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Biomedical subjects

D J Newton

Publications and source records attributed to D J Newton.

At least 19 recordsLinked to original sources
PubMed1999

Disruption of skin perfusion following longitudinal groin incision for infrainguinal bypass surgery.

OBJECTIVE: The objective of our study was to investigate whether such an incision results in a reduction in blood flow, and therefore haemoglobin oxygen saturation, across the wound. DESIGN: Microvascular oxygenation was measured with lightguide spectrophotometry in 21 patients undergoing femoropopliteal or femorodistal bypass procedures. A series of measurements were made in the groin, medial and lateral to the surface marking of the femoral artery. The mean oxygen saturation on each side was calculated, and the contra-lateral groin was used as a control. The measurements were repeated at 2 and 7 days postop. RESULTS: Oxygen saturation in the skin of the operated groins was increased significantly from baseline at 2 days postop (f = 25.80, p < 0.001) and had begun to return to normal by day 7. The rise was more marked on the lateral side of the wound than on the medial (f = 12.32, p < 0.001). There was no such difference in the control groins. All wounds healed at 10 days. CONCLUSIONS: These results show a significant difference in skin oxygenation between the lateral and medial sides of the groin following longitudinal incision. This may contribute to the relatively high incidence of postoperative infection in these wounds.

Aged↗
PubMed1999

Inhibitory anti-CYP3A4 peptide antibody: mapping of inhibitory epitope and specificity toward other CYP3A isoforms.

An antipeptide antibody has been produced that recognizes CYP3A4 and exhibits greater than 90-95% inhibition on CYP3A4-mediated reactions [Wang RW and Lu AYH (1997) Drug Metab Dispos 25:762-767]. The inhibitory epitope of the 21-amino acid peptide, corresponding to residues 253 to 273 of CYP3A4, has been identified to reside in a 7-amino acid sequence (LEDTQKH: residues 261-267 of CYP3A4). This conclusion was based on the reversal of antibody inhibition of testosterone 6beta-hydroxylation when peptides with overlapping sequence in this region were preincubated with the antibody. In immunoblotting analysis, this antibody did not recognize CYP3A5 or CYP3A7 in microsomes prepared from baculovirus-infected cells containing these two expressed isoforms. In addition, the antipeptide antibody did not inhibit testosterone 6beta-hydroxylation or midazolam 1'- and 4-hydroxylation in microsomes containing expressed CYP3A5 and CYP3A7. Because the corresponding sequence in CYP3A5 (LNDKQKH) and CYP3A7 (LKETQKH) differs from CYP3A4 by only two amino acids, six peptides with either one or two amino acid changes were used to determine which amino acid is essential for antibody-antigen interaction. Our data indicate that Glu, Asp, and Thr in the 7-amino acid sequence of CYP3A4 are critical determinants of selectivity among CYP3A isoforms.

Amino Acid Sequence↗
PubMed1998

A novel isoform of human membrane cofactor protein (CD46) mRNA generated by intron retention.

The reverse transcription polymerase chain reaction (RT-PCR) with primers specific for each of the 14 exons of the human complement regulatory protein membrane cofactor protein (MCP;CD46) has been utilized to determine MCP mRNA transcript expression in peripheral blood mononuclear cells (PBMC). An additional transcript of a larger size than predicted was consistently detected in reactions with a sense primer for exon 7, that encodes the first alternatively spliced serine-threonine-rich region (ST-A), together with an antisense exon 12 primer, RT-PCR with primers for other exons both 5' and 3' of exon 7 further showed that these MCP transcripts contain additional sequences immediately both 5' and 3' to the exon 7-encoded sequence. Comparison of genomic DNA with cDNA by PCR, in combination with sequence analysis, demonstrated the presence of the complete invariant sequences of both introns adjacent to exon 7, i.e. intron 6 (411 bp) and intron 7 (127 bp). RT-PCR using primers specific for the intron 6 sequence, together with Southern and Northern blotting using an intron 6-specific probe, confirmed retention of this intron within a novel 4.8-kb mRNA transcript in human PBMC. Due to the presence of a stop codon within intron 6, translation would result in a novel truncated MCP isoform (MCPi) containing the four invariant short consensus repeat (SCR) regions and a unique C-terminal 39 amino acid transmembrane and cytoplasmic tail region that may promote endoplasmic reticulum retention.

Alternative Splicing↗
PubMed1998

TNFB gene polymorphism in insulin-dependent diabetes mellitus: association with HLA-DR alleles.

A polymorphism of the TNF-beta gene can be detected by restriction digestion of a PCR product with NcoI. In this study we look at the risk associated with this polymorphism in a study of 69 insulin-dependent diabetes patients and 119 healthy controls. The risk was further characterized by comparison to the HLA type of the individual, since the TNF polymorphism is in linkage disequilibrium with HLA genes.

Alleles↗
PubMed1997

Human cytochrome P450 3A4-catalyzed testosterone 6 beta-hydroxylation and erythromycin N-demethylation. Competition during catalysis.

Cytochrome P450 3A4 is known to catalyze the metabolism of both endogenous substrates (such as the 6 beta-hydroxylation of testosterone) and many important therapeutic agents, including the N-demethylation of erythromycin. However, erythromycin and testosterone have been reported to have little or no effect on the metabolism of each other by recombinant CYP3A4. In an effort to understand the basis of these observations, we studied the N-demethylation of erythromycin and the 6 beta-hydroxylation of testosterone in human liver microsomes and in microsomes from cells containing recombinant human CYP3A4 and P450 reductase under a variety of experimental conditions. In both human liver microsomal and recombinant CYP3A4 systems, erythromycin inhibited testosterone 6 beta-hydroxylation in a concentration dependent manner, and vice versa. However, the inhibition mechanism was complex. At low substrate concentrations, testosterone and erythromycin acted as competitive inhibitors to each other. Under these experimental conditions, an apparent competitive inhibition of testosterone 6 beta-hydroxylation by erythromycin was observed, with Ki values similar to that of the K(m) values for erythromycin. When the rates of testosterone 6 beta-hydroxylation and erythromycin N-demethylation were determined in microsomal incubations containing both substrates at lower concentrations, the observed rates for each reaction were in good agreement with the calculated rates based on the rate equation describing simultaneous metabolism of two substrates by a single enzyme. However, at high substrate concentrations, the kinetic results could be best explained by a mechanism involving partial competitive inhibition. We conclude from these studies that testosterone and erythromycin mutually inhibit the metabolism of each other, consistent with the fact that CYP 3A4 catalyzes the metabolism of both substrates.

Aryl Hydrocarbon Hydroxylases↗
PubMed1997

Use of lightguide spectrophotometry to investigate the effect of postural changes on skin oxygenation in deep venous insufficiency.

BACKGROUND: Postural changes play an important role in the development of venous disease. The aim of this study was to investigate skin oxygenation in patients with deep venous insufficiency. METHODS: Haemoglobin oxygen saturation (SO2) was measured with macro- and micro-lightguide spectrophotometry in clinically normal skin at the gaiter area with the leg in the supine position, raised to 45 degrees and on standing. Similarly, transcutaneous oxygen tension (PtcO2) and laser Dopper flux (LDF) values were obtained. SO2 was also measured after heating the skin to 44 degrees C in the supine and standing positions. RESULTS: SO2 and PtcO2 decreased on leg raising (P < 0.05 and P < 0.01 respectively). SO2 fell on standing (P < 0.05) while PtcO2 rose (P < 0.05). On heating the skin, SO2 increased (P < 0.05) on standing. LDF fell on standing (P < 0.005) but increased on leg raising (P < 0.05). CONCLUSION: Lightguide spectrophotometry showed a profound decrease in skin oxygenation on standing and leg raising; PtcO2 measurements may miss the role of vasoconstriction with postural changes.

Adult↗
PubMed1995

Use of lightguide spectrophotometry to quantify skin oxygenation in a variable model of venous hypertension.

A variable model of venous hypertension was used to examine the immediate effect on skin oxygenation and blood flow. Measurements were carried out using laser Doppler flowmetry, transcutaneous oximetry and both macro- and micro-lightguide spectrophotometry to measure haemoglobin saturation (SO2). Assessments were performed on 20 normal subjects and in ten patients with deep venous insufficiency lying supine with a pressure cuff left uninflated (P0), then inflated to 40 mmHg (P40), 60 mmHg (P60) and 80 mmHg (P80). All measurements were performed at the gaiter area of the leg. In normal subjects median Doppler values fell significantly at P40 (P < 0.001) but further pressure increases had no more effect. Mean values of transcutaneous partial pressure of oxygen (PtcO2) fell steadily (P < 0.001), although the initial fall at P40 was small. Median SO2 fell with increasing occlusion pressure with both macro- and micro-lightguide spectrophotometry (P < 0.001), although the main reduction occurred predominantly at lower occlusion pressures. Higher levels of SO2 were obtained with local heating up to 44 degrees C, with a pattern similar to that of PtcO2. In patients, the same pattern of response to increased venous pressure occurred in areas of clinically normal skin. These data suggest that small increases in venous hypertension have an immediate and profound influence on skin blood flow and oxygen availability in normal skin.

Adult↗
PubMed1995

Amputation level assessment using lightguide spectrophotometry.

The aim of this experimental study was to investigate whether lightguide spectrophotometry in the visible wavelength range in skin could be used to predict stump healing viability in patients with critical lower limb ischaemia. Remission spectra recorded at two sites (medial and lateral) on the line of a proposed trans-tibial amputation (TTA) and at 10mm intervals along the leg were analysed to give haemoglobin oxygenation (SO2). Degree of tissue hypoxia (DTH) along the leg was defined as the percentage of values along the leg less than 10% SO2. DTH and mean SO2 values were compared with skin blood flow values ((I125) 4-Iodoantipyrine clearance technique) and clinical outcome of trans-tibial amputation, (TTA) or trans-femoral amputation (TFA), in 41 patients. SO2 histograms were also measured in 12 normal subjects for comparison. The results of the study allowed the establishment of criteria for the accurate prediction of flap healing potential. Successful TTAs all displayed a minimum mean SO2 at the medial and lateral measurement sites of 30%, together with a maximum degree of tissue hypoxia of 15% along the limb. The combination of these criteria gave a sensitivity and selectivity of 1.0 for prediction of a successful outcome of TTA.

Amputation, Surgical↗
PubMed1995

Cytochrome P450 inhibitors. Evaluation of specificities in the in vitrometabolism of therapeutic agents by human liver microsomes.

Identifying selective inhibitors of cytochrome P450 isoforms is a useful tool in defining the role of individual cytochrome P450s in the metabolism process. In this study, nine chemical inhibitors were selected based on literature data and were examined for their specificity toward cytochrome P450-mediated reactions in human liver microsomes. Furafylline was a potent, mechanism-based inhibitor for CYP1A2-mediated phenacetin O-deethylation. The probes sulfaphenazole (CYP2C9) and quinidine (CYP2D6) selectively inhibited tolbutamide methylhydroxylation and bufuralol 1'-hydroxylation, respectively. Additionally, the CYP2E1-catalyzed chlorzoxazone 6-hydroxylation was significantly inhibited by diethyldithiocarbamate. Of the CYP3A4 inhibitor probes used, troleandomycin proved to be the most specific for testosterone 6 beta-hydroxylation.

Adult↗