Analysis of 4-phosphopantetheinylation of polyhydroxybutyrate synthase from Ralstonia eutropha: generation of beta-alanine auxotrophic Tn5 mutants and cloning of the panD gene region.
The postulated posttranslational modification of the polyhydroxybutyrate (PHA) synthase from Ralstonia eutropha by 4-phosphopantetheine was investigated. Four beta-alanine auxotrophic Tn5-induced mutants of R. eutropha HF39 were isolated, and two insertions were mapped in an open reading frame with strong similarity to the panD gene from Escherichia coli, encoding L-aspartate-1-decarboxylase (EC 126.96.36.199), whereas two other insertions were mapped in an open reading frame (ORF) with strong similarity to the NAD(P)+ transhydrogenase (EC 188.8.131.52) alpha 1 subunit, encoded by the pntAA gene from Escherichia coli. The panD gene was cloned by complementation of the panD mutant of R. eutropha Q20. DNA sequencing of the panD gene region (3,312 bp) revealed an ORF of 365 bp, encoding a protein with 63 and 67% amino acid sequence similarity to PanD from E. coli and Bacillus subtilis, respectively. Subcloning of only this ORF into vectors pBBR1MCS-3 and pBluescript KS- led to complementation of the panD mutants of R. eutropha and E. coli SJ16, respectively. panD-encoded L-aspartate-1-decarboxylase was further confirmed by an enzymatic assay. Upstream of panD, an ORF with strong similarity to pntAA from E. coli, encoding NAD(P)+ transhydrogenase subunit alpha 1 was found; downstream of panD, two ORFs with strong similarity to pntAB and pntB, encoding subunits alpha 2 and beta of the NAD(P)+ transhydrogenase, respectively, were identified. Thus, a hitherto undetermined organization of pan and pnt genes was found in R. eutropha. Labeling experiments using one of the R. eutropha panD mutants and [2-14C]beta-alanine provided no evidence that R. eutropha PHA synthase is covalently modified by posttranslational attachment of 4-phosphopantetheine, nor did the E. coli panD mutant exhibit detectable labeling of functional PHA synthase from R. eutropha. (+info)
H+-zwitterionic amino acid symport at the brush-border membrane of human intestinal epithelial (CACO-2) cells.
Transport of a number of dipolar amino acids (and the orally active antibiotic D-cycloserine) across the apical membrane of human intestinal epithelial (Caco-2) cell monolayers is mediated by a Na+-independent, pH-dependent transport mechanism. Relatively little is known about the mode of action of this transport system so to differentiate between pH dependence and proton coupling three experimental protocols were designed and tested. The results demonstrate, firstly, that it is the transapical pH gradient and its maintenance (rather than apical acidity alone) that is important in amino acid uptake. Secondly, Na+-independent uptake of seven dipolar amino acids (with pKa (-log of acid dissociation constant) values between 1 50 and 4 23) showed a similar dependence on apical pH (half-maximal uptake being observed at pH 5 99-6 20). Thirdly, the pattern of pH-dependent amino acid ([beta]-alanine) uptake is similar irrespective of whether the cationic substrate concentration is varied or constant, demonstrating no relationship between uptake and concentration of the cationic form of the amino acid. These observations demonstrate that the transport mechanism is a H+-zwitterionic amino acid symporter and suggest that the presence of a H+ gradient at the apical surface of the human small intestine (in the form of the acid microclimate) may be important in driving nutrient absorption. (+info)
Decreased insulin-stimulated GLUT-4 translocation in glycogen-supercompensated muscles of exercised rats.
It was recently found that the effect of an exercise-induced increase in muscle GLUT-4 on insulin-stimulated glucose transport is masked by a decreased responsiveness to insulin in glycogen-supercompensated muscle. We evaluated the role of hexosamines in this decrease in insulin responsiveness and found that UDP-N-acetyl hexosamine concentrations were not higher in glycogen-supercompensated muscles than in control muscles with a low glycogen content. We determined whether the smaller increase in glucose transport is due to translocation of fewer GLUT-4 to the cell surface with the 2-N-4-(1-azi-2,2,2-trifluroethyl)-benzoyl-1, 3-bis(D-mannose-4-yloxy)-2-propylamine (ATB-[2-3H]BMPA) photolabeling technique. The insulin-induced increase in GLUT-4 at the cell surface was no greater in glycogen-supercompensated exercised muscle than in muscles of sedentary controls and only 50% as great as in exercised muscles with a low glycogen content. We conclude that the decreased insulin responsiveness of glucose transport in glycogen-supercompensated muscle is not due to increased accumulation of hexosamine biosynthetic pathway end products and that the smaller increase in glucose transport is mediated by translocation of fewer GLUT-4 to the cell surface. (+info)
Alterations of intratumoral pharmacokinetics of 5-fluorouracil in head and neck carcinoma during simultaneous radiochemotherapy.
The kinetics of local drug uptake and metabolism of the anticancer drug 5-fluorouracil (5-FU) has been monitored by means of 19F nuclear magnetic resonance spectroscopy in 17 patients with neck tumors during concurrent radiochemotherapy. All of the patients underwent an accelerated hyperfractionated, concomitant-boost radiochemotherapy with 5-FU [600 or 1000 mg/m2 of body surface (b.s.)] and carboplatin (70 mg/m2 of b.s.). Serial 19F nuclear magnetic resonance spectra were obtained during and after the administration of 5-FU in a 15-T scanner with the use of a 5-cm diameter surface coil positioned on a cervical lymph node metastasis. Examinations were performed at day 1 of therapy and, in 13 patients, also after 43.5 Gy of irradiation at day 1 of the second chemotherapy cycle. Resonances of 5-FU and the catabolites 5,6-dihydro-5-fluorouracil (DHFU) and alpha-fluoro-beta-alanine (FBAL) were resolved in the tumor spectra. The median of the 5-FU and FBAL levels was significantly higher (more than 2-fold) at the second compared with the first examination, whereas the level of DHFU did not change. This effect could indicate an increased delivery of 5-FU into the interstitial space of the tumor in the course of the combined treatment, which would result in an enhanced exposure of the tumor cells to the drug. A potential mechanism for synergy between radio- and chemotherapy is discussed, but alternative mechanisms are also being considered. The findings indicate that a method is available to rationally address the design of dosing schedules in concurrent therapy regimens. (+info)
Role of tyrosine 265 of alanine racemase from Bacillus stearothermophilus.
Tyrosine 265 (Y265) of Bacillus stearothermophilus is believed to serve as a catalytic base specific to the L-enantiomer of a substrate amino acid by removing (or returning) an alpha-hydrogen from (or to) the isomer on the basis of the X-ray structure of the enzyme [Stamper, C.G., Morollo, A.A., and Ringe, D. (1998) Biochemistry 37, 10438-10443]. We found that the Y265-->Ala mutant (Y265A) enzyme is virtually inactive as a catalyst for alanine racemization. We examined the role of Y265 further with beta-chloroalanine as a substrate with the expectation that the Y265A mutant only catalyzes the alpha,beta-elimination of the D-enantiomer of beta-chloroalanine. However, L-beta-chloroalanine also served as a substrate; this enantiomer was rather better as a substrate than its antipode. Moreover, the mutant enzyme was as equally active as the wild-type enzyme in the elimination reaction. These findings indicate that Y265 is essential for alanine racemization but not for beta-chloroalanine elimination. (+info)
Effect of hypertonic stress on amino acid levels and system A activity in rat peritoneal mesothelial cells.
OBJECTIVE: Peritoneal mesothelial cells (PMC) are exposed to a hypertonic environment during peritoneal dialysis. When exposed to a hypertonic medium, many types of cells accumulate small osmotically active organic solutes, which are called osmolytes, to match the higher external osmolality. However, no information has been available concerning the osmolytes in PMC. To investigate osmoregulation in rat PMC, the levels of amino acids in the cells and the activity of system A, a major neutral amino acid transport, were measured after switching to a medium made hypertonic by the addition of NaCl. System A was measured by Na+-dependent [14C]-2-methylamino-isobutyric acid (MeAIB) uptake. RESULTS: Total amount of 20 amino acids increased from 306 to 757 nmol/mg protein after 12 hours of hypertonicity. The amount of neutral amino acids accounted for 81% of the increase in total amino acids. Glutamine, alanine, glycine, threonine, and serine were the major neutral amino acids that accumulated in the hypertonic mesothelial cells. The amount of neutral amino acids increased 2.9-fold after 12 hr of hypertonicity, and decreased thereafter. MeAIB uptake increased 36-fold relative to the uptake in isotonic cells after 4-8 hr of hypertonicity. When the culture medium was made hypertonic by adding raffinose or glucose, the activity of system A was also stimulated (raffinose > glucose > NaCl). System A was located on both the apical and basal sides of isotonic PMC, and extracellular hypertonicity stimulated the MeAIB uptake on both sides. CONCLUSIONS: These data indicate that neutral amino acids and system A transport play an important role in early-phase osmoregulation in rat peritoneal mesothelial cells. (+info)
Fibrinogen receptor antagonist-induced thrombocytopenia in chimpanzee and rhesus monkey associated with preexisting drug-dependent antibodies to platelet glycoprotein IIb/IIIa.
Most clinical trials with fibrinogen receptor antagonists (FRAs) have been associated with thrombocytopenia. This report describes the occurrence of thrombocytopenia in one chimpanzee and one rhesus monkey upon administration of potent FRAs. Chimpanzee A-264 experienced profound thrombocytopenia on two occasions immediately upon intravenous administration of two different potent FRAs, L-738, 167 and L-739,758. However, an equally efficacious antiaggregatory dose of another potent antagonist, L-734,217, caused no change in platelet count. These compounds did not affect platelet count in five other chimpanzees or numerous other nonhuman primates. Flow cytometric analysis showed drug-dependent antibodies (DDAbs) in the plasma of chimpanzee A-264 that bound to platelets of chimpanzees, humans, and all other primates tested only in the presence of the compounds that induced thrombocytopenia. Rhesus monkey 94-R021 experienced thrombocytopenia upon administration of a different antagonist, L-767,679, and several prodrugs that are converted into the active form, L-767,679, in the blood. More than 20 other FRAs, including those that induced thrombocytopenia in chimpanzee A-264, had no effect on platelet count in this monkey. Flow cytometric measurements again identified DDAbs that reacted with platelets of all primates tested and required the presence of L-767,679. Screening for DDAbs in the plasma of 1,032 human subjects with L-738, 167 and L-739,758 demonstrated that the incidence of these preexisting antibodies in this population was 0.8% +/- 0.6% and 1.1% +/- 0.6%, respectively. (+info)
Investigation of the alpha(1)-glycine receptor channel-opening kinetics in the submillisecond time domain.
The activation and desensitization kinetics of the human alpha(1)-homooligomeric glycine receptor, which was transiently expressed in HEK 293 cells, were studied with a 100-microseconds time resolution to determine the rate and equilibrium constants of individual receptor reaction steps. Concentration jumps of the activating ligands glycine and beta-alanine were initiated by photolysis of caged, inactive precursors and were followed by neurotransmitter binding, receptor-channel opening, and receptor desensitization steps that were separated along the time axis. Analysis of the ligand concentration-dependence of these processes allows the determination of 1) the rate constants of glycine binding, k(+1) approximately 10(7) M(-1) s(-1), and dissociation, k(-1) = 1900 s(-1); 2) the rates of receptor-channel opening, k(op) = 2200 s(-1), and closing, k(cl) = 38 s(-1); 3) the receptor desensitization rate, alpha = 0.45 s(-1); 4) the number of occupied ligand binding sites necessary for receptor-channel activation and desensitization, n >/= 3; and 5) the maximum receptor-channel open probability, p(0) > 0.95. The kinetics of receptor-channel activation are insensitive to the transmembrane potential. A general model for glycine receptor activation explaining the experimental data consists of a sequential mechanism based on rapid ligand-binding steps preceding a rate-limiting receptor-channel opening reaction and slow receptor desensitization. (+info)