ABSTRACT
Six genes in the 1.5 Mb region of chromosome 22 deleted in DiGeorge/22q11 deletion syndrome-Mrpl40, Prodh, Slc25a1, Txnrd2, T10, and Zdhhc8-encode mitochondrial proteins. All six genes are expressed in the brain, and maximal expression coincides with peak forebrain synaptogenesis shortly after birth. Furthermore, their protein products are associated with brain mitochondria, including those in synaptic terminals. Among the six, only Zddhc8 influences mitochondria-regulated apoptosis when overexpressed, and appears to interact biochemically with established mitochondrial proteins. Zdhhc8 has an apparent interaction with Uqcrc1, a component of mitochondrial complex III. The two proteins are coincidently expressed in pre-synaptic processes; however, Zdhhc8 is more frequently seen in glutamatergic terminals. 22q11 deletion may alter metabolic properties of cortical mitochondria during early post-natal life, since expression complex III components, including Uqcrc1, is significantly increased at birth in a mouse model of 22q11 deletion, and declines to normal values in adulthood. Our results suggest that altered dosage of one, or several 22q11 mitochondrial genes, particularly during early post-natal cortical development, may disrupt neuronal metabolism or synaptic signaling.
Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 22/genetics , DiGeorge Syndrome/genetics , Mitochondria/genetics , Mitochondrial Proteins/metabolism , Acyltransferases/genetics , Acyltransferases/metabolism , Animals , Brain/cytology , Brain/metabolism , Cells, Cultured , Computational Biology , Fibroblasts/cytology , Fibroblasts/physiology , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Synapses/metabolism , Two-Hybrid System TechniquesABSTRACT
Thymidylate synthase catalyzes the reductive methylation of dUMP to dTMP and is essential for the synthesis of DNA. Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy. In the cell, 5-FU is metabolized to 5-fluoro-2'-deoxyuridine 5'-monophosphate, a tight binding covalent inhibitor of thymidylate synthase. Recent studies have identified 5-fluoro-2'-deoxyuridine (5-FdUR) and antifolate-resistant mutants of human thymidylate synthase (TS) that contain single residue substitutions within the highly conserved Arg50-loop, which binds the pyrimidine substrate (Y. Tong et al., J. Biol. Chem. 273: 11611-11618, 1998). We have used random sequence mutagenesis to gain structure-function information about the TS and to create novel drug-resistant mutants for gene therapy. A library of 1.5 million mutants of the Arg50-loop and the nearby residue Tyr 33 was selected to identify mutants of the human enzyme with the ability to complement a thymidylate synthase-deficient Escherichia coli strain and form colonies in the presence of 5-FdUR. E. coli-harboring plasmids that were encoding TS with single, double, and triple amino acid substitutions were identified that survive at dosages of 5-FdUR clearly lethal to E. coli harboring either wild-type thymidylate synthase or constructs encoding previously characterized drug resistant mutants. Four 5-FdUR-resistant mutants were purified to apparent homogeneity. Kinetic studies indicate that these enzymes are highly efficient. Inhibition constants (Ki) for the double mutant K47Q;D48E and the triple mutant D48E;T51S;G52C in the presence of 5-fluoro-2'-deoxyuridine 5'-monophosphate were determined to be 75 to 100 times higher, respectively, than that of the wild-type enzyme. These mutant TSs, or others similarly created and selected, could be used to protect bone marrow cells from the cytotoxic side effects of 5-FU chemotherapy.
Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Arginine/genetics , Drug Resistance , Floxuridine/pharmacology , Mutation , Thymidylate Synthase/genetics , Cloning, Molecular , Dose-Response Relationship, Drug , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/growth & development , Gene Library , Genetic Complementation Test , Humans , Kinetics , Mutagenesis , Thymidylate Synthase/chemistry , Thymidylate Synthase/metabolismABSTRACT
Three cases of intracerebral venous angioma, a rare vascular malformation, were studied by radionuclide brain scan, transmission computed tomography (TCT) and angiography. In each case, the radionuclide flow study demonstrated a typical area of abnormal increase in activity during the venous phase; in two of the cases the arterial phase was also abnormal. Each contrast angiogram demonstrated a normal arterial distribution and a characteristic network of abnormal veins that converged to a large transcerebral draining vein. The TCT scans showed enhancing, curvilinear densities; while not specific, this finding should suggest the possibility of venous angioma in the brain.
