Biomedical subjects

C R Mueller

Publications and source records attributed to C R Mueller.

At least 37 records · Page 2Linked to original sources

PubMed1997

Casein kinase II-mediated phosphorylation of the C terminus of Sp1 decreases its DNA binding activity.

We have previously observed that Sp1, a ubiquitous zinc finger transcription factor, is phosphorylated during terminal differentiation in the whole animal, and this results in decreased DNA binding activity (Leggett, R. W., Armstrong, S. A., Barry, D., and Mueller, C. R. (1995) J. Biol. Chem. 270, 25879-25884). In this study, we demonstrate that casein kinase II (CKII) is able to phosphorylate the C terminus of Sp1 and results in a decrease in DNA binding activity. This suggests that CKII may be responsible for the observed regulation of Sp1. Mutation of a consensus CKII site at amino acid 579, within the second zinc finger, eliminates phosphorylation of this site and the CKII-mediated inhibition of Sp1 binding. Phosphopeptide analysis confirms the presence of a CKII site at Thr-579 as well as additional sites within the C terminus. No gross changes in CKII subunit levels were seen during de-differentiation associated with liver regeneration. The serine/threonine phosphatase PP1 was identified as the endogenous liver nuclear protein able to dephosphorylate Sp1 but again no gross changes in activity were observed in the regenerating liver. Okadaic acid treatment of K562 cells increases Sp1 phosphorylation and inhibits its DNA binding activity suggesting that steady state levels of Sp1 phosphorylation are established by a balance between kinase and phosphatase activities.

Amino Acid Sequence↗

PubMed1997

A genome wide search for susceptibility loci in three European malignant hyperthermia pedigrees.

Malignant hyperthermia (MH) is an autosomal dominant disorder which is potentially lethal in susceptible individuals on exposure to commonly used inhalational anaesthetics and depolarising muscle relaxants. Crises reflect the consequences of disturbed skeletal muscle calcium homeostasis. Susceptibility was first localised to chromosome 19q13.1 and the skeletal muscle ryanodine receptor, RYR1 (the calcium release channel of the sarcoplasmic reticulum). Defects in this gene have been identified which cosegregate with the MHS phenotype and evidence as to their potential causal roles has accumulated. MH has, however, been shown to be genetically heterogeneous, additional loci on chromosomes 3q, 17q and 7q being proposed. Pedigrees remain in Europe where linkage status is still unclear. In a collaborative search of the human genome conducted with three pedigrees whose disease status was classified according to the European IVCT protocol we have evidence to suggest that at least two further loci exist for MH susceptibility. One of these locates to chromosome 1q, the site of a candidate gene, CACNL1A3, encoding the alpha-subunit of the dihydropyridine receptor. The second region resides on chromosome 5p to where no known candidate has been mapped to date. The third family exhibited inconclusive results which suggests the existence of at least one other locus. This study adds to the evidence for considerable genetic heterogeneity in MH and will provide a route to further our understanding of the molecular pathology of the condition.

Calcium Channels↗

PubMed1996

Genomic structure of the human D-site binding protein (DBP) gene.

The human gene for the D-Site Binding Protein (DBP) has been sequenced and characterized. This gene is a member of the b/ZIP family of transcription factors and is one of three genes forming the PAR subfamily. DBP has been implicated in the diurnal regulation of a variety of liver-specific genes. Examination of the genomic structure of DBP reveals that the gene is divided into four exons and is contained within a relatively compact region of approximately 6 kb. These exons appear to correspond to functional divisions of the DBP protein. Exon 1 contains a long 5' UTR, and conservation between the rat and the human genes of the presence of small open reading frames within this region suggests that it may play a role in translational control. Exon 2 contains a limited region of similarity to the other PAR domain genes, which may be part of a potential activation domain. Exon 3 contains the PAR domain and differs by only 1 of 71 amino acids between rat and human. Exon 4, containing both the basic and the leucine zipper domains, is likewise highly conserved. The overall degree of homology between the rat and the human cDNA sequences is 82% for the nucleic acid sequence and 92% for the protein sequence. Comparison of the rat and human proximal promoters reveals extensive sequence conservation, with two previously characterized DNA binding sites being conserved at the functional and sequence levels.

Amino Acid Sequence↗

PubMed1996

Trocar site seeding of inapparent gallbladder carcinoma during laparoscopic cholecystectomy.

There are increasing reports of seeding of tumor at the trocar sites following laparoscopic cholecystectomy in patients with unexpected or inapparent gallbladder carcinoma. A patient is reported here whose primary tumor appeared controlled by surgery and radiation, but who died of the disease after developing implant metastases at three untreated trocar sites. The second case report illustrates the difficulty in identifying gallbladder cancer during laparoscopic cholecystectomy, and the importance of a diligent preoperative effort to establish the diagnosis. Current literature suggests that tumor implantation occurring during laparoscopic cholecystectomy for inapparent carcinoma adversely affects prognosis, and, until the effect of laparoscopy on the spread of this tumor is better understood and controlled, open operation should be performed when carcinoma of the gallbladder is suspected. When laparoscopic cholecystectomy is done for inapparent gallbladder cancer, surgical and adjuvant radiotherapy to the trocar sites appears to improve outcome in association with extended treatment to the gallbladder bed and adjacent areas. Recent reports suggest that progress in diagnostic, surgical, and adjuvant techniques could substantially improve survival in carcinoma of the gallbladder.

Adenocarcinoma↗

PubMed1996

Binding of the Ets factor GA-binding protein to an upstream site in the factor IX promoter is a critical event in transactivation.

Factor IX is an essential vitamin K-dependent serine protease that participates in the intrinsic pathway of coagulation. The protein is expressed exclusively in the liver. The rare Leyden form of hemophilia B (inherited factor IX deficiency) results from point mutations in three proximal promoter elements that decrease factor IX expression. Recovery of expression occurs following puberty, with factor IX protein levels rising into the normal range. We have previously implicated the PAR domain D-site-binding protein (DBP) as well as an upstream element, site 5, as playing important roles in the phenotypic recovery of hemophilia B Leyden. Here we demonstrate that site 5 binds both the CCAAT/enhancer-binding protein (C/EBPalpha) and the ubiquitous Ets factor GA-binding protein (GABPalpha/beta). Transactivation of the factor IX promoter by the PAR proteins DBP and hepatic leukemia factor (HLF) is dependent on the binding of GABPalpha/beta to site 5, and coexpression of these two factors is required for optimal activation of this promoter. The binding of C/EBPalpha to site 5 also augments the activity of GABPalpha/beta. Analysis of the developmental regulation of site 5-binding proteins in rat liver has shown that C/EBPalpha and the GABPbeta subunit increase markedly in the 2 weeks after birth. These observations establish a functional association between the Ets factor GABPalpha/beta and C/EBPalpha and indicate that the two PAR proteins, DBP and HLF, may play complementary roles in factor IX activation. Given the developmental changes exhibited by these proteins, it is likely that they play a role in regulation of the normal factor IX promoter as well as promoters carrying hemophilia B Leyden mutations.

Aging↗

PubMed1996

Role of the liver-enriched transcription factor hepatocyte nuclear factor 1 in transcriptional regulation of the factor V111 gene.

Coagulation factor VIII is an essential cofactor required for normal hemostatic function. A deficiency in factor VIII results in the bleeding disorder hemophilia A. Despite the fact that the factor VIII gene was cloned a decade ago, the mechanisms which control its transcription remain unresolved. In our studies, we have characterized 12 protein binding sites within the factor VIII promoter by DNase I protection assays performed with rat liver nuclear extracts. Three of these elements (sites 1 to 3) are situated within the 5' untranslated region of the gene, while three other sites (sites 4 to 6) lie within the first 100 bp upstream of the transcriptional start site. We have identified an additional site (site 7) approximately 300 bp upstream from site 6, as well as a cluster of five sites in a 250-bp region which terminates approximately 1 kb from the transcriptional start site. Seven of these binding sites (sites 2, 3, 4, 6, 7, 9, and 10) bind members of the C/EBP family of transcription factors. DBP also binds to five of these sites (sites 3, 4, 6, 7, and 9). Utilizing transient transfection studies in HepG2 cells, we have shown that deletion of the factor VIII promoter sequences distal to nucleotide -44 results in a significant but small increase in promoter activity. The activity of each of the various 5' deletion constructs is significantly enhanced by cotransfection of C/EBPalpha and D-site-binding protein expression plasmids, while cotransfection of both C/EBPalpha and C/EBPbeta plasmids resulted in a further enhancement of transactivation. These studies also provide evidence of a repressor element located between nucleotides -740 and -1002. Since the minimal promoter sequence (-44 to +148) maintains the transcriptional activity of the full-length promoter sequence, we proceeded to identify additional factors binding to sites 1 to 4. Competition studies revealed that a ubiquitous transcription factor, NF-Y, binds to site 4, while the liver-enriched transcription factor hepatocyte nuclear factor I (HNF-1) binds to site 1. Mutation analysis of the minimal promoter demonstrated that HNF-1 is critical for activating transcription of the factor VIII gene in vitro. Our results also suggest that the multiple upstream elements that we have identified may act as a backup regulatory region in the event of disruption of the HNF-1 element in the 5' untranslated region.

Animals↗

PubMed1995

Sp1 is phosphorylated and its DNA binding activity down-regulated upon terminal differentiation of the liver.

Using nuclear extracts prepared from rat liver it was demonstrated that binding of a transcription factor to site II of the D-site binding protein promoter could be induced by dephosphorylation of these extracts. Competition band shifts and supershift assays reveal this protein to be the general transcription factor Sp1. Phosphorylation of Sp1 appears to occur as a result of terminal differentiation of the liver. Proteins from both 1-day-old rat liver and adult liver undergoing regeneration have less of the phosphorylated form of Sp1 present with consequent increased DNA binding activity. Sp1 is similarly phosphorylated in brain, kidney, and spleen with phosphatase treatment of the extracts significantly increasing the level of DNA binding activity. Dephosphorylation of Sp1 results in a 10-fold increase in the affinity of Sp1 for its cognate site. Two-dimensional gel electrophoresis reveals that approximately 20% of the detectable protein appears to be in the phosphorylated form in adult liver extracts. Another protein with similar characteristics also appears to be present in the liver. Decreasing Sp1 DNA binding activity by phosphorylation may be an important mechanism for regulating gene expression, and possibly bringing about growth arrest during terminal differentiation.

Animals↗

PubMed1995

Treatment of metastatic carcinoid tumors using multimodality therapy of octreotide acetate, intra-arterial chemotherapy, and hepatic arterial chemoembolization.

BACKGROUND: Overall survival and quality of life in patients with metastatic carcinoid tumors depend on control of tumor growth and suppression of amine-induced symptoms. METHODS: We report on a series of 10 patients with carcinoid tumors metastatic to the liver who were treated with long-term octreotide acetate therapy (100 to 500 micrograms three times a day), sequential intra-arterial 5-fluorouracil (5-FU) infusions, and hepatic tumor chemoembolization. RESULTS: All 10 patients remained asymptomatic or had extremely mild symptoms after combined modality therapy (mean follow-up duration of 51.5 months). Sixty percent of the patients had a > 50% reduction of their tumor size (mean duration 42 months). An additional 30% experienced stabilization of tumor growth for 6 months or longer. Five of the 10 patients are currently alive. The mean group survival is 58 months since diagnosis (range 33 to 115) and 40 months since starting therapy (range 12 to 65). CONCLUSIONS: Combining octreotide acetate, intra-arterial 5-FU, and tumor chemoembolization effectively retards tumor growth while providing excellent symptom control.

Aged↗

PubMed1994

Transcriptional control of the factor IX gene: analysis of five cis-acting elements and the deleterious effects of naturally occurring hemophilia B Leyden mutations.

Hemophilia B Leyden is a rare form of inherited factor IX deficiency in which patients experience spontaneous postpubertal recovery of factor IX levels. The mutations resulting in this disorder are localized in a 40-nucleotide region encompassing the major transcriptional start site for factor IX. Here we report the further characterization of five cis-acting elements in the factor IX promoter and the effects on protein binding and transcriptional activation of five Leyden mutations (at nucleotides +13, -5, -6, -20, and -26) that occur within the proximal three elements (sites 1 through 3). Bandshift studies using nuclear extracts from four different rat tissues have shown that at least some of the proteins binding to each of the five sites are ubiquitous in nature. The pattern of DNA binding at site 1 suggests that this element plays an important role in mediating the liver-specific expression of factor IX. Additional studies with liver nuclear extracts obtained at several different points in development have shown an increase in DNA binding at sites 1, 4, and 5 between 1 day and 1 week. Using DNase I footprint analysis and competition bandshift studies, we have shown that the binding of nuclear proteins to each of the mutant sites is disrupted to a variable extent. There appears to be some, although reduced, protein binding to all of the mutant oligonucleotides apart from the -26 mutant. In vitro transcription assays have shown that each of the mutations reduces the global proximal promoter activity by approximately 40%. Two double mutant promoters did not show any additional downregulation in the in vitro transcription assay. In experiments designed to assess the relative transcriptional activity mediated from each of the five sites independently, we have tested artificial homopolymer promoters of each site in the in vitro transcription assay. These studies show that sites 4 and 5 are the strongest activators and that transactivation from site 5 is further enhanced by the albumin D site-binding protein. In summary, these investigations show deleterious effects of each of the Leyden mutations tested on the binding of trans-acting factors and also show disruption of transcriptional activation in a functional in vitro transcription assay. Our results also show that cis-acting elements 4 and 5 are the principal activators of this locus.

Animals↗

PubMed1994

Pharmacokinetics and results of dose escalation in cis-platin hyperthermic isolation limb perfusion.

BACKGROUND: We analyzed prospectively collected data on 145 cis-platin hyperthermic isolation limb perfusion (HILPs) for melanoma and soft-tissue sarcoma to determine the pharmacokinetics and maximum tolerable dose of cis-platin. There were 70 melanoma and 75 sarcoma patients. Dosages ranged from 26 to 265 mg/m2. Perfusate and systemic cis-platin levels were measured in patients perfused at doses of 190-200 mg/m2. Tissue levels were measured in patients perfused at 123-209 mg/m2. METHODS: Cis-platin HILP was well tolerated up to doses of 250 mg/m2 for lower extremities. Higher doses produced toxicities of rhabdomyolysis, myoglobinuria, hyponatremia, and neuropathy. Systemic levels of cis-platin were equivalent to those of routine intravenous administration, while perfusate levels were 33 times higher. Tissue levels of cis-platin were five to six times higher than effective intravenous levels. RESULTS: Six melanoma patients have developed local recurrences. All were perfused at doses < 120 mg/m2. However, regional nodal recurrences have occurred in six other patients perfused at doses < or = 200 mg/m2. Four sarcomas have recurred locally, but three of them were present at the time of perfusion. CONCLUSIONS: We conclude that 250 mg/m2 is the maximum tolerable dose of cis-platin for lower-extremity HILPs. Neoadjuvant cis-platin HILP may improve local control rates for sarcomas. However, no tolerable dose of cis-platin provides control of nodal metastases from melanoma.

Adult↗

PubMed1994

Retinoblastoma control element binding proteins are induced during in vivo terminal differentiation of the liver and bind to the DBP promoter.

Growth regulation of terminal differentiation was studied by analysis of the promoter of the transcription factor D-site binding protein (DBP) using nuclear extracts from rat liver undergoing regeneration or from normal liver early in development. Four proximal binding sites for nuclear proteins were identified, three of which showed significant quantitative or qualitative changes in associated factors upon growth induction. Two of these sites bound factors common to the retinoblastoma control element and these proteins were induced upon terminal differentiation of the liver. These factors were also rapidly down-regulated upon induction of regenerative growth and consequent dedifferentiation. These results provide a link between the regulation of growth and differentiation in an in vivo model system.

Animals↗

PubMed1993

Synergy between transcription factors DBP and C/EBP compensates for a haemophilia B Leyden factor IX mutation.

Haemophilia B Leyden is characterized by low childhood levels of factor IX which gradually increase after puberty, eventually resulting in a return to health. The disease is the result of single nucleotide substitutions within a 40 bp region encompassing the major transcriptional start site. We have characterized transcription factor binding sites within the factor IX promoter. Five sites were identified and a Leyden mutation at nucleotide -5 was shown to interfere with the binding of proteins to one of three newly identified sites. The correlation between the post-pubertal recovery of these mutants and the induction of the transcription factor DBP led us to the discovery of a synergistic interaction between DBP and C/EBP responsible for the recovery of normal transcriptional activity of the -5 mutant promoter and may play a role in the resolution of other Leyden mutants.

Adolescent↗

PubMed1992

The carbamyl phosphate synthetase promoter contains multiple binding sites for C/EBP-related proteins.

The promoter of the gene (CPS) encoding rat carbamyl phosphate synthetase I has been mapped 5' to a segment of about 525 nucleotides upstream from the transcription start point and, when analyzed in liver nuclear extracts, contained six well-defined protein-recognition elements, designated CPS sites I-VI. All six elements were recognized, with varying affinities, by CAAT and enhancer-binding protein (C/EBP alpha) produced in bacteria. Oligodeoxyribonucleotides corresponding to CPS site II or to the C/EBP alpha-recognition element of the ALB promoter, site D, competed with the six CPS-promoter elements in footprinting assays. However, mutagenesis of the C/EBP alpha-recognition element, 5'-GTTGCAAC, at the core of site II was sufficient to abolish transactivation of the CPS promoter by C/EBP alpha in co-transfected HepG2 cells. These findings indicate that the CPS promoter contains multiple recognition elements for factors with DNA-binding specificities similar to C/EBP proteins. Activation by C/EBP alpha, however, requires promoter site II.

Animals↗

PubMed1992

Effective palliative treatment of metastatic carcinoid tumors with intra-arterial chemotherapy/chemoembolization combined with octreotide acetate.

Survival in patients with metastatic carcinoid tumors is dependent on control of tumor growth and adequate palliation of vasoactive amine-induced symptoms of flushing, diarrhea, wheezing, and valvular heart disease. Eight patients with carcinoid tumors metastatic to the liver were treated with long-term octreotide acetate therapy (100 to 500 micrograms three times a day), intra-arterial 5-fluorouracil infusion (2 g/day x 5 days), and hepatic tumor chemoembolization. All eight patients became asymptomatic and have remained so with a mean follow-up duration of 22 months from the time of first infusion. Following institution of subcutaneous octreotide acetate, intra-arterial infusion, and tumor chemoembolization, all patients are alive with a mean survival of 40 months from the time of diagnosis of carcinoid syndrome (range: 2 to 108 months). Four patients had greater than a 50% decrease in tumor size after therapy (mean follow-up duration: 10.6 months), and the other four patients have had stable disease after institution of therapy. It appears that combinations of long-term subcutaneous administration of octreotide acetate, intra-arterial 5-fluorouracil, and tumor chemoembolization effectively control progressive liver metastasis and provide excellent symptomatic palliation in patients with hepatic metastasis from functional carcinoid tumors.

Carcinoid Tumor↗

PubMed1992

The down-regulation of albumin transcription during regeneration is due to the loss of HNF-1 and the D-site transcription factors.

Liver regeneration has served as an important in vivo model for studying the control of differentiation. The molecular mechanisms responsible for the negative regulation of the albumin promoter during regeneration have not been examined previously. Changes in the proteins interacting with the albumin promoter were characterized using nuclear extracts prepared from the livers of rats undergoing chemically induced regeneration. Reduction in the activities of both hepatocyte nuclear factor-1 (HNF-1) and factors binding to the D site are responsible for the loss of albumin transcription during regeneration. No evidence for the involvement of a transcriptional repressor in this process was found.

Albumins↗

PubMed1991

Acute gastric pH changes alter intraluminal but not plasma peptide levels.

Gastric acidity is influenced by systemic and local peptide effects. Previous work by others has shown that intraluminally secreted peptides may have a role in local control of gastric acidity; however, the response of these peptides to acute changes in gastric pH is unknown. To determine the effects of acute changes in pH on systemic and intraluminal peptide levels, 14 normal volunteers underwent placement of a nasogastric tube after an overnight fast. Blood and gastric fluid were analyzed on a control day, 2 hours after completion of 24 hours of aluminum-magnesium antacid therapy and after 24 hours of H2 blockade. Plasma and acid-alcohol-extracted gastric peptide levels were measured with specific radioimmunoassays. Specimens were subdivided into two groups: 28 gastric fluid specimens with a pH less than 4 and 10 specimens with a pH greater than 4. In the patients with a pH greater than 4, the luminal peptides, motilin, neurotensin, pancreatic polypeptide, somatostatin, substance P, and gastrin, were decreased by 50% to 90% and gastrin-releasing peptide was decreased by 36% compared with specimens with a pH less than 4. Conversely, intraluminal vasoactive intestinal polypeptide and calcitonin levels were elevated by 60% and 27%, respectively, in the samples with a pH greater than 4. Intraluminal peptide concentrations are responsive to changes in intragastric pH; however, this response was not seen in plasma peptide levels.

Antacids↗

PubMed1990

DBP, a liver-enriched transcriptional activator, is expressed late in ontogeny and its tissue specificity is determined posttranscriptionally.

The full-length cDNA for a transcriptional activator, DBP, that binds to the D site of the albumin promoter has been cloned. DBP belongs to a family of related transcription factors including Fos, Jun, CREB, and C/EBP, which share a conserved basic domain. However, unlike most other members of this family, DBP does not contain a "leucine zipper" structure. Among several rat tissues tested, significant levels of its protein are only observed in liver; yet, with the exception of testis, DBP mRNA is present in all of the examined tissues. DBP as well as its mRNA accumulate to significant levels only in adult animals. During chemically induced liver regeneration, DBP expression is rapidly down-regulated, suggesting that DBP may be involved in the proliferation control of hepatocytes. This cell growth-dependent expression of DBP, in contrast to its tissue specificity, appears to be controlled at the level of mRNA accumulation.

Aging↗

PubMed1990

Differential in vitro transcription from the promoter of a rat alpha 2u globulin gene in liver and spleen nuclear extracts.

When used in an in vitro transcription assay, the promoter of a cloned alpha 2u globulin gene is much more active in liver than in spleen nuclear extracts. Promoter deletion experiments suggest that both positive and negative regulatory mechanisms may be involved in the differential in vitro transcription from the alpha 2u globulin promoter in these two nuclear extracts. Interestingly, removal of promoter elements upstream from position -74 results in a significant increase of in vitro transcription in spleen but not in liver nuclear extracts, and thus reduces the difference in transcription observed with longer alpha 2u promoters in these two extracts. Deletion of additional nucleotides to position -43 strongly reduces the in vitro transcription efficiency of the promoter in extracts from both tissues. None of the examined promoters containing between 3000 and 22 nucleotides of 5' flanking regions are differentially transcribed in liver nuclear extracts from either male or female rats. Thus, in contrast to cell-type specificity, sex-specificity could not be observed in our in vitro transcription experiments. DNase I protection experiments with crude nuclear extracts and partially or highly purified nuclear proteins suggests the presence of six recognition sites for DNA-binding factors between the TATA element and position -210. Some of these factors could be identified as proteins that also bind to elements within the albumin gene promoter.

Alpha-Globulins↗