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Aceruloplasminaemia is a rare recessive disorder caused by mutations in the gene encoding the multicopper ferroxidase ceruloplasmin, thought to be involved in cellular iron export. Primary intracellular iron accumulation characterises this disorder. We investigated a case of aceruloplasminaemia early in the course of the disease by structural and functional neuroimaging and correlated the results with the clinical findings. The patient, a diabetic 59 year old lady, presented with perioral dyskinesia. Magnetic resonance imaging (MRI) revealed massive iron accumulation in the basal ganglia, notably sparing the pallidum, and along the cortical surface. However, most of these structures had preserved metabolic activity as evaluated by fluorodeoxyglucose positron emission tomography (FDG-PET). Voxel based analysis of FDG-PET data showed a significant hypometabolism only in the heads of the caudate nuclei. Molecular genetic analysis revealed compound heterozygosity for two null mutations in the ceruloplasmin gene, a rather surprising finding for a very rare recessive disease, suggesting that aceruloplasminaemia could be somewhat more frequent than is commonly thought and could therefore be underdiagnosed.

Aceruloplasminemia is a newly recognized autosomal recessive disorder of iron metabolism that causes neurodegeneration of the retina and basal ganglia as well as diabetes mellitus. Our previous studies suggested that increased susceptibility to plasma lipid peroxidation secondary to iron accumulation may contribute to the pathogenesis in this disease. We now have identified increases in the very long-chain fatty acids cis-17-hexacosenoic(C26:1) and hexacosanoic (C26:0) acid in the erythrocyte membranes of three family members affected with aceruloplasminemia. All of them had elevated C26:1/C22:0 and C26:0/C22:0 ratios. These findings suggest that free radicals generated in persons with aceruloplasminemia may interrupt the peroxisomalβ-oxidation of fatty acids.

Long-Evans Cinnamon rats develop a necrotizing hepatitis characterized by excessive hepatic copper accumulation, defective holoceruloplasmin biosynthesis and impaired biliary copper excretion. To elucidate the molecular basis of this defect, a cDNA clone encoding the rat Wilson disease gene was isolated and used to examine gene expression in selected tissues from normal and Long-Evans Cinnamon rats. Although this cDNA readily detects Wilson transcripts in liver and other tissues from normal rats, such transcripts are entirely absent from tissues derived from the Long-Evans Cinnamon rat strain. These data therefore identify the Long-Evans Cinnamon rat as the first bona fide animal model of Wilson disease and suggest that this rat strain may be a valuable resource in the study of this genetic disorder.

To determine the mechanisms of expression of the rat caeruloplasmin gene, the promoter region was analysed by DNAase I footprinting. Using nuclear extract from rat liver, a prominent site of protein-DNA interaction was detected from -93 to -48 upstream of the caeruloplasmin gene transcription start and sequence analysis of this region revealed three potential CCAAT/enhancer-binding protein (C/EBP) consensus elements. Mobility-shift analysis using an oligonucleotide encoding this region identified specific binding of proteins from rat liver nuclear extract, and some of these complexes were supershifted using antisera to the C/EBP alpha and beta family members. Mobility-shift studies using a polypeptide encoding the DNA-binding domain of C/EBP alpha also revealed a specific interaction with this region of the caeruloplasmin promoter, and DNAase I footprinting using this polypeptide protected the identical region from -93 to -48. Co-transfection of expression plasmids encoding C/EBP alpha or a related leucine-zipper factor D-binding protein (DBP) revealed a C/EBP-specific increase in reporter gene activity in HepG2 cells transfected with caeruloplasmin-chloramphenicol acetyltransferase containing the -93 to -48 region. A similar result was obtained when these constructs were co-transfected into mouse L cells which were shown not to express the endogenous caeruloplasmin gene. Taken together, these data indicate a role for C/EBP alpha and beta in mediating transcription from the caeruloplasmin gene promoter and suggest that this region of the promoter is not responsible for tissue-specific expression.

We report the case of a 52 year old male with diabetes mellitus and long standing evidence of hepatic iron excess. Initially considered to have haemochromatosis, this patient was reevaluated when hepatic iron stores were found to be unaffected by a prolonged course of weekly phlebotomy. The development of neurological disease prompted diagnostic consideration of aceruloplasminaemia, which we confirmed by demonstration of a novel frameshift mutation in the ceruloplasmin gene. Our inability to resolve the patient's iron overload by regular phlebotomy is consistent with recent animal studies indicating an essential role for ceruloplasmin in cellular iron efflux. Evaluation of this case underscores the clinical relevance of aceruloplasminaemia in the differential diagnosis of hepatic iron overload and provides insight into the pathogenetic mechanisms of hepatocellular iron storage and efflux. Keywords: aceruloplasminaemia; ceruloplasmin; diabetes; haemochromatosis; iron

Using a ceruloplasmin cDNA clone in RNA blot analysis, a single 3.7 kb ceruloplasmin-specific transcript was detected in rat mammary gland tissue from pregnant and lactating animals. Ceruloplasmin gene expression in the mammary gland was tissue-specific, with no evidence of expression in brain, heart or other extrahepatic tissues. Ceruloplasmin mRNA was also detected in mammary gland tissue from male, virgin female and non-pregnant/multiparous animals, and the abundance of ceruloplasmin-specific transcripts in virgin female rats was independent of their stage of oestrus. In virgin female mammary gland the content of ceruloplasmin mRNA was 20% of that in hepatic tissue from these animals and approx. 2-3-fold greater than that found in mammary gland tissue of pregnant or lactating animals. Development studies revealed ceruloplasmin gene expression in male and female mammary gland by only 2 weeks of age, prior to the onset of puberty. Biosynthetic studies indicated that the ceruloplasmin mRNA in mammary gland tissue was translated into a 132 kDa protein qualitatively similar to that synthesized in liver. By in situ hybridization, ceruloplasmin gene expression was localized to the epithelium lining the mammary gland alveolar ducts, without evidence of expression in the surrounding mesenchyme. Ceruloplasmin gene expression was also detected in a human breast adenocarcinoma cell line and in biopsy tissue from women with invasive ductal carcinoma. Taken together, these data indicate that the mammary gland is a prominent site of extrahepatic ceruloplasmin gene expression and add to the evidence that ceruloplasmin biosynthesis is associated with growth and differentiation in non-hepatic tissues.

The patterns of dipeptidyl peptidase (DPP) IV activity and protein amount in different rat organs during development were compared. In order to elucidate the molecular basis for these patterns, total RNA was isolated from lung and kidney at different stages of development and analysed by Northern-blot hybridization using an oligonucleotide derived from the DPP IV cDNA sequence. This oligonucleotide hybridized to two distinct mRNAs of approx. 3.2 and 4.8 kb respectively. During kidney development, the pattern for DPP IV mRNA paralleled that of DPP IV activity and protein amount, suggesting that, in kidney, the expression of DPP IV is primarily controlled at the transcriptional level. In contrast, the magnitude of DPP IV activity during lung development compared with that of DPP IV mRNA in lung suggests that post-transcriptional mechanisms are involved in regulating the expression of DPP IV in lung. Organ-specific regulation of DPP IV expression may provide a useful model for further comparative studies of transcriptional and post-transcriptional mechanisms of DPP IV expression within the same organism.

To study the molecular mechanisms controlling the rapid increase in myometrial gap junctions observed in the parturient uterus, we have isolated a full-length cDNA clone corresponding to a rat uterine gap junction protein. Nucleotide sequence analysis of the cDNA clone reveals complete identity of the coding region with that of a previously reported heart gap junction protein (connexin43). Southern blot analysis suggests that the gene encoding this gap junction protein exists as a single copy in the rat haploid genome and contains no introns within the coding region. RNA blot analysis with this gap junction cDNA reveals a single 3.0-kb mRNA in uterine tissue without changes in transcript size throughout gestation. When normalized to the amount of 28S rRNA, the relative abundance of the connexin43 transcript in uterine tissue is quite constant between the nonpregnant state, during gestation, intrapartum, and postpartum. Similar size transcripts are shown by RNA blot analysis to be present in heart, lung, liver, brain, and skeletal muscle, and these transcripts are identified by the same 3'-nontranslated sequence probe. The results of these studies suggest that rat connexin43 is encoded by a single gene that is transcribed to identical transcripts in heart, uterus, and other tissues. They further suggest that changes in the abundance of connexin43 transcript are unlikely to be responsible for the abrupt increase in connexin43-containing myometrial gap junctions at term.

Copper is an essential transition metal that permits the facile transfer of electrons in a series of critical biochemical pathways. Menkes disease and Wilson's disease are inherited disorders of copper metabolism resulting from the absence or dysfunction of homologous copper-transporting ATPases that reside in the trans-Golgi network of all cells. Despite striking differences in the clinical presentation of these two diseases, the respective ATPases function in precisely the same manner within the cell and the unique clinical features of each disease are entirely the result of the tissue-specific expression of each protein. Elucidation of the basic defect in these rare genetic disorders has provided a valuable heuristic paradigm for understanding the mechanisms of cellular copper homeostasis.

Aceruloplasminemia is a disorder of iron metabolism characterized by degeneration of the retina and basal ganglia. CSF from affected patients showed a threefold increased iron concentration that was associated with increased superoxide dismutase activity and lipid peroxidation products. These findings support the hypothesis that iron-mediated lipid peroxidation contributes to neurodegeneration in patients with aceruloplasminemia. Such measurements may have value in assessing disease progression as well as the results of iron chelation and other therapeutic interventions.

Aceruloplasminemia is an autosomal recessive disorder resulting in neurodegeneration of the retina and basal ganglia in association with iron accumulation in these tissues. To begin to define the mechanisms of central nervous system iron accumulation and neuronal loss in this disease, cDNA clones encoding murine ceruloplasmin were isolated and characterized. RNA blot analysis using these clones detected a 3.7-kb ceruloplasmin-specific transcript in multiple murine tissues including the eye and several regions of the brain. In situ hybridization of systemic tissues revealed cell-specific ceruloplasmin gene expression in hepatocytes, the splenic reticuloendothelial system and the bronchiolar epithelium of the lung. In the central nervous system, abundant ceruloplasmin gene expression was detected in specific populations of astrocytes within the retina and the brain as well as the epithelium of the choroid plexus. Analysis of primary cell cultures confirmed that astrocytes expressed ceruloplasmin mRNA and biosynthetic studies revealed synthesis and secretion of ceruloplasmin by these cells. Taken together these results demonstrate abundant cell-specific ceruloplasmin expression within the central nervous system which may account for the unique clinical and pathologic findings observed in patients with aceruloplasminemia.

The pulmonary epithelium is a derivative of the foregut endoderm. Proliferation and differentiation of the primitive pulmonary epithelium result in an array of epithelial cell phenotypes that determine lung function and the response of the lung to injury, infection, or neoplastic transformation. The establishment of a cell phenotype requires the presence of transcription factors that activate or repress expression of specific genes. Members of the forkhead family of transcription factors, in particular HNF-3 alpha, HNF-3 beta, HFH-4; the homeodomain protein TTF-1; and N-myc, are all expressed in the developing pulmonary epithelium and may play important regulatory roles during development. Two genes specific to the pulmonary epithelium, the surfactant protein A and Clara cell secretory protein genes, serve as useful paradigms for understanding the mechanisms regulating cell-specific gene expression in the pulmonary epithelium.

The 5′ flanking region of the Clara cell secretory protein (CCSP) gene contains two cis-acting elements which bind hepatocyte nuclear factor (HNF)-3 alpha and HNF-3 beta in vitro. To determine the role of these proteins in mediating CCSP gene expression in the bronchiolar epithelium, chimeric CCSP-reporter gene constructs containing various regions of the CCSP 5′ flanking region were co-transfected into H-441 cells with HNF-3 alpha or HNF-3 beta expression plasmids. These studies indicate that each of these transcription factors positively regulates CCSP gene expression and revealed that CCSP region I (-132 to -76) is sufficient to mediate this effect. Gel-mobility-shift assays with oligonucleotides corresponding to CCSP region I, nuclear extract from bronchiolar epithelial cells and HNF-3-specific antibodies indicate that HNF-3 alpha and HNF-3 beta are the only proteins in bronchiolar epithelial cells which directly interact with this region. Consistent with these observations, HNF-3 alpha and HNF-3 beta transcripts were found to be enriched in this cell population and in situ hybridization of adult lung revealed HNF-3 gene expression in non-ciliated bronchiolar epithelial cells expressing the CCSP gene. Finally, experiments with CCSP region I and a heterologous promoter indicate that this region acts in a promoter-specific context, suggesting that additional factors interacting via the minimal CCSP promoter region are essential in determining the effects of HNF-3 on cell-specific CCSP gene expression in the bronchiolar epithelium.

Expression of a transgene containing 2.25 kb of the 5' flanking region of the rat Clara cell secretory protein gene and the human growth hormone gene was examined in developing mice. Despite an absolute preservation of tissue specificity based on RNA blot analysis, transgene-specific transcripts were detectable as early as 12.5 days of gestation, at least 4 days prior to endogenous Clara cell secretory protein gene expression. As differentiation proceeded, in situ hybridization revealed an increasingly restricted pattern of transgene expression in the developing pulmonary epithelium, such that by day 16.5 of gestation endogenous and transgene expression were confined to identical cells within the bronchiolar epithelium. The temporal discordance in transgene expression suggests the presence of unique cis-acting elements within the Clara cell secretory protein gene, not present in the transgene, which transduce developmental timing within pulmonary epithelium by actively repressing Clara cell secretory protein gene expression during early development. The unique expression of this transgene serves as a lineage marker in the respiratory epithelium and unmasks a temporal and spatial pattern of gene expression not observed in any pulmonary genes.

To determine the mechanisms of cell-specific gene expression in the developing pulmonary epithelium the Clara cell secretory protein (CCSP) gene promoter was analysed by DNAase I footprinting. A prominent site of protein-DNA interaction was detected from nucleotides -132 to -76 using nuclear extract from mouse lung and human H441 cells. Mobility shift analysis revealed that an oligonucleotide corresponding to this region interacted with multiple proteins from lung and H441 cell nuclear extracts. Analysis of the nucleotide sequence of this region identified two potential binding sites for hepatocyte nuclear factor 3 (HNF-3), and consistent with this finding binding to this CCSP oligonucleotide was specifically competed for by an oligonucleotide corresponding to the HNF-3-binding site from the mouse transthyretin gene. Mobility shift of the CCSP oligonucleotide was supershifted using antisera specific to HNF-3 alpha and HNF-3 beta, and HNF-3 alpha and HNF-3 beta translated in vitro were found to bind specifically to this same oligonucleotide. Co-transfection of HNF-3 alpha- and HNF-3 beta-expression plasmids increased cell-specific reporter gene activity in H441 cells transfected with a CCSP-CAT gene chimeric construct containing this -132 to -76 region. Taken together, these results suggest a role for HNF-3 in mediating cell-specific CCSP gene expression within the bronchiolar epithelium. These findings support the hypothesis that members of the HNF-3 ‘forkhead’ family of transcription factors determine gene expression and cell fate in multiple cell lineages derived from the primitive gut endoderm.

To characterize the mechanisms determining tissue-specific ceruloplasmin gene expression during development, the rat ceruloplasmin gene was isolated in a series of overlapping phage clones. The 5'-flanking region was characterized and the transcription initiation site was identified by primer extension and RNase protection. Nucleotide sequence analysis of this region revealed a typical eukaryotic promotor structure, but no obvious homology with cis-acting elements previously characterized as determining tissue-specific gene expression. Transient expression of chimeric ceruloplasmin-reporter gene constructs containing up to 5200 base pairs (bp) of the 5'-flanking region revealed that sequences 732 bp upstream of the start nucleotide were sufficient to confer hepatocyte-specific expression. The region from -393 to -348 was determined by deletion analysis to contain a positive-acting element, and includes sequence partially homologous to the rat albumin D site. Mobility shift analysis revealed that this region specifically binds a heat-labile nuclear protein from rat liver and from newborn but not adult rat lung. Binding to this region was competed by oligonucleotides corresponding to the albumin D site, but not by oligonucleotides corresponding to binding sites for the hepatocyte transcription factors HNF-1, HNF-3, HNF-4, and C/EBP. These data indicate that ceruloplasmin gene expression is determined in part by a cis-acting region 393 bp upstream of the transcription start site, which binds a previously uncharacterized nuclear protein. The tissue distribution of this nuclear protein suggests that it plays a role in directing ceruloplasmin gene expression in lung and liver during development.

Decreased amounts of the serum copper-binding protein caeruloplasmin (hypocaeruloplasminaemia) are one of the characteristic abnormalities of copper metabolism that are found in all neonatal mammals. In the present study we have investigated the mechanism responsible for hypocaeruloplasminaemia found in neonatal guinea pigs. Northern-blot analysis of guinea-pig liver RNA revealed the presence of two caeruloplasmin mRNA species. A marked developmental change in expression was observed, with no detectable mRNA before birth, when hepatic copper is elevated. Expression was initiated soon after birth, when copper levels in the liver are falling, and was closely linked to the rise in enzymic activity and protein levels in serum. There was no differential regulation of the two mRNA species; however, a marked inter-animal variation in mRNA levels was observed. Using gel-filtration chromatography we were able to show that, before birth, serum copper was associated with a low-molecular-mass species and that, with the advent of expression of the caeruloplasmin gene after birth, copper was increasingly associated with caeruloplasmin.

cDNA clones corresponding to rat ceruloplasmin were isolated from newborn rat lung and liver cDNA libraries and the nucleotide sequence was obtained. The derived amino acid sequence of rat ceruloplasmin is 93% homologous to the corresponding human sequence and contains a 19-amino acid leader peptide plus 1040 amino acids of mature protein. Southern blot analysis indicates that the ceruloplasmin gene exists as a single copy in the rat haploid genome. Using these cDNA clones in RNA blot analysis, a single 3.7-kilobase ceruloplasmin-specific transcript is detected in fetal rat liver and lung by day 15 of gestation. During fetal development the abundance of this transcript increases selectively in these two tissues and at birth is 60% of that found in the adult liver. Postnatally the temporal pattern of ceruloplasmin gene expression in lung and liver differs. Within the first 3 weeks postpartum ceruloplasmin mRNA content decreases in lung to undetectable levels, while that in the liver reaches adult levels. Primer extension reveals a single identical start site of ceruloplasmin gene transcription in lung and liver and biosynthetic studies indicate that each tissue synthesizes a ceruloplasmin protein which is qualitatively similar to that synthesized by adult liver. Ceruloplasmin mRNA is also detected in human fetal lung explant and a human lung adenocarcinoma cell line suggesting that a similar pattern of expression occurs in the developing human lung. These data indicate that lung is the predominant extrahepatic site of ceruloplasmin gene expression during fetal development and suggest that this protein may play a previously unappreciated role in lung development or pulmonary antioxidant defense.

Pages L68–L74: R. E. Fleming, I. P. Whitman, and J. D. Gitlin. “Induction of ceruloplasmin gene expression in rat lung during inflammation and hyperoxia.” The vertical axes of Figs. 2, 3B, and 5C should read: % over control.

Human peripheral blood phagocytes ingest Escherichia coli 026:B6 lipopolysaccharide (LPS)-coated paraffin oil droplets containing Oil red O only if fresh serum deposits C3 on the surfaces of the particles (opsonizes them), by reactions involving the properdin system. The rate of binding of purified [125-I]C3 in serum to LPS-coated particles correlated precisely with the rate of acquisition of ingestibility assayed spectrophotometrically. Once opsonized, LPS-coated particles remained fully ingestible and retained fixed [125-I]C3 radioactivity even after exposure to extremes of temperature, pH, ionic strength, phospholipases, urea or guanidine, some nonionic and ionic detergents, and organic solvents. Trypsin, human conglutinogen-activating factor, another heat-stable activity found in human serum, and sodium dodecyl sulfate removed radioactivity and diminished ingestibility of the opsonized particles. Alkylation, reduction plus alkylation and F(ab')2 from anti-C3 blocked ingestibility but did not alter particle-bound radioactivitymelectrophoretic and tryptic peptide autoradiographic analysis of dodecyl sulfate eluates of opsonized particles, cleansed of many contaminating proteins by boiling with 2 M NaCl (yet still opsonized), revealed that the polypeptide with C3-derived radioactivity had a mol wt of approximately 140,000 and was composed of 70,000 mol wt subunits linked by disulfide bonds. Immunochemical analysis and comparison of the peptide structure of the eluate with that of C3 indicated that the opsonic fragment is not the fragment defined as C3b but a smaller derivative of C3.

We conducted a prospective, randomized, unblinded, controlled trial of exogenous bovine surfactant (surfactant TA) in premature infants requiring ventilator support for the treatment of severe hyaline membrane disease. Forty-one low birth weight infants with severe hyaline membrane disease were randomly assigned to saline or surfactant therapy and treated within eight hours of birth. Significant improvements in oxygenation (increased arterial/alveolar PO2) and respiratory support (decreased mean airway pressure) were seen in the group receiving surfactant within four hours after treatment. These improvements were maintained in the surfactant-treated infants, who also had fewer pneumothoraces and fewer number of days in environments of fractional inspiratory oxygen greater than 0.4 mm Hg. No problems were associated with administration of surfactant, and no acute side effects were detected. We conclude that exogenous surfactant, administered early in the course of severe hyaline membrane disease, is an effective therapy that can diminish the amount of respiratory support required during the first 48 hours of life.

Exogenous surfactant therapy appears to offer promise in the treatment and possible prevention of HMD. Laboratory investigations have begun to reveal the molecular basis for surfactant metabolism and the relationship of this complex process to alveolar stability and pulmonary function. There is every reason to encourage clinical investigation with surfactant therapy in parallel with further basic research. Nevertheless, pediatricians must proceed in small steps with carefully designed studies to address specific questions regarding both efficacy and toxicity. Results from various studies must be shared and discussed and every attempt must be made to eventually provide standardized, readily available preparations of known efficacy and toxicity. Efforts by many investigators make it seem probable that this goal will be achieved in the near future.

Mixed sequence oligonucleotides were used to isolate a series of acute-phase human liver cDNA clones corresponding to the serum alpha 2-globulin ceruloplasmin. These clones were characterized, sequenced, and used to analyze changes in hepatic ceruloplasmin mRNA content during inflammation. In all species examined, hepatic ceruloplasmin mRNA content increased approximately 6-10-fold over control values within 24 h following the induction of inflammation. The mechanisms leading to this increase in hepatic ceruloplasmin mRNA content were studied following turpentine-induced inflammation in Syrian hamsters. Nuclear run-on assays demonstrated an increase in the relative rate of transcription of the ceruloplasmin gene within 3 h following induction, reaching maximum values by 18 h. Hepatic ceruloplasmin mRNA content increased 2-fold within 12 h following induction, reached maximum values by 24 h, and returned to control within 72 h. In contrast, serum ceruloplasmin concentration did not increase until 36 h, reached maximal levels by 120 h, and remained elevated for the course of the study. These data indicate that inflammation leads to a rapid increase in hepatic ceruloplasmin mRNA content. This increase is largely the result of increased ceruloplasmin gene transcription, but comparison of the relative rate of transcription and mRNA accumulation suggests that changes in ceruloplasmin mRNA turnover are also involved. In addition, translational and/or post-translational mechanisms must account for the observed changes in serum ceruloplasmin concentration seen during inflammation.

The complement genes and many other acute phase protein genes are expressed in liver and in extrahepatic tissues (Alper et al. 1980; Kushner and Feldmann 1978; Kushner 1982). The hepatocyte is a major source of the plasma proteins encoded by these genes. The original definition of an acute phase reponse included changes in plasma protein concentration but recent data suggest that changes in expression of these genes occur at the local site of tissue injury or inflammation as well as in liver (reviewed in Colten and Dowton 1986). These observations raise important questions about the mechanisms accounting for tissue specific control of acute phase protein synthesis following tissue injury or inflammation.