Mapping, cloning and genetic characterization of the region containing the Wilson disease gene.

Wilson disease (WD) is an autosomal recessive disorder of copper transport which map to chromosome 13q14.3. In pursuit of the WD gene, we developed yeast artificial chromosome and cosmid contigs, and microsatellite markers which span the WD gene region. Linkage disequilibrium and haplotype analysis of 115 WD families confined the disease locus to a single marker interval. A candidate cDNA clone was mapped to this interval which, as shown in the accompanying paper, is very likely the WD gene. Our haplotype and mutation analyses predict that approximately half of all WD mutations will be rare in the American and Russian populations.

Hormonal and dietary regulation of hepatic enzymes in tumor-bearing rats.

Enzymes in the histologically normal liver of hosts of mammary carcinomas were examined for their responsiveness to endocrine and dietary modulations. Treatments with the developmental stimuli of alanine aminotransferase (glucocorticoids) and of pyruvate kinase (thyroid hormone) which had no effect in control adult rats raised the levels of these enzymes in the tumor-bearing rats. The latter also showed a greater percentage of increase in malic enzyme upon thyroid hormone administration than did control animals. The tumor-induced increase in hexokinase remained unaltered by the various dietary treatments; enzymes at subnormal levels were raised (glucokinase, malic enzymes, and pyruvate kinase) or further decreased (alanine aminotransferase and ornithine aminotransferase) by excessive carbohydrate intake in immature and adult experimental rats. The normal upsurge of glucokinase and malic enzyme upon weaning to the standard solid diet (from the relatively low-carbohydrate-containing milk) was prevented by cancerous growth in the organism. Similarly, the standard diet, which reversed within 2 days the partial loss of these enzymes in normal adult rats fasted for 48 hr, had no restorative effect on the essentially complete loss of the glucokinase and the very low malic enzyme activity in the fasted tumor bearers. The results suggest that failure in the dietary adaptations of hepatic enzymes as well as diminutions of their basal levels contributes to the clinically observed abnormalities in the glucose metabolism of cancer subjects.

Enzyme activities and isozyme patterns in human lung tumors.

The isozyme patterns and activities of six enzymes were determined in surgical biopsy samples of lung tumors and non-neoplastic pulmonary areas. Fetal lungs were also examined. No tissue differences were found in the isozyme pattern of hexokinase or alkaline phosphatase; small differences in pyruvate kinase isozyme proportions were observed. The tumors exhibited significant deviations with respect to the lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) isozyme patterns. Despite the diversity of cell types, the proportions of the M-subunit of LDH in each tumor and that of the mitochondrial isozyme of MDH in all but one tumor were higher than in control samples from the same lung. In contrast, the normal fetal lung showed a higher LDH-H proportion than did adult lung and a mature MDH isozyme pattern. The alpha-glycerophosphate dehydrogenase and adenylate kinase activities of the tumors were about one-tenth and one-fourth, respectively, of those of nonneoplastic adult lung. These lower activities (evident also in normal fetal lung) were accompanied by 3- to 5-fold increases in the LDH, MDH, pyruvate kinase, and hexokinase activities of the tumors; fetal lungs had lesser increases (2- to 3-fold) for the first 3 enzymes. The common features of tumors with different cell types and histological grade identified here point to several enzymes the quantitation or isozyme analysis of which may be of practical use in distinguishing cancerous from nonneoplastic human lung samples. A combination of different indicators, such as opposite changes in LDH and alpha-glycerophosphate dehydrogenase activity, coupled with elevated proportions of LDH-M, may be used to diagnose neoplasia most reliably.

A single gene for human TRAF-3 at chromosome 14q32.3 encodes a variety of mRNA species by alternative polyadenylation, mRNA splicing and transcription initiation.

Human TRAF-3 is a signaling molecule that interacts with the cytoplasmic tails of CD40 and other TNF-receptor family members. TRAF-3 mRNA is expressed as two major classes of approximately 2 and 8 kb and a number of TRAF-3 encoding cDNA clones differ in discrete gene segments. Because this variety of mRNA species could result from mRNA processing events and/or multiple genes, the structure and localization of TRAF-3 encoding gene elements were determined. FISH and radiation hybrid mapping demonstrated that TRAF-3 is located at chromosome 14q32.3, approximately 1 Mb centromeric to the Ig heavy chain gene complex. Physical mapping of four overlapping genomic PAC clones established that TRAF-3 transcripts are encoded by a single gene, comprised of 13 exons and spanning 130 kb. Alternative polyadenylation in the mRNA segment encoded by exon 12 accounts for the difference between the 2 kb and the 8 kb classes of transcripts. Alternative mRNA splicing in the coding region (encoded by exons 3-12) generates transcripts which delete exons 8 (75 nt), 7+8 (156 nt) or 8+9 (168 nt) and that encode distinct protein isoforms (delta25, delta52 and delta56 aa, respectively). Alternative splicing of exon 2 (139 nt) and alternative transcriptional initiation result in mRNA species with distinct 5'UTRs. Together, these data indicate that a single TRAF-3 gene encodes a variety of mRNA species by a combination of alternative polyadenylation, alternative mRNA splicing and/or alternative initiation.

Effect of tumors with different growth rates on enzymes in host liver.

In response to extrahepatic neoplasms, ornithine aminotransferase, malic enzyme, alanine aminotransferase and glucokinase activity of the 'uninvolved' liver is diminished and that of hexokinase is increased. Comparison of rats at various times after the implantation of ascites tumor, mammary carcinoma, fibrosarcoma and Morris hepatomas indicate that the faster the growth rate of tumors, the earlier the onset of these hepatic changes. The results also show that, when the different tumors are the same size, the magnitude of the enzymic deviations in the liver is directly related to characteristic growth rate of the tumor lines. These and previous observations on other host tissues suggest that tumor-doubling time, which is a known factor in metastatic spread and survival, may also be a variable in the production of systemic agents through which neoplasms affect the metabolic state of the cancer host.

Phosphotransferases and lysosomal enzymes in fetal human and rat lung.

The present investigations on rat lung show that metabolic changes occurring around the 20th gestational day are accompanied by multiple alterations in the quantitative pattern of enzymes. This involves increases in two lysosomal enzymes (N-acetyl beta-glucosaminidase and beta-galactosidase) and a rise and fall in pyruvate kinase and alpha-glucosidase. The striking transient upsurge of adenylate kinase, however, is postponed until after birth. The normal diminution of thymidine kinase and peptidylproline hydroxylase is drastically enhanced by an injection of cortisol to fetal rats. Studies on human pulmonary tissues consisted in determining enzyme concentration from the ninth to the 21st week of gestation and an histologically normal adult lungs. The results show that the 15th to the 21st week of gestation is the period of increase in pyruvate kinase, adenylate kinase and alpha-glucosidase. The rise during the development of several enzymes (e.g., 5'-nucleotidase, alkaline phosphatase, and gamma-glutamyl transpeptidase) and the decline in thymidine kinase and peptidylproline hydroxylase, however, dose not begin until after the 21st week of gestation.

Adenylate kinase isoenzyme patterns in normal and neoplastic human lung, and in various adult as compared to fetal rat tissues.

The isozyme pattern and total activity of adenylate kinase were studied in normal adult and fetal human and rat tissues using starch gel electrophoresis. Three adenylate kinase isoenzymes were identified in human tissues. Although normal adult lung exhibited higher adenylate kinase activity than did its fetal or neoplastic variant, isozyme patterns in the three types of tissues were indistinguishable from each other and from that in fetal human liver. The pattern of these three isozymes in rat lung (as in spleen) also did not change between fetal and adult life. However, adult kidney and heart of this species did appear to contain isozymes not present in fetal life. Brain (both adult and fetal) was striking different from all the other tissues in that it contained only one adenylate kinase isozyme. The total adenylate kinase activity per gram of adult rat liver, kidney and lung was significantly higher than in the cognate fetal organs, whereas that in brain or spleen did not change with age. The activity in adult heart (similar to the fetal one) was higher than in any other tissue examined.

Evidence for differences in the steroid binding domains of the glucocorticoid receptor versus the idiotype antibody.

Triamcinolone acetonide (TA), coupled to bovine serum albumin, was used to obtain a polyclonal anti-TA antibody in the rabbit. This idiotype differed from rat glucocorticoid receptor and transcortin in several respects. RU 38486, a synthetic antagonist with high affinity for the receptor, could neither bind the anti-TA antibody nor displace the idiotype bound 3H-TA. Similarly, corticosterone, the natural rodent ligand, had no affinity for the idiotype. These results imply differences in the conformation and topology of the corticoid binding domains, contrary to the current notion where all agonists and antagonists would saturate an identical configuration.

Cell-free transcription and translation of Na,K-ATPase alpha and beta subunit cDNAs.

Synthetic mRNAs (i.e. cRNA alpha and cRNA beta) were obtained by cell-free transcription of M13 KS(+) (Bluescript) expression vectors which contained the entire coding region of the alpha or beta subunits of lamb kidney Na,K-ATPase. Translation in reticulocyte lysates of cRNA alpha yielded full length alpha polypeptide, as well as a limited array of immunoprecipitable lower molecular weight products. cRNA beta yielded a single immunoprecipitable full length polypeptide. Association of the alpha polypeptide with the microsomal membranes was obtained only co-translationally. Fifteen to 50% of the membrane-associated alpha subunit was resistant to extraction with alkali. The resistance of a 29-kDa fragment to trypsinolysis indicated that the alpha subunit was inserted into microsomal membranes. In the presence of dog pancreatic microsomes, the beta polypeptide was glycosylated as indicated by the appearance of three higher molecular weight polypeptides that were sensitive to endoglycosidase H and bound to Concanavalin A. The beta subunit was predominantly translocated into the lumen of the endoplasmic reticulum since 90% of the mass of the membrane-associated beta polypeptide was resistant to trypsin (i.e. reduced in size from 40 kDa to 37.5 kDa), and 95% of all of the beta chains were resistant to extraction with alkali. Neither the alpha nor the beta subunits have NH2-terminal leader signal sequences, but both may require the signal recognition receptor for membrane insertion, as evidenced by inhibition of incorporation of both subunits into microsomes pretreated with N-ethylmaleimide. Simultaneous translation of cRNA alpha and cRNA beta did not enhance membrane insertion of either the alpha or beta polypeptide.

The effect of delta-aminolevulinic acid on the synthesis and metabolism of GABA in rabbit brain homogenates.

The porphyrin precursor delta-aminolevulinic acid (delta-ALA) is a structural analogue of the putative amino acid neurotransmitter, gamma-aminobutyric acid (GABA). This study has demonstrated that delta-ALA has no effect on glutamate decarboxylase activity and only a small inhibitory effect of GABA aminotransferase activity. This would suggest that if accumulation of delta-ALA is related to development of the acute attack of porphyria, it is not via an effect on GABA synthesis and metabolism.

Reduced ferrochelatase activity in fibroblasts from patients with porphyria variegata.

Ferrochelatase deficiency has been shown in both porphyria variegata (PV) and erythropoietic protoporphyria (EPP). It has been suggested that in PV there is a decrease in the enzyme, whereas in EPP the enzyme is unstable. In the present study ferrochelatase activity was measured in skin fibroblasts from three patients with PV and three normal subjects. The enzymatic activity in the patients with PV (17.5 +/- 4.5 pmoles heme formed per 10(7) fibroblasts per hour) was 50% of that of the control group (31.0 +/- 3.2 pmoles heme formed per 10(7) fibroblasts per hour). This supports the contention that the enzyme is deficient in PV and that an inactive ferrochelatase is the primary deficiency in this type of porphyria.

Identification of an epitope shared by the DNA-binding domain of glucocorticoid receptor and the B chain of insulin.

A monoclonal antibody (8G11-C6) generated by an auto-anti-idiotypic route and directed to a site near the ligand-binding site of the glucocorticoid receptor also binds to native insulin and the B chain of insulin but not to the A chain of insulin. The glucocorticoid receptor and the B chain of insulin, therefore, share a cross-reacting epitope. Examination of the primary sequences of the two proteins revealed a limited number of regions of identity or close homology. Several peptides representative of those regions were synthesized. A heptapeptide sequence of the B chain of insulin with homology to a sequence in the first "zinc finger" of the DNA-binding domain of the glucocorticoid receptor was identified as the cross-reactive epitope. This heptapeptide sequence is restricted to and highly conserved among insulins of various species. Homologous sequences are found in the DNA-binding domains of most steroid receptors and related DNA-binding proteins. Consistent with this is the finding that 8G11-C6 inhibits the binding of glucocorticoid receptor to DNA-cellulose.

Generation of an auto-anti-idiotypic antibody that binds to glucocorticoid receptor.

A monoclonal antibody (8G11-C6) that is directed to a region near the ligand-binding site of the glucocorticoid receptor was obtained by an auto-anti-idiotypic route, using a derivative of triamcinolone coupled to thyroglobulin to immunize a mouse. The resulting hybridomas were screened for anti-idiotypic antibody (anti-antisteroid) with Fab fragments of affinity-purified polyclonal rabbit anti-triamcinolone antibody. The anti-idiotypes were then screened for binding to rat cytosol glucocorticoid receptor by a depletion procedure, yielding a clone, 8G11-C6, whose specificity for receptor was verified by sucrose density and Western blot analyses. Depletion was not significantly reduced by prelabeling the cytosol with [3H]triamcinolone acetonide. The anti-idiotype (8G11-C6) bound to Fab fragments of antisteroid and to partially purified receptor in a concentration-dependent manner. Both binding reactions were inhibited only by rabbit serum albumin conjugates of steroids known to bind to the glucocorticoid receptors. Triamcinolone derivatives of lysine and of oligopeptides containing up to six amino acids inhibited the binding of the anti-idiotype to the Fab fragments but not to the receptor, implying that the target epitope of the antisteroid antibody may be closer to its glucocorticoid-binding site than the cross-reacting epitope of the receptor. Our findings demonstrate further the versatility of the auto-anti-idiotypic route for the preparation of anti-receptor antibodies.

Serum independence of low K+ induction of Na,K-ATPase: possible role of c-fos.

Cultured ARL15 cells respond to abnormally low extracellular K+ concentrations by increasing the abundance of Na,K-ATPase (the Na/K pump). This response is preceded by significant increases in the mRNAs of the alpha 1 and beta 1 subunits of this enzyme, implying transcriptional or post-transcriptional regulation in the response. The present study concerned the possible participation of serum factors in low K+ induction of Na,K-ATPase. In normal K+ (4.5 mM) or low K+ (0.68 mM) the presence of 10% calf serum had no effect on Na,K-ATPase activity. The serum independence of the response to low K+ raised the possibility that low K+ may itself elicit a "growth" response. Accordingly, the effect of low K+ on mRNA abundances of four proto-oncogenes (c-fos, c-myc, c-jun and c-ski) was evaluated in the early phase of the response by quantitative Northern blot analysis. The mRNA for c-fos was transiently elevated by low K+, with a peak at 30 min. In contrast, low K+ had no measurable effect on the abundances of c-myc, c-jun and c-ski, for up to 2 hr of exposure. The early elevation of c-fos mRNA makes it a candidate mediator in this signal-transduction pathway. Induction of c-fos mRNA by the phorbol ester, PMA, or by dioctanoyl glycerol, however, had no effect on Na,K-ATPase activity. These results indicate that an increase in c-fos mRNA alone is not sufficient to induce Na,K-ATPase. Whether induction of c-fos is necessary for the response to low K+ remains to be determined in future studies.

Haemolytic anaemia associated with glucosephosphate isomerase (GPI) deficiency in a Black South African child.

Haemolytic anaemia in a Black South African child was found to be associated with reduced glucosephosphate isomerase activity in the red cells. Apart from the haemolytic anaemia, there was no other clinical evidence of dysfunction. Family studies pointed to an autosomal recessive mode of inheritance, with the symptomatic homozygous propositus having a marked enzyme deficiency and the asymptomatic heterozygotes showing intermediate levels of activity. Biochemical characterization showed that, apart from being thermolabile, the electrophoretic mobility and the kinetic properites of the variant enzyme were similar to those of the normal wild type.

Glucose-6-phosphate dehydrogenase Porbandar: a new slow variant with slightly reduced activity in a South African family of Indian descent.

A new variant of the red cell enzyme glucose-6-phosphate dehydrogenase has been detected in a South African male of Indian descent and in several of his relatives. The enzyme variant is characterized by slow electrophoretic mobility, low Michaelis constants for the substrates glucose-6-phosphate and NADP, and increased utilization of the substrate analogues 2-deoxyglucose-6-phosphate and deamino-NADP relative to the normal (B+) enzyme. There is no evidence that the enzyme variant, for which the name "G6PD Porbandar" is suggested, is associated with any hematological abnormality.

G6PD hillbrow: a new variant of glucose-6-phosphate dehydrogenase associated with drug-induced haemolytic anaemia.

A new genetic variant of the red cell enzyme glucose-6-phosphate dehydrogenase is described. It was observed in a patient presenting with severe haemolytic anaemia and renal failure following ingestion of an overdose of Beserol (paracetamol and chlormezanone). The enzyme in the red cell had 12% of the activity of a normal B+ control, but only slightly lower activity in the kidney compared with a normal control. The red cell enzyme showed normal electrophoretic mobility and thermostability, a biphasic pH optimum curve, higher than normal utilization of the substrate analogues 2-deoxy-glucose-6-phosphate and deamino-NADP, and lower than normal Michaelis constants for both substrates, glucose-6-phosphate and NADP. The enzyme was strongly inhibited in vitro by high concentrations of paracetamol and chlormezanone. The extent of inhibition was similar to that for the enzyme from a normal B+ individual.

Regional localization of 32 NotI-HindIII fragments from a human chromosome 13 library by a somatic cell hybrid panel and in situ hybridization.

Relatively few DNA probes have been regionally assigned to chromosome 13 bands, and these tend to be clustered in the proximal end of the chromosome in the regions where intensive mapping efforts have been focused because of the retinoblastoma and Wilson disease genes. We have developed a chromosome 13-specific half-linking library of NotI-HindIII fragments and regionally mapped 32 of these on chromosome 13, using a somatic cell hybrid panel that subdivides chromosome 13 into 11 regions and nonisotopic in situ hybridization with silver amplification of a digoxigenin-labeled probe.