ABSTRACT
The candidate tumor suppressor gene, FHIT, encompasses the common human chromosomal fragile site at 3p14.2, the hereditary renal cancer translocation breakpoint, and cancer cell homozygous deletions. Fhit hydrolyzes dinucleotide 5',5"'-P1,P3-triphosphate in vitro and mutation of a central histidine abolishes hydrolase activity. To study Fhit function, wild-type and mutant FHIT genes were transfected into cancer cell lines that lacked endogenous Fhit. No consistent effect of exogenous Fhit on growth in culture was observed, but Fhit and hydrolase "dead" Fhit mutant proteins suppressed tumorigenicity in nude mice, indicating that 5',5"'-P1, P3-triphosphate hydrolysis is not required for tumor suppression.
Subject(s)
Acid Anhydride Hydrolases , Genes, Tumor Suppressor , Neoplasm Proteins , Proteins/genetics , Proteins/metabolism , Animals , Cell Division/genetics , Cell Division/physiology , Chromosome Fragile Sites , Chromosome Fragility , Chromosomes, Human, Pair 3/genetics , Dinucleoside Phosphates/metabolism , Humans , Mice , Mice, Nude , Mutation , Neoplasm Transplantation , Phenotype , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Transfection , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
The binding of ethanol to rat liver microsomes is shown to be saturable at clinically relevant ethanol concentrations, whereas this effect is not observed in extracted microsomal phospholipids. Brief exposure of the microsomes to heat abolishes saturable ethanol binding. Equilibrium binding data analysis, although only approximate in this context, suggests the presence of at least two groups of specific sites: high capacity sites with affinities near the pharmacological range and low capacity sites at lesser levels. The results indicate that the specificity of ethanol for tissue is considerably greater than previously recognized.
Subject(s)
Ethanol/metabolism , Microsomes, Liver/metabolism , Animals , Binding Sites , Dose-Response Relationship, Drug , Liposomes/metabolism , Membranes/metabolism , Phospholipids/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Implicit within the concept of membrane-buffer partition coefficients of solutes is a nonspecific solvation mechanism of solute binding. However, (2)H NMR studies of the binding of (2)H(6)-ethanol and [1-(2)H(2)] n-hexanol to phosphatidylcholine vesicles have been interpreted as evidence for two distinct alcohol binding modes. One binding mode was reported to be at the membrane surface. The second mode was reported to be within the bilayer interior. An examination of the (2)H NMR binding studies, together with direct radiolabel binding assays, shows that other interpretations of the data are more plausible. The results are entirely consistent with partitioning (nonspecific binding) as the sole mode of alcohol binding to liposomes, in accord with our previous thermodynamic interpretation of alcohol action in phosphatidylcholine liposomes.
Subject(s)
Ethanol/chemistry , Hexanols/chemistry , Liposomes/chemistry , Phosphatidylcholines/chemistry , Deuterium , Lipid Bilayers/chemistry , Magnetic Resonance Spectroscopy/methods , Models, Biological , Molecular Conformation , Radioisotope Dilution TechniqueABSTRACT
Different phospholipids, except the choline-containing phospholipids phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, formed complexes with the dye Victoria blue R, which selectively partitioned into the chloroform phase of chloroform/ethylene glycol/glycerol biphasic solvent system, and were quantitatively estimated at 590 nm. Considerable amounts of water, alcohols, nonlipid phosphates, neutral lipids, free fatty acids, and some detergents did not interfere with the formation of phospholipid-dye complexes. This special advantage of the method described allowed combined phospholipid extraction and estimation procedures in one test tube. Because of its high sensitivity (about 24.00 OD units/mumol of phosphatidic acid and about 10.25 OD units/mumol of other phospholipids), specificity, and simplicity, the proposed phospholipid assay appears to be very useful for rapid analyses of lipid extracts as well as TLC spots or suspensions of biological materials, as demonstrated for membranes and cells of Micrococcus lysodeikticus. The applicability of the dye Victoria blue B to the quantitative determination of phospholipids, except phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, at 605 nm using chloroform/ethylene glycol/glycerol/water and pentane (hexane)/ethyl acetate/isopropanol/water biphasic solvent systems with similar sensitivities and of sodium dodecyl sulfate in the pentane-containing system with high sensitivity (22.96 OD units/mumol) is also shown. The adaptation of this phospholipid assay to the determination of phospholipases C and D and to the differential quantitation of choline-containing phospholipids using additional phospholipid estimation techniques is discussed.
