Effect of extracellular matrix and growth arrest on the alkaline phosphatase activity of F9 embryonal carcinoma cells.
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Alkaline Phosphatase↗
Biomedical subjects
S M Hay
Publications and source records attributed to S M Hay.
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↗
Tibial fractures--a useful plastering technique.
The management of the acute tibial fracture is controversial, since no single method is appropriate in all circumstances and treatment indications often overlap. Given the potential problems in the management of this often difficult fracture, it is important that the attending clinician be familiar with both operative and conservative methods of treatment. In recent years, operative intervention has become increasingly popular, particularly the use of the intra-medullary locking nail, but such techniques are not without complications, including delayed union, non-union, malunion and infection. The authors strongly believe that conservative treatment remains an important option in the treatment armamentarium. A plastering technique is therefore described which is easy to perform, maintains good reduction and avoids the need for skilled assistance, excessive analgesia or an anaesthetic. It can be performed when the patient is initially assessed in the accident department, and it has a definitive role in the treatment of closed tibial fractures.
Casts, Surgical↗
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↗
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↗
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↗
Acute compartment syndromes resulting from anticoagulant treatment.
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Acute Disease↗
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↗
Spinal manipulation and mobilisation for back and neck pain.
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Back Pain↗
Denervation increases clenbuterol sensitivity in muscle from young rats.
Clenbuterol has been shown to ameliorate denervation-induced atrophy and, therefore, clearly has therapeutic potential in the treatment of muscle wasting conditions in man. Previous studies have used dosages in rats which would be unacceptable in clinical practice, but the present results show that denervated muscle has a greater sensitivity to the drug than innervated or cardiac muscle. Fiber hypertrophy and an increase in protein and RNA content could be observed in denervated muscles but not in innervated muscles at a dose of 10 micrograms/kg body weight. When considered on a metabolic body weight basis, the effective dose in rats and the "safe" dose in man are surprisingly comparable. The observations imply that there is good reason to suppose that clenbuterol could be effective in ameliorating similar wasting conditions in man.
Animals↗
Case report. Bee sting brachial block.
A case of brachial plexus block is presented, following a bee sting in the posterior triangle of the neck. The onset of neurological symptoms was rapid as was their subsequent resolution. Delayed peripheral neurological symptoms believed to have an immunological basis have been reported in response to stings from bees and other Hymenoptera both in the central and peripheral nervous systems (Goldstein et al., 1960; Means et al., 1973; Bachman et al., 1982; Weatherall et al., 1987; Van Antwerpen et al., 1988), but to the authors' knowledge no similar case of immediate peripheral block has been reported.
Adult↗
Tissue specific responses to clenbuterol; temporal changes in protein metabolism of striated muscle and visceral tissues from rats.
The beta-adrenoceptor agonist, clenbuterol, was administered orally to male weanling rats for a period of up to 10 days. The composition and fractional rate of protein synthesis (Ks) in skeletal and cardiac muscle, gut and liver were determined. There were few changes in the visceral tissues, but there was marked protein accretion in the muscles. The results suggested that in skeletal muscles there is an increase in both Ks and the amount of protein synthesised per unit RNA. In cardiac muscle, the results indicated that there was only a very transient increase in Ks and that changes in translational capacity (RNA/prot) may account in part for the increase in protein content. It is concluded that the mechanistic basis for the increased protein gain may be different between skeletal and cardiac muscles.
Animals↗
Effects of bovine pituitary growth hormone alone or in combination with the beta-agonist clenbuterol on muscle growth and composition in veal calves.
Twenty-three British Friesian bull calves at approximately 7 d of age were allocated to one of four treatments: controls untreated (five calves), a group (Clen) given 1 mg clenbuterol/kg diet (five calves), a group (GH) given a daily subcutaneous injection of 3.5 mg bovine pituitary growth hormone (GH) (five calves) and a group (Clen + GH) given a combination of clenbuterol as in the Clen group with GH as in the GH group (seven calves). All calves were given milk-substitute at levels adjusted weekly according to metabolic live weight. The animals were slaughtered over the weight range 150-170 kg. Samples of semimembranosus and triceps muscles were excised at slaughter. Treatment with GH produced approximately a threefold increase in mean daily serum GH concentration. Calves given Clen + GH were heaviest at slaughter and the combined treatment produced a significantly higher (P less than 0.01) feed conversion ratio. Administration of clenbuterol either alone or in combination with GH increased the cross-sectional area of both fast twitch glycolytic (FG), and fast twitch oxidative glycolytic (FOG) fibres in both muscles. In contrast GH produced little change in fibre size in semimembranosus muscle, although FOG fibres in triceps were slightly larger than in controls. Neither Clen nor GH resulted in any change in fibre percentage frequency in either muscle. Treatments involving clenbuterol produced a significant decrease in muscle glycogen concentration. Muscles from all three treatment groups tended to show small increases in protein and RNA concentration compared with the controls. Muscles from animals treated with GH alone exhibited an increase in DNA concentration not seen in muscles from the two other treatment groups. Overall, the differential response to the two agents suggested that clenbuterol does not mediate its effects via the GH axis, and that an additive response in terms of protein anabolism may be achieved from the use of a combination of clenbuterol plus GH.
Animals↗
The effect of clenbuterol administration in utero and throughout lactation on pre- and post-natal muscle development in the rat.
The beta-adrenergic agonist, clenbuterol was fed to pregnant rats during gestation and throughout lactation. Changes in muscle morphology and composition were studied in foetal and weanling rats. Drug treatment did not affect mean foetal number, but mean foetal body weight was significantly reduced. Heart weights were increased and both muscle weight and secondary to primary fibre ratios were decreased in foetuses exposed to clenbuterol in utero. In animals exposed to clenbuterol throughout gestation and lactation, muscle weights, and protein, RNA and DNA content and total fibre numbers were reduced. In addition a drug induced anabolism was observed in the muscles of the dams. The data are discussed in terms of a direct drug effect on immature and differentiated muscle together with a possible indirect action through the repartitioning action of clenbuterol.
Adenosine Triphosphatases↗
The action of the beta-agonist clenbuterol on protein metabolism in innervated and denervated phasic muscles.
1. Clenbuterol treatment in innervated and denervated phasic extensor digitorum longus, plantaris and gastrocnemius muscles from rats caused a significant increase in RNA and protein contents in all muscles except denervated extensor digitorum longus. 2. All muscles showed an increase in the fractional rate of protein synthesis (Ks) with clenbuterol, but the temporal response varied. 3. The data suggest that the effect of clenbuterol on protein metabolism in innervated muscles is muscle-type specific, and demonstrate the homology of response for denervated muscles.
Animals↗
Evidence that the hypertrophic action of clenbuterol on denervated rat muscle is not propranolol-sensitive.
1. The effect of propranolol on the clenbuterol-induced protein anabolism in innervated and denervated soleus and plantaris muscles of the rat was studied. 2. The response to the beta-agonist, clenbuterol, in both innervated and denervated muscles, was not significantly inhibited by the beta-antagonist, propranolol. 3. The results provide further evidence to suggest that the action of clenbuterol on skeletal muscle protein accretion may not be directly mediated by beta-adrenoceptors.
Animals↗
The effect of beta-agonists and antagonists on muscle growth and body composition of young rats (Rattus sp.).
1. The addition of the beta-selective adrenergic agonist clenbuterol to the diet was associated with an increase in the protein and RNA of skeletal and cardiac muscle, a reduction in fat deposition and an increase in energy expenditure. 2. Neither propranolol nor atenolol blocked the effect of clenbuterol on muscle protein but both reduced its effect on cardiac and fat mass and energy expenditure. 3. Five other beta-agonists were tested. All increased the interscapular brown fat mass and lowered body fat but only two increased skeletal muscle protein. 4. It is concluded that the anabolic and anti-lipogenic actions of certain beta-agonists are mechanistically distinct.
Adrenergic beta-Agonists↗
Clenbuterol, a beta agonist, induces growth in innervated and denervated rat soleus muscle via apparently different mechanisms.
Dietary administration of the anabolic agent, clenbuterol, has already been shown to inhibit or reverse denervation-induced atrophy in rat soleus muscles. We now show that the ameliorative effects of clenbuterol in denervated rat muscles are due principally to a large increase in protein synthesis. This results from both an increase in protein synthetic capacity and a normalised translational efficiency. The responses of innervated and denervated muscles are therefore fundamentally different, the changes in denervated muscles being reminiscent of the classical pleiotypic response of cells to growth factors.
Animals↗