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Biomedical subjects

W D Rees

Publications and source records attributed to W D Rees.

At least 19 recordsLinked to original sources
PubMed1995

The biosynthesis of threonine by mammalian cells: expression of a complete bacterial biosynthetic pathway in an animal cell.

The coding regions for the Escherichia coli gene for aspartokinase I/homoserine dehydrogenase I (thrA) and the Corynebacterium glutamicum gene for aspartic semialdehyde dehydrogenase (asd) have been subcloned into a Simian Virus 40 (SV40)-based mammalian expression vector. Both enzyme activities are expressed in mouse 3T3 cells after transfer of the corresponding chimaeric gene. The kinetic parameters are similar to those of the native bacterial enzymes, and aspartokinase I/homoserine dehydrogenase I retains its allosteric regulation by threonine. An extract of the cells expressing aspartokinase I/homoserine dehydrogenase I, mixed with one from cells expressing aspartic semialdehyde dehydrogenase, produced homoserine when the mixture was incubated with aspartic acid, ATP and NADPH. The thrA and asd expression cassettes were combined into a single plasmid which, when transfected into 3T3 cells, enabled them to produce homoserine from aspartic acid. Homoserine-producing 3T3 cells were transfected with the plasmid pSVthrB/C (homoserine kinase and threonine synthase) and selected for growth on homoserine. Cell lines isolated from these cells expressed the complete bacterial threonine pathway, were independent of threonine for growth and could be maintained in medium which contained no free threonine. The threonine in the proteins of these cells became enriched in 15N when the culture medium contained [15N]aspartic acid. The production of homoserine and the growth of cells was at a maximum when there was more than 2.5 mM aspartate in the medium. Below this concentration the high Km of aspartokinase limited the flux through the pathway. In the presence of additional aspartic acid the new pathway could sustain a cell cycle time close to that of the same cells cultured in threonine-containing medium.

3T3 Cells↗
PubMed1995

Neural influences on human esophageal and salivary alkali secretion.

Esophageal secretion HCO3- ions occurs in opossum and man and may contribute to mucosal defense. Using a perfusion technique, neuroregulatory influences on esophageal and salivary HCO3- secretion were investigated in 24 healthy human subjects. The sight and smell of food increased median salivary HCO3- output from 424 to 573 mumol/15 min (P = 0.014), without significantly altering esophageal HCO3- secretion (74-105 mumol/15 min, P = 0.24). Atropine reduced both salivary (610 to 68, 17, 10, and 3 mumol/15 min in successive periods; P < 0.028) and esophageal HCO3- output (108 to 78, 35, 18, and 7 mumol/10 cm/15 min; P < 0.028, respectively. Following atropinization, cholinergic stimulation failed to increase salivary secretion but did "unmask" a small rise in esophageal alkali output (7 to 27 mumol/10 cm/15 min, P = 0.036), implicating a noncholinergic mechanism. Cold-induced pain activated sympathetic reflexes and reduced esophageal HCO3- output (91 to 64 mumol/10 cm/15 min, P = 0.041) without influencing salivary secretion. These observations support a role for the autonomic nervous system in modulating human esophageal and salivary HCO3- secretion.

Adolescent↗
PubMed1995

Review article: factors protecting the oesophagus against acid-mediated injury.

Reflux of gastric acid and pepsins into the lower oesophagus causes symptoms such as heartburn and nausea, and tissue injury leading to erosive oesophagitis and stricture formation. This article reviews the mechanisms involved in protecting the oesophagus against acid-mediated injury, including the role of the lower oesophageal sphincter, secondary oesophageal peristalsis and swallowed saliva. The oesophageal mucosa has inherent abilities to resist acid damage, and recent data from three laboratories suggest a secretory function with local production of bicarbonate and mucus responsive to local acidification. The evidence for these putative oesophageal defence mechanisms is discussed.

Animals↗
PubMed1995

Effect of topical oesophageal acidification on human salivary and oesophageal alkali secretion.

Recent human studies suggest that oesophageal HCO3- secretion, in conjunction with salivary HCO3- secretion and secondary oesophageal peristalsis, is important for the protection of oesophageal mucosa from refluxed gastric contents. This study evaluated simultaneously the responsiveness of oesophageal and salivary HCO3- secretion to oesophageal acidification in eight healthy subjects. A 10 cm segment of oesophagus was perfused at a constant rate of 5 ml/min with a specially designed tube assembly. Saline was used initially, and then 10 mM and 100 mM HCl. The perfusates contained 3H-polyethylene glycol (PEG) as a concentration marker to determine volumes. Corrections were applied for a small degree of contamination by swallowed saliva and refluxed gastric alkali. Oesophageal perfusion with 10 mM HCl did not cause symptoms (nausea and heartburn), but tripled the oesophageal HCO3- output from a baseline of 51 mumol/10 cm/10 min (p = 0.021), while doubling the rate of salivary HCO3- secretion from a median basal value of 140 mumol/10 min (p = 0.021). Oesophageal perfusion with 100 mM HCl was associated with symptoms of nausea and heartburn in all subjects. The median oesophageal HCO3- output increased 32 fold to 1659 mumol/10 cm/10 min (interquartile range 569 to 3373; p = 0.036), and salivary HCO3- secretion approximately tripled from basal values (p = 0.036). In conclusion, oesophageal acidification stimulates both salivary and oesophageal HCO3- secretion, responses which may be protective to the oesophageal epithelium.

Administration, Topical↗
PubMed1994

The expression of Escherichia coli diaminopimelate decarboxylase in mouse 3T3 cells.

We have subcloned the coding sequence for the Escherichia coli lysA gene coding for diaminopimelic acid decarboxylase (DAP decarboxylase) into a eukaryotic expression vector based on the SV40 early promoter. The activities of a series of constructs with different lengths of non-coding DNA at the 5' and 3' ends of the coding region have been compared by measuring the synthesis of lysine from diaminopimelic acid (DAP) in mouse 3T3 cells. A short non-coding sequence at the 3' end reduced the expression of enzyme activity. Stable lines of 3T3 cells have been produced by co-transfection of the chimeric gene with a plasmid coding for G-418 resistance. Cells were grown in medium containing G-418 and resistant clones were screened for an ability to synthesise lysine from DAP. [3H]Lysine produced from [3H]DAP was incorporated into cell proteins. An enzyme extract from a cell line which had incorporated two copies of the gene synthesised 0.082 nmol of lysine/min per mg protein. In the intact cell the rate of lysine synthesis is limited by the uptake of DAP which is taken up at only 5% of the rate of lysine. lysA has a potential as a reporter gene in studies of gene expression in mammalian cells.

3T3 Cells↗
PubMed1994

Threonine synthesis from homoserine as a selectable marker in mammalian cells.

The plasmid pSVthrBC expresses the Escherichia coli thrB (homoserine kinase) and thrC (threonine synthase) genes in mouse cells and enables them to synthesize threonine from homoserine. After transfection with pSVthrBC and culture in medium containing homoserine, only cells that have incorporated pSVthrBC survive. Homoserine at concentrations greater than 1 mM is toxic to mammalian cells. Mouse cells selected from medium containing 5 mM homoserine had incorporated 20-100 copies of the plasmid per cell and had homoserine kinase activities of 0.001-0.012 nmol/min per mg of protein per copy. Cells selected from medium containing 10 mM homoserine had incorporated one or two copies of the plasmid per cell and had homoserine kinase activities of 0.06-0.39 nmol/min per mg of protein per copy. By using high concentrations of homoserine, it is possible to use pSVthrBC to select and isolate cell lines that have one or two copies of the plasmid incorporated into an active region of chromatin. CHO and HeLa cells have also been successfully transfected with pSVthrBC. COS-7 cells are naturally resistant to homoserine as they are able to metabolize homoserine.

3T3 Cells↗
PubMed1993

The expression of Escherichia coli threonine synthase and the production of threonine from homoserine in mouse 3T3 cells.

We have subcloned the coding sequence for the Escherichia coli threonine synthase gene into a eukaryotic expression vector based on the simian-virus-40 early promoter. When mouse 3T3 cells which already expressed homoserine kinase were transfected with the new plasmid, the cells were able to incorporate radioactivity from [14C]homoserine into their cell proteins. Stable cell lines were established by co-transfecting 3T3 cells with the plasmid coding for threonine synthase and another coding for homoserine kinase and G-418 (Geneticin) resistance. Cells were selected for G-418 resistance and then screened for an ability to synthesize threonine from homoserine and incorporate it into the cell protein. A cell line which expressed both the homoserine kinase and threonine synthase genes was capable of growth in a threonine-deficient medium containing homoserine.

3T3 Cells↗
PubMed1993

Measurement of bicarbonate output from the intact human oesophagus.

Injury of the oesophageal mucosa can result from exposure to refluxed gastric acid and pepsin. Competence of the lower oesophageal sphincter and peristaltic activity serve to reduce contract time between luminal acid and oesophageal mucosa, but intraluminal neutralisation of residual acid by bicarbonate may also be important in preventing oesophageal mucosal injury. Whereas swallowed saliva contains bicarbonate, recent experiments have also demonstrated alkali secretion from the mammalian oesophagus. Bicarbonate secretion from the human oesophagus was therefore examined with an intubation technique and perfusion of the oesophagus with a non-absorbable marker. Saliva, gastric, and oesophageal aspirates were collected and bicarbonate concentrations determined by measurements of pH and pCO2 or by back titration. In 32 normal subjects (17 women, 15 men) median basal oesophageal bicarbonate secretion determined by a pH/pCO2 method was 416 (range 139-1050) mumol/hour/10 cm. In a subgroup of 15 experiments median oesophageal bicarbonate output was 489 (range 157-1033) mumol/hour/10 cm (pH/pCO2 method) compared with a median alkali output of 563 (range 135-799) mumol/hour/10 cm as determined by back titration. The difference was not significant. Salivary contamination of the oesophagus accounted for 25% of all bicarbonate measured within the oesophagus and refluxed gastric bicarbonate accounted for 2.5%. Bicarbonate secretion from the normal human oesophagus may, in combination with swallowed salivary bicarbonate, play a part in preventing oesophageal mucosal damage due to refluxed gastric acid and pepsin.

Adolescent↗
PubMed1992

Expression of Escherichia coli homoserine kinase in mouse 3T3 cells.

The Escherichia coli gene for homoserine kinase (thrB) has been cloned into a simian-virus-40-based eukaryotic expression vector which also includes a neomycin-resistance gene. Mouse 3T3 cells transfected with this plasmid were selected for resistance and screened for homoserine kinase activity. It has thus been possible to isolate clones which are capable of accumulating homoserine O-phosphate when supplied with homoserine. In broken-cell preparations the kinetic constants for the production of homoserine O-phosphate were similar to those of the wild-type E. coli enzyme. These experiments demonstrate that E. coli homoserine kinase can be expressed in an animal cell and that it can successfully phosphorylate L-homoserine in the intact cell utilizing endogenous ATP.

3T3 Cells↗
PubMed1992

Mucosal adaptation to indomethacin induced gastric damage in man--studies on morphology, blood flow, and prostaglandin E2 metabolism.

The effect of 28 days' continuous administration of oral indomethacin on gastroduodenal morphology, gastric mucosal blood flow, and gastric mucosal prostaglandin E2 (PGE2) metabolism in man was studied to define further the mechanisms of mucosal injury induced by indomethacin. Indomethacin caused acute gastroduodenal damage in all cases, which was maximal at 24 hours of administration. With continued intake, mucosal adaptation occurs resulting in resolution of endoscopic mucosal damage. At the time of maximal mucosal damage, gastric mucosal blood flow was significantly reduced compared with values before treatment (p less than 0.001 in fundus and p less than 0.002 in antrum), with good correlation between the severity of damage and the magnitude of the reduction in blood flow (r = 0.76). Mucosal recovery was associated with a return of the blood flow to normal. PGE2 in mucosal homogenate was significantly reduced by indomethacin in both the fundus (p less than 0.01) and antrum (p less than 0.01) after 24 hours but there was no correlation between the magnitude of this reduction and the severity of mucosal damage (r = -0.34). Despite mucosal recovery by 28 days, PGE2 values remained significantly below those before treatment in both the fundus (p less than 0.01) and antrum (p less than 0.01). The PGE2 degradation capacity was not influenced by indomethacin. In conclusion, mucosal adaptation to acute damage by indomethacin occurs in man and seems independent of local PGE2 metabolism.

Adaptation, Physiological↗
PubMed1992

Prevention of peptic ulcer relapse by sucralfate: mechanisms of action.

Sucralfate has a complex effect on the luminal and mucosal environment of the stomach and duodenum. Some of the actions are important in ulcer healing whilst others are important in preventing subsequent ulcer relapse. Although sucralfate has little direct effect on acid secretion, there is evidence that after ulcer healing with this drug, parietal cell responsiveness is reduced. This may in part be mediated by increased somatostatin release from gastric D cells and may be important in reducing ulcer relapse. Sucralfate has been shown to increase mucosal resistance to damaging agents, such as ethanol and aspirin. Studies have shown that this protective action may be related to the drugs effect on various protective zones such as the 'mucous-bicarbonate' barrier, mucosal hydrophobicity, epithelial cell function and morphology, and mucosal blood flow. These complex actions of sucralfate are in part related to direct interaction between the drug or its components and gastroduodenal tissues, and in part related to effects on various mediators of tissue injury and repair.

Gastric Mucosa↗
PubMed1991

Mechanisms of gastroduodenal protection by sucralfate.

Over the past 5-10 years, a number of studies have shown that topical sucralfate enhances a number of gastric and duodenal mechanisms, e.g., the "mucus-bicarbonate barrier," mucosal hydrophobicity, mucosal blood flow, cell viability, and local production of prostaglandins, as well as endogenous mediators of tissue injury and repair. It seems likely that the complex actions of sucralfate are in part related to direct interaction between the drug or its components (aluminum, sucrose, and sulfate) and gastric mucosal tissues, and in part related to effects of the drug on the various mucosal mediators of tissue injury and repair. Local actions may play a role in accelerating healing of ulcer-damaged mucosa, but this does not explain the protective actions of sucralfate on normal mucosa. Thus sucralfate appears to enhance the protective function of the "mucus-bicarbonate" barrier by actions on both components. This may depend in part on an interaction with the unstirred layer overlying gastric epithelium. Sucralfate has also been shown to increase the hydrophobicity of mucus gel. There is little doubt that sucralfate increases local production and release of protective prostaglandins (PGs), but the precise role played by these agents in mediating mucosal protection and in chronic ulcer healing remains uncertain. Currently, the mechanism of action of sucralfate on vascular integrity remains unknown and the role of PGs in this protective function is unclear. There is little evidence that epidermal growth factor plays any role in mediating mucosal protection by sucralfate, but it may be important in its ulcer-healing action. Sucralfate has been shown to be truly "cytoprotective" in that it protects isolated epithelial cells from damage by noxious agents. In animals treated with sucralfate, the surface epithelial cells were disrupted, but necrotic lesions in the deep proliferative zone were virtually absent. It seems likely that investigations of the actions of sucralfate and its components will move ever closer to defining the target cells, the intracellular events, and the mediators that bring about its protective and ulcer-healing activity.

Animals↗
PubMed1991

Sarcosine kinetics in pigs by infusion of [1-14C]sarcosine: use for refining estimates of glycine and threonine kinetics.

To investigate in vivo the interconversion between glycine (Gly) and its N-methyl product sarcosine (Sar), [1-13C]Gly and [1-14C]Sar were infused into hourly fed pigs receiving diets with low- and high-threonine levels. An open two-pool model was developed to calculate Sar demethylation (DM) and Gly methylation (GM). During [1-14C]Sar infusion, intracellular Gly specific radioactivities (SA) in the liver and kidney were higher than plasma Gly SA, suggesting that demethylation of Sar occurred in those tissues. DM estimated by using hippuric acid (HA) as the production pool had a mean value of 1.55 mumol.kg-1.h-1, similar to the Sar production rate (mean 1.85 mumol.kg-1.h-1). GM was undetectable (less than 0.5 mumol.kg-1.h-1). These results suggest that, in fed pigs, Sar is produced mainly from choline catabolism and is degraded only to Gly in liver and kidney. On the assumption that Sar degradation gave rise only to Gly, the production rate of Gly (Gly PR) was calculated from [1-13C]Gly and [1-14C]Sar infusions using either the primary pools (plasma Gly and HA, respectively) or the secondary pools (HA and plasma Gly, respectively). The results were explained by a liver-plasma Gly exchange model. The whole body Gly irreversible loss, i.e., direct loss from plasma and liver, was calculated from this model to be 832 +/- 58 mumol.kg-1.h-1, showing that the estimation of Gly PR with [1-13C]Gly infusion and plasma Gly enrichment (599 +/- 56 mumol.kg-1.h-1) was a significant underestimate of the true value.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗