Identification of the Human Mitochondrial Oxodicarboxylate Carrier

In Saccharomyces cerevisiae, the genes ODC1 and ODC2 encode isoforms of the oxodicarboxylate carrier. They both transport C5-C7 oxodicarboxylates across the inner membranes of mitochondria and are members of the family of mitochondrial carrier proteins. Orthologs are encoded in the genomes of Caenorhabditis elegans and Drosophila melanogaster, and a human expressed sequence tag (EST) encodes part of a closely related protein. Information from the EST has been used to complete the human cDNA sequence. This sequence has been used to map the gene to chromosome 14q11.2 and to show that the gene is expressed in all tissues that were examined. The human protein was produced by overexpression in Escherichia coli, purified, and reconstituted into phospholipid vesicles. It has similar transport characteristics to the yeast oxodicarboxylate carrier proteins (ODCs). Both the human and yeast ODCs catalyzed the transport of the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a counter-exchange mechanism. Adipate, glutarate, and to a lesser extent, pimelate, 2-oxopimelate, 2-aminoadipate, oxaloacetate, and citrate were also transported by the human ODC. The main differences between the human and yeast ODCs are that 2-aminoadipate is transported by the former but not by the latter, whereas malate is transported by the yeast ODCs but not by the human ortholog. In mammals, 2-oxoadipate is a common intermediate in the catabolism of lysine, tryptophan, and hydroxylysine. It is transported from the cytoplasm into mitochondria where it is converted into acetyl-CoA. Defects in human ODC are likely to be a cause of 2-oxoadipate acidemia, an inborn error of metabolism of lysine, tryptophan, and hydroxylysineAJ278148AJ289714. In Saccharomyces cerevisiae, the genes ODC1 and ODC2 encode isoforms of the oxodicarboxylate carrier. They both transport C5-C7 oxodicarboxylates across the inner membranes of mitochondria and are members of the family of mitochondrial carrier proteins. Orthologs are encoded in the genomes of Caenorhabditis elegans and Drosophila melanogaster, and a human expressed sequence tag (EST) encodes part of a closely related protein. Information from the EST has been used to complete the human cDNA sequence. This sequence has been used to map the gene to chromosome 14q11.2 and to show that the gene is expressed in all tissues that were examined. The human protein was produced by overexpression in Escherichia coli, purified, and reconstituted into phospholipid vesicles. It has similar transport characteristics to the yeast oxodicarboxylate carrier proteins (ODCs). Both the human and yeast ODCs catalyzed the transport of the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a counter-exchange mechanism. Adipate, glutarate, and to a lesser extent, pimelate, 2-oxopimelate, 2-aminoadipate, oxaloacetate, and citrate were also transported by the human ODC. The main differences between the human and yeast ODCs are that 2-aminoadipate is transported by the former but not by the latter, whereas malate is transported by the yeast ODCs but not by the human ortholog. In mammals, 2-oxoadipate is a common intermediate in the catabolism of lysine, tryptophan, and hydroxylysine. It is transported from the cytoplasm into mitochondria where it is converted into acetyl-CoA. Defects in human ODC are likely to be a cause of 2-oxoadipate acidemia, an inborn error of metabolism of lysine, tryptophan, and hydroxylysineAJ278148AJ289714. oxodicarboxylate carrier protein base pair(s) expressed sequence tag fluorescence in situ hybridization kilobase(s) nucleotide polyacrylamide gel electrophoresis reverse transcription polymerase chain reaction In mammals, 2-oxoadipate is produced from lysine in the cytosol of cells via the saccharopine and the pipecolic acid pathways. Catabolites of hydroxylysine and tryptophan enter these pathways as 2-aminoadipic-δ-semialdehyde and 2-oxoadipate, respectively. In the matrix of mitochondria, 2-oxoadipate is decarboxylated to glutaryl-CoA by the 2-oxoadipate dehydrogenase complex and then converted to acetyl-CoA. Over the years, several patients with 2-oxoadipate acidemia, an in-born error of metabolism of lysine, tryptophan, and hydroxylysine, have been reported, most of them slightly to deeply mentally retarded with hypotonia or seizures (1Przyrembel H. Bachmann D. Lombeck I. Becker K. Wendel U. Wadman S.K. Bremer H.J. Clin. Chim. Acta. 1975; 58: 257-269Crossref PubMed Scopus (55) Google Scholar, 2Wendel U. Rudiger H.W. Przyrembel H. Bremer H.J. Clin. Chim. Acta. 1975; 58: 271-276Crossref PubMed Scopus (21) Google Scholar, 3Wilson R.W. Wilson C.M. Gates S.C. Higgins J.V. Pediatr. Res. 1975; 9: 522-526Crossref PubMed Scopus (31) Google Scholar, 4Wilson R.W. Wilson C.M. Higgins J.V. Clin. Chim. Acta. 1976; 69: 323-332Crossref PubMed Scopus (9) Google Scholar, 5Fischer M.H. Brown R.R. Am. J. Med. Genet. 1980; 5: 35-41Crossref PubMed Scopus (21) Google Scholar, 6Duran M. Beemer F.A. Wadman S.K. Wendel U. Janssen B. J. Inherit. Metab. Dis. 1984; 7: 61Crossref PubMed Scopus (19) Google Scholar, 7Vianey-Liaud C. Divry P. Cotte J. Teyssier G. J. Inherit. Metab. Dis. 1985; 8: 133-134Crossref PubMed Scopus (10) Google Scholar). It was speculated without experimental verification that their abnormal levels of serum and urinary 2-oxoadipate might arise from a defect in 2-oxoadipate dehydrogenase. As this enzyme is found in the matrix of mitochondria, and 2-oxoadipate is produced in the cytosol (8Nishizuka Y. Ichiyama A. Hayaishi O. Methods Enzymol. 1970; 17A: 463-491Crossref Scopus (32) Google Scholar), impaired transport of 2-oxoadipate into the organelle provides an alternative explanation, but until the present work no such transport function had been demonstrated in man. In this paper, the identification of the human 2-oxoadipate mitochondrial carrier (ODC)1is described. It is based on two isoforms, ODC1 and ODC2 encoded in the genome of Saccharomyces cerevisiae, that transport the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate across the inner membranes of mitochondria (9Palmieri L. Agrimi G. Runswick M.J. Fearnley I.M. Palmieri F. Walker J.E. J. Biol. Chem. 2001; 276: 1916-1922Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). However, yeast and man are too distant phylogenetically for these isoforms to provide a basis for direct cloning of the mammalian counterpart. Therefore, orthologs were sought and detected in Caenorhabditis elegans and Drosophila melanogaster and used to bridge between yeast and man. In this way, a human EST was identified that encodes a fragment of a related protein. It provided information to complete the human cDNA sequence. The encoded protein is 299 amino acids long and has the characteristic features of the family of mitochondrial carrier proteins (10Krämer R. Palmieri F. Ernster L. Molecular Mechanisms in Bioenergetics. Elsevier Science Publishers B. V., Amsterdam1992: 359-382Google Scholar, 11Walker J.E. Curr. Opin. Struct. Biol. 1992; 2: 519-526Crossref Scopus (105) Google Scholar, 12Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 13Palmieri F. van Ommen B. Papa S. Guerrieri F. Tager J.M. Frontiers of Cellular Bioenergetics. Kluwer Academic/Plenum Publishers, New York1999: 489-519Crossref Google Scholar). It was overexpressed in Escherichia coli, reconstituted into phospholipid vesicles, and shown to have transport specificity and other biochemical properties similar to those of the recombinant yeast ODC isoforms, including transport of 2-oxoadipate. Data bases of the genomes ofC. elegans and D. melanogaster at the Sanger Center (Hinxton, United Kingdom) and at the National Center for Biotechnology Information (Washington, D. C.) were screened with the sequences of the S. cerevisiae ODC proteins (9Palmieri L. Agrimi G. Runswick M.J. Fearnley I.M. Palmieri F. Walker J.E. J. Biol. Chem. 2001; 276: 1916-1922Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) using the program BLASTP. The NCBI nonredundant EST human data base was probed with the program TBLASTN. Amino acid sequences were aligned with ClustalW (version 1.7). Touchdown PCRs (14Don R.H. Cox P.T. Wainwright B.J. Baker K. Mattick J.S. Nucleic Acids Res. 1991; 19: 4008Crossref PubMed Scopus (2240) Google Scholar) were performed with adaptor-ligated double-stranded human liver cDNA (1 ng, CLONTECH), as described previously (15Indiveri C. Iacobazzi V. Giangregorio N. Palmieri F. Biochem. J. 1997; 321: 713-719Crossref PubMed Scopus (127) Google Scholar, 16Fiermonte G. Palmieri L. Dolce V. Lasorsa F.M. Palmieri F. Runswick M.J. Walker J.E. J. Biol. Chem. 1998; 273: 24754-24759Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The full-length cDNA sequence was obtained in two PCR reactions of 5′- and 3′-extension using the adaptor primers AP1 and AP2 (CLONTECH) and a primer set 1F, 2F, 1R, and 2R (Fig. 1) designed from the nucleotide sequence of the human EST, R29313. The PCR products were identified, recovered from agarose gels, cloned into the pCR2.1 topo-vector (Invitrogen), and sequenced. The cDNA for rat ODC was amplified similarly with oligonucleotides based on the human cDNA sequence. Human metaphase chromosome spreads were obtained from phytohemagglutinin-stimulated peripheral lymphocytes. A probe for FISH analysis was made by PCR amplification of human genomic DNA using primers corresponding to nt 916–939 (forward sense) and nt 1191–1212 (reverse sense) of the human cDNA for ODC (see Fig. 1). The resulting 4.2-kb fragment was cloned in the pCR2.1 topo-vector and sequenced to confirm its identity. The FISH experiments were performed as described previously (17Pannone E. Fiermonte G. Dolce V. Rocchi M. Palmieri F. Cytogenet. Cell Genet. 1998; 83: 238-239Crossref PubMed Google Scholar). Total RNAs (2 μg) were extracted and reverse-transcribed with the Gene Amp RNA PCR Core kit (PerkinElmer Life Sciences) using either random hexamers or oligo(dT)16 as primers (final volume, 40 μl). A 230-bp fragment of the ODC cDNA was then amplified from the reverse transcription reaction products (20 μl) by 35 cycles of PCR using oligonucleotides RT1F (nt 662–682) and RT1R (nt 869–892) as forward and reverse primers, respectively (Fig. 1). The products were probed with the radiolabeled oligonucleotide RT1P (nt 804–825; Fig. 1). As a control, a 384-bp β-actin fragment was amplified from the remainder of the reverse transcription products (18Dolce, V., Fiermonte, G., Runswick, M. J., Palmieri, F., and Walker, J. E. (2001) Proc. Natl. Acad. Sci. U. S. A., 98, in press.Google Scholar). The coding regions for the human and rat ODCs were amplified from human and rat liver cDNAs (1 ng) by 35 cycles of PCR. The forward and reverse primers in these reactions corresponded to nt 311–331 and 1188–1210 of the human ODC cDNA (see Fig. 1) and to nt 100–120 and 976–996 of the rat ODC cDNA (deposited as GenBankTMaccession number AJ289714). The forward and reverse primers carried anNdeI and a XhoI site, respectively, at their 5′-ends. The 0.9-kb products were gel-purified and cloned into the pRUN expression vector. Transformants of E. coli DH5α cells were selected on ampicillin (100 μg/ml) and screened by direct colony PCR and restriction digestion of plasmids. The sequences of inserts were verified. The overproduction of the ODC as inclusion bodies in the cytosol of E. coli was accomplished as described first for the bovine oxoglutarate-malate carrier (19Fiermonte G. Walker J.E. Palmieri F. Biochem. J. 1993; 294: 293-299Crossref PubMed Scopus (182) Google Scholar), except that the host cells were E. coli CO214(DE3) (20Palmieri L. De Marco V. Iacobazzi V. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1997; 410: 447-451Crossref PubMed Scopus (80) Google Scholar). Control cultures with the empty vector were processed in parallel. Inclusion bodies were isolated, and ODC was purified by centrifugation and washing steps as described previously (19Fiermonte G. Walker J.E. Palmieri F. Biochem. J. 1993; 294: 293-299Crossref PubMed Scopus (182) Google Scholar, 21Palmieri L. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1996; 399: 299-302Crossref PubMed Scopus (105) Google Scholar). The recombinant protein in Sarkosyl was reconstituted into liposomes in the presence of substrates, as described previously (9Palmieri L. Agrimi G. Runswick M.J. Fearnley I.M. Palmieri F. Walker J.E. J. Biol. Chem. 2001; 276: 1916-1922Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). External substrate was removed from proteoliposomes on a Sephadex G-75 columns. The transport activity at 25 °C was determined by measuring the uptake (forward exchange) or the efflux (backward exchange) of [14C]oxoglutarate in exchange for unlabeled counter-substrates (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (228) Google Scholar). For backward exchange measurements the proteoliposomes containing 1 mm internal oxoglutarate were prelabeled, after reconstitution, by carrier-mediated exchange equilibration by adding 10 μm[14C]oxoglutarate (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (228) Google Scholar). After 30 min, the residual external radioactivity was removed by passing the proteoliposomes through a column of Sephadex G-75. In forward exchange reactions, transport was started by adding [14C]oxoglutarate to the proteoliposomes and in the backward exchanges by adding nonradioactive substrate. In both cases, transport was stopped after 1 min (in the initial linear range of substrate exchange) by addition of 10 mm pyridoxal 5′-phosphate and 10 mmbathophenanthroline (the “inibitor-stop” method (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (228) Google Scholar)). In controls, the inhibitors were added at the beginning together with the external substrate. Finally, the external substrate was removed, and the radioactivity in the liposomes was measured (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (228) Google Scholar). In forward exchange measurements, the experimental values were corrected by subtracting control values, and the rate was calculated in millimoles/min/gram of protein. In the case of backward exchanges, the rate in Δcpm/min was obtained from the decrease of internal radioactivity in 1 min. The reconstituted protein was for other exchange by the method (22Palmieri F. Indiveri C. Bisaccia F. Iacobazzi V. Methods Enzymol. 1995; 260: 349-369Crossref PubMed Scopus (228) Google Scholar). were by and with was carried as described previously L. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1996; 399: 299-302Crossref PubMed Scopus (105) Google Scholar). The of ODC was by of L. Palmieri F. Runswick M.J. Walker J.E. FEBS Lett. 1996; 399: 299-302Crossref PubMed Scopus (105) Google Scholar). The of ODC into liposomes was measured as described previously G. Dolce V. Palmieri F. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) and between and of the protein added to the was carried as described previously F. L. G. Palmieri F. 1996; Scopus Google Scholar) with a the expressed rat ODC protein. data and and were found with and of the of their encoded proteins respectively, to the yeast ODC protein A human EST data base was with these and a of was identified that encoded a protein sequence that was and respectively, to regions of the C. elegans and the D. melanogaster proteins. The human EST was in the 5′- and by two PCR the products of the of with primer sequence the The 3′-extension a of sequence the The human cDNA sequence of (Fig. 1) of a by an of a containing a at nt 1976; PubMed Scopus Google Scholar), and a (see Fig. 1). The at nt is by an and is likely to be the The of the human protein is at the as in the and D. melanogaster this The encoded a of 299 amino acids with a calculated of and a of The cDNA for the rat ODC was cloned in a similar This sequence number of with a by an of and a containing a a The encoded a of amino acids with a of The human ODC is and to the R. C. melanogaster, and S. cerevisiae ODC1 and ODC2 respectively. The gene for human ODC was found on chromosome at 14q11.2 by FISH experiments on human metaphase was no in this for a gene an and no isoforms were detected in of data bases of and The of for the human and rat ODCs was by performed on RNA using primers and from regions of between the human and the rat nucleotide The ODC was detected in all tissues that were The from RNA was in other as the control in Fig. A similar of expression was by analysis of rat mitochondria (see Fig. The human ODC was overexpressed in E. coli (see in the of inclusion The purified protein a by (Fig. with an of The protein was not detected in of expression (Fig. 1 in cells after but the coding sequence in the expression vector The sequence of of the purified protein was to that for of the human ODC (Fig. 1). 35 of purified protein were obtained of reconstituted with recombinant ODC catalyzed a counter-exchange of external [14C]oxoglutarate for internal oxoglutarate with first not The exchange reaction was by a of pyridoxal 5′-phosphate and In the of substrate in the or the protein was the into was no uptake of external substrate. no exchange was detected by of from cells either the expression vector for ODC or of the proteoliposomes not for and 10 external 1 The substrate specificity of human ODC was in by measuring the uptake of [14C]oxoglutarate into proteoliposomes that had been with a of (Fig. or the efflux of [14C]oxoglutarate from proteoliposomes in the presence of external nonradioactive (Fig. [14C]oxoglutarate transport were glutarate, and were used as counter-substrates on both of the were also found with external and a lesser extent, internal pimelate, and as as both internal and external 2-aminoadipate, and oxaloacetate, also for was found with and (Fig. A and The uptake of mm [14C]oxoglutarate by proteoliposomes containing mm oxoglutarate 1 was by 5′-phosphate and by mmbathophenanthroline and mm and of also the ODC activity and was with mm of other mitochondrial and 10 of the information the the of the exchange rate on substrate was at of added [14C]oxoglutarate at a internal of The transport and the activity values for oxoglutarate exchange at 25 calculated from a set of 35 were mm and of respectively. The activity was calculated by into the of ODC recovered in the proteoliposomes after The of several added are in of them the without the exchange as In the exchange was by external addition of of the that are transported by human ODC (Fig. and and it was not by of other mitochondrial such as and However, is not transported by the ODC the exchange activity that it to the substrate of ODC without values for with [14C]oxoglutarate values were calculated from of the rate of exchange of external were as mm internal 2-oxoglutarate and mm external The were added with [14C]oxoglutarate at the The values are of at in a The values were calculated from of the rate of exchange of external were as mm internal 2-oxoglutarate and mm external The were added with [14C]oxoglutarate at the The values are of at The between S. cerevisiae and man the of yeast sequences to human orthologs with The is the protein in is a of a family with but related as are the two isoforms of the yeast oxodicarboxylate carrier (9Palmieri L. Agrimi G. Runswick M.J. Fearnley I.M. Palmieri F. Walker J.E. J. Biol. Chem. 2001; 276: 1916-1922Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), is a identified of the family of mitochondrial transport proteins. family members are in the of and across the inner membranes of the in the cloning the rat carrier G. Palmieri L. Dolce V. Lasorsa F.M. Palmieri F. Runswick M.J. Walker J.E. J. Biol. Chem. 1998; 273: 24754-24759Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), is to the yeast sequence to orthologs in phylogenetically intermediate where the genome sequence is such as C. elegans and D. melanogaster, and then to these orthologs to orthologs in man. the yeast ODC isoforms were used to in the C. elegans and D. melanogaster and were used to a related protein sequence in a human This sequence to be part of the human ODC and provided the to the complete human and rat ODC Both have the and the sequence that are characteristic of the mitochondrial carrier family (10Krämer R. Palmieri F. Ernster L. Molecular Mechanisms in Bioenergetics. Elsevier Science Publishers B. V., Amsterdam1992: 359-382Google Scholar, 11Walker J.E. Curr. Opin. Struct. Biol. 1992; 2: 519-526Crossref Scopus (105) Google Scholar, 12Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 13Palmieri F. van Ommen B. Papa S. Guerrieri F. Tager J.M. Frontiers of Cellular Bioenergetics. Kluwer Academic/Plenum Publishers, New York1999: 489-519Crossref Google Scholar). The transport characteristics of the human ODC are similar to the yeast ODC The main differences are the of the human protein to transport 2-aminoadipate, and its to transport The properties of the human ODC from those of the oxoglutarate-malate has for and oxodicarboxylates and (19Fiermonte G. Walker J.E. Palmieri F. Biochem. J. 1993; 294: 293-299Crossref PubMed Scopus (182) Google Scholar, F. E. M. J. Biochem. PubMed Scopus Google Scholar, F. Indiveri C. Palmieri F. Acta. 1985; PubMed Scopus Google Scholar), whereas the human ODC the C5-C7 (Fig. and In to the bovine oxoglutarate-malate the human ODC not transport and but it transport at The sequence between the ODC and oxoglutarate-malate carrier is also with their properties and The for the human both on the external and the internal are 2-oxoadipate and Therefore, the of the human ODC is most likely to be to the uptake of 2-oxoadipate into the mitochondrial matrix in exchange for internal a in catabolism of lysine, hydroxylysine, and ODC by a counter-exchange the carrier-mediated uptake of 2-oxoadipate the efflux of a the basis of transport measurements, 2-oxoglutarate as the of ODC for 2-oxoadipate. The efflux of 2-oxoglutarate is by the in the first of lysine lysine and 2-oxoglutarate into In with its in the human ODC is expressed in all tissues that were for the human ODC be to the uptake of 2-aminoadipate into the mitochondrial matrix this amino acid is not to 2-oxoadipate in the for after in amino acids that cause a decrease in 2-oxoglutarate in the cytosol and an of the 2-aminoadipate In this it is that and are present both in the cytosol and the mitochondria M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, F. H. Y. Acta. PubMed Scopus Google Scholar). The of ODC also its in 2-oxoadipate acidemia, is by and of of 2-oxoadipate, 2-aminoadipate and in and by the of and and (1Przyrembel H. Bachmann D. Lombeck I. Becker K. Wendel U. Wadman S.K. Bremer H.J. Clin. Chim. Acta. 1975; 58: 257-269Crossref PubMed Scopus (55) Google Scholar, 2Wendel U. Rudiger H.W. Przyrembel H. Bremer H.J. Clin. Chim. Acta. 1975; 58: 271-276Crossref PubMed Scopus (21) Google Scholar, 3Wilson R.W. Wilson C.M. Gates S.C. Higgins J.V. Pediatr. Res. 1975; 9: 522-526Crossref PubMed Scopus (31) Google Scholar, 4Wilson R.W. Wilson C.M. Higgins J.V. Clin. Chim. Acta. 1976; 69: 323-332Crossref PubMed Scopus (9) Google Scholar, 5Fischer M.H. Brown R.R. Am. J. Med. Genet. 1980; 5: 35-41Crossref PubMed Scopus (21) Google Scholar, 6Duran M. Beemer F.A. Wadman S.K. Wendel U. Janssen B. J. Inherit. Metab. Dis. 1984; 7: 61Crossref PubMed Scopus (19) Google Scholar, 7Vianey-Liaud C. Divry P. Cotte J. Teyssier G. J. Inherit. Metab. Dis. 1985; 8: 133-134Crossref PubMed Scopus (10) Google Scholar). The for this have not been of patients with this are to and acid to U. Rudiger H.W. Przyrembel H. Bremer H.J. Clin. Chim. Acta. 1975; 58: 271-276Crossref PubMed Scopus (21) Google Scholar, 6Duran M. Beemer F.A. Wadman S.K. Wendel U. Janssen B. J. Inherit. Metab. Dis. 1984; 7: 61Crossref PubMed Scopus (19) Google Scholar) to Therefore, it was that the be to 2-oxoadipate but no such defect has been The alternative that ODC might provide the basis for this human be