Knockout of a putative ergothioneine transporter in Caenorhabditis elegans decreases lifespan and increases susceptibility to oxidative damage.

In addition to excretion of metabolic waste products, organic ionic transporters facilitate uptake of specific compounds of physiological importance. In animals, the organic cation transporter, OCTN1 was found to enable the specific uptake of the unique amino acid, ergothioneine (EGT). EGT can accumulate in the body at up to millimolar concentrations and is believed to function as a physiological antioxidant. However the main function of EGT and the reasons for its active accumulation in the body remain obscure. Through bioinformatic approaches, we identified an analogous EGT transporter in the nematode, Caenorhabditis elegans. The present study investigated and characterized deletion mutants of this gene, OCT-1, in the nematodes. Gene deletion mutations of the OCT-1 transporter were shown to decrease overall lifespan of the worms and increase oxidative damage. However the absence of impaired EGT uptake and the inability of excess EGT to rescue the debilitating phenotype indicate that EGT transport does not explain the deleterious effects of the gene deletion.

Human tumor fluorouracil trapping: clinical correlations of in vivo 19F nuclear magnetic resonance spectroscopy pharmacokinetics.

We previously reported that fluorouracil (5FU) accumulation and metabolism in human livers and tumors can be studied by in vivo nuclear magnetic resonance spectroscopy (NMRS). We have extended these observations by evaluating the pharmacokinetics of 5FU in the tumors of 11 patients with carcinoma of the breast, colon, endometrium, cervix, and kidney, using 19F-NMRS in a 1.5 Magnetom (Siemens Medical Systems, Cerrito, CA) magnetic resonance imaging unit (MRI). These NMRS measurements detected a long-lived tumor pool of 5FU in six of 11 tumors in our patients including carcinomas in the pelvis, breast, lung, and liver. The half-life (T1/2) of this tumor pool of "trapped" 5FU was 0.33 to 1.3 hours (20 to 78 minutes), much longer than the T1/2 of 5FU in blood (5 to 15 minutes). Neither the anabolites of 5FU (fluorinated nucleosides, nucleotides, 5FU-RNA, or 5FU-thymidylate synthase) nor the catabolites (eg, fluorobetaalanine [FBAL]) were detectable by 19F NMRS. Patient response to chemotherapy appeared to correlate with the extent of trapping of free 5FU in the human tumors: in the seven patients receiving 5FU, or 5FU or FUdR plus leucovorin, four of four patients whose tumors trapped 5FU responded to fluorinated pyrimidine chemotherapy, whereas three patients in whom there was a failure to detect tumor trapping were resistant to 5FU. We conclude that NMRS is clinically feasible, and enables investigators to study 5FU pharmacokinetics and metabolism in tumors in vivo. 19F-NMRS of 5FU allows for in vivo evaluation of 5FU metabolic modulation and might be able to guide therapeutic decisions.

Association-dissociation studies of bovine and rat liver glutamic dehydrogenase by high-performance liquid chromatography gel filtration.

The concentration-dependent association-dissociation tendency of purified bovine liver and rat liver glutamic dehydrogenase (GDH) has been demonstrated by high-performance liquid chromatographic gel filtration. In the concentration range of 100 to 1.0 micrograms bovine GDH/ml molecular species ranged from dimer and unimer to subunimeric forms. The dissociation process of the unimeric hexapeptide, consisting of six polypeptide chains, to the subunimeric tripeptide, consisting of three polypeptide chains, was irreversible without added ionic support, but reversible with added ionic support. In dilute Tris-HCl bovine liver GDH was dispersed to subunimeric sizes. Increasing the ionic strength in 20 mM phosphate as the mobile phase increased dissociation to a subunimeric tripeptide while sustaining as much as 80% of its activity. Activity of a eluting subunimer was verified by the inclusion of reaction substrates (NAD and glutamute) in the mobile phase and quantification of reaction products (NADH) in chromatograms. Gel filtration of GDH in the presence of GTP with NADH rendered a subunimeric tripeptide, largely independent of ionic strength or GDH concentration. Rat liver GDH, differing from bovine liver GDH, was dissociated by gel filtration to an active tripeptide independent of ionic or buffer conditions.

Adenine nucleotide metabolism in hearts of diabetic rats. Comparison to diaphragm, liver, and kidney.

High-performance liquid chromatography analysis of acid-extracted tissues revealed decreases of high-energy nucleotides and increases in low-energy nucleotides and metabolites in heart, diaphragm, and liver but not in kidneys of diabetic rats. In comparison with nondiabetic rats, the total adenine nucleotide content of diabetic rat heart and diaphragm but not liver decreased, indicating an increase in catabolism of AMP. Maximal initial rates of the AMP catabolic enzymes 5'-nucleotidase, adenosine deaminase, and AMP deaminase were elevated in the hearts of BB/Wistar and streptozocin-induced diabetic rats. Nucleotide salvage enzymes adenylosuccinate synthetase and adenylosuccinate lyase were elevated above normal in the diabetic heart, whereas hypoxanthine-guanine phosphoribosyl transferase was not altered. Cytosolic-to-mitochondrial ratios from maximal initial rates after correction for mitochondrial breakage were increased above controls in diabetic hearts for nucleoside diphosphokinase and aspartate aminotransferase. Nucleotide levels, degradation rates, and substrate compartmentation between cytosol and mitochondria are discussed in relation to concurrent diabetes.

Myocardial fuel and energy balance, acute ischemia and diabetes.

When glucose-insulin-potassium (GIK) is infused, glucose supplies most of the energy demands of the heart. Fatty acid becomes the major substrate during fasting, pathologically increased work loads or insulin deficiency. Myocardial purine breakdown reflects myocardial energy status and influences coronary tone. Ischemia accelerates breakdown of ATP to AMP, which is further metabolized to adenosine, which causes vasodilatation and a blunted response to catecholamines. If normal circulation is restored, ADP and AMP are rapidly converted to ATP and purine metabolism is changed from degradation to salvage and de novo synthesis of purines. Ischemia impairs mitochondrial function, causing decreased capacity to oxidize fatty acids once aerobic conditions return. Thus, reperfusion with elevated plasma free fatty acids results in acyl-CoA accumulation in the heart. In diabetic animals, phosphorylation of AMP to ATP is defective in the heart, and AMP degradation is increased. Therefore, careful regulation of the blood sugar with concomitant lowering of plasma free fatty acids in diabetics with ischemic heart disease should improve myocardial salvage by preserving and repleting myocardial ATP. Thus, along with reestablishment of coronary flow and reduction in myocardial oxygen demands, may significantly reduce the morbidity of acute ischemia in diabetics.

Substrate specificity of iodothyronine 5'-deiodinase in rat liver homogenates and its requirements of divalent cations in vitro.

Studies were carried out to compare the 5'-deiodination reactions of thyroxine (T4) and 3,3'-5'-triiodothyronine (rT3) in 2.5% rat liver homogenates. The 5'-deiodinase activity was assayed by the 3,5,3'-triiodothyronine (T3) produced from T4 or by 125I-rT3. Under our experimental conditions, the two 5'-monodeiodination reactions resulted in similar apparent KMs: 1.5 microM for T4 and 1.1 microM for rT3. However, the apparent Vmax values of T4 and rT3 deiodination reactions were, respectively, 0.91 and 222 pmol/mg protein/min. Both reactions were stimulated by thiol reagents but only rT3 deiodination showed complete thiol dependence. The inhibitory effect of 6-propyl-2-thiouracil on the 5'-deiodination of rT3 was at least 50 fold greater than that of T4. The divalent ion requirement of the deiodination system was tested with CaCl2, MgCl2, and ZnCl2 at a range of concentrations. Zinc ion appeared to be a potent inhibitor in both T4 and rT3 deiodination systems. Only the 5'-deiodination of rT3 was inhibited slightly by low concentrations of calcium and magnesium ions. Our results suggest that based on their apparently distinct regulation mechanisms, the 5'-monodiodination of T4 and rT3 in rat liver homogenates is likely mediated by more than one enzyme, despite the similarity of observed KMs.

23Na-NMR studies of Na+ interaction with human red cell membranes from normotensives and hypertensives.

Na+ interaction with unsealed human red cell ghosts has been studied by 23Na-NMR relaxation rate (R1) measurements. Data on a total of nine subjects including seven volunteer normotensives (NBP) and two untreated hypertensives (HBP) are presented. Qualitative treatment of the data gives information on the dynamic behavior of Na+ undergoing fast exchange between the free and bound states. The excess longitudinal relaxation rate (delta R)-1 plotted against total [Na+], known as the James-Noggle plot, exhibits different behavior for NBP and HBP ghosts, with a relatively low binding constant of approx. 100 M-1 for HBP (p less than 0.025) compared to a high constant of 500-1000 M-1 for NBP. To associate our NMR data with membrane-bound (Na+ + K+)-ATPase, 23Na relaxation rates were measured in the presence of 5 mM ouabain. James-Noggle plots constructed for ouabain-sensitive excess relaxation rates show the binding for NBP to be even high affinity (greater than 10(3) M-1) but low capacity. These data may suggest that for a given amount of intracellular Na+, the binding affinity could determine the distribution of Na+ between the membrane and cytoplasm, and that the (Na+ + K+)-ATPase which is primarily responsible for the Na+ affinity might assume an abnormal transport mechanism in HBP membranes.

1H-NMR assignments of GM1-oligosaccharide in deuterated water at 500 MHz by two-dimensional spin-echo J-correlated spectroscopy.

The 1H-NMR spectra of the oligosaccharide derived from monosialoganglioside GM1 (GM1 = beta-D-galactosyl-(1-3)-beta-D-N-acetylgalactosaminyl-(1-4)- [alpha-N-acetylneuraminyl-(2-3)-]-beta-D-galactosyl-(1-4)-b eta-D-glucosylceramide) (GM1OS) and its reduced form (GM1OS-R) have been obtained at 500 MHz in D2O. Through the combined use of one-dimensional and homonuclear two-dimensional spin-echo J-correlated (2D SECSY) spectra of GM1OS-R, the assignments for the ring protons of GM1OS are made. Data on chemical shifts and coupling constants of GM1OS including the alpha-linked neuraminic acid protons, in aqueous solution, are tabulated. Due to the very small coupling constants (less than 2 Hz) and the closeness in chemical shifts (less than 0.04 ppm) for the pair of correlated peaks in the two-dimensional spectrum, the information on the connectivities of the H5 ring protons of the neutral sugar residues is missing. Second-order coupling also blurs this information. Data are compared with those obtained for ganglioside GM1 in dimethyl sulfoxide (DMSO; the actual composition therein was 97% DMSO-d6 and 3% D2O) by T. A. W. Koerner, J. H. Prestegard, P. C. Demou, and R. K. Yu (1983, Biochemistry 22, 2676). While the heterogeneity of chemical shifts for the H5, H6a, and H6b protons diminishes in D2O, that for A-9a and A-9b remains. The latter suggests an intraneuraminic acid conformation involving the glycerol side chain unaffected by the solvent. Moreover, the chemical shifts of the III-1, III-2, and A-4 protons (and perhaps the II-4, IV-2, and A-8 protons) in D2O exhibit unusual upfield shifts compared with those in DMSO. This indicates that the intramolecular interactions between GalNAc residue III and neuraminic acid present in DMSO are weakened in D2O. The effect of temperature on the conformation is also examined and appears to be minimal (less than 0.02 ppm) in the range 22-50 degrees C.

Redefined role of radiation in combined treatment of Ewing's sarcoma.

Eighty-six patients with Ewing's sarcoma were analyzed as to the role of radiation therapy. Fifty-eight patients (P group) had removal of the involved part of the bone after preoperative chemotherapy, and 28 (NP group) had local treatment either at the same time or before chemotherapy. Thirty-six of 58 P-group patients (61%) and 13 of the 28 NP-group patients (48%) are alive. Five of 48 patients in P-group, who had postoperative radiation, had local recurrence, as did 6 of 11 without postoperative radiation, a statistically significant difference (p = 0.001).

Proton nuclear magnetic resonance assignments of thyroid hormone and its analogues.

1H NMR data of a series of thyroid hormone analogues, e.g., thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), 3-monoidothyronine (3-T1), 3'-monoiodothyronine (3'-T1), and thyronine (TO) in dimethylsulfoxide (DMSO) have been obtained on a 300 MHz spectrometer. The chemical shift and coupling constant are determined and tabulated for each aromatic proton. The inner tyrosyl ring protons in T4, T3, and 3,5-T2 have downfield chemical shifts with respect to those of the outer phenolic ring protons. Four-bond cross-ring coupling has been observed in all the monoiodinated rings. However, this long-range coupling does not exist in T4, diiodinated on both rings, and T0, containing no iodines on the rings. There is no evidence that at 30 degrees C these iodothyronines have any motional constraint in DMSO solution. In addition to identification of the hormones, the potential use of some characteristic peaks as probes in binding studies is discussed.

31P and 19F NMR studies of glycophorin-reconstituted membranes: preferential interaction of glycophorin with phosphatidylserine.

Glycophorin A, a major glycoprotein of the erythrocyte membrane, has been incorporated into small unilamellar vesicles composed of a variety of pure and mixed phospholipids. Nuclear spin labels including 31P and 19F have been used at natural abundance or have been synthetically incorporated in lipids to act as probes of lipid-protein interaction. Interactions produce broadening of resonances in several cases and it can be used to demonstrate preferential interaction of certain lipids with glycophorin. 31P and 19F probes show a strong preferential interaction of glycophorin with phosphatidylserine over phosphatidylcholine. There is some evidence that interactions are more pronounced at the inner surface of the bilayer and these results are rationalized in terms of the asymmetric distribution of protein and lipid.

Interaction of ganglioside GM1 and myelin basic protein studied by carbon-13 and proton nuclear magnetic resonance spectroscopy.

The interaction of the myelin basic protein (MBP) and the major endogenous ganglioside GM1 in myelin of the central nervous system has been investigated using both 500-MHz 1H and 67.89 MHz 13C NMR. Titration of MBP by GM1 resulted in 13C NMR signal shifts for the I1e and His residues of MBP at a GM1/MBP mole ratio of one or less. The carbohydrate head group of GM1 was also found to be perturbed. 1H NMR results obtained in a similar manner demonstrated the perturbation of His and Phe residues. At a GM1/MBP mole ratio of 0.5, small perturbation of Trp #116 was observed, and at mole ratios of two and beyond significant involvement of Phe residues and methylated Arg #107 was found. Met #167 was more perturbed than Met #20; hence, more extensive interaction of the lipid is occurring with the C-terminus of the protein than with the N-terminus. No resonances from GM1 bound to MBP at mole ratios of up to one appeared in the spectra. However, as the GM1/MBP mole ratio was increased to eight or greater a major conformational change of MBP was detected. An upfield shift of the GM1 midchain methylene resonance was observed for the GM1/MBP complex. This observation provides strong evidence that the state of GM1 interacting with MBP is different from that of GM1 micelles. The number of saturable GM1 binding sites on MBP is estimated to be four. The data also favor a rapid exchange between bound GM1 and GM1 micelles. Interaction of MBP with the oligosaccharide derived from GM1 was found to be weaker than with GM1. Based on our data, a model for the interaction can be proposed: the first GM1 molecule is bound to the protein molecule through its head group and hydrocarbon chains, followed by the formation of a GM1/MBP complex with a concomitant conformational change of MBP as more GM1 is added.

High-resolution 13C nuclear magnetic resonance studies of small unilamellar vesicles containing glycophorin A.

Glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, has been incorporated in small unilamellar vesicles containing phosphatidylcholine and phosphatidylethanolamine in varying proportions. Hydrocarbon chains of these two lipids have been selectively enriched with 13C and 13C-NMR spin relaxation parameters have been monitored in the presence and absence of protein. Perturbations to 13C line-widths and spin-lattice relaxation times are found to be small and consistent with relatively weak interactions. The perturbations, though small, show some specificity. The carbonyl carbons in both phosphatidylcholine and phosphatidylethanolamine are broadened, but in addition the olefinic carbons in phosphatidylethanolamine are broadened.

Small unilamellar vesicles containing glycophorin A. Chemical characterization and proton nuclear magnetic resonance studies.

Glycophorin A, a major glycoprotein of the red blood cell, is reconstituted in small lipid vesicles (250-300 A in diameter) by using cholate detergent solubilization followed by rapid removal of cholate on a molecular sieve column. The extent of glycophorin incorporation is found to be critically dependent on the amount of cholate used, with higher amounts yielding vesicles with higher percentages of glycophorin. Vesicles with as much as 1 molecule of protein per 20 molecules of lipid can be prepared. Data on the vesicles obtained by using hydrolytic enzymes such as neuraminidase and trypsin, combined with amino acid analysis, suggest that glycophorin is incorporated in a transbilayer fashion with a high fraction of the molecules oriented with the carbohydrate-containing amino terminus to the vesicle exterior. Interaction of the protein with the hydrophobic portion of the bilayer is apparent in proton nuclear magnetic resonance spectra, and lipid line-width increases have been used to characterize the strength and stoichiometry of interaction. Glycophorin is found to affect directly as many as 40 lipid molecules per molecule of protein; however, the magnitude of the effects is not large.