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Biomedical subjects

J E Davies

Publications and source records attributed to J E Davies.

At least 19 recordsLinked to original sources
PubMed2000

Engineering three-dimensional bone tissue in vitro using biodegradable scaffolds: investigating initial cell-seeding density and culture period.

New three-dimensional (3D) scaffolds for bone tissue engineering have been developed throughout which bone cells grow, differentiate, and produce mineralized matrix. In this study, the percentage of cells anchoring to our polymer scaffolds as a function of initial cell seeding density was established; we then investigated bone tissue formation throughout our scaffolds as a function of initial cell seeding density and time in culture. Initial cell seeding densities ranging from 0.5 to 10 x 10(6) cells/cm(3) were seeded onto 3D scaffolds. After 1 h in culture, we determined that 25% of initial seeded cells had adhered to the scaffolds in static culture conditions. The cell-seeded scaffolds remained in culture for 3 and 6 weeks, to investigate the effect of initial cell seeding density on bone tissue formation in vitro. Further cultures using 1 x 10(6) cells/cm(3) were maintained for 1 h and 1, 2, 4, and 6 weeks to study bone tissue formation as a function of culture period. After 3 and 6 weeks in culture, scaffolds seeded with 1 x 10(6) cells/cm(3) showed similar tissue formation as those seeded with higher initial cell seeding densities. When initial cell seeding densities of 1 x 10(6) cells/cm(3) were used, osteocalcin immunolabeling indicative of osteoblast differentiation was seen throughout the scaffolds after only 2 weeks of culture. Von Kossa and tetracycline labeling, indicative of mineralization, occurred after 3 weeks. These results demonstrated that differentiated bone tissue was formed throughout 3D scaffolds after 2 weeks in culture using an optimized initial cell density, whereas mineralization of the tissue only occurred after 3 weeks. Furthermore, after 6 weeks in culture, newly formed bone tissue had replaced degrading polymer.

Animals↗
PubMed2000

No longer an exclusive club: eukaryotic signalling domains in bacteria.

Reversible phosphorylation of serine, threonine and tyrosine residues by the interplay of protein kinases and phosphatases plays a key role in regulating many different cellular processes in eukaryotic organisms. A diversity of control mechanisms exists to influence the activity of these enzymes and choreograph the correct concert of protein modifications to achieve distinct biological responses. Such enzymes and their adaptor molecules were long thought to be specific to eukaryotic cellular processes. However, there is increasing evidence that many prokaryotes achieve regulation of key components of cellular function through similar mechanisms.

Bacterial Physiological Phenomena↗
PubMed2000

Profile of plasma N-terminal proBNP following acute myocardial infarction; correlation with left ventricular systolic dysfunction.

AIMS: The aims of this study were to describe the temporal pattern of plasma N-terminal pro-brain natriuretic peptide, to examine the optimum time of sampling and to compare plasma N-terminal pro-brain natriuretic peptide to clinical criteria in terms of identification of impaired left ventricular systolic function following acute myocardial infarction. METHODS AND RESULTS: Measurements of N-terminal pro-brain natriuretic peptide were made in 60 patients at 14-48 h, 49-72 h, 73-120 h, 121-192 h following myocardial infarction and at 6 weeks in survivors. Left ventricular wall motion index was assessed during hospitalization (WMI-1) and at 6 weeks (WMI-2). N-terminal pro-brain natriuretic peptide levels were elevated at all time points, to a greater extent in anterior compared to inferior infarction (P < 0.05). A biphasic profile of plasma concentration was observed in anterior infarction with peaks at 14-48 h and 121-192 h. This was sustained at 6 weeks. N-terminal pro- brain natriuretic peptide at 73-120 h was the best independent predictor of WMI-1 (P < 0.005). N-terminal pro-brain natriuretic peptide was higher at all times in patients who received ACE inhibitor therapy compared to those who did not (P < 0.005). N-terminal pro-brain natriuretic peptide at 73-120 h (R(2) = 17.7%, P = 0.005) and previous myocardial infarction (R(2) = 5.3%, P < 0.05) were independent predictors of poor outcome (WMI-2 < or = 1.2 or death by 6 weeks). CONCLUSIONS: A biphasic pattern of plasma N-terminal pro-brain natriuretic peptide is seen after anterior myocardial infarction. Plasma level is strongly correlated to wall motion index soon after and remote from acute myocardial infarction. Plasma N-terminal pro-brain natriuretic peptide measured later in hospitalization better predicts poor outcome following myocardial infarction than when it is measured in the immediate post infarction period.

Adult↗
PubMed2000

Conjugative junctions in RP4-mediated mating of Escherichia coli.

The physical association of bacteria during conjugation mediated by the IncPalpha plasmid RP4 was investigated. Escherichia coli mating aggregates prepared on semisolid medium were ultrarapidly frozen using copper block freezing, followed by freeze substitution, thin sectioning, and transmission electron microscopy. In matings where the donor bacteria contained conjugative plasmids, distinctive junctions were observed between the outer membranes of the aggregates of mating cells. An electron-dense layer linked the stiffly parallel outer membranes in the junction zone, but there were no cytoplasmic bridges nor apparent breaks in the cell walls or membranes. In control experiments where the donors lacked conjugative plasmids, junctions were not observed. Previous studies have shown that plasmid RP4 carries operons for both plasmid DNA processing (Tra1) and mating pair formation (Tra2). In matings where donor strains carried Tra2 only or Tra2 plus the pilin-processing protease TraF, junctions were found but they were shorter and more interrupted than the wild type. If the donor strain had the pilin gene knocked out (trbC), junctions were still found. Thus, it appears that the electron-dense layer between the outer membranes of the conjugating cells is not composed of pilin.

Bacterial Proteins↗
PubMed2000

DNA-Based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III.

Bacteria of the Burkholderia cepacia complex consist of five discrete genomic species, including genomovars I and III and three new species: Burkholderia multivorans (formerly genomovar II), Burkholderia stabilis (formerly genomovar IV), and Burkholderia vietnamiensis (formerly genomovar V). Strains of all five genomovars are capable of causing opportunistic human infection, and microbiological identification of these closely related species is difficult. The 16S rRNA gene (16S rDNA) and recA gene of these bacteria were examined in order to develop rapid tests for genomovar identification. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA revealed sequence polymorphisms capable of identifying B. multivorans and B. vietnamiensis but insufficient to discriminate strains of B. cepacia genomovars I and III and B. stabilis. RFLP analysis of PCR-amplified recA demonstrated sufficient nucleotide sequence variation to enable separation of strains of all five B. cepacia complex genomovars. Complete recA nucleotide sequences were obtained for 20 strains representative of the diversity of the B. cepacia complex. Construction of a recA phylogenetic tree identified six distinct clusters (recA groups): B. multivorans, B. vietnamiensis, B. stabilis, genomovar I, and the subdivision of genomovar III isolates into two recA groups, III-A and III-B. Alignment of recA sequences enabled the design of PCR primers for the specific detection of each of the six latter recA groups. The recA gene was found on the largest chromosome within the genome of B. cepacia complex strains and, in contrast to the findings of a previous study, only a single copy of the gene was present. In conclusion, analysis of the recA gene of the B. cepacia complex provides a rapid and robust nucleotide sequence-based approach to identify and classify this taxonomically complex group of opportunistic pathogens.

Burkholderia Infections↗
PubMed2000

Plasma N terminal pro-brain natriuretic peptide and cardiotrophin 1 are raised in unstable angina.

OBJECTIVE: To compare circulating concentrations of N terminal pro-brain natriuretic peptide (N-BNP) and cardiotrophin 1 in stable and unstable angina. DESIGN AND SETTING: Observational study in a teaching hospital. PATIENTS: 15 patients with unstable angina, 10 patients with stable angina, and 15 controls. MAIN OUTCOME MEASURES: Resting plasma N-BNP and cardiotrophin 1 concentrations. RESULTS: N-BNP concentration (median (range)) was 714 fmol/ml (177-3217 fmol/ml) in unstable angina, 169.5 fmol/ml (105.7-399.5 fmol/ml) in stable angina (p = 0.005 v unstable angina), and 150.5 fmol/ml (104. 7-236.9 fmol/ml) in controls (p < 0.0001 v unstable angina; NS v stable angina). Cardiotrophin 1 concentration was 142.5 fmol/ml (42. 2-527.4 fmol/ml) in unstable angina, 73.2 fmol/ml (41.5-102.1 fmol/ml) in stable angina (p < 0.05 v unstable angina), and 27 fmol/ml (6.9-54.1 fmol/ml) in controls (p < 0.0005 v stable angina; p < 0.0001 v unstable angina). Log cardiotrophin 1 correlated with log N-BNP in unstable angina (r = 0.93, p < 0.0001). CONCLUSIONS: Both circulating N-BNP and cardiotrophin 1 are raised in unstable angina, while cardiotrophin 1 alone is raised in stable angina. The role of cardiotrophin 1 and the relation between cardiotrophin 1 and N-BNP in myocardial ischaemia remain to be defined.

Aged↗
PubMed2000

Na+/H+ exchanger activity and phosphorylation in temperature-sensitive immortalized proximal tubule cell lines derived from the spontaneously hypertensive rat.

Freshly isolated proximal tubules from the spontaneously hypertensive rat (SHR) demonstrate elevated Na(+)/H(+) exchanger (NHE) activity, but the underlying mechanism is unclear. Because of the difficulties in preparing sufficient numbers of proximal tubule cells for detailed biochemical studies, we have generated cell lines from SHR and Wistar-Kyoto rat (WKY) proximal tubule cells. Cell lines were obtained by transforming the cells with an origin-defective mutant of simian virus 40 encoding a heat-labile T antigen (tsA58 transformant). Such cells proliferate at the permissive temperature of 33 degrees C, but growth is abolished at the restrictive temperature of 39 degrees C. The predominant NHE isoform expressed was isoform 1, as determined by sensitivity to HOE-694 (3-methylsulphonyl-4-piperidinobenzoyl guanidine) and Western blotting using specific polyclonal antisera to NHE-1. NHE-3 protein was also present. Northern blots of poly(A) mRNA extracts of the cell lines revealed a low abundance of transcripts for NHE-2, -3 and -4, with no systematic difference between the lines. Although the intracellular pH was similar in the SHR and WKY lines, HOE-694-sensitive H(+) efflux due to NHE-1 was substantially elevated in SHR lines compared with WKY lines (95.0+/-2.8 and 39. 9+/-5.7 mmol.min(-1).l(-1) respectively; P<0.001; n=6). H(+) efflux due to non-Na(+)-dependent mechanisms were similar in lines from the two strains. Western blotting revealed that NHE-1 density was also very similar in SHR and WKY lines, and subcellular fractionation of homogenates indicated that NHE-1 was localized predominantly to plasma membranes. Thus the turnover number of NHE-1 was increased. Immunoprecipitation of (32)P-labelled phosphoproteins from these lines demonstrated an approximately 2-fold higher degree of phosphorylation of NHE-1 in SHR compared with WKY lines. These cell lines form a useful model for defining the biochemical mechanisms leading to the NHE-1 phenotype in the SHR kidney, in addition to investigations of other SHR phenotypic markers.

Animals↗
PubMed2000

Elevated circulating cardiotrophin-1 in heart failure: relationship with parameters of left ventricular systolic dysfunction.

Cardiotrophin-1 (CT-1) is a cytokine that has been implicated as a factor involved in myocardial remodelling. The objective of the present study was to establish the relationship between circulating levels of CT-1 and measures of left ventricular size and systolic function in patients with heart failure. We recruited 15 normal subjects [six male; median age 60 years (range 30-79 years)] and 15 patients [11 male; median age 66 years (range 43-84 years)] with a clinical diagnosis of heart failure and echocardiographic left ventricular systolic dysfunction (LVSD). Echocardiographic variables (left ventricular wall motion index, end-diastolic and -systolic volumes, stroke volume, fractional shortening) and plasma CT-1 levels were determined. In patients with LVSD [median wall motion index 0.6 (range 0.3-1.4)], CT-1 was elevated [median 110.4 fmol/ml (range 33-516 fmol/ml)] compared with controls [wall motion index 2 in all cases; median CT-1 level 34.2 fmol/ml (range 6.9-54.1 fmol/ml); P<0.0001]. Log CT-1 was correlated with log wall motion index (r=-0.76, P<0.0001), log left ventricular end-systolic volume (r=0.54, P<0.05), stroke volume (r=-0.60, P=0.007) and log fractional shortening (r=-0.70, P=0.001). In a multivariate model of the predictors of log wall motion index, the only significant predictor was log CT-1 (R(2)=56%, P=0.006). This is the first assessment of the relationship between plasma CT-1 levels and the degree of LVSD in humans, and demonstrates that CT-1 is elevated in heart failure in relation to the severity of LVSD.

Adult↗
PubMed1999

In vitro degradation of a novel poly(lactide-co-glycolide) 75/25 foam.

Macroporous poly(lactide-co-glycolide) PLGA 75/25 foams were prepared for application in bone tissue engineering. Their in vitro degradation behaviour was followed over a 30 week period at 37 degrees C and at one of three pHs: (1) pH 5.0, which mimics the acidic environment produced by activated macrophages, (2) pH 7.4, which reproduces normal physiological conditions and (3) an intermediate pH 6.4. The degradation of the PLGA 75/25 foams was studied by measuring changes in mass, molecular weight and morphology. The degradation profile of foams maintained at pH 5.0, 6.4 and 7.4 was similar until week 16, after which foams maintained at pH 6.4 and 7.4 had comparable degradation patterns whereas foams maintained at pH 5.0 degraded faster. For example, mass loss was less than 3% for foams maintained at all three pHs until week 16; however, by week 30, foams maintained at pH 6.4 and 7.4 had lost 30% of their mass whereas foams maintained at pH 5.0 had lost 90% of their mass. Foams maintained at pH 6.4 and 7.4 showed a similar constant decrease in molecular weight over the entire degradation study. Foams maintained at pH 5.0 had a similar rate of molecular weight loss as those maintained at pH 6.4 and 7.4 until week 16, after which the rate of molecular weight loss of foams maintained at pH 5.0 was accelerated. The morphology of the foams maintained at pH 6.4 and 7.4 was unchanged for 25 weeks. Foams maintained at pH 5.0 collapsed after week 18. Thus the PLGA 75/25 foams, described herein, maintained their 3-D morphology at physiological pH for over 6 months, which is an important feature for tissue engineering applications.

Bone Substitutes↗
PubMed1999

Plasma N-terminal pro-brain natriuretic peptide and the ECG in the assessment of left-ventricular systolic dysfunction in a high risk population.

AIMS: To examine the value of N-terminal pro-brain natriuretic peptide, abnormal electrocardiogram and other baseline clinical and laboratory variables in identifying patients with left ventricular systolic dysfunction in a high risk population. METHODS AND RESULTS: We studied 243 patients (129 male, median age 73 years, range 20-94) referred for echocardiography. The relationship between left ventricular wall motion index and log N-terminal pro-brain natriuretic peptide, log creatinine, electrocardiogram, age, history of hypertension, history of ischaemic heart disease, gender, valvular disease and current drug therapy was examined using regression analysis. There was a strong correlation between N-terminal pro-brain natriuretic peptide and left ventricular wall motion index for the whole population (r=-0.624, P<0.001) and in those receiving diuretic +/- angiotensin converting enzyme inhibitor (r= -0.661, P<0.005) and in those receiving neither (r=-0.584, P<0. 005). On multiple regression analysis, log N-terminal pro-brain natriuretic peptide (P<0.001), age (P=0.015), current diuretic (P=0. 002) or angiotensin converting enzyme inhibitor use (P=0.001) and male gender (P=0.026) were independently associated with a low left ventricular wall motion index. Log N-terminal pro-brain natriuretic peptide alone (R(2)=39%) was a better predictor of left ventricular wall motion index than any other single or combination of factors. Plasma N-terminal pro-brain natriuretic peptide>275 pmol l(-1)predicted left ventricular wall motion index < or =1.2 with a sensitivity of 93.8%, a specificity of 55% and a negative predictive value of 93%. Left ventricular function was impaired in 18/36 patients with a normal electrocardiogram, in all of whom N-terminal pro-brain natriuretic peptide was >275 fmol ml(-1). CONCLUSION: Of the variables studies, N-terminal pro-brain natriuretic peptide had the strongest correlation with reduced left ventricular wall motion index. The electrocardiogram had a poor predictive value for left ventricular systolic dysfunction in this population. Plasma N-terminal pro-brain natriuretic peptide can usefully predict patients with a reduced left ventricular wall motion index in whom echocardiographic examination may be appropriate.

Adult↗
PubMed1999

An immunoluminometric assay for N-terminal pro-brain natriuretic peptide: development of a test for left ventricular dysfunction.

Measurement of plasma levels of brain natriuretic peptide (BNP) has been used to assess left ventricular dysfunction and prognosis. Levels of the N-terminus of the precursor of BNP (NT-proBNP) have been reported to be elevated to a greater extent than BNP in left ventricular dysfunction. We have devised a non-radioactive sensitive and specific assay for NT-proBNP based on a competitive ligand binding principle. The chemiluminescent label 4-(2-succinimidyloxycarbonylethyl)phenyl-10-methylacridinium 9-carboxylate fluorosulphonate was used to label peptides representing domains in the middle and C-terminal sections of NT-proBNP. Assay of the C-terminal section of NT-proBNP (amino acids 65-76) in patients with proven left ventricular dysfunction [left ventricular wall motion index median 0.9 (range 0.3-1.4)] revealed elevated values [median 639 (386-911) fmol/ml] compared with normal controls [left ventricular wall motion index of 2 in all, NT-proBNP median 159 (120-245) fmol/ml, P<0.001]. Measurement of the middle section of NT-proBNP (amino acids 37-49) was not a discriminating test. It is thus possible to derivatize small peptides with a methyl acridinium label and preserve immunodetection with specific antibodies. Such methodology may allow non-radioactive immunoluminometric assays to be devised.

Adult↗
PubMed1999

Assessment of the stability of N-terminal pro-brain natriuretic peptide in vitro: implications for assessment of left ventricular dysfunction.

Plasma concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP) are raised in patients with left ventricular dysfunction. Measurement of this peptide has a potential diagnostic role in the identification and assessment of patients with heart failure. The stability of this peptide over time periods and conditions pertaining to routine clinical practice has not been reported previously. Blood samples were obtained from 15 subjects. One aliquot was processed immediately, and the remaining portions of the blood samples were stored for 24 h or 48 h at room temperature or on ice prior to processing. Plasma concentrations of NT-proBNP were measured with a novel immunoluminometric assay developed within our laboratory. Mean plasma concentrations of NT-proBNP were not significantly different whether blood samples were centrifuged immediately and stored at -70 degrees C or kept at room temperature or on ice for 24 h or 48 h. The mean percentage differences from baseline (reference standard) were +5.2% (95% confidence interval +18.2 to -7.8%) and +0.8% (+15.2 to -13.7%) after storage for 24 h at room temperature or on ice respectively, and +8.9% (+24.2 to -6. 5%) and +3.2% (+15.1 to -0.9%) for storage for 48 h at room temperature or on ice respectively. Pearson correlation coefficients for baseline NT-proBNP concentrations compared with levels at 48 h at room temperature or on ice were r=0.89 and r=0.83 respectively (both P<0.0001). Thus NT-proBNP extracted from plasma samples treated with EDTA and aprotinin is stable under conditions relevant to clinical practice.

Adult↗