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1. The digitonin fractionation procedure [Zuurendonk, P. F. and Tager, J. M. (1974) Biochim. Biophys. Acta, 333, 393 -3991 was used to determine the concentrations of adenine nucleotides and of inorganic phosphate in the mitochondrial and cytosolic compartments of hepatocytes isolated from the livers of starved rats.2. Using glycerol 3-phosphate as a cytosolic marker, it was concluded that leakage of cytosolic metabolites was complete after 15 s exposure of the hepatocytes to 2 mM digitonin. The recovery of the adenine nucleotides after digitonin fractionation was measured. The recovery of ATP was about 100% and that of ADP and AMP 88-109%. Radioactively labelled ATP or ADP were added to the fractionation medium in order to test whether redistribution or metabolism of adenine nucleotides occurs during the fractionation procedure. No significant redistribution of adenine nucleotides occurs during the fractionation. Although slight metabolic activity appears to occur in the cytosolic fraction this leads to a change of only 20% in ADP and no change in ATP. It is concluded that the digitonin fractionation procedure can be used to obtain an accurate estimation of the content of adenine nucleotides in the cytosolic and mitochondrial fractions of isolated rat-liver cells. 3. In isolated hepatocytes from fasted rats, 60% of total adenine nucleotides is found in the cytosolic fraction.4. When isolated hepatocytes from fasted rats are incubated with alanine, the ATPjADP ratio is 8.76 in the cytosolic fraction and 1.77 in the mitochondrial fraction. On addition of oleate there is no change in cytosolic ATP/ADP. 5. In hepatocytes incubated with alanine or alanine + oleate, the concentration of inorganic phosphate is 5-times higher in the mitochondrial fraction than in the cytosol.6. It can be calculated that in isolated hepatocytes incubated with alanine the phosphorylation state ([ATP]/[ADP] x [P,]) in the mitochondrial fraction is 105 M-' (AC, = 12.0 kJ/mol) (2.87 kcal/ mol) and that in the cytosolic fraction 2630 M-' (AG, = 20.3 kJ/mol) (4.85 kcal/mol). The addition of oleate did not alter the value of the cytosolic phosphorylation state whereas that in the mitochondria was increased by about 20%.7. The concentrations of the complexed and uncomplexed forms of the adenine nucleotides and inorganic phosphate in the cytosol were calculated assuming [K+Ifree = 100 mM, [M$+Ifree =0.4 mM, pH = 7.0 and 1=0.25 M. The cytosolic concentration of uncomplexed ATP was calculated to be 170 pM, and that of uncomplexed ADP 115 pM. The latter value is about 10-times greater than the K, for ADP that has been reported for the adenine nucleotide translocator at low temperatures.Adenine nucleotides play a central role in metabolism, acting as a link between energy-yielding and energy-utilizing processes in the cell. The rate of these processes can be controlled by the adenine nucleotides directly, since they are substrates and products of the reactions. Furthermore, studies with isolated enzymes have shown that adenine nucleotides can regula...

Experiments were conducted with intact rat hepatocytes to identify inhibitors and incubation conditions that cause selective inhibition of alanine aminotransferase or aspartate aminotransferase. Satisfactory results were obtained by preincubating cells with L-cycloserine or L-2-amino-4-methoxy-trans-but-3-enoic acid in the absence of added substrates. When cells were incubated for 20 min with 50 microM-L-cycloserine, alanine aminotransferase activity was decreased by 90%, whereas aspartate aminotransferase was inhibited by 10% or less. On subsequent incubation, synthesis of glucose and urea from alanine was strongly inhibited, but glucose synthesis from lactate was unaffected. L-2-Amino-4-methoxy-trans-but-3-enoic acid (400 microM) in hepatocyte incubations caused 90-95% inactivation of aspartate aminotransferase, but only 15-30% loss of alanine aminotransferase activity. After preincubation with the inhibitor, glucose synthesis from lactate was almost completely blocked; with alanine as the substrate, gluconeogenesis was unaffected, and urea synthesis was only slightly decreased. By comparison with preincubation with inhibitors, simultaneous addition of substrates (alanine; lactate plus lysine) and inhibitors (cycloserine; aminomethoxybutenoic acid) resulted in smaller decreases in aminotransferase activities and in metabolic rates. Other compounds were less satisfactory as selective inhibitors. Ethylhydrazinoacetate inactivated the two aminotransferases to similar extents. Vinylglycine was almost equally effective in blocking the two enzymes in vitro, but was a very weak inhibitor when used with intact cells. Concentrations of DL-propargylglycine (4 mM) required to cause at least 90% inhibition of alanine aminotransferase in hepatocytes also caused a 16% decrease in aspartate aminotransferase. When tested in vitro, alanine aminotransferase was, as previously reported by others, more sensitive to inhibition by amino-oxyacetate than was aspartate aminotransferase, but in liver cell incubations the latter enzyme was more rapidly inactivated by amino-oxyacetate.

Lactic acid bacterial strains have received interest for their immunomodulating activities and potential use in probiotic products. A wide variety of strain-dependent properties have been reported, but comparative studies at the species level are scarce. The objective of this study was to assess the immunomodulatory effect of Lactobacillus species on the cytokine profiles and proliferative response of human peripheral blood mononuclear cells (hPBMC), and in particular, on the comparison between the species Lactobacillus acidophilus and Lactobacillus plantarum. hPBMC from healthy donors were stimulated in the presence or absence of the lactic acid bacteria, and cytokine production, surface marker staining, proliferation and cell death were determined after 1 and 4 days of culture. All Lactobacillus strains tested were capable of inducing the production of interleukin (IL)-1beta, IL-10, interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). The bacterial strains did not differentially influence the amount of proliferating, viable, apoptotic and necrotic cells. Generally, L. plantarum showed a significantly higher induction capacity of IFN-gamma, IL-12 and TNF-alpha compared with L. acidophilus. We conclude that the variation in immunomodulatory effects between species is even larger than the variation between the strains of the same species. In addition, we demonstrate that L. plantarum strains are most potent in skewing the T-cell differentiation toward a putative Th1 response.

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