A central role for Bid in granzyme B-induced apoptosis

Granzyme B, a protease released from cytotoxic lymphocytes, has been proposed to induce target cell death by cleaving and activating the pro-apoptotic Bcl-2 family member Bid. It has also been proposed that granzyme B can induce target cell death by activating caspases directly, by cleaving caspase substrates, and/or by cleaving several non-caspase substrates. The relative importance of Bid in granzyme B-induced cell death has therefore remained unclear. Here we report that cells isolated from various tissues of Bid-deficient mice were resistant to granzyme B-induced cell death. Consistent with the proposed role of Bid in regulating mitochondrial outer membrane permeabilization, cytochrome c remained in the mitochondria of Bid-deficient cells treated with granzyme B. Unlike wild type cells, Bid-deficient cells survived and were then able to proliferate normally, demonstrating the critical role for Bid in mediating granzyme B-induced apoptosis.     

Post-mortem glycolysis in ox skeletal muscle. Effect of pre-rigor freezing and thawing on the intermediary metabolism

1. Ox sternomandibularis muscle was ;slow-frozen' by placing it in air at -22 degrees or ;fast-frozen' by immersion in liquid air or acetone-solid carbon dioxide. In all cases muscles were frozen pre-rigor. Changes in length, pH and the concentrations of P(i), creatine phosphate, hexose monophosphate (glucose 1-phosphate+glucose 6-phosphate+fructose 6-phosphate), fructose diphosphate (fructose 1,6-diphosphate+(1/2) triose phosphate), lactate, ATP, ADP, AMP and NAD(+) during freezing and during subsequent thawing were determined. In addition some measurements were made of the changes in alpha-glycerophosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate and pyruvate concentrations during slow freezing. 2. Appreciable shortening and marked changes in chemical composition took place during slow freezing but not during fast freezing. 3. During slow freezing the hexose monophosphate concentration fell and fructose 1,6-diphosphate and triose phosphate increased substantially. Increases also took place in 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate, but not in pyruvate. 4. On thawing, most of the chemical changes were similar to those in unfrozen muscle post mortem, but took place much more rapidly; loss of NAD(+) was particularly rapid. Fast-frozen muscle metabolized at a faster rate on thawing than did slow-frozen muscle. 5. The overall changes in length during freezing and thawing were about the same in slow-frozen as in fast-frozen muscle.     

Post-mortem glycolysis in ox skeletal muscle. Effect of temperature on the concentrations of glycolytic intermediates and cofactors

1. Post-mortem changes in the concentrations of the following compounds in ox sternomandibularis muscles stored in nitrogen at 1 degrees , 5 degrees and 15 degrees are reported: P(i) creatine phosphate, hexose monophosphates, fructose diphosphate, triose phosphates, alpha-glycerophosphate, phosphoglycerates, lactate, ATP, ADP, AMP, NAD(+) and total nucleotides. Some results obtained with muscles stored at 37 degrees are included. 2. At the time the muscles were placed at controlled temperatures (about 1.5hr. post mortem) the phosphorus in the compounds measured accounted for 91+/-6% (s.d.) of the total acid-soluble phosphate. 3. The results indicated that at all temperatures the activities of the phosphorylase and phosphofructokinase steps limited the rate and the extent of post-mortem glycolysis. 4. The large variations in hexose monophosphate concentrations during storage indicated that the ratio of phosphorylase to phosphofructokinase activity varied considerably with time and temperature. 5. Between 3.5 and 7hr. post mortem the rates of glycolysis and of ATP turnover were not slower at 5 degrees that at 15 degrees , and were probably faster at 1 degrees . The significance of this finding is discussed.     

Effects of the ionophores monensin and tetronasin on simulated development of ruminal lactic acidosis in vitro.

A continuous coculture of four ruminal bacteria, Megasphaera elsdenii, Selenomonas ruminantium, Streptococcus bovis, and Lactobacillus sp. strain LB17, was used to study the effects of the ionophores monensin and tetronasin on the changes in ruminal microbial ecology that occur during the onset of lactic acidosis. In control incubations, the system simulated the development of lactic acidosis in vivo, with an initial overgrowth of S. bovis when an excess of glucose was added to the fermentor. Lactobacillus sp. strain LB17 subsequently became dominant as pH fell and lactate concentration rose. Both ionophores were able to prevent the accumulation of lactic acid and maintain a healthy non-lactate-producing bacterial population when added at the same time as an excess of glucose. Tetronasin was more potent in this respect than monensin. When tetronasin was added to the culture 24 h after glucose, the proliferation of lactobacilli was reversed and a non-lactate-producing bacterial population developed, with an associated drop in lactate concentration in the fermentor. Rises in culture pH and volatile fatty acid concentrations accompanied these changes. Monensin was unable to suppress the growth of lactobacilli; therefore, in contrast to tetronasin, monensin added 24 h after the addition of glucose failed to reverse the acidosis. Numbers of lactobacilli and lactate concentrations remained high, whereas pH and volatile fatty acid concentrations were low.