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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.

Carbonic anhydrase (CA) is implicated in the acidification of epididymal fluid and thereby in the regulation of sperm maturation and motility. Among the CA isoenzymes, CA IV and II have been shown to be present in the rat epididymal duct epithelium. In the present study, we examined the expression and androgen regulation of CA IV and II mRNAs along the epididymal duct. Northern blot analysis revealed the presence of CA II mRNA in all regions of the epididymis with the strongest signal in the corpus region, while CA IV mRNA was expressed predominantly in the corpus epididymidis. Three days after bilateral castration, CA IV and II mRNAs were decreased by 80-90% in the corpus epididymidis. Testosterone (T) replacement maintained the expression of CA mRNAs at 50-60% of the control levels, indicating that circulating androgens alone are not sufficient to recover the CA expression in the corpus region. However, unilateral castration did not affect the mRNA levels of CA IV and II, suggesting that factors in testicular fluid do not play a major role in the regulation of CA expression in the corpus epididymidis. Immunoblot analysis showed that CA IV protein levels decreased 3 days after castration, while T administration maintained the protein expression virtually at the precastration levels. These data demonstrate that mRNAs for CA IV and II are predominantly expressed in the corpus region of the rat epididymis and can be regulated by androgens in that region. The present data suggest that the regulation of CA expression in the corpus epididymidis by androgens contributes to the known androgen effects on epididymal acidification.

We have mapped 11 novel, anonymous genetic markers to rat chromosome 2. The rat ceruloplasmin gene (Cp) had been previously mapped to chromosomes 2 and 7q11--q13 by two different methods. To resolve the assignment and to localize the Cp gene on the rat genetic linkage map, we used linkage analysis to confirm that rat Cp lies on chromosome 2.

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.

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.

Of five patients in who sciatic paresis developed as the result of hemorrhage and hematoma following hip surgery, four were receiving prophylactic or therapeutic anticoagulants. The patient who was managed expectantly still had disabling motor and sensory deficity at follow-up. Three patients who had early operative decompression showed more complete return of nerve function. The fifth patient died three weeks after onset with the neuropathy still present. Severe low-back and buttock pain, ecchymosis over these regions, marked swelling in the thigh, sciatic-nerve tenderness, and a distal sciatic neural deficit in the ipsilateral lower limb of a patient who has had hip surgery are evidence of hemorrhage in the vicinity of the sciatic nerve. Early recognition and prompt surgical decompression can prevent irreversible nerve damage.

Of five patients in who sciatic paresis developed as the result of hemorrhage and hematoma following hip surgery, four were receiving prophylactic or therapeutic anticoagulants. The patient who was managed expectantly still had disabling motor and sensory deficity at follow-up. Three patients who had early operative decompression showed more complete return of nerve function. The fifth patient died three weeks after onset with the neuropathy still present. Severe low-back and buttock pain, ecchymosis over these regions, marked swelling in the thigh, sciatic-nerve tenderness, and a distal sciatic neural deficit in the ipsilateral lower limb of a patient who has had hip surgery are evidence of hemorrhage in the vicinity of the sciatic nerve. Early recognition and prompt surgical decompression can prevent irreversible nerve damage.