Search PubMed⌕ Search

Biomedical subjects

D J Newman

Publications and source records attributed to D J Newman.

At least 55 records · Page 3Linked to original sources
PubMed1999

Discovery and development of antineoplastic agents from natural sources.

Nature has provided many effective anticancer agents in current use, such as the microbially derived drugs; dactinomycin; bleomycin and doxorubicin; and the plant-derived drugs, vinblastine, irinotecan, topotecan, etoposide, and paclitaxel. The search for novel antitumor agents from natural sources continues through collaboration among scientists worldwide in the investigation of coral reefs, rainforests, and deep subsurface thermal vents for novel bioactive compounds. The potential for drug discovery is being further enhanced by recent advances in procedures for microbial cultivation and the extraction of nucleic acids from environmental samples, resulting in the identification of novel microbes that provide a vast untapped reservoir of genetic and metabolic diversity. Manipulation of the biosynthetic pathways of microbial polyketides through genetic engineering permits the biosynthesis of bioactive polyketides not generated naturally.

Animals↗
PubMed1999

Performance of a reagent strip device for quantitation of the urine albumin: creatinine ratio in a point of care setting.

AIM: We have evaluated the performance of a reagent strip (Clinitek, Bayer plc, Newbury) incorporating a novel dip and read device for the quantitation of the albumin: creatinine ratio together with their individual concentrations in urine. METHODS: The performance was compared with that of a lateral flow device for the semi-quantitation of albumin (Micral II, Roche Diagnostics, Lewes) and also a laboratory based procedure. The device employs novel methods for both analytes, using a sulphonephthalein dye binding at pH 1.5 for albumin and the peroxidase-like activity of copper creatinine complexes. The color yields of the separate reaction pads are monitored with the Clinitek 50TM bench top urine chemistry analyzer and compared to a pre-programmed calibration algorithm. RESULTS: The imprecision of the device was assessed by observing the discrepancy between duplicates in a total of 144 urine samples collected from patients with diabetes and/or renal disease; there were 10 discrepancies in the case of the albumin estimation (6.9%), 12 in the case of the creatinine estimation (12.5%) and 23 in the case of the albumin: creatinine ratio (16.0%). In the case of the Micral II, where 96 of the urines were analyzed there were 12 discrepancies (12.5%). When considered as a two-class test for albumin with a cut off of 20 mg/L the Clinitek gave a sensitivity of 95.4% with specificity of 78.9% and a positive predictive value of 87.4%. When analyzing four urines (two controls, two patient pools) with replicate analysis on the Clinitek system we found 100% agreement for the albumin estimation, 95% for creatinine and 96.7% for the albumin: creatinine for 60 analysis within a day and 100, 95 and 97.5% on single analyses each day for 20 days for the two urine controls. The discrepant results were always within one color block. When considering the albumin: creatinine ratio with a cut off of < 30 mg/g the Clinitek gave a sensitivity of 76.3% with a specificity of 89.1% and a positive predictive value of 89.7%. CONCLUSION: The Clinitek system provides a reliable means to screen for microalbuminuria with the opportunity of a semi-quantitative assessment when microalbuminuria is found.

Albuminuria↗
PubMed1999

Impact of antibody specificity and calibration material on the measure of agreement between methods for cardiac troponin I.

BACKGROUND: Available assays for cardiac troponin I (cTnI) yield numerically different results. The aim of this study was to compare patient values obtained from four cTnI immunoassays. METHODS: We studied the Stratus(R) II assay, the Opus(R) II assay, the Access(R) assay, and a research-only cTnI heterogeneous immunoassay that uses the Dade Behring aca(R) plus immunoassay system equipped with two new noncommercial monoclonal antibodies. Because the aca plus cTnI assay is for research only, we first evaluated and analytically validated it for serum and citrated plasma. Initially, each method was calibrated using the method-specific calibrator supplied by each manufacturer; however, the aca plus cTnI assay was calibrated using patient serum pools containing cTnI and selected on the basis of increased creatine kinase MB isoenzyme and with values assigned by use of the Stratus cTnI assay. For method comparisons, individual patient sample cTnI values were determined and compared with the Stratus II assay. RESULTS: Passing and Bablock regression analysis yielded slopes of 1.44 (r = 0.96; n = 72) for the Opus II vs Stratus II assays; 0.07 (r = 0.91; n = 72) for the Access vs Stratus II assays; and 0.90 (r = 0.91, n = 72) for the aca plus vs Stratus II assays. The recalibration of each method with a Stratus II-assigned serum pool improved, but did not entirely eliminate, the slope differences between the different assays (range, 1.00-1.16). The observed scatter in the correlation curves remained. CONCLUSION: There is a need to further explore the specificities of these assays with respect to the different circulating forms of cTnI.

Antibodies, Monoclonal↗
PubMed1998

Real-time analysis of cell surface HLA class I interactions.

Characterisation of the kinetics of assembly and dissociation of the HLA class I heterotrimeric complex provides valuable insights into the relative contributions of each element to complex stability. However, to date there has been no real-time binding analysis on whole cells. Here we have developed an optical biosensor model to investigate the binding of class I HLA complexes on whole cells to human beta 2-microglobulin (beta 2m) and the effects of different HLA-specific peptides on this binding. We immobilised beta 2m on an IAsys biosensor surface and established conditions to analyse the binding of this to HLA-A2 expressing cells (T2 cells). Using 721.221 cells as an HLA negative control we showed that HLA-A2 binding was optimal using a) a carboxymethylated dextran surface and b) no growth factors or supplements in the culture medium at the binding event. Using these conditions we verified specificity of binding by inhibition of the reaction with free beta 2m and determined the dissociation rate constant for T2 cell binding to beta 2m (0.03 s-1). In addition, we demonstrated the ability of different HLA specific peptides to modulate cellular HLA-A2 binding to beta 2m. This is the first time that interactions of cell surface HLA class I molecules has been investigated using real-time analysis. Furthermore, our peptide analysis has shown that this model can be used to characterise peptide specific HLA-binding responses on the whole cell surface in real-time.

Cell Line↗
PubMed1998

The matrix effects on kinetic rate constants of antibody-antigen interactions reflect solvent viscosity.

This study describes the influence of different matrices on two model antibody-antigen interactions; that between beta2microglobulin and anti beta2microglobulin, and that of rabbit anti mouse Fc fragment (RAMFc) with mouse IgG. The matrices investigated were; phosphate-buffered saline pH 7.4 containing 0.05% Tween 20 detergent, horse serum, a 50:50 mixture of phosphate-buffered saline/Tween 20 and horse serum, and four glycerol solutions of differing concentrations. A recently developed optical biosensor, the IAsys, was used to monitor the interactions in real-time and provide precise determinations of k(ass), k(diss) and KA values. The results show that the rates of association and dissociation for the two different antibody:antigen models are significantly affected by the surrounding matrix. Glycerol of known viscosity was used as a matrix in both models to show that this effect is attributable to the viscosity as opposed to proteins present in the matrix. The viscosity of the matrix has also been shown to have an apparent influence upon the overall equilibrium/affinity constant for the interaction, with measurements of KA tending to increase with viscosity. The significant effects of matrix on kinetic rate constants for antibody-antigen interactions shown here have important implications in the use of immunoassays where non-equilibrium measurements are made in serum matrices.

Animals↗
PubMed1998

The influence of exogenous peptide on beta2-microglobulin exchange in the HLA complex: analysis in real-time.

We used an optical biosensor to determine the relative binding affinity of peptides to purified HLA class I molecules. In this assay we monitor beta2-microglobulin (beta2m) exchange within the HLA-A2 molecule, whereby native beta2m in the complex is replaced by beta2m immobilized at the surface of the biosensor. Quantitative kinetic measurements permit us to obtain association rate (kass), dissociation rate (kdiss) and affinity constants (KA) for the beta2m exchange reaction, alone, (control) and in the presence of exogenous peptide. We tested a panel of six peptides which had been designed and synthesized with an HLA-A2 binding motif, and had also been tested by the T2-cell binding assay, along with control peptides. The biosensor results demonstrate that exogenous peptide influences the dynamics of beta2m exchange in a sequence-specific manner. Five of six peptides increased the association rate, decreased the dissociation rate, and significantly increased the affinity (KA=1. 55-1.88x10(9) M-1) of HLA-A2 for immobilized beta2m compared with the control (KA =1.14+/-0.04x10(9)M-1), demonstrating stabilization of the complex. One peptide was unable to stabilize the complex, as also shown in the T2 binding assay. However, analysis of peptide sequences demonstrated that the HLA-A2 secondary motif as well as primary motif residues are required for HLA-A2 stabilization. Further experiments demonstrated that beta2m exchange alone cannot stabilize the HLA class I complex at the cell surface until a peptide of sufficient binding affinity is bound. Hence kinetics equal to or below the control values in our biosensor assay probably represent an unstable complex in vivo. Unlike other methods described for the analysis of peptide stabilization, this approach is significantly faster, provides full kinetic analysis, and is simpler, since it requires no labeling of peptides. Furthermore, this may have important implications in the assessment of peptide vaccines.

Biosensing Techniques↗
PubMed1998

Measurement of the kinetics of protein uptake by proximal tubular cells using an optical biosensor.

BACKGROUND: The affinity and specificity of protein reabsorption by proximal tubular cells have been investigated using techniques for monitoring endocytosis, demonstrating a high capacity but low affinity process. It is not known whether uptake is through binding to a single binding site/receptor with differing affinities, or if there are several classes of binding sites receptors, each specific for differing proteins or groups, such as, high or low molecular weight proteins. METHODS: We have developed a novel technique for analyzing the kinetics of protein binding to tubular cells using a optical biosensor system. We have studied the binding of cultured LLCPK cells to albumin and RBP immobilized onto the sensor. By adding increasing concentrations of competing proteins [varying in molecular weight from 66,000 to 11,800 D and pI from 4.6 to 9.2 as represented by albumin, alpha1-microglobulin (alpha1M), retinol binding protein (RBP), cystatin C and beta2-microglobulin (beta2m)], specific and inhibitable cell binding was demonstrated. RESULTS: Equilibrium constants, KA, could be calculated from the reciprocal of the protein concentration causing 50% inhibition in binding rate. These were: albumin = 8.0 x 10(4) M(-1), alpha1M = 2.0 x 10(5) M(-1), RBP = 2.7 x 10(4) M(-1), cystatin C = 2.0 x 10(4) M(-1), beta2m = 4.2 x 10(3) M(-1). There were no significant differences between the measured KA's whether RBP or albumin were immobilized on the surface. CONCLUSIONS: All the proteins gave similar shaped inhibition profiles, suggesting that there is one binding site/receptor for all proteins studied, regardless of molecular weight or charge, but there are differing affinities for each protein.

Alpha-Globulins↗
PubMed1998

Development and validation of an automated latex-enhanced immunoassay for prealbumin.

The measurement of circulating prealbumin has been shown to be clinically useful in the assessment of nutritional status, both as an initial screen and in the monitoring of nutritional recovery. We describe a fully automated, noncompetitive, homogeneous, light-scattering immunoassay that has been developed for this analyte on a Dimension (Dade) analyzer. A sheep anti-prealbumin IgG fraction was covalently coupled to 40-nm chloromethyl styrene particles and, after the addition of sample, polyethylene glycol-assisted immunoagglutination was monitored by turbidimetry. The prealbumin working assay range was 8-550 mg/L at a sample volume of 2 microL and a reaction time of 6.5 min. When data were analyzed using ANOVA, total and within-run assay imprecision values (CVs) were 1-5%, and calibration and reagent stabilities were in excess of 40 days. Mean analytical recoveries were 102% +/- 4% (SD), and there was no lack of parallelism. Hemolysis, lipemia, and bilirubin did not interfere. Both plasma anticoagulated with heparin or EDTA and serum from plain or serum-separation tubes were acceptable as sample matrices. Comparison with the Beckman Array method gave a Passing and Bablok regression of: Dimension analyzer = 1.01Beckman + 7.1 (n = 103), using a common calibrator. We conclude that the prealbumin method is appropriate for clinical use according to the analytical criteria used in this study.

Agglutination↗
PubMed1997

Altered astronaut lower limb and mass center kinematics in downward jumping following space flight.

Astronauts exposed to the microgravity conditions encountered during space flight exhibit postural and gait instabilities upon return to earth that could impair critical postflight performance. The aim of the present study was to determine the effects of microgravity exposure on astronauts' performance of two-footed jump landings. Nine astronauts from several Space Shuttle missions were tested both preflight and postflight with a series of voluntary, two-footed downward hops from a 30-cm-high step. A video-based, three-dimensional motion-analysis system permitted calculation of body segment positions and joint angular displacements. Phase-plane plots of knee, hip, and ankle angular velocities compared with the corresponding joint angles were used to describe the lower limb kinematics during jump landings. The position of the whole-body center of mass (COM) was also estimated in the sagittal plane using an eight-segment body model. Four of nine subjects exhibited expanded phase-plane portraits postflight, with significant increases in peak joint flexion angles and flexion rates following space flight. In contrast, two subjects showed significant contractions of their phase-plane portraits postflight and three subjects showed insignificant overall changes after space flight. Analysis of the vertical COM motion generally supported the joint angle results. Subjects with expanded joint angle phase-plane portraits postflight exhibited larger downward deviations of the COM and longer times from impact to peak deflection, as well as lower upward recovery velocities. Subjects with postflight joint angle phase-plane contraction demonstrated opposite effects in the COM motion. The joint kinematics results indicated the existence of two contrasting response modes due to microgravity exposure. Most subjects exhibited "compliant" impact absorption postflight, consistent with decreased limb stiffness and damping, and a reduction in the bandwidth of the postural control system. Fewer subjects showed "stiff" behavior after space flight, where contractions in the phase-plane portraits pointed to an increase in control bandwidth. The changes appeared to result from adaptive modifications in the control of lower limb impedance. A simple 2nd-order model of the vertical COM motion indicated that changes in the effective vertical stiffness of the legs can predict key features of the postflight performance. Compliant responses may reflect inflight adaptation due to altered demands on the postural control system in microgravity, while stiff behavior may result from overcompensation postflight for the presumed reduction in limb stiffness inflight.

Acceleration↗
PubMed1997

A simple, automated, nonextraction assay for serum digoxin using particle-enhanced immunoassay.

We have adapted a latex particle-enhanced immunoassay for serum digoxin to a centrifugal analyser. A 4-microliter serum sample (without pretreatment) inhibits the monoclonal antibody induced aggregation of digoxin-coated latex particles. The total assay time is 10 min and mean analytical recoveries were 98.1%. Intra and interassay precision were < 4.2 and < 15.0%, respectively. Method comparison with an established fluorescence polarisation immunoassay (FPIA) gave R = 0.97, and a Deming regression analysis of particle-enhanced turbidimetric inhibition immunoassay (PETINIA) = FPIA x 0.78 + 0.007 microgram/L (n = 91). There was no evidence of significant interference from digoxin-like immunoreactive compounds in patients with chronic renal failure. This assay can be adapted to most photometric analysers used in routine laboratories and is a significant advance in the sensitivity of latex particle-enhanced immunoassays in a serum matrix.

Cardiotonic Agents↗
PubMed1997

Measurement of albumin and low molecular weight proteins in the urine of newborn infants using a cotton wool ball collection method.

OBJECTIVE: The aim of this study was to investigate the inter-relationship between urinary excretion of alpha-1-microglobulin (A1M), retinol-binding protein (RBP) and albumin in term and premature neonates, with urine collected into cotton wool balls and extracted by a novel method. SUBJECTS AND METHODS: Sixty-four infants were studied on the first day of life; 26 had been born at term (37-42 weeks gestation) and 38 prematurely (24-28 weeks n = 16, 29-36 weeks n = 22). Urine collected into cotton wool balls was analysed following a new detergent extraction method, which resulted in a recovery rate of 94-107% for albumin. A1M, RBP and creatinine. RESULTS: Urinary protein excretion, expressed as a ratio to urinary creatinine, decreased significantly with increasing gestational age (24-28 weeks, 29-36 weeks, 37-42 weeks: albumin:creatinine ratio mg/mmol mean 96.9, 31.7, 19.3; A1M:creatinine ratio mg/mmol mean 99.3, 37.0, 7.8; RBP:creatinine ratio mg/mmol mean 16.2, 3.8, and < 0.01, below the limit of detection, respectively). When results were corrected for birthweight, this gestation-associated effect was still present for A1M and RBP, but not for albumin. In premature infants there was a significant positive correlation between A1M:creatinine ratio and RBP:creatinine ratio (r = 0.85), and also between albumin and both A1M and RBP (r = 0.82 and 0.77). CONCLUSION: Increased excretion of A1M, RBP and albumin at earlier gestational ages is probably due to proximal tubular immaturity, although tubular damage and also glomerular dysfunction cannot be excluded as possible explanations.

Albuminuria↗
PubMed1997

Adaptation of latex-enhanced assay for percent glycohemoglobin to a Dade Dimension analyzer.

At present no method for glycohemoglobin (%HbA1c) is automated on a main-line analyzer to allow joint measurement with other indicators of diabetic control such as glucose and cholesterol. We describe an adaptation of a latex-enhanced competitive immunoassay for quantifying %HbA1c to the Dade International Dimension analyzer. After a manual hemolysis step, HbA1c and total hemoglobin (Hb) are determined separately. The concentration of glycated beta-subunit is obtained from the immunoassay, whereas Hb is assessed colorimetrically from a derivatized form. Both reactions were fully optimized for accuracy, precision, and specificity on the Dimension; stabilities of reagents and calibration were established; and potential interferences were assessed. The analyzer gave reliable results over the required clinical range of 1-15% HbA1c. Within-run and total assay variation were within 5% of the target CV limits, as determined by ANOVA with three representative sample pools across 20 days. Close agreement with an established HPLC procedure and a commercially available enzyme immunoassay was observed for 140 samples from clinically defined patient groups. Additional samples from patients with hemoglobinopathies (n = 20) demonstrated a more complex relationship between methods. We conclude that adaptation of the method for use with the Dimension analyzer is a valid method for quantifying %HbA1c.

Autoanalysis↗
PubMed1997

Development and validation of a particle-enhanced turbidimetric inhibition assay for urine albumin on the Dade aca analyzer.

The measurement of urine albumin now has a well-established role in the monitoring of patients with diabetes mellitus. We have developed a particle-enhanced immunoturbidimetric inhibition assay for urine albumin on the Dade aca analyzer. The inhibition approach removes any of the potential antigen excess difficulties that could be expected from the wide clinical range of urine albumin, but retains the sensitivity advantages of latex-enhanced immunoturbidimetry. Human serum albumin (HSA) is covalently attached to 40-nm poly(chloromethyl)styrene-modified latex particles. This reagent, along with monoclonal antibody to HSA, is aliquoted into the aca reagent pack along with polyethylene glycol 8000 in a tablet form (giving a final reaction concentration of 15 g/L). A 150 mmol/L phosphate buffer, pH 7.8, is used to fill the reagent pack in the instrument and the agglutination reaction is monitored at 340 nm. The sample volume is 100 microL and the calibration curve covers the range 2-250 mg/L. Evaluation of commercial scale reagents against the Beckman Array nephelometric immunoassay system gave a Deming regression correlation of aca = 0.87 x Beckman + 8.5, r = 0.995, n = 145. Mean analytical recovery was 104+/-4.5%, n = 20, and there was no evidence of a lack of parallelism. Interassay precision was 8.8% at 10.0 mg/L and <2.5% at >65 mg/L. Calibrator stability was in excess of 60 days. A small reference range study (24-h urine collections, n = 27) gave a mean of 5.6 mg/L with a range of 0.5-16.2 mg/L. Analytical sensitivity (2.5 SD from zero) was 0.40 mg/L.

Albuminuria↗