in the chaperone-like activity of the site-directed mutants were demonstrated by the three assays. The results suggested that both charge-charge and hydrophobic interactions are important in protein binding by alphaB-crystallin and that the conserved RLFDQFF region is vital for chaperone-like activity.

Amino Acid Sequence↗
PubMed1996

Accumulation of xylitol in the mammalian lens is related to glucuronate metabolism.

Cataract remains the major cause of blindness worldwide and a common complication of diabetes. Polyol accumulation in the lens is associated with cataract formation. Here we present evidence for a novel pathway for xylitol production in the lens involving glucuronate metabolism. Xylitol can be produced in rat and bovine lens from glucose, via the enzymes myo-inositol-oxygen oxidoreductase, D-glucuronate reductase, L-gulonate NAD(+)-3-oxidoreductase and L-iditol-NAD(+)-5-oxidoreductase, which have been found in the mammalian lens for the first time. Glucuronate reductase has been purified and was inhibited by thiol quenching reagents. UDP-glucuronyl transferase is also present in mammalian lenses; this enzyme may be an anti-toxic defense mechanism in the lens.

Animals↗
PubMed1995

Modelling molecular stability in gamma B crystallin.

Molecular modelling and dynamics simulations of gamma B crystallin and a number of its adducts were used to simulate effects of post-translational modifications. N-terminal modifications which disrupted the surface charge network of gamma-crystallin did not produce significant unfolding in any part of the gamma-crystallin molecule (P > 0.05) consistent with the results of in vitro aggregation studies. There were no significant changes to the molecular volume of the protein, suggesting that adduct formation did not result in molten globule formation. Adduct formation did however increase the vulnerability of particular gamma-crystallin cysteine residues to sulphydryl bond formation and so increased the possibility of mixed disulphide formation with glutathione. Modelling of such secondary modification products (containing both N-terminal modifications and glutathione adducts) demonstrated significant (P < 0.05) structural changes. This suggests that a post-translational modification pathway leading to crystallin cross-linking and cataractogenesis proceeds via a number of co-operative steps involving both modification at the N-terminus and mixed glutathione formation.

Computer Simulation↗
PubMed1995

Protein folds and functional similarity; the Greek key/immunoglobulin fold.

Investigations on a number of proteins in the Greek key/immunoglobulin superfold group using computer methods suggested that proteins containing modified Greek keys might exhibit some protein-protein interactions similar to those seen with immunoglobulins. The two domain beta-sheet modified Greek key structure of the beta/gamma-crystallins is shared by other proteins, for example the chaperone protein PapD. Based on computer analysis, we tested for protein-protein interactions using protein A from Staphylococcus aureus, and showed that gamma-crystallin, unlike alpha- or gamma s-crystallin, exhibits weak protein A binding. beta-Crystallin exhibits much weaker binding than gamma-crystallin. Protein A interaction may involve hydrophobic interactions around the interconnecting peptide region. We discuss the implications for the molecular evolution of the crystallins, the superfold, and for the molecular interactions in the mammalian lens that could be important in maintaining transparency.

Amino Acid Sequence↗
PubMed1994

Heterologous expression of gamma E-crystallin produces protein with an aberrant tertiary structure.

Expression of complete rat gamma E-crystallin cDNA in Saccheromyces cerevisiae and in Escherichia coli at 25 degrees C produced soluble proteins similar to rat gamma E-crystallin but with an altered tertiary structure as judged by tryptophan fluorescence. Expression of rat gamma E-crystallin cDNA in E. coli at 37 degrees C produced insoluble inclusion bodies. Refolding of denatured rat gamma E-crystallin from these inclusion bodies produced a protein similar to rat gamma E-crystallin but with altered secondary and tertiary structure, judged by tryptophan fluorescence and circular dichroism. The secondary structure of the refolded gamma-crystallin was similar in beta-sheet content to the native gamma-crystallin structure but was somewhat shifted from the native spectrum, suggesting some alteration in the relative position of the beta-sheets in the refolded structure. These results have implications for crystallin folding, in particular the importance of lens-specific factors (alpha-crystallin, low water content, redox potential) and crystallin hydration.

Animals↗
PubMed1994

Survival in patients with recurrent glioma as a measure of treatment efficacy: prognostic factors following nitrosourea chemotherapy.

The assessment of efficacy of treatment in patients with recurrent glioma is notoriously difficult, and survival is the most objective endpoint. Between 1970 and 1992, a cohort of 211 patients with recurrent glioma received nitrosourea-based chemotherapy at the time of disease progression. The median survival from the start of chemotherapy was 7 months, with 30% 1-year and 10% 2-year survival probabilities. One-year survival was 22% in 147 patients with recurrent high-grade astrocytoma, 41% in 37 patients with low-grade astrocytoma and 45% in 24 patients with oligodendroglioma. Age, histological grade and Karnofsky performance status (KPS) at recurrence were independent prognostic factors for survival on multivariate analysis. Based on patients' age, tumour grade and KPS, it was possible to define three distinct prognostic groups with 1-year survival probabilities of 60, 21 and 17% (P < 0.005). Response to chemotherapy was difficult to assess but correlated with prognostic subgroup, with highest response rate (46%) in the most favourable group and lowest (13%) in the poor prognostic group. In patients with recurrent glioma, patient and tumour parameters are the major determinants of outcome which are identical to prognostic factors at the time of primary diagnosis. They can be used to provide prognostic information for the individual patient, and to stratify patients particularly in trials assessing the efficacy of novel treatments.

Adolescent↗