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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
PubMed1992

Bleomycin-induced unscheduled DNA synthesis in non-permeabilized human and rat hepatocytes is not paralleled by 8-oxo-7,8-dihydrodeoxyguanosine formation.

The genetic toxicity of the antitumour antibiotic bleomycin (BLM) is thought to involve the formation of a reactive oxygen intermediate. 8-Oxo-7,8-dihydrodeoxyguanosine (oxo8dG), an oxidation product of deoxyguanosine, is one of the major products formed when isolated DNA is exposed to oxygen radical generating systems. Gamma-irradiation (10-500 Gy 60Co; 10 Gy/min) or BLM and Fe2+ (37.5-150 U/L and 0.5 mM, respectively) treatment of isolated DNA (0.25 mg/mL) increased oxo8dG above background. In the latter case, the effect was greater than that with Fe2+ (0.5 mM) alone and was dependent on the dose of BLM. When DNA was irradiated with 500 Gy60Co, deoxyguanosine oxidation was inhibited by antioxidants (ethanol: 37.5 and 98% inhibition at 2 and 20 mM, respectively; mannitol: 20.5, 60 and 92% inhibition at 0.1, 1.0 and 10 mM, respectively). Similarly the BLM-induced production of oxo8dG was inhibited (64%) by mannitol (10 mM). BLM also caused production of base propenals on interaction with isolated DNA. In contrast, oxo8dG was not induced above background concentration (27 mol oxo8dG/10(6) mol dG) in permeabilized (37 degrees) and non-permeabilized (4 degrees and 37 degrees) rat hepatocytes treated with BLM (260 U/L). Despite this, there was extensive BLM-induced unscheduled DNA synthesis (10 and 100 U/L) in non-permeabilized rat and human hepatocytes in the absence of hydroxyurea. These findings, in accord with other observations, draw into question the role of .OH in BLM-induced DNA damage and the mimicry of ionizing radiation in cellular systems.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1992

Intracellular free-magnesium levels in vascular smooth muscle and striated muscle cells of the spontaneously hypertensive rat.

In humans with essential hypertension and in spontaneously hypertensive rats (SHR), insulin resistance may be present even in lean individuals. As the basis for this abnormality is unknown, we have used a newly developed fluorophore to measure intracellular free-Mg2+ concentrations in cultured aortic vascular smooth muscle cells and striated muscle cells from SHR and normotensive Wistar Kyoto (WKY) rats. Intracellular free-Mg2+ levels were lower in both striated muscle cells (SHR, 0.423 +/- 0.077 mmol.L-1 v WKY, 0.559 +/- 0.068 mmol.L-1; P less than .001) and vascular smooth muscle cells (SHR, 0.406 +/- 0.067 mmol.L-1 v WKY, 0.625 +/- 0.077 mmol.L-1; P less than .001) from hypertensive animals. This widespread, intrinsic defect in the regulation of intracellular Mg2+ may explain the increased vascular resistance and reduced insulin sensitivity present in hypertension.

Animals↗
PubMed1992

Fibroblastic regulation of osteoblast function by prostaglandins.

The effects of osteogenic inhibitory factors secreted by human periodontal ligament fibroblasts were studied in rat bone marrow stromal cell cultures. Serum-free conditioned medium from cultures of fibroblasts strongly depressed formation of mineralized tissue by bone marrow cell cultures. The inhibitory activity was reduced by treatment of fibroblast cultures with indomethacin or by pretreatment of conditioned medium with specific antibodies to prostaglandins (PGs) E2 and F2 alpha. Passage of conditioned medium over octadecyl columns enriched PGs four-fold and significantly increased inhibitory activity. Inhibition of mineralization was replicated by treatment of bone-cell cultures with PGs B2, D2, E2, F2 alpha and I2 at concentrations of 350 ng/ml to 350 pg/ml. All combinations of these agents were inhibitory but PGE2 and PGF2 alpha exhibited the greatest inhibition at low concentrations (350 pg/ml). These experiments indicate that fibroblasts secrete PGs which can inhibit bone formation, and this may be one mechanism whereby fibroblasts can modulate osteogenesis at the interfaces of soft and mineralizing connective tissues.

Animals↗
PubMed1992

Lipids and cellular Na+/H+ antiport activity in diabetic nephropathy.

Increased cellular Na+/H+ antiport activity has been documented in various cell types from hypertensive humans and rats. This membrane abnormality may be associated with the thickening of the vascular media of resistance vessels. Such an abnormality has also been demonstrated in cells from type I diabetic patients with nephropathy, and may indicate the predisposition to essential hypertension in such patients. We now demonstrate the importance of the rate-limiting enzyme for cholesterol and isoprenoid synthesis, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, in determining cellular Na+/H+ antiport activity. This finding may have application in the future treatment of diabetic patients with proteinuria.

Carrier Proteins↗
PubMed1992

Intracellular pH and Na+/H+ antiport activity of cultured skin fibroblasts from diabetics.

Increased leucocyte Na+/H+ antiport activity has previously been demonstrated in both hypertensive subjects and Type 1 diabetic patients with nephropathy and may indicate a predisposition to hypertension in such diabetic patients. We have studied intracellular pH and Na+/H+ antiport activity in cultured skin fibroblasts from diabetic patients with and without nephropathy, together with non-diabetic controls to assess if such differences persisted in cultured cells. Fibroblasts from diabetic patients with nephropathy were significantly more alkaline [median (range): 6.90 (6.82 to 7.07)] compared to both normoalbuminuric diabetic patients [6.81 (6.75 to 6.89)] or normal controls [6.82 (6.77 to 6.93)] (P < 0.001 for both). This was associated with a raised Na+/H+ antiport activity in cells from patients with nephropathy when intracellular pH (pHi) was clamped to pH 6.5, without any differences in the maximal transport capacity of the antiport at pHi 6.2. Using both intracellular pH and Na+/H+ antiport activity at pHi 6.5, patients with nephropathy were separated from uncomplicated subjects with a sensitivity of 92% and a specificity of 100%. In conclusion, the raised Na+/H+ antiport activity in cells from patients with diabetic nephropathy persists despite passaging in vitro, thus indicating a heritable component, and results mainly from an increased apparent affinity of the antiport for intracellular H+.

Adult↗
PubMed1992

Intracellular free magnesium in lymphocytes from patients with congestive cardiac failure treated with loop diuretics with and without amiloride.

Cellular Mg2+ depletion has been reported in patients on long term diuretic therapy. We therefore measured lymphocyte free Mg2+ concentrations in patients with congestive cardiac failure treated with loop diuretics or a frusemide/amiloride mixture (Frumil) and compared them with control subjects. There was no correlation between lymphocyte free Mg2+ levels and plasma Mg2+ concentrations. Patients treated with loop diuretics did not show lymphocyte intracellular free Mg2+ depletion compared with normal controls, and those on Frumil showed higher intracellular free Mg2+ levels than normal.

Amiloride↗
PubMed1992

Global changes in gene expression related to antibiotic synthesis in Streptomyces hygroscopicus.

Two-dimensional gel electrophoresis was used to follow changes in gene expression associated with antibiotic (bialaphos) biosynthesis in Streptomyces hygroscopicus. Cultures were pulse-labelled with [35S]-methionine before, during, and after the switch from primary to secondary metabolism in order to compare kinetic profiles of bialaphos (antibiotic) production (bap) genes during this metabolic transition. Separation of gene products on two-dimensional gels revealed that 27 were dependent on brpA for optimal expression and were activated as the culture approached stationary phase. Genes which encoded 10 brpA-dependent proteins were mapped to a 10 kb SstI fragment of the 35 kb bap gene cluster by expressing them in Streptomyces lividans using the thiostrepton-inducible tipA promoter. N-terminal amino acid sequences of two brpA-dependent proteins, obtained by direct microsequencing of protein spots excised from two-dimensional gels, identified them as gene products mapping to the same region and involved in secondary metabolic conversions of the bap pathway. The kinetics of synthesis of 16 brpA-dependent gene products were characterized using QUEST computer software. Cluster analysis performed on the kinetics of synthesis of 346 of the most highly expressed gene products of HP5-29, including 16 brpA-dependent ones, identified 75 families having distinct patterns of expression. Many brpA-dependent proteins were clustered together; 10 were found in one kinetic family. These kinetic families also included brpA-independent gene products perhaps subject to similar regulatory mechanisms and thus possibly involved in bialaphos biosynthesis. The activation/derepression of bap expression took place as cultures approached stationary phase and was temporally related to synthesis of ppGpp.

Amino Acid Sequence↗
PubMed1992

Abnormalities in Na+/H+ antiporter activity in diabetic nephropathy.

In hypertensive humans and the spontaneously hypertensive rat, increased cellular Na+/H+ antiport activity has been demonstrated in leukocytes, platelets, skeletal muscle, and vascular smooth muscle cells. This membrane abnormality may be associated with medial thickening of resistance vessels. A similar membrane transport abnormality has also been demonstrated in leukocytes and fibroblasts from type 1 diabetic patients with nephropathy. This membrane transport marker of hypertension may indicate a predisposition to essential hypertension in such patients and may lead to diabetic nephropathy, possibly from mesangial expansion.

Biomarkers↗
PubMed1992

Infrared spectroscopic method for analysis of precipitates on a cell culture dish.

A new quick infrared spectroscopic method was developed and applied to analyze precipitates formed by cultured cells on a Petri dish. This IR method allows a dish to be placed directly on the sample beam window of a normal double beam IR spectrometer. Placing a blank dish on the reference beam window of the spectrometer compensates for the high background absorption due to the dish on the sample beam window and enables an identifiable spectrum to be obtained. Using this technique with a Petri dish, a high correlation factor of 0.998 was found between the PO4 peak absorbance and the weight of synthetic hydroxyapatite in the range of 0.5-2.5 mg/cm2. The IR pattern, however, showed a shift in the positions of the absorption band and the appearance of ghost peaks. Using a thin based Petriperm dish, these difficulties were overcome producing a correlation factor of 0.992 at the lower range of 0.05-1.45 mg/cm2. Application of this technique to the precipitates formed on a Petri dish by rat bone marrow derived cells showed the presence of collagen and apatite-like substance.

Absorption↗
PubMed1991

Evidence for altered Na+/H+ antiport activity in cultured skeletal muscle cells and vascular smooth muscle cells from the spontaneously hypertensive rat.

1. Intracellular pH and Na+/H+ antiport activity were determined by a fluorimetric method in cultured skeletal muscle cells (myoblasts) and aortic vascular smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats. 2. The intracellular pH was significantly more alkaline at three different extracellular pH values in both myoblasts and vascular smooth muscle cells from the spontaneously hypertensive rats than in those from the normotensive control rats. 3. A kinetic analysis of the Na+/H+ antiport activity in these cells showed that the raised activity in the spontaneously hypertensive rats was due to an increased maximal transport capacity in vascular smooth muscle cells and to an increase in the affinity of the antiport for internal H+ in the myoblasts. 4. When the extracellular pH was reduced in the skeletal muscle cells of both types of rat, the intracellular pH fell. However, in vascular smooth muscle cells, a reduction in the extracellular pH was not associated with a fall in the intracellular pH. This resistance of the intracellular pH to changes in the extracellular pH differentiates vascular smooth muscle cells from other cells that have been studied in this way.

Animals↗
PubMed1991

Intracellular free magnesium in human lymphocytes and the response to lectins.

1. Intracellular free [Mg2+] was measured in human peripheral blood lymphocytes using a fluorimetric method based on the dye furaptra. It was necessary to correct for the extracellular leakage of the dye by using either 10 mmol/l EDTA or 0.05 mmol/l Mn2+. 2. As the proliferative response of lymphocytes to mitogenic lectins has been linked to a dependence on extracellular Mg2+, the intracellular [Mg2+] was studied in lymphocytes stimulated with various mitogenic and non-mitogenic lectins. 3. Only lymphocytes treated with phytohaemagglutinin-L, a leucoagglutinin from Phaseolus vulgaris that binds to tri- and tetra-antennary complex glycoproteins, showed a marked increase in intracellular [Mg2+]. This effect was partially inhibited by N-acetylgalactosamine. The stimulation by different lectins of the incorporation of [3H]-thymidine into lymphocytes was not correlated to the changes in intracellular [Mg2+]. 4. The proliferative response of lymphocytes to lectins is therefore not wholly dependent on a rise in intracellular [Mg2+].

Benzofurans↗
PubMed1991

Chloride influx in human leucocytes: a triple-isotope technique for the assessment of chloride transporters.

1. A novel triple-isotope method using 3H2O, [14C]-sucrose and 36Cl- for measuring initial Cl- uptake rates in human leucocytes that had been preincubated in 10% (v/v) autologous serum is described. 2. There was a marked dependence of Cl- influx on intracellular pH, the flux at an intracellular pH of 7.20 being about 11.2% of that at an intracellular pH of 7.56. 3. This Cl- influx was inhibited by 4,4-di-isothiocyanatostilbene-2,2-disulphonic acid in a dose-dependent manner. Half-maximal inhibition by 4,4-di-isothiocyanatostilbene-2,2-disulphonic acid at an internal pH of 7.56 in the presence or absence of HCO3- was 0.30 or 0.35 mmol/l, respectively. 4. Depletion of intracellular Cl- resulted in a 70.7% decrease in Cl- influx, whereas depletion of cellular ATP led to a 37.7% decrease in Cl- influx. 5. The phorbol ester 12-O-tetradecanoyl phorbol acetate at a concentration of 0.1 mumol/l reduced Cl-influx, especially the 4,4-di-isothiocyanatostilbene-2,2-disulphonic acid-sensitive component. This effect was independent of intracellular pH changes or Na+/H+ antiport activity.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Leukocyte intracellular pH and Na/H antiporter activity in uraemia and type I diabetes mellitus.

The leukocyte Na/H antiporter has been studied in patients with end-stage renal failure on maintenance haemodialysis. Thirteen non-diabetic haemodialysis patients (CRF group) and eight haemodialysis patients with diabetic nephropathy (CRF-DM group) were investigated. Measurements were made using the pH-sensitive fluorescent dye bis (carboxyethyl) carboxyfluorescein (BCECF). The initial intracellular pH (pHi), intracellular buffering capacity, and Na/H antiporter Vmax (at pHi = 6.0) have been recorded in bicarbonate-free solutions. The mean initial intracellular pH in the CRF group was 7.34 (SD 0.05, P less than 0.004) and this was significantly less than the CRF-DM group (7.42, SD 0.07) and normal controls (7.43, SD 0.09, n = 25). The mean intracellular buffering capacity was normal in the CRF and CRF-DM groups. The mean Na/H antiporter Vmax was also normal in the CRF and CRF-DM groups (56.5, SD 9.9; and 56.8, SD 12.8, mmol/l per min respectively compared to 55.2, SD 8.8, mmol/l per min in controls). These data are discussed with reference to the reported high values of Na/H antiporter Vmax in diabetic patients with early nephropathy. This abnormality does not appear to be present in end-stage diabetic nephropathy.

Adolescent↗
PubMed1991

Obstructive sleep apnoea in adults presenting with snoring.

Snoring is a common disorder, and may be associated with obstructive sleep apnoea, although there is little published information on the incidence of apnoea in snorers. This study aimed to assess the upper airway and to relate the findings to sleep study data in a population of patients referred by their general practitioners with loud snoring. Each patient had a full history, weight and height measurements, nasal examination, rhinomanometry, peroral grading of the oropharyngeal features, and fibreoptic pharyngoscopy with a modified Muller manoeuvre, followed by a sleep study. The results in our group of 35 patients demonstrate a high incidence of obstructive sleep apnoea (46%). Factors which correlated well with apnoea were excessively loud snoring, a narrow oropharynx, and marked obesity; 94% of patients with one or more of these features had evidence of sleep apnoea.

Adult↗
PubMed1991

Intermediate hearing tests as predictors of hearing aid acceptance.

This prospective study analyses the ability of several different intermediate hearing screening techniques to predict hearing aid acceptance in a pre-retirement population. In addition to the traditional methods of using sensitivity and specificity to analyse test performance, signal detection theory has been used to obtain a single detectability rate for each test. Questionnaires were able to identify individuals with hearing difficulties but included a significant group of people who ultimately would not accept a hearing aid. This group could be eliminated either clinically using free field speech testing, or audiometrically using warble tone thresholds, without the need for formal pure tone audiometry.

Aged↗