Biomedical subjects
Publications and source records attributed to S Barker.
Moyamoya disease was diagnosed as the cause of cerebral infarction in an 18 year old white female. The clinical features, pathology and treatment of this occlusive cerebrovascular disorder are discussed.
The angiotensin II (Ang II)-binding sites in rat adrenal gland membranes were characterized using 125I-radiolabelled Ang II. While Scatchard analysis identified a single population of Ang II receptor sites, isoelectric focusing (IEF) on polyacrylamide gels revealed four peaks of specific Ang II binding which migrated to isoelectric points (pI values) 6.8, 6.7, 6.5 and 6.3. In binding assays in the presence of an excess of the Ang II receptor AT1 subtype antagonist DuP 753, a monophasic dose-dependent displacement of 125I-labelled Ang II binding by the Ang II receptor AT2 subtype antagonist CGP42112A was observed, and vice versa. In this system, reduction of disulphide bridges using 1 mmol dithiothreitol (DTT)/l markedly increased the number of binding sites in the adrenal zona glomerulosa without affecting receptor affinity. Using IEF, it was found that both DuP 753 and CGP42112A were able to reduce specific binding of each of the four peaks to some extent. However, the predominant effect of DuP 753 was to reduce the labelling of the isoform at pI 6.7 substantially, while CGP42112A significantly inhibited the specific 125I-labelled Ang II binding to the pI 6.3 isoform. When DuP 753 and CGP42112A were used together, specific binding of 125I-labelled Ang II to the isoforms of pI values 6.8, 6.7 and 6.3 was completely eliminated. These data suggest that the four peaks of specific binding found may be composed of different isoforms of both AT1 and AT2 receptor subtypes and that the Ang II receptor isoforms which migrated to pI 6.7 and pI 6.3 are predominantly composed of AT1 and AT2 receptor subtypes respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Oestrogen receptors (ERs) in breast tumours are highly heterogeneous. In previous studies we have shown that at least four isoforms may exist. These migrate in isoelectric focusing (IEF) gels to isoelectric points (pI values) 6.1, 6.3, 6.6 and 6.8. Of these the first (pI 6.1) corresponds to the 8S isoform as detected by sucrose gradient fractionation, while the others all sediment at 4S. In a series of 66 breast tumours it was found that those at pI 6.3 and pI 6.8 were significantly correlated with the presence of progesterone receptors. To characterize the isoforms more fully, ER isoforms labelled by [3H]oestradiol binding were fractionated by IEF. The results were compared with those obtained after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the H222 anti-ER monoclonal antibody. In other experiments, tumour ER isoforms were covalently labelled with [ring-3H] tamoxifen aziridine and separated by IEF. The individual isoforms were electroeluted from the IEF gel and further analysed by SDS-PAGE and non-denaturing PAGE. In summary, the evidence shows that the isoforms of pI values 6.3, 6.8 and 6.6 have molecular masses of 50, 65 and 70 kDa respectively. In addition, all three of these isoforms, i.e. the pI 6.3, 6.8 and 6.6 isoforms, could form dimers. We conclude that the three isoforms sedimenting at 4S have the capacity to form dimers and thus may have the potential for binding to oestrogen response elements in the genome.
We have generated hybridomas which secrete monoclonal antibodies to the AT1 subtype of the angiotensin II receptor (AT1 receptor). These were obtained after immunization of Balb C/c mice with synthetic peptides representing sequences from either the extracellular domain (residues 8-17) or the intracellular domain (residues 229-237) of the AT1 receptor. Hybridoma populations were first screened for the production of antibodies which bound to rat liver cells. Further selection, and cloning by limiting dilution, was carried out for antibodies which bound specifically to rat adrenal glomerulosa cells. Confirmation that the antibody designated 6313/G2 interacted with the angiotensin II receptor was obtained using COS-7 cells transfected with AT1A receptor cDNA. In particular, the initial characterization of 6313/G2 showed specific immunofluorescence of vascular endothelium.
Elements of the muscarinic signal transduction pathway were compared in submandibular acinar cells of 1-day-old, 1-week-old, and adult rats after exposure to concentrations of acetylcholine ranging from 0.05 to 10 microM. Formation of inositol trisphosphate (IP3) and intracellular Ca2+ were comparable in cells from the three age groups after exposure to agonist concentrations < 1 microM. At higher agonist concentrations, IP3 generation and peak initial changes in [Ca2+]i were significantly greater in cells of newborn animals. In cells of 1-week-old animals, increased peak [Ca2+]i responses were seen even at low agonist concentrations, although IP3 production was not increased when compared with fully mature cells. Increased initial [Ca2+]i peaks, but comparable subsequent plateau [Ca2+]i values, were seen in the immature cells in both Ca(2+)-containing and Ca(2+)-free solutions. Permeabilized cells of early postnatal animals took up less 45Ca2+ into nonmitochondrial Ca2+ pools in the presence of 1.5 mM ATP and also released less tracer in response to intermediate IP3 concentrations than adult cells. Developing salivary cells thus show differences in important functional linkages of the muscarinic signal transduction pathway, including those between receptor activation and phosphoinositide turnover and between IP3 and release of internally stored Ca2+. Differences in the Ca2+ stores or in their sensitivity to IP3 may account for the latter observation. Immature salivary cells seem to have adequate mechanisms for Ca2+ entry.
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Platelet factor 4 (PF4) exhibits high affinity for heparin and exists as a tetramer in solution under physiologic conditions. Reduction of the two disulfide bridges in PF4 increases the protein's dissociation constant for heparin approximately 20-fold and shifts the highest apparent aggregation state from tetramer to dimer as evidenced by gel filtration, chemical cross-linking, and 1H-NMR studies. 1H-NMR spectra of reduced PF4 monomers generally show narrower, less dispersed, upfield-shifted NH and alpha H resonances, suggesting the presence of an unfolded monomer state. Reduced PF4 monomer folding, however, is evidenced by the presence of about 12 relatively long-lived backbone NHs and by CD spectra that indicate conservation of overall secondary structure. These data suggest the presence of a molten globule-type state. Urea denaturation shifts this apparent molten globule to a fully unfolded state characterized by more random coil-like resonance shifts. The reduced PF4 dimer state yields NMR and CD data consistent with preservation of tertiary structural folds found for the native species. In this regard, the reduced PF4 folding transition is thermodynamically linked with dimer formation which stabilizes tertiary structure. Monomer-dimer association equilibria for reduced PF4 essentially follow the same pH and salt titration trends as reported previously for native PF4 dimers [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469-9478], indicating that that dimer interface is generally conserved in the absence of disulfide constraints. Reduced PF4 tetramers are not apparent under any conditions investigated, suggesting that disulfides are necessary for efficient antiparallel beta-sheet alignment between dimer pairs.
The effects of graded doses (5 x 10(-8) to 10(-5)) acetylcholine on intracellular Ca2+ and on 86Rb and 36Cl efflux were compared in submandibular cell clusters of 1 and 7 day-old and adult rats. Initial Ca2+ peaks were similar at agonists concentrations lower than 10(-7) M but the release of Rb+ and Cl- were smaller in cells of young animals. At higher agonist concentrations, Ca2+ peaks were higher in immature cells; however, initial Cl- (but not Rb+) efflux was similar to that of mature cells. Plateau Ca2+ levels were independent of age and agonist concentrations but the content of Cl- and Rb+ varied greatly and differences between age groups were less evident. These data confirm a dissociation between intracellular Ca2+ levels and Ca(2+)-mediated ion transport in immature salivary cells.
Data were obtained on soluble oestrogen (ER) and progesterone (PR) receptors from 273 primary breast tumours, using an enzyme immunoassay (EIA) and ligand binding assay with dextran-coated charcoal (DCC) or isoelectric focussing separations. The p29 and total cathepsin D content was also assayed in the same samples. Tumours expressing ER (by either steroid binding assay or EIA) had higher levels of p29 than those which did not express the receptor (P < 0.0001). Moreover, tumours co-expressing ER, PR and p29 appeared to have higher levels of cathepsin D than those which were negative for at least one protein (P < 0.0001). Twenty out of the 273 human breast cancer samples, containing a level of ER positive by EIA, which did not bind labelled oestradiol, were identified; isoelectric focussing showed that such a receptor was also unable to bind hydroxytamoxifen. These tumours did not significantly differ from those in the whole population in their capacity to express ER (positive by EIA), PR, p29 and cathepsin D. It was concluded that the EIA can detect both a ligand binding ER and a receptor which is able to initiate PR transcription but does not bind radio-labelled ligands in vitro; such a receptor could have a bearing on the variability of tumour response to endocrine therapy.
Uptake of this isotopic tracer was essentially the same as in cells derived from salivary glands of adult animals, both in the absence and in the presence of 1 microM acetylcholine, 10 microM monensin or 10 microM A23187. Tracer accumulation in resting cells was inhibited in the immature cells by 1 mM amiloride but the inhibition was significantly smaller than in fully mature cells (16 and 33%, respectively). Bumetanide inhibited tracer uptake by 28% in mature cells not exposed to agonist, but had essentially no effect in those of newborn rats. The tracer content of adult cells exposed to acetylcholine in Ca(2+)-free incubation solutions was significantly reduced when compared to that of cells exposed to agonist in solutions containing 1.0 mM CaCl2. A similar but significantly smaller reduction in tracer content was observed in cells of newborn animals incubated in the Ca(2+)-free medium. The inhibition of 22Na uptake observed in the presence of amiloride was the same in both types of cells in the presence or absence of external Ca2+. The findings suggest that Na entry into immature salivary cells of newborn rats is quantitatively similar to that in mature cells, but that the contribution of the loop diuretic-sensitive co-transporter and of the amiloride-sensitive Na/H exchange to Na+ entry is less in the immature cells. Na entry in the immature cells is likely to occur, therefore, primarily by other mechanisms such as Ca(2+)-regulated channels.
Dispersed salivary acini isolated from the rat submandibular gland were incubated in a HEPES-buffered Krebs-Ringer solution or in the same buffer containing 20 mM NH4Cl and the accumulation and efflux of K+ were measured with the radiotracer 86Rb+, in the presence and absence of acetylcholine and of transport inhibitors. Exposure to NH4Cl caused a significant (greater than 50%) reduction in tracer accumulation. This effect was blocked by 0.1 mM bumetanide, but not by 1 mM ouabain. The effect of NH4Cl was, on the other hand, nearly additive with that of 1 microM acetylcholine. In cells preincubated with tracer, acute addition of NH4Cl caused a significant net efflux of isotope, so that the tracer content fell to 45% of the control value within 10 min. Bumetanide added to preloaded cells in the same fashion had no effect on tracer content and did not modify the efflux of 86Rb+ induced by 1 microM acetylcholine. However, this inhibitor essentially abolished the NH4Cl-induced tracer efflux. Exposure to NH4Cl during tracer loading did not appear to affect subsequent agonist-stimulated tracer efflux. These results suggest that: (1) the inhibition of K+ entry by NH4Cl is due to an effective competition by the NH4+ ion with Rb+ (and K+) for uptake via a bumetanide-sensitive Na+/K+/2Cl- contransporter.(ABSTRACT TRUNCATED AT 250 WORDS)
Using high resolution isoelectric focusing we have been able to identify a low affinity/high capacity oestrogen binding protein, which exhibits an apparent pI of 7.0. Using this system it can be separated from the previously described high affinity oestrogen receptor (ER) isoforms which focus at pI 6.1, 6.3, 6.6 and 6.8. The pI 7.0 protein was detected in 30/30 breast tumours analysed and had the binding characteristics of the cytoplasmic Type II ER (Kd = 88 +/- 8 nM). The concentration of this protein was shown to be significantly correlated with the concentration of the pI 6.6 species, which represents the major 4S isoform. It is not related to any other isoform of ER, and is expressed independently of the progesterone receptor. The importance of this observed relationship with respect to ER function remains obscure, but it may provide new insights into the role of the Type II oestrogen binding site in breast cancer.
We have previously reported that clinical trials relating to the use of danazol in the management of benign breast disease show a positive correlation between favourable clinical response and an induction of progesterone receptors in the affected tissue which is maintained for a period of at least 6 months subsequent to the cessation of treatment. Further studies designed at elucidating more clearly the actions of danazol at the cellular and molecular levels have confirmed that progesterone receptors are down-regulated by short-term progestin action at the level of the mRNA transcript, but that danazol is subsequently able to produce an enhanced cellular response, inducing progesterone receptors in the presence of oestrogenic agents. Uteri from danazol-treated rats showed a doubling of progesterone receptor concentrations compared with the control uteri. In the mammary cancer cell line T-47D, cells treated with danazol had increased progesterone receptor concentrations of 558.4 +/- 32.0 compared with 152.6 +/- 7.0 fmol/mg protein in the control cells. In both cases, these inductions were observed following a period of progesterone receptor suppression. Short-term molecular studies on T-47D cells indicated that progesterone and danazol initially inhibit mRNA transcription, but that 24 h after treatment an induction is observed. This is especially marked in the danazol-treated cells.
Narratives of the delusions of 83 schizophrenic and 55 nonschizophrenic psychiatric subjects were categorized as of one or more of 12 types. Narratives were also independently assessed along dimensional scales of bizarreness and mood theme. Schneiderian and grandiose types were found to be more common in schizophrenics and mood psychotics, respectively. Dimensional measures showed that the delusions of schizophrenics were more unlikely and that those of mood psychotics had a stronger mood theme. Regression analysis determined that Schneiderian delusions and a dimensional estimate of mood theme best differentiated schizophrenics from mood psychotics. Assessments along dimensions of other parameters, particularly those represented by Schneiderian delusions, may further discriminate the functional psychoses.
Oestrogen receptors (ER) in breast cancer tumours are highly heterogeneous. In this study, the variability in the profile of ER isoforms and its relation to progesterone receptor (PgR) levels in breast tumours has been studied. Using high resolution isoelectric focusing (IEF) 4 ER isoforms can be detected with pI values of 6.1 (corresponding to the 8S ER), and 6.3, 6.6 and 6.8 (all of which have a sedimentation pI values of 6.1 (corresponding to the 8S ER), and 6.3, 6.6 and 6.8 (all of which have a sedimentation coefficient of approximately 4S in sucrose density gradients). Data were obtained on the soluble receptors from supernatants of 66 ER-positive primary breast tumour homogenates using high resolution IEF. In 43 of these samples PgR levels were also measured. The isoform at pI 6.6 was present in 97.0% of tumours, the isoform at pI 6.1 in 83.3%, the pI 6.3 isoform 39.4% of tumours and the pI 6.8 isoform in only 33.3% of tumours. Only 12.1% of tumours studied contained the full complement of ER isoforms (pI 6.1, 6.3, 6.6 & 6.8). The ER isoforms at pI 6.1 & 6.8 were only found in PgR-positive (> 10 fmol PgR/mg protein) tumours. Some tumours contained only a single ER isoform at pI 6.6 or 6.1, but those at pI 6.3 and 6.8 were never found singly. Tumours containing 3 or 4 ER isoforms had significantly higher levels of PgR (> 90 fmol/mg protein) than those with only 1 or 2 (P < 0.001). The presence of ER isoforms at pI 6.3 and pI 6.8 also significantly correlated with high levels of PgR (P < 0.001). This variability in the ER isoform profile of breast tumours and their correlation with PgR levels may have a bearing on prognosis and tumour response to endocrine therapy.
Erythromelalgia is an unusual condition characterized by attacks of burning pain in the hands and feet with local congestion and increased skin temperature. We report a case of erythromelalgia, with transient hypertension and elevated urinary catecholamines successfully treated by hypnotherapy. Such an association has not to our knowledge been previously reported in English language publications.
Transmembrane movements of K+ and Cl- in salivary acinar cells are important in the formation of saliva, and may be affected by changes in intracellular pH (pHi). Exposure to NH4Cl increases pHi transiently, but NH4+ may have effects independent of pHi. To investigate how Cl- transport may be altered under these conditions, rat submandibular acini were exposed to NH4Cl, and transmembrane Cl- transport was studied with 36Cl-. NH4Cl increased intracellular Cl- in these cells. The initial phase of this increase was partially HCO(3-)-dependent and was inhibited by 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), while the sustained phase was inhibited by 0.1 mM bumetanide. NH4Cl also inhibited acetylcholine-induced Cl- efflux from tracer preloaded cells. Changes in pH did not always correlate in time or extent with those of Cl- transport. We conclude that 1) exposure to NH4Cl increases Cl-uptake primarily by a bumetanide-sensitive transport system that did not reach steady state during the experiment, 2) exposure to NH4Cl also stimulates Cl- uptake by a DIDS-sensitive mechanism, and 3) only the latter is pHi sensitive.
The intracellular mechanisms of action of alpha-MSH in rat adrenocortical cells were examined. When rat adrenal capsule (largely glomerulosa) cells were stimulated with a range of concentrations of alpha-MSH there was significant stimulation of aldosterone secretion at 10(-10) mol/l, although cyclic AMP was not increased until high concentrations of alpha-MSH were used (10(-6) mol/l and above). However, cells incubated with ACTH showed an increase in aldosterone secretion at 10(-11) mol/l and levels of cyclic AMP were elevated at 10(-9) mol ACTH/l. When rat adrenal whole capsules were incubated with alpha-MSH, membrane-bound protein kinase C (PKC) activity was increased and cytosolic enzyme activity decreased, showing PKC activation. Stimulation with angiotensin II also induced translocation of PKC activity, but ACTH did not. When [3H]inositol-loaded glomerulosa cells were stimulated with alpha-MSH there was significant generation of [3H]inositol trisphosphate (IP3) at concentrations of alpha-MSH which stimulated secretion of aldosterone. Significantly increased levels of [3H]IP3 were also measured when loaded cells were exposed to angiotensin II. ACTH did not cause any significant stimulation of [3H]IP3 production at any concentration used. These results indicate that activation of PKC and phospholipase C is important in modulating the steroidogenic effect of alpha-MSH.