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Biomedical subjects

D Boyd

Publications and source records attributed to D Boyd.

At least 19 recordsLinked to original sources
PubMed1992

Expression of urokinase and its receptor in invasive and non-invasive prostate cancer cell lines.

We previously reported that extracellular matrix invasion by the prostate cancer cell lines, PC-3 and DU-145 was contingent on endogenous urokinase being bound to a specific cell surface receptor. The present study was undertaken to characterize the expression of both urokinase and its receptor in the non-invasive LNCaP and the invasive PC-3 and DU-145 prostate cells. Northern blotting indicated that the invasive PC-3 cells, which secreted 10 times more urokinase (680 ng/ml per 10(6) cells per 48 h) than DU-145 cells (63 ng/ml per 10(6) cells per 48 h), had the most abundant transcript for the plasminogen activator. This, at least, partly reflected a 3 fold amplification of the urokinase gene in the PC-3 cells. In contrast, urokinase-specific transcript could not be detected in the non-invasive LNCaP cells previously characterized as being negative for urokinase protein. Southern blotting indicated that this was not a consequence of deletion of the urokinase gene. Crosslinking of radiolabelled aminoterminal fragment of urokinase to the cell surface indicated the presence of a 51 kDa receptor in extracts of the invasive PC-3 and DU-145 cells but not in extracts of the non-invasive LNCaP cells. The amount of binding protein correlated well with binding capacities calculated by Scatchard analysis. In contrast, the steady state level of urokinase receptor transcript was a poor predictor of receptor display. PC-3 cells, which were equipped with 25,000 receptors per cell had 2.5 fold more steady state transcript than DU-145 cells which displayed 93,000 binding sites per cell.

Blotting, Northern↗
PubMed1992

Decreased urokinase receptor expression by overexpression of the plasminogen activator in a colon cancer cell line.

There is now ample evidence that the proteolytic action of urokinase (UK) is potentiated by a specific cell surface receptor. The present study was undertaken to determine the role of UK as a modulator of its binding site. GEO colonic cells, which secrete low levels of UK (approximately 2.5 ng/ml per 72 h per 10(6) cells) and display approx. 10(4) receptors per cell, the majority of which are vacant, were transfected with an exogenous UK gene driven by the RSV long terminal repeat (LTR) promoter (pRSVUK). Several UK-overexpressing pRSVUK clones were identified by an e.l.i.s.a., Northern blotting and Southern blotting, and analysed for receptor numbers after an acid pretreatment which dissociates receptor-bound UK. pRSVUK GEO clones, expressing high levels of UK, consistently bound 50-75% less radioactive di-isopropylfluorophosphate (DFP)-UK than clones harbouring the selectable marker gene neo only or control GEO cells. Cross-linking experiments with a radioactive N-terminal fragment of UK which binds to the receptor showed a decreased amount of a binding protein of approx. 51 kDa in representative pRSVUK-transfected cells. Saturation and Scatchard analysis indicated that this reduction in radioligand binding reflected a 40-70% decrease in the number of UK receptors, rather than a change in the dissociation constant. The reduction in receptor display could be accounted for by a decrease in the amount of steady-state mRNA encoding the receptor. Radioactive DFP-UK binding to pRSVUK GEO clones, which display two-thirds less receptors than their neo counterparts, could be restored to control levels (untreated cells harbouring neo) by cultivating them in the presence of an antibody which inhibits the interaction of UK with its receptor. These data suggest that for one colonic cell line at least, UK reduces the expression of its own binding site via an autocrine stimulation of its cell surface receptor.

Blotting, Northern↗
PubMed1992

The dynamics of assembly of a cytoplasmic membrane protein in Escherichia coli.

The topology of integral cytoplasmic membrane proteins can be analyzed using alkaline phosphatase fusions by determining which constructs have low and which have high specific activity. We show that in all cases the enzymatic activity is due to the fraction of the alkaline phosphatase moiety of the fusion protein localized to the periplasm. We present evidence that these fusions can also be used to analyze the process of assembly of cytoplasmic proteins into the membrane. The rate of acquisition of protease resistance of the alkaline phosphatase moiety of such hybrid proteins is compared for fusions to periplasmic and cytoplasmic domains. We show that this process, which is assumed to be representative of export of alkaline phosphatase, is significantly slower for fusions to cytoplasmic and certain periplasmic domains than for most periplasmic domains. These results are discussed in the context of the normal assembly of integral membrane proteins.

ATP-Binding Cassette Transporters↗
PubMed1991

Regulation of the urokinase receptor by its plasminogen activator.

There is now ample evidence that the proteolytic action of urokinase (UK) is potentiated by a specific cell surface receptor. The present study was undertaken to assess the role of UK as a modulator of its receptor. GEO colonic cells, which secrete relatively low levels of UK (congruent to 0.1 nM/72 h per 10(6) cells) and display approximately 10(4) receptors per cell, 10% of which are "tagged" with the endogenous plasminogen activator (PA), was selected for the study. A 90% reduction in the specific binding of radioactive DFP-UK was observed for cells cultivated in the presence of two-chain (TC) UK (Mr 55,000). This only partly reflected occupation of the receptors with UK supplied in the culture medium, since the specific binding of the radioligand was still reduced by 60% after an acid pretreatment, which dissociates receptor-bound UK. The reduction in radioactive DFP-UK binding to cells treated with high molecular weight UK, either in the single or two-chain form, was both concentration and time dependent. Maximum reductions (70%) were achieved by treatment of the cells for 24 h with 1 nM of the plasminogen activator. In contrast, low molecular weight UK, which lacks part of the UK A chain, had no effect on ligand binding. Attenuation of radioactive DFP-UK binding to UK treated GEO cells was a consequence of a 60% reduction in the number of binding sites. Treatment of GEO cells with an antibody, which blocks the binding of endogenous UK to its receptor, augmented radioactive DFP-UK binding by two-fold. These data indicate that for one colonic cell line, at least, UK down-regulates its own binding site subsequent to it being bound to the receptor.

Amino Acid Sequence↗
PubMed1991

Role of the urokinase receptor in facilitating extracellular matrix invasion by cultured colon cancer.

There is increasing evidence that urokinase secreted by tumor cells can be bound to a cell surface receptor retaining its full potential to activate plasminogen and subsequently cleave basement membrane constituents. This study was undertaken to discriminate between soluble and cell surface bound urokinase as a potential mediator of in vitro invasion by cultured colon cancer. Extracellular matrix invasion by a colon cancer cell line GEO, characterized as being a poor secretor of urokinase and having few receptors (less than 10(4) receptors/cell) was not augmented when these cells were made to secrete up to 8 times as much urokinase, in response to an exogenous urokinase gene driven by the Rous sarcoma virus long terminal repeat promoter. The majority of the plasminogen activator (greater than 95%) appeared in the culture medium, this reflecting the low numbers of binding sites displayed by GEO cells. In contrast, the cell line HCT 116 equipped with 10 times as many binding sites, (greater than 10(5)/cell), the majority of which are occupied with endogenous ligand, was an efficient invader of the extracellular matrix. Inhibition of urokinase binding to the cell surface receptors using an antibody to the A chain of the plasminogen activator reduced invasion by 65%. The cell line RKO is equipped with 3 x 10(5) receptors/cell, 15% of which are tagged with endogenous urokinase. Pretreatment of these cells with a concentration range of urokinase known to result in the majority of these binding sites being charged with the plasminogen activator led to a dose dependent increase in extracellular matrix invasion. Together, these data suggest that for cultured colon cancer, at least, invasion is a function of the amount of cell surface receptor bound urokinase.

Colonic Neoplasms↗
PubMed1991

A new qualitative variant of the RhE antigen revealed by heterogeneity among anti-E sera.

We have recently detected a new qualitative variant of the RhE antigen characterised by negative reactions with a minority of anti-E sera, and an inability of the relevant cells to completely absorb most, if not all, anti-E samples that we tested. In tube tests, using a two-stage enzyme technique, cells from the proposita (V.R.) were completely agglutinated by 8 out of 12 undiluted polyclonal sera, weakly agglutinated (2+ and 3+ reactions) by 2 sera, but not agglutinated by the 2 remaining sera. Cross-absorption tests using 1 non-reacting, 1 weakly reacting and 4 strongly reacting polyclonal anti-E sera showed that V.R.'s cells only slightly reduced their anti-E titres versus r"r cells, whilst completely removing their reactivity against her own cells. Absorptions with R2R2 cells abolished activity against both cell types. The cells of V.R. failed to absorb or elute anti-Ew, and had normal expression of c. Three monoclonal human anti-E reagents were tested by fluorescence flow cytometry; one reagent failed to react with the cells of V.R., a second bound as strongly with V.R.'s cells as with R1R2 controls while the third bound much more weakly with V.R. than with any other E+ sample. The variant of RhE in the V.R. family is therefore distinct from Ew, and lacks an epitope recognised by a major constituent of most anti-E sera. This epitope is unlikely to be ET, since all of 91 samples from E-positive Australian aborigines (many of which are expected to lack ET) were agglutinated by a serum that failed to react with V.R.'s cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Agglutination Tests↗
PubMed1991

Sleep and nocturnal acid reflux in normal subjects and patients with reflux oesophagitis.

Nocturnal gastro-oesophageal reflux may be important in the pathogenesis of reflux oesophagitis. This study aimed to determine whether: (1) gastro-oesophageal reflux occurs during sleep in patients with reflux oesophagitis and, if so, to explore the mechanism, and (2) the sleep pattern of patients with oesophagitis is different from that of control subjects. After a standard evening meal, simultaneous manometric, oesophageal pH, and polysomnographic recordings were obtained in 11 patients with endoscopic oesophagitis and 11 control subjects. Patients with gastrooesophageal reflux disease had significantly more total reflux episodes throughout the nocturnal monitoring period than control subjects (105 v 6). Ninety two of 105 episodes of gastro-oesophageal reflux in patients occurred during the awake state and 10 during sleep stage II. A number of reflux episodes occurred during brief periods of arousal from the various sleep stages. Of the 105 reflux events recorded in patients, 42 were induced by transient lower oesophageal sphincter relaxation, 20 by stress reflux, 22 by free reflux mechanisms, and in 21 the mechanism was unclear. The sleep pattern and the time spent in each sleep stage was not different between the two groups. It is concluded that the awake state is crucial for the occurrence of nocturnal reflux episodes in normal subjects as well as in patients with reflux oesophagitis and that the difference between the frequency of gastro-oesophageal reflux between normal subjects and patients cannot be explained by different sleep patterns.

Adult↗
PubMed1979

Propranolol in thyrotoxicosis: II. Serum thyroid hormone concentrations during subtotal thyroidectomy.

Propranolol alone was used to prepare 20 thyrotoxic patients, 19 women and 1 man, for subtotal thyroidectomy. Serum thyroxine (T4) and triiodothyronine (T3) concentrations were measured immediately before, at several stages during and after the surgical procedure. As judged primarily by the cardiovascular response, an average of 80 mg (range 40 to 120 mg) of propranolol qid for 8 days (range 3 to 18 days) was required to prepare the patients. During the various stages of surgical removal there was no change from the initial mean (+/- SEM) T4 concentration of 25.0 +/- 2.5 microgram/dl (321.8 +/- 32.2 nmol/l) or T3 concentration of 4.2 +/- 0.6 microgram/l (6.45 +/- 0.92 nmol/l) (P greater than 0.2). At discharge on the fifth postoperative day values were significantly lower, 12.9 +/- 1.5 microgram/dl (166.0 +/- 19.3 nmol/l) and 1.9 +/- 0.2 microgram/l (2.9 +/- 0.31 nmol/l), respectively (P less than 0.001). There were no operative complications but four patients had transient hypoparathyroidism. After 1 year 2 of 18 patients had permanent hypoparathyroidism and 4 of the 18 followed up for 1 year had permanent hypothyroidism requiring thyroid hormone replacement. There was no instance of recurrent thyrotoxicosis. The authors conclude that during surgical manipulation of the gland no release of thyroid hormones into the circulation was detected and that, using propranolol as the sole agent, thyrotoxic patients can be rapidly and safely prepared for subtotal thyroidectomy.

Adult↗
PubMed1978

Emotional adjustment and chronic pain.

Previous work has suggested that patients with organic lesions causing pain may show as much emotional disturbance as patients with pain but without lesions. This study examined 141 chronic pain patients for their life experience, both currently and premorbidly, in terms of upbringing, neurotic traits and personality disturbance. Patients with an organic cause for pain reported significantly less family disturbance in childhood, less premorbid personality problems and less neurotic traits than patients who did not have any organic cause for their pain. The data provide support for the view that a significant proportion of the emotional disturbance associated with chronic pain is a secondary effect. Adjectives used to describe pain and factors causing exacerbation and relief of pain, although overlapping, also differed in the two groups.

Adolescent↗
PubMed1978

Relative detectability of intracranial calcifications on computed tomography and skull radiography.

Phantom studies comparing skull radiography and cranial computed tomography (CT) show that CT is 5 to 15 times more sensitive than skull radiography in the detection of intracranial calcifications. The difference in detectability varies with object size. The relative sensitivity of CT increases with increased object size within the constraints of the section thickness. The relationship of these findings to the detectability of pineal and choroid calcifications is discussed.

Brain↗
PubMed1978

Measurement of left ventricular cast volume by computed tomography.

Angiocardiographic methods available for cardiac-chamber volume measurements are neither consistently accurate nor precise. To explore the capability of computed tomography for left ventricular volume measurement, Silastic casts of 24 normal human left ventricles were measured by a displacement method, a conventional angiocardiographic biplane volume method, and computed tomography. The displacement method used degassing to remove air trapped in the casts; displacement was measured by Archimedes' principle. Cast volumes measured by biplane methods displayed spread around the regression line, caused by the chamber's irregular shape and its variations in orientation. Computed tomographic measurements were independent of chamber orientation and significantly more accurate.

Angiocardiography↗