As compared with interferon alfa, temsirolimus improved overall survival among patients with metastatic renal-cell carcinoma and a poor prognosis. The addition of temsirolimus to interferon did not improve survival. (ClinicalTrials.gov number, NCT00065468 [ClinicalTrials.gov].).

No abstract

1. Pyruvate strongly inhibited aspartate production by mitochondria isolated from Ehrlich ascites-tumour cells, and rat kidney and liver respiring in the presence of glutamine or glutamate; the production of (14)CO(2) from l-[U-(14)C]glutamine was not inhibited though that from l-[U-(14)C]glutamate was inhibited by more than 50%. 2. Inhibition of aspartate production during glutamine oxidation by intact Ehrlich ascites-tumour cells in the presence of glucose was not accompanied by inhibition of CO(2) production. 3. The addition of amino-oxyacetate, which almost completely suppressed aspartate production, did not inhibit the respiration of the mitochondria in the presence of glutamine, though the respiration in the presence of glutamate was inhibited. 4. Glutamate stimulated the respiration of kidney mitochondria in the presence of glutamine, but the production of aspartate was the same as that in the presence of glutamate alone. 5. The results suggest that the oxidation of glutamate produced by the activity of mitochondrial glutaminase can proceed almost completely through the glutamate dehydrogenase pathway if the transamination pathway is inhibited. This indicates that the oxidation of glutamate is not limited by a high [NADPH]/[NADP(+)] ratio. 6. It is suggested that under physiological conditions the transamination pathway is a less favourable route for the oxidation of glutamate (produced by hydrolysis of glutamine) in Ehrlich ascites-tumour cells, and perhaps also kidney, than the glutamate dehydrogenase pathway, as the production of acetyl-CoA strongly inhibits the first mechanism. The predominance of the transamination pathway in the oxidation of glutamate by isolated mitochondria can be explained by a restricted permeability of the inner mitochondrial membrane to glutamate and by a more favourable location of glutamate-oxaloacetate transaminase compared with that of glutamate dehydrogenase.

1. Rat kidney mitochondria oxidize glutamate very slowly. Addition of glutamine stimulates this respiration two- to three-fold. Addition of glutamate also stimulates respiration in the presence of glutamine. 2. By measuring mitochondrial swelling in iso-osmotic solutions of glutamine or of ammonium glutamate it was shown that glutamine penetrates the mitochondrial membrane rapidly whereas ammonium glutamate penetrates very slowly. 3. Experiments in which reduction of NAD(P)(+) was measured in preparations of intact and broken mitochondria indicated that glutamate dehydrogenase shows the phenomenon of ;latency'. On the addition of glutamine rapid reduction of nicotinamide nucleotides in intact mitochondria was obtained. 4. During the action of glutaminase there is an accumulation of glutamate inside the mitochondria. 5. When the mitochondria were suspended in a medium containing glutamine, P(i) and rotenone the rate of production of ammonia was stimulated by the addition of a substrate, e.g. succinate. Addition of an uncoupler or antimycin A abolished this stimulation. 6. The effects of succinate and uncoupler were especially pronounced in the presence of glutamate, which is an inhibitor of glutaminase activity by competition with P(i). 7. Determination of the enzyme activity in media at different pH values showed that the optimum pH for glutaminase activity in the preparation of broken mitochondria was 8, whereas for intact mitochondria it was dependent on the energy state. In the presence of succinate as an energy source it was pH 8.5, but in the presence of uncoupler or antimycin A it was 9. This displacement of the pH optimum to a higher value was especially pronounced in the presence of both glutamate and uncoupler. 8. If nigericin was present in potassium chloride medium the pH optimum for enzyme activity in intact non-respiring mitochondria was nearly the same as in the preparation of broken mitochondria; however, its presence in K(+)-free medium displaced the pH optimum for glutaminase activity to a very high value. 9. It is postulated that because of low permeability of the kidney mitochondrial membrane to glutamate the latter accumulates inside the mitochondria, and that this leads to the inhibition of the enzyme by competition with P(i) and also by lowering the pH of the intramitochondrial space. With succinate as substrate for respiration there is an outward translocation of H(+) ions, which together with accumulation of P(i) increases glutaminase activity. Translocation of K(+) ions inward increases the enzyme activity, perhaps by increasing the pH of the internal spaces and causing an accumulation of P(i). 10. The importance of the location of the enzyme in the mitochondria in relation to its biological function and conditions for activity is discussed.

Intensive oxidation of glutamine in Ehrlich ascites tumor cells results in a high intracellular accumulation of aspartate. Since K , of aspartate aminotransferase for aspartate is high it was assumed that the level of aspartate may be the rate-limiting step in the operation of the malate-aspartate shuttle. I n that case the oxidation of glutamine would stimulate or activate the shuttle. However, it was found that if aerobic glycolysis takes place in the presence of glutamine the production of lactate was the same whereas the accumulation of aspartate by glutamine oxidation was strongly inhibited. Preincubation of the cells with glutamine and the addition of glucose a t the moment when the level of aspartate was high and the level of glutamate was low brought about significant decrease in lactate production suggesting activation of the shuttle. This was supported by the experiments with an oxygen electrode. Namely, if the cells were preincubated with glutamine the addition of lactate stimulated oxygen consumption in the presence of arsenite. This effect of lactate was lacking or abolished in the presence of pyruvate, aminooxyacetate or rotenone.These and other experimental findings suggest that low level of aspartate is the limiting step in the operation of the malate-aspartate shuttle in the strain of Ehrlich ascites cells studied. Analysis of all experimental data indicates that the shuttle probably does not normally operate in these cells since in the presence of glucose the accumulation of aspartate was strongly inhibited. This together with high level of glutamate strongly suppresses the activity of aspartate aminotransferase in the direction of oxaloacetate production in the cytosol.Since the mitochondrial membrane is not permeable to the pyridine nucleotides [I -51, several mechanisms were proposed in order to explain aerobic oxidation of cytoplasmic NADH. The malateaspartate shuttle was originally proposed by Sacktor [6] as a malate-oxaloacetate shuttle. To avoid the difficulty posed by the impermeability of the mitochondrial membrane to oxaloacetate, Borst included double transamination in the scheme [7]. Borst's substrate cycle involves malate dehydrogenase and aspartate aminotransferase both inside and outside the mitochondria. It is believed that the shuttle does function in vivo. It has helped to explain some experimental results [8,9] and it has been demonstrated to operate in a reconstructed system [lo]. However, experimental evidence for the operation of this cycle in intact cells is still lacking.