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S Barker

Publications and source records attributed to S Barker.

At least 127 records · Page 7Linked to original sources
PubMed1992

Characterization of a rat adrenocortical inner zone-specific antigen and identification of its putative precursor.

We have previously reported the production of a monoclonal antibody (IZAb) which interacts with an antigen, found predominantly in rat adrenal inner zone tissue, which may have a role in steroidogenesis. Here we describe initial studies on its characterization. Immunoblot analysis of rat adrenocortical proteins obtained from fresh tissue and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, showed that the IZAb interacted with a protein with a molecular mass of approximately 30,000 Da (IZAg1). This protein was found predominantly in rat adrenal inner zone tissue. Small amounts were seen in the zona glomerulosa, while no corresponding protein was seen in rat ovary, heart, liver, testis or kidney tissue. Subcellular fractionation of rat adrenocortical inner zone tissue and immunoblot analysis showed that the IZAg1 was present in the microsomal and mitochondrial fractions of the cell, but was absent from the cytosol. In-vivo treatment with ACTH (100 micrograms/day) for more than 5 days also increased the expression of this protein by rat adrenal inner zone tissue, and this was coincident with increased corticosterone and 18-hydroxydeoxycorticosterone (18-OH-DOC) production in incubations of inner zone tissue in vitro. In experiments involving the short-term culture of rat adrenal inner zone cells, IZAb interacted with two protein bands. IZAg1 was detected as a minor band in untreated control cells, while another protein with a molecular mass of approximately 60,000 Da, designated IZAg2, was present in greater amounts. Treatment of cells for 48 h with either ACTH (1 mumol/l) or dibutyryl-cAMP (100 mumol/l) resulted in apparent increased expression of IZAg1 and diminished levels of IZAg2.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1991

Significance of the 8S complex in oestrogen receptor recognition.

Previous studies from this laboratory have drawn attention to discrepancies between enzyme-linked immunoassay (EIA) and steroid binding assay (SBA) in the analysis of oestrogen receptors (ER) in breast tumours. In particular, EIA values were at least 3-fold higher than SBA values in tumours which also contained progesterone receptors (PR) when both 4 and 8S isoforms of the ER are present. To test the influence of these isoforms on the two assay systems, the relationships between the oestrogen receptor (ER) values obtained by EIA and SBA were examined in tumour cytosols prepared in the presence of molybdate and protease inhibitors to prevent degradation of the 8S form. Under these conditions, values for ER were the same by EIA and SBA (slope = 1.08, r = 0.886, n = 25) when EIA was performed using low salt phosphate buffer instead of the high salt-containing Abbott-diluent provided with the kit. However, after disruption of the 8S assembly using high K+ concentration, the slope of the regression was 6.37, r = 0.865, n = 25. Using ER from rat uterus, EIA was also performed on intact 8S oligomers, on 8S ER dissociated by high salt, and on glycerol density gradient-fractionated 4S ER. The identity of the ER oligomers and components was confirmed by glycerol density gradient fractionation, and by isoelectric focussing. For the 4S ER, EIA gave similar values whether using low or high salt phosphate buffer. However EIA values for the 8S form were 2-fold higher when the supplied diluent was used than when the assay was performed in low salt buffer. The amount of oestradiol which could be extracted was affected by the different conditions used. Addition of KCl or trypsin to disrupt the 8S ER caused an increase in the amount of extractable oestradiol compared with control values (control = 52 +/- 4.0, high KCl = 91 +/- 4.4, trypsin = 152 +/- 7.5, pg oestradiol/mg protein). We conclude that further antibody binding sites are revealed from the 8S ER form after its disaggregation by high salt. The steroid extraction data also suggests the possibility that tightly bound steroid is retained within the 8S ER structure, and released by 8S disaggregation. Both of these may contribute to the differences between EIA and SBA values.

Animals↗
PubMed1991

Identification of a gene encoding an HPr-like protein in Aspergillus fumigatus.

The gene encoding a histidine-containing protein (HPr)-like protein was identified in a cDNA library of Aspergillus fumigatus. The predicted amino acid sequence of the fungal HPr showed greater homology with HPr from Gram-positive bacteria than from Gram-negative bacteria. Since other components of the phosphoenolpyruvate: carbohydrate phosphotransferase system have not been identified in eukaryotes, this raises the question of what regulatory function the HPr-like protein might have evolved in this fungus.

Amino Acid Sequence↗
PubMed1991

Torsion of the gall-bladder: rare, unrecognized or under-reported?

Four cases of torsion of the gall-bladder are reported. The clinical features closely mimic those of acute cholecystitis. Although conservative management was initially instituted, close monitoring of our patients led to appropriate surgical intervention. Review of the literature suggests that torsion is uncommon. Four surgical registrars at busy district general hospitals seeing four cases in one year suggests that it is under-reported. Acute torsion can develop after a period of recurrent abdominal pain--retrospectively diagnosed as 'chronic torsion'. A prospective study using currently available imaging procedures to assess gall-bladder mobility, itself associated with torsion, could reveal whether this leads to chronic pain.

Acute Disease↗
PubMed1991

Multiple forms of angiotensin II receptors in rat tissues.

Angiotensin II (AII) receptors were identified in rat tissue membranes by specific binding of 125I-labelled AII. Using an isoelectric focusing technique, two forms of the high-affinity AII receptor were identified in rat adrenal zona glomerulosa and liver membranes. These migrated to isoelectric points (pI) 6.8 and 6.7. Two low-affinity forms migrated to pI 6.5 and 6.3. The two high-affinity forms were in greatest abundance in the zona glomerulosa, while the low-affinity pI 6.5 isoform was predominant in liver membranes. In uterine membranes both low-affinity isoforms were observed, but there was only one of the high-affinity forms (pI 6.7). Concentrations of AII receptor isoforms were increased in the zona glomerulosa of sodium-deprived rats. Reduction of disulphide bridges with dithiothreitol (DTT) had different effects on the various AII receptor isoforms. Thus 1 mmol DTT/1 caused a twofold increase in 125I-labelled AII binding in zona glomerulosa membranes. DTT produced no appreciable differences in specific AII binding in uterine membranes, whereas there was a 50% reduction of binding in liver membranes. At 20 mmol/l, DTT greatly decreased AII binding in all tissues. The data suggest the existence of multiple forms of AII receptors which may have different functions.

Animals↗
PubMed1991

4 S oestrogen receptor isoforms and their distribution in breast cancer samples.

The variability in the profile of oestrogen receptor (ER) isoforms in breast tumours has been studied. Using low-resolution isoelectric focussing (IEF), two major ER isoforms with isoelectric point (pI) values of 6.1 and 6.6 could be identified, with corresponding sedimentation coefficients in sucrose density gradients of 8 S and 4 S respectively. Using high-resolution IEF or immunoblotting, the pI 6.6 form (4 S) was shown to be composed of three different species, with pI values of 6.3, 6.6 and 6.8, while the oligomeric pI 6.1 protein (8 S) did not show charge heterogeneity. Data were obtained on the soluble receptors from supernatants of 42 ER-positive primary breast tumour homogenates using high-resolution IEF to obtain ER isoform profiles. It was found that 54.7% of tumours contained the isoforms at pI 6.6 and 6.1, while only 11.9% contained the full complement of isoforms (pI 6.1, 6.3, 6.6 and 6.8). Of the tumours studied, 11.9% contained isoforms of pI 6.1, 6.6 and 6.8, with 14.3% containing isoforms with pI 6.1, 6.6 and 6.3. Very few tumours contained only one isoform, with 4.8% of tumours containing a single isoform at pI 6.1 and 2.4% of tumours containing only the isoform at pI 6.6. All four ER isoforms were also shown to be present in some tumours by immunoblotting using antibody H222 and, in addition, high-resolution IEF indicated that all isoforms bind oestradiol, diethylstilboestrol and tamoxifen. The variability in the ER isoform profile may have a bearing on the known variability of tumour response to endocrine therapy and prognosis.

Breast Neoplasms↗
PubMed1990

Discrepancies between antibody (EIA) and saturation analysis of oestrogen receptor content in breast tumour samples.

The use of different techniques for assay of oestrogen receptors (ER) in breast cancer raises the question of their relative effectiveness in measuring concentrations of functional receptors. Data were obtained on soluble receptors from supernatants from 58 primary breast tumour homogenates, using the ligand ([3H]oestradiol) binding assay with dextran-coated charcoal (DCC) separation, either at a single saturating ligand dose, or by Scatchard analysis, and by using the Abbott enzyme immunoassay (EIA) kit. As previous reports have shown, the two methods gave reasonably good correlation (r = 0.8), but EIA values were systematically higher than DCC (slope = 3.0). Similar values were obtained when the ER + ve/progesterone receptor (PR) + ve subgroup were examined separately (n = 34, r = 0.86, slope = 3.0). However the two sets of data were in much better agreement in the ER + ve/PR - ve subgroup (n = 10, r = 0.98, slope = 1.24). When analysed by isoelectric focusing on polyacrylamide gels (IEF), two major specific binding components were identified, at pI 6.1 and at pI 6.6. Both isoforms were present in 50/66 ER + ve PR + ve breast tumour samples, but only the pI 6.6 (4S) was present in most ER + ve/PR - ve samples (13/20). It appears that, compared with DCC, the EIA method gives much higher values for the 8S isoform, whereas the two methods detect the 4S isoform with similar sensitivity. In assays on the tumour cell lines, T47D and MCF-7, still greater discrepancies, at least 10-fold, were found between EIA and DCC data.

Breast Neoplasms↗
PubMed1990

Effects of acetylcholine and monensin on 22Na uptake and cytosolic Ca2+ in rat submandibular salivary cells.

Cells isolated by enzymatic digestion of gland fragments were incubated in solutions with or without Ca2+ or Na+ and exposed to monensin (10 microM) or acetylcholine (1 microM). Effects on accumulation of 22Na and on cell Ca2+ (measured with fura-2) were compared. In Ca2(+)-containing medium, accumulation of 22Na was increased by the 2 drugs (23 and 20%, respectively) and their effects were additive. Tracer accumulation was also increased by the ionophore A23187. The effect of monensin was not inhibited by 1 mM amiloride, but partially inhibited by 1 mM furosemide. Acetylcholine caused a rapid increase (peak) in cell Ca2+, followed by a gradual decline, while monensin caused a gradual increase with no initial peak. In Ca2(+)-free medium, acetylcholine failed to enhance 22Na accumulation, but still caused a rapid peak in cell Ca2+, followed by a more rapid decrease to resting levels. Monensin enhanced 22Na uptake 16% and caused a gradual increase in cell Ca2+ in this medium. In a medium with no Na+ but containing 1 mM Ca2+, acetylcholine increased cell Ca2+ but no initial peak was observed; monensin caused a slight decrease in cell Ca2+ and then an increase to resting levels. These results suggest important interactions between Na+ and Ca2+ movements in salivary cells. Ca2+ mobilization may activate Na+ uptake and changes in cell Na+ may, in turn, influence Ca2+ mobilization from cell pools. Some of these interactions may involve a Na/Ca co- or counter-transport system.

Acetylcholine↗
PubMed1990

Heterotopic brain tissue: a rare cause of adult recurrent meningitis.

Heterotopic brain tissue is a rare cause of symptoms in neonates in whom it may present with respiratory obstruction. In infants it may produce unilateral nasal obstruction due to a nasal 'polyp' or cerebrospinal fluid rhinorrhoea with an associated risk of meningitis. It is exceedingly rare in adults but may nevertheless be a cause of significant morbidity as this case demonstrates.

Brain↗
PubMed1990

Intermittent hypercalcaemia and vitamin D sensitivity in Hodgkin's disease.

A patient with Hodgkin's disease spontaneously developed steroid-responsive hypercalcaemia during two consecutive summers. Administration of 3000 U/day of vitamin D, while he was normocalcaemic, caused a sharp increase in serum 1,25(OH)2D3 (from 59 pg/ml to 142 pg/ml) and subsequently hypercalcaemia while serum 25(OH)D3 rose moderately within the normal range (from 2.8 ng/ml to 10 ng/ml). During a spontaneous episode of hypercalcaemia which was accompanied by increased circulating 1,25(OH)2D3 concentrations, administration of hydrocortisone decreased serum 1,25(OH)2D3 rapidly (from 115 pg/ml to 62 pg/ml) and eventually led to normocalcaemia while serum 25(OH)D3 remained unchanged. Thus the disturbances of mineral metabolism found in this patient with Hodgkin's disease are very similar to those previously described in sarcoidosis.

Aged↗
PubMed1989

Amiloride inhibits 22Na uptake and [3H]QNB binding in rat submandibular cells.

Dispersed acini isolated by collagenase digestion of the rat submandibular gland were used to compare the effects of amiloride and furosemide on the uptake of the isotopic tracer 22Na and on the binding of [3H]quinuclidinyl benzylate ([3H]QNB). In mM concentrations, both inhibitors reduced 22Na uptake in resting cells 34 and 25-29%, respectively. Acetylcholine (1 microM) enhanced uptake 23% and this effect was reduced 45% by amiloride and 26% by furosemide. Amiloride inhibited the binding of [3H]QNB to crude membranes prepared from fresh submandibular glands in a dose-dependent fashion (IC50 = 8 x 10(-6) M). Furosemide (3 x 10(-8) to 10(-3) M) did not inhibit radioligand binding. Na influx into resting salivary acini thus appears to occur by both amiloride-sensitive and furosemide-sensitive transport systems. The similar inhibition by furosemide of unstimulated and stimulated uptake of 22Na suggests that acetylcholine does not significantly activate the cotransport system within the time frame (i.e., 2 min) of the experiments. Acetylcholine appears to activate an amiloride-sensitive Na/H antiport, but amiloride blocks cholinergic receptors and may thus affect Na transport by receptor blockade. Other actions of amiloride, such as its ability to penetrate into cells and to act as a weak base which alters intracellular pH, may also contribute to the inhibition of Na entry into salivary cells.

Acetylcholine↗
PubMed1989

Inhibition of 36Cl uptake by stilbene sulphonic acid derivatives and loop diuretics in rat submandibular salivary acini.

Acini were isolated from submandibular glands by enzymatic digestion and incubated in HCO3-containing and HCO3-free [hydroxyethyl-1-piperazine-ethane sulphonic acid (HEPES) buffered] solutions to compare the accumulation of the isotopic tracer 36Cl in the absence and presence of stilbene sulphonic acid derivatives and of loop diuretics. Tracer accumulation was similar in both solutions in the absence of inhibitors and reached 17-19 nmol/mg protein after 5 min of incubation. Exposure to 10(-3) M 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid (DIDS) or to 10(-4) M bumetanide in HCO3-buffered medium resulted in significant reductions in both the initial phase (from 0 to 5 min) and the steady-state phase (5-30 min) of 36Cl accumulation. By contrast, in HCO3-free, HEPES-buffered incubation solution, DIDS was significantly less effective in reducing 36Cl accumulation, while bumetanide was somewhat more effective than in HCO3-buffered medium. At a higher dose (10(-3) M), furosemide was less effective than bumetanide in reducing the steady-state tracer content of the cells in the HCO3-buffered medium (22% versus 35% reduction), although the effects were similar in the initial phase of tracer uptake. Exposure to 10(-4) M 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) caused an increase in the steady-state 36Cl content of the acini of 24 +/- 9%. Exposure to bumetanide and DIDS caused an additive reduction of tracer content, while exposure to bumetanide and SITS resulted in a smaller reduction than with bumetanide alone.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Modulation of myelomonocytic U937 cells by vitamin D metabolites.

Investigation of the effects of 1,25(OH)2D3 and 24,25(OH)2D3 on the proliferation and differentiation of the human myelomonocytic cell line U937 has been complemented with studies of the effect of the same metabolites on the number of nuclear receptors for 1,25(OH)2D3. Both 1,25(OH)2D3 and 24,25(OH)2D3 inhibit the proliferation of U937 cells in a dose-dependent manner. The concentrations of 24,25(OH)2D3 required to produce this effect were 100-times greater than those of 1,25(OH)2D3. Inhibition of proliferation was associated with increased expression of the CD14 and 200 kDa 63D3 antigens thus confirming differentiation of U937 towards a more mature cell type. Studies of the nuclear receptor for 1,25(OH)2D3 showed that pre-treatment of the cells with 1,25(OH)2D3 resulted in an apparent 40% decrease in the number of detectable 1,25(OH)2D3 receptors as compared to control U937 cells. This is due to the fact that the 1,25(OH)2D3 binds to U937 cell nuclei during culture and thus blocks the subsequent binding of radiolabelled 1,25(OH)2D3 used to measure the number of 1,25(OH)2D3 receptors. Measurement of the binding of unlabelled 1,25(OH)2D3 by radioimmunoassay indicated that pre-treatment of the cells with 1,25(OH)2D3 increased the capacity of U937 to bind the hormone, although measurement of these receptors by whole cell assay was prevented by the binding of 1,25(OH)2D3 itself. This effect was not observed with 24,25(OH)2D3 which was more easily displaced from binding sites by radiolabelled 1,25(OH)2D3 and it appears to act through low affinity binding to the 1,25(OH)2D3 receptor.

24,25-Dihydroxyvitamin D 3↗
PubMed1989

Epidermal growth factor receptor and oestrogen receptors in the non-malignant part of the cancerous breast.

Fifty-one samples of non-malignant tissue from four mastectomies were analysed to assess oestrogen receptor (ER) and epidermal growth factor receptor (EGFR) status across the cancerous breast. No significant relationship was found between the presence of EGFR and ER. Eighty-four per cent of these samples were EGFR positive and 29% expressed both receptor types. EGFR and ER expression was not affected by histological sub-group. In contrast, analysis of 44 primary cancers showed, in agreement with the literature, a significant inverse relationship between the presence of ER and EGFR (Fisher's exact test P less than 0.002). The difference between malignant and non-malignant tissue appeared to result from the prevalence of co-expression of EGFR and ER in the non-malignant specimens. This suggests different regulation of receptor expression in malignant and non-malignant tissue.

Adult↗