Transport of amino acids and ammonium in mycelium of Agaricus bisporus.
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Mycelium of Agaricus bisporus took up methylamine (MA), glutamate, glutamine and arginine by high-affinity transport systems following Michaelis-Menten kinetics. The activities of these systems were influenced by the nitrogen source used for mycelial growth. Moreover, MA, glutamate and glutamine uptakes were derepressed by nitrogen starvation, whereas arginine uptake was repressed. The two ammonium-specific transport systems with different affinities and capacities were inhibited by NH(+)(4), with a K(i) of 3.7 microM for the high-velocity system. The K(m) values for glutamate, glutamine and arginine transport were 124, 151 and 32 microM, respectively. Inhibition of arginine uptake by lysine and histidine showed that they are competitive inhibitors. MA, glutamate and glutamine uptake was inversely proportional to the intracellular NH(+)(4) concentration. Moreover, increase of the intracellular NH(+)(4) level caused by PPT (DL-phosphinotricin) resulted in an immediate cessation of MA, glutamine and glutamate uptake. It seems that the intracellular NH(+)(4) concentration regulates its own influx by feedback-inhibition of the uptake system and probably also its efflux which becomes apparent when mycelium is grown on protein. Addition of extracellular NH(+)(4) did not inhibit glutamine uptake, suggesting that NH(+)(4) and glutamine are equally preferred nitrogen sources. The physiological importance of these uptake systems for the utilization of nitrogen compounds by A. bisporus is discussed. (+info)
Taurocholate-induced inhibition of hepatic lysosomal degradation of horseradish peroxidase.
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Endocytosed proteins in hepatocytes are transported to lysosomes for degradation. Metabolites accumulating in these organelles are released into bile by exocytosis, a process that seems to be regulated by the bile salt taurocholate (TC). In this study we examined if TC is also involved in the control of the lysosomal degradation of endocytosed proteins. We used [(14)C]sucrose-labeled horseradish peroxidase ([(14)C]S-HRP), a probe suitable to evaluate lysosomal proteolysis. TC-infused rats as well as isolated rat hepatocytes exposed to TC showed a significant inhibition in the lysosomal degradation of [(14)C]S-HRP (approximately 30%), with no change in either the uptake or the amount of protein reaching lysosomes. Under these conditions, the in vitro assay of lysosomal cathepsins B, L, H, and D revealed no change in their activities, suggesting that a reversible inhibition (lysosomal alkalinization?) was taking place in hepatocytes. Nevertheless, lysosomal pH measured using fluorescein isothiocyanate-dextran was shown not to be altered by TC. In addition, TC was unable to inhibit proteolysis in [(14)C]S-HRP loaded lysosomes or interfere in cathepsin assays. The results suggest that TC inhibits the lysosomal degradation of endocytosed proteins in hepatocytes and that the mechanism does not involve an effect of the bile salt per se or a rise in lysosomal pH. (+info)
Transport of D-xylose in Lactobacillus pentosus, Lactobacillus casei, and Lactobacillus plantarum: evidence for a mechanism of facilitated diffusion via the phosphoenolpyruvate:mannose phosphotransferase system.
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We have identified and characterized the D-xylose transport system of Lactobacillus pentosus. Uptake of D-xylose was not driven by the proton motive force generated by malolactic fermentation and required D-xylose metabolism. The kinetics of D-xylose transport were indicative of a low-affinity facilitated-diffusion system with an apparent K(m) of 8.5 mM and a V(max) of 23 nmol min(-1) mg of dry weight(-1). In two mutants of L. pentosus defective in the phosphoenolpyruvate:mannose phosphotransferase system, growth on D-xylose was absent due to the lack of D-xylose transport. However, transport of the pentose was not totally abolished in a third mutant, which could be complemented after expression of the L. curvatus manB gene encoding the cytoplasmic EIIB(Man) component of the EII(Man) complex. The EII(Man) complex is also involved in D-xylose transport in L. casei ATCC 393 and L. plantarum 80. These two species could transport and metabolize D-xylose after transformation with plasmids which expressed the D-xylose-catabolizing genes of L. pentosus, xylAB. L. casei and L. plantarum mutants resistant to 2-deoxy-D-glucose were defective in EII(Man) activity and were unable to transport D-xylose when transformed with plasmids containing the xylAB genes. Finally, transport of D-xylose was found to be the rate-limiting step in the growth of L. pentosus and of L. plantarum and L. casei ATCC 393 containing plasmids coding for the D-xylose-catabolic enzymes, since the doubling time of these bacteria on D-xylose was proportional to the level of EII(Man) activity. (+info)
Evidence for the presence of the reductive pentose phosphate cycle in a filamentous anoxygenic photosynthetic bacterium, Oscillochloris trichoides strain DG-6.
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Studies on autotrophic CO2 fixation by the filamentous anoxygenic photosynthetic bacterium Oscillochloris trichoides strain DG-6 demonstrated that, unlike other green bacteria, this organism metabolized CO2 via the reductive pentose phosphate cycle. Both key enzymes of this cycle--ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase--were detected in cell extracts. The main product of ribulose 1,5-bisphosphate-dependent CO2 fixation was 3-phosphoglyceric acid. KCN, which is known to be a competitive inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase, completely inhibited the CO2 assimilation by whole cells as well as by cell extracts of O. trichoides. The 13C/12C carbon isotope fractionation during photoautotrophic growth of O. trichoides was -19.7/1000, which is close to that obtained for autotrophic organisms that use ribulose-1,5-bisphosphate carboxylase as the primary carboxylation enzyme. Cell extracts of O. trichoides contained all the enzymes of the tricarboxylic acid cycle except 2-oxoglutarate dehydrogenase. No activity of isocitrate lyase, a key enzyme of the glyoxylate shunt, was found in cell extracts of O. trichoides DG-6. (+info)
Blood glycerol is an important precursor for intramuscular triacylglycerol synthesis.
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The utilization of blood glycerol and glucose as precursors for intramuscular triglyceride synthesis was examined in rats using an intravenous infusion of [2-(14)C]glycerol and [6-(3)H]glucose or [6-(14)C]glucose. In 24-h fasted rats, more glycerol than glucose was incorporated into intramuscular triglyceride glycerol in soleus (69 +/- 23 versus 4 +/- 1 nmol/micromol triglyceride/h, respectively, p = 0.02 glycerol versus glucose) and in gastrocnemius (25 +/- 5 versus 9 +/- 2 nmol/micromol triglyceride/h, respectively, p = 0.02). Blood glucose was utilized more than blood glycerol for triglyceride glycerol synthesis in quadriceps. In fed rats, the blood glycerol incorporation rates (4 +/- 2, 8 +/- 3, and 9 +/- 3 nmol/micromol triglyceride/h) were similar (p > 0.3) to those of glucose (5 +/- 2, 8 +/- 2, and 5 +/- 2 nmol/micromol triglyceride/h for quadriceps, gastrocnemius, and soleus muscle, respectively). Glucose incorporation into intramuscular triglycerides was less with [6-(3)H]glucose than with [6-(14)C]glucose, suggesting an indirect pathway for glucose carbon entry into muscle triglyceride. The isotopic equilibrium between plasma and intramuscular free glycerol ([U-(13)C]glycerol) was complete in quadriceps and gastrocnemius, but not soleus, within 2 h after beginning the tracer infusion. We conclude that blood glycerol is a direct and important precursor for muscle triglyceride synthesis in rats, confirming the presence of functionally important amounts of glycerol kinase in skeletal muscle. (+info)
Caco-2 cells and human fetal colon: a comparative analysis of their lipid transport.
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Caco-2 cells and human colonic explants were compared for their ability to esterify lipid classes, synthesize apolipoproteins and assemble lipoproteins. Highly differentiated cells and colonic explants were incubated with [(14)C]oleic acid or [(35)S]methionine for 48 h. Caco-2 cells demonstrated a higher ability to incorporate [(14)C]oleic acid into cellular phospholipids (13-fold, P<0.005), triglycerides (28-fold, P<0.005) and cholesteryl ester (2-fold, P<0. 01). However, their medium/cell lipid ratio was 11 times lower, indicating a limited capacity to export newly synthesized lipids. De novo synthesis of apo B-48 and apo B-100 was markedly increased (7%0 and 240%, respectively), whereas the biogenesis of apo A-I was decreased (60%) in Caco-2 cells. The calculated apo B-48/apo B-100 ratio was substantially diminished (107%), suggesting less efficient mRNA editing in Caco-2 cells. When lipoprotein distribution was examined, it displayed a prevalence of VLDL and LDL, accompanied along with a lower proportion of chylomicron and HDL. In addition, differences in lipoprotein composition were evidenced between colonic explants and Caco-2 cells. Therefore, our findings stress the variance in the magnitude of lipid, apolipoprotein and lipoprotein synthesis and secretion between the two intestinal models. This may be due to various factors, including the origin of Caco-2 cell line, i.e., colon carcinoma. (+info)
Salicylhydroxamic acid inhibits delta6 desaturation in the microalga Porphyridium cruentum.
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Treatment of the microalga Porphyridium cruentum with salicylhydroxamic acid (SHAM) inhibited growth and affected fatty acid composition. At a relatively low concentration (40 microM) SHAM predominantly inhibits Delta6 desaturation. The effect of the inhibitor was most intense in phosphatidylcholine (PC) and phosphatidylethanolamine, in which the proportions of the downstream products of the Delta6 desaturase were reduced, whereas that of the substrate, 18:2, increased. As a result of the availability of 18:2, 18:3omega3, which under normal conditions is not observed, appeared predominantly in chloroplastic lipids. Pulse labeling with linoleic acid has shown that SHAM inhibits Delta6 desaturation almost immediately, suggesting an apparent inhibition of the activity of the desaturase, rather than its synthesis or that of its cofactors. Furthermore, the addition of gamma-linolenic acid to SHAM-inhibited cultures relieved the inhibition. Following exposure to the inhibitor, 18:3omega3 appeared first in chloroplastic glycolipids and only later in PC, indicating that the former are the substrates for the first dedicated step of the proposed omega3 pathway in this alga. (+info)
Detection of muscarinic receptors in the human lung using PET.
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The characterization of pulmonary muscarinic receptors with PET is still in its infancy. Because approximately 70% of the lungs consists of air and pulmonary muscarinic receptor densities are low, ligands with high receptor affinity are required to obtain reasonable signal-to-noise ratios on PET images. Therefore, the potent 11C-labeled muscarinic antagonist N-methyl-piperidin-4-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate methiodide ([R]-VC-002) was developed. We administered this radioligand to four healthy human volunteers to examine its suitability for studying pulmonary muscarinic receptors in vivo. METHODS: [11C]VC-002 (185 MBq, specific activity > 7.4 TBq/mmol) was intravenously injected on 2 separate days, with an interval of at least 1 wk. On the first day the volunteers were not pretreated, but on the second day they received the anticholinergic glycopyrronium bromide (Robinul; 2 x 0.1 mg intravenous) 25 and 30 min before the injection of the radiopharmaceutical. C[15O]O scans (approximately 740 MBq [20 mCi] by inhalation) were acquired before the receptor scan to calculate pulmonary blood volume. RESULTS: On PET images of the thorax, the lungs were clearly visible. After the volunteer was pretreated with glycopyrronium bromide, pulmonary uptake of the radioligand was reduced to 32%+/-12% of the control value at 60 min postinjection and the lungs could no longer be seen. (R)-[11C]-VC-002 was rapidly cleared from plasma and was slowly metabolized during the time course (60 min) of the PET scan. The fraction of radioligand representing parent compound decreased from 99.9% at the time of injection to 82% at 40-60 min postinjection, both in the presence and absence of Robinul. Pulmonary tissue-to-plasma ratios, calculated on a count-per-minute-per-gram basis, reached a plateau value of 17.8+/-1.2 at 40-50 min postinjection. CONCLUSION: [11C]VC-002 appears to be suitable for in vivo studies of pulmonary cholinoceptors. (+info)