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Biomedical subjects

R E Fleming

Publications and source records attributed to R E Fleming.

29 records · Page 2Linked to original sources
PubMed1993

Interaction of CCAAT/enhancer-binding protein alpha and beta with the rat caeruloplasmin gene promoter.

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.

Animals↗
PubMed1993

Pulmonary carbonic anhydrase IV: developmental regulation and cell-specific expression in the capillary endothelium.

To investigate the pulmonary expression of membrane-associated carbonic anhydrase (CA IV), the full-length rat lung CA IV cDNA was isolated and characterized. The 1,205-bp cDNA was found to encode a 309-amino acid protein, including a 27-amino acid hydrophobic COOH-terminal extension, presumably cleaved prior to transfer of the protein to its glycosyl-phosphatidylinositol membrane anchor. The tissue-specific pattern of CA IV gene expression was found to be distinct from that of other CAs, with highest expression in lung, colon, kidney, brain, and heart. Pulmonary CA IV expression was found to be developmentally regulated, with a marked postnatal increase in mRNA content and immunoreactive protein concentration. CA IV localized by immunohistochemistry to the luminal side of the alveolar capillary endothelium, but was absent from endothelium of larger pulmonary vessels. Selective expression of CA IV in the alveolar capillaries provides evidence for specialized differentiation of this endothelial cell subpopulation and places CA IV in a strategic location to participate in carbon dioxide exchange and local pH regulation.

Aging↗
PubMed1992

Structural and functional analysis of the 5'-flanking region of the rat ceruloplasmin gene.

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.

Albumins↗
PubMed1991

Induction of ceruloplasmin gene expression in rat lung during inflammation and hyperoxia.

To determine the effect of inflammation on extrahepatic ceruloplasmin gene expression we examined the ceruloplasmin mRNA content of adult rat tissues after endotoxin injection. Within 8 h of a dose of endotoxin ceruloplasmin mRNA content increased in the liver as expected and was also detectable in the lung. The effect of endotoxin was tissue specific because ceruloplasmin mRNA was not consistently detected in other extrahepatic tissues. The kinetics of ceruloplasmin mRNA accumulation in lung and liver tissue were similar with a maximum seven- to ninefold increase in ceruloplasmin mRNA content in each tissue within 24 h. The relative rate of ceruloplasmin gene transcription was increased in both tissues within 3 h of endotoxin, suggesting similar mechanisms of regulation of ceruloplasmin gene expression during inflammation. One cellular site of ceruloplasmin production in the inflamed lung was found to be the alveolar macrophage, which expressed the ceruloplasmin gene and synthesized ceruloplasmin protein in response to endotoxin in vitro. Because of these findings we also examined the effects of hyperoxia on ceruloplasmin gene expression. Exposure of adult rats to 95% O2 resulted in a five- to sixfold induction of ceruloplasmin mRNA in lung tissue within 46 h, and this response was time dependent, reaching maximum values at 86 h. Hyperoxic induction of ceruloplasmin mRNA was specific to the lung and not the result of systemic inflammation because hepatic ceruloplasmin mRNA content remained constant. These data indicate that the lung is a prominent site of ceruloplasmin gene expression during inflammation and hyperoxia and suggest that this protein may play a previously unappreciated role in pulmonary injury or repair.

Animals↗
PubMed1990

Primary structure of rat ceruloplasmin and analysis of tissue-specific gene expression 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.

Aging↗
PubMed1986

Primary lymphatic dysplasia in children: chylothorax, chylous ascites, and generalized lymphatic dysplasia.

Primary lymphatic "dysplasia", a congenital maldevelopment, interferes with function of the lymphatic system and causes effusion of chyle or lymph into the limbs and pleural or peritoneal cavity. Between 1955 and 1982, 38 Mayo Clinic patients were found to have a chylous effusion or dysplasia of the lymphatic system. In 22, the condition was secondary to surgery or other medical problems and in 16 it was primary. These cases were separated into three categories: chylothorax, chylous ascites, and generalized lymphatic dysplasia. Conservative therapy, such as a restricted fat diet or total parenteral nutrition with repeated thoracentesis or paracentesis, was effective in the children with isolated abnormalities of the lymphatic system (75% resolution rate, no deaths). All five children with documented generalized dysplasia reported in the literature had died; of the three reported here, one has died and two have become progressively worse.

Adolescent↗
PubMed1979

Sciatic paralysis. A complication of bleeding following hip surgery.

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.

Adolescent↗
PubMed1978

An operative repair of acromioclavicular separation.

Sixteen patients underwent an open reduction for complete acromioclavicular separation with a loop of woven Dacron arterial graft. This technique of internal fixation permits an early range of shoulder motion and eliminates the need for a second procedure to remove wire or pins. Experience in vascular reconstruction suggests that fibroblastic ingrowth might occur to substitute for the disrupted ligament complex. All but two of the 16 patients showed excellent results: full restoration of shoulder function, satisfactory cosmesis, and complete relief of pain. One required reoperation for an improperly placed loop, and one had definite over-reduction of the clavicle on the acromion but remained asymptomatic. In several, X-rays showed evidence of pressure resorption and periosteal new-bone formation along the clavicle at the site of the loop, but no loss of fixation. This approach was used successfully in acute trauma and as late as one year following injury.

Acromioclavicular Joint↗
PubMedDate not supplied

Posterior problems in the knee. Posterior cruciate insufficiency and posterolateral rotatory insufficiency.

During a recent three-year period, 39 patients underwent surgical procedures for lesions of the posterior complex of the knee. The results in 28 followed for greater than six months (average followup, 16 months) are reported; eight are presently in the early rehabilitative phase and three have been lost to followup. In reconstruction for chronic laxity, a semitendinosus replacement of the posterior cruciate ligament with anterior advancement of the lateral arcuate complex is recommended. Use of the medial gastrocnemius tendon is still advised in selected cases. Reconstruction for posterolateral rotatory laxity with an intact posterior cruciate is performed with anterior advancement of the popliteus tendon and the arcuate complex. Pes anserinus transfer, imbrication of the posterior oblique ligament, and biceps tenodesis may be performed as supplemental procedures.

Follow-Up Studies↗
PubMedDate not supplied

Lateral reconstruction for anterolateral rotatory instability of the knee.

Sixty patients underwent surgery for correction of anterolateral rotatory instability using an extraarticular approach. Forty procedures were performed utilizing Hughston's technique which is outlined. This approach is shown to provide effective control of pivot shift and anterolateral rotatory laxity.

Athletic Injuries↗