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Biomedical subjects

E Moran

Publications and source records attributed to E Moran.

At least 55 records · Page 3Linked to original sources
PubMed1996

A potential role for cell cycle control proteins in regulation of the cyclic adenosine 5'-monophosphate-responsive glycoprotein hormone alpha subunit gene.

The production of chorionic gonadotropin is coupled to the differentiation of the placenta. Expression of the alpha subunit of chorionic gonadotropin [glycoprotein hormone alpha (GPH-alpha)] is also known to be stimulated by treatment of placental cells with either cAMP or DNA synthesis inhibitors. Given these features, we used adenovirus E1A as a molecular probe to investigate a potential role for cell cycle regulatory proteins and kinases in the regulation of GPH-alpha expression. The E1A protein contains well-characterized domains that interact with a variety of cell cycle regulatory proteins. The E1A conserved regions 1 and 2 bind proteins that regulate cell cycle progression, including pRB, p107, and p130. The amino-terminal region of E1A binds several high molecular weight proteins and inhibits the transcriptional coactivator function of p300 and the homologous cAMP response element (CRE)-binding protein. We found that coexpression of E1A13S activated the GPH-alpha promoter, whereas E1A12S caused marked repression. Deletion mutants and point mutations revealed that repression by E1A12S required the CRE of the GPH-alpha promoter. Several distinct domains in E1A12S were necessary for maximal repression. A mutation of the E1A amino terminus (RG2), which inhibits binding of p300 and related high molecular weight proteins, reduced 12S repression by 40%. Mutation of the pocket protein-binding domains reduced repression by 20%, and mutations of both domains reduced repression by 80%. Overexpression of p300 or the pocket proteins (pRB, p130, and p107) induced GPH-alpha promoter activity 2-4-fold. Because the E1A amino terminus and pocket protein-binding domains together induce p34cdc2 kinase activity, the effect of p34cdc2 kinase expression on GPH-alpha activity was also assessed. Coexpression of p34cdc2 kinase or the activating p34cdc2 kinase mutant (T14AY15F) inhibited GPH-alpha promoter activity and acted through the CRE. We conclude that the GPH-alpha gene CRE is subject to regulation by cell cycle regulatory kinases and proteins.

Adenoviridae↗
PubMed1996

Behavior of a novel monoclonal antibody for investigation of ovarian cancer and with possible involvement in multiple drug resistance.

Monoclonal antibodies (MAbs) were raised to an ovarian cancer cell line, OAW42, derived from a patient with a histology of serous cystadenocarcinoma of the ovary, in an attempt to identify novel antigens with a possible role in cancer medicine. One antibody P1H10, subclass IgG1, with a high titer was isolated and shown to recognize an antigen of 48 kDa. Enzyme-linked immunosorbent assay and immunocytochemical studies showed the presence of the target antigen in a number of carcinoma cell lines, including lung and breast, and in two out of three frozen breast tissue specimens. The antigen was not detected in normal human lymphocytes and there was minimal binding of the antibody to normal buccal cells. The antigen was not secreted by the OAW42 or the HepG2 cell lines and was not detected in the sera of a number of ovarian cancer patients. Indirect immunofluorescence studies confirmed the localization of the antigen to be intracellular. The binding of the antibody P1H10 to a number of multidrug resistant variants of the OAW42 cell line showed that the presence and the localization of the antigen in the drug-sensitive parental line and resistant variant cell lines was distinctly different and varied with the degree of drug resistance. The relative specificity of the antibody suggests it may be a possible diagnostic agent in human cancer. A possible role of the antigen in multiple drug resistance (MDR) is also suggested.

Animals↗
PubMed1995

Relief of YY1 transcriptional repression by adenovirus E1A is mediated by E1A-associated protein p300.

YY1 represses transcription when bound upstream of transcriptional initiation sites. This repression can be relieved by adenovirus E1A. Here, we present genetic evidence that the ability of E1A to relieve YY1 repression was impaired by mutations that affect E1A binding to its associated protein p300. This suggests that E1A may modulate the repressor activity of YY1 by binding to p300, which may be physically complexed with YY1. A YY1/p300 protein complex in vivo was demonstrated by several independent approaches, and the YY1-interacting domain was mapped to the carboxy-terminal region of p300, distinct from the E1A-binding site. Unlike E2F/RB, the YY1/p300 complex is not disrupted by E1A. Functional studies using recombinant p300 demonstrated unequivocally that p300 is capable of mediating E1A-induced transcriptional activation through YY1. Taken together, these results reveal, for the first time, a YY1/p300 complex that is targeted by E1A and demonstrate a function for p300 in mediating interactions between YY1 and E1A. Our data thus identify YY1 as a partner protein for p300 and uncover a molecular mechanism for the relief of YY1-mediated repression by E1A.

Adenoviridae Infections↗
PubMed1995

Arthritis not immobilization causes bone loss in the carrageenan injection model of inflammatory arthritis.

One suggested cause of the high turnover osteopenia of experimental inflammatory arthritis is disuse of affected joints. To compare the influence of immobilization or disuse, or both, with that of inflammatory arthritis on bone turnover, rabbits were placed into four groups. In group 1, arthritis was induced in the right knee by seven intra-articular injections of 1% carrageenan, over 49 days; in group 2, a plaster cast was applied to immobilize the right hindlimb in flexion; in group 3, arthritis was induced and the hindlimb was immobilized; and in group 4, nothing was done (control). The fluorescent label calcein was administered in drinking water (0.05%) ad libitum to all groups on days 22-36. On day 49, specimens were prepared for analysis of bone volume and new bone volume at a near site (right femur) and at remote sites (contralateral femur and ipsilateral humerus). The data were analysed by multiple regression and Bonferroni tests. In group 1, new bone volume was three times higher than in group 2 or 4 (p < 0.05 for each comparison); this indicated increased bone remodeling in the right femur. This contrasted with group 2, in which neither index of bone remodeling was changed. The combination of immobilization with arthritis resulted in more intense osseous effects of inflammatory arthritis, with a one-quarter decrease in bone volume (group 3, 30.99 +/- 2.50; group 4, 42.07 +/- 2.38, p < 0.05), as well as a 4-fold increase in new bone volume (p < 0.001) compared with group 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1995

E1A promotes association between p300 and pRB in multimeric complexes required for normal biological activity.

The oncogenes of the small DNA tumor viruses encode transforming proteins with multiple domains that influence the cell cycle and aspects of the transformed phenotype. Like other gene products of this type, the adenovirus E1A proteins influence the cell by binding to specific cell growth control proteins. These include members of the retinoblastoma gene product (pRB) family, which are bound by the E1A region 2-specific site, and p300, which is bound at the E1A amino terminus. Binding at these two sites is largely independent, and discrete transcription-regulating functions remain intact in E1A products when only one or the other binding site is functional. In this report, immunoprecipitation with p300 antibodies reveals the presence of the pRB family proteins in p300 complexes when E1A is expressed in host cells, indicating that E1A can mediate physical contact between p300 and the pRB-related proteins. The ability of E1A to induce proliferation efficiently in quiescent primary cells correlates closely with the ability to bind p300 and individual members of the pRB family simultaneously in multimeric complexes, even though the E1A active sites can bind their target proteins efficiently when separated on different molecules. Conservation of a spacer region between the two binding sites that is required for simultaneous binding and efficient induction of proliferation supports the concept that the E1A protein structure has evolved to facilitate simultaneous binding. These results indicate that the E1A proteins are designed not merely to sequester these cellular products, but also to bring them into proximal association with each other in biologically significant complexes.

Adenoviridae↗
PubMed1994

Reliability and validity of the grading of heterotopic ossification.

The purpose of this study is to assess the reliability and validity of the Brooker grading of heterotopic ossification. Anteroposterior hip radiographs of 77 patients, taken 6 months following total hip arthroplasty, were blindly evaluated twice by two surgeons using the Brooker scale. Patients were also evaluated with the Harris hip scale, and range of motion of the hip was determined. All inconsistencies in heterotopic ossification grading were reviewed by the same two surgeons who developed six points of clarification to minimize ambiguities in the Brooker grading criteria. A second set of 76 radiographs were reviewed by the two surgeons using this modified Brooker index. The intraobserver reliability of the two readers was good, with agreements of 77 and 86% and weighted kappa values of 0.63 and 0.69, respectively. The two surgeons demonstrated fair interobserver reliability on the first reading (agreement of 68% and weighted kappa of 0.57) that improved to a good level of reliability (agreement of 77% and weighted kappa of 0.68) using the revised Brooker index. The grade of heterotopic ossification was correlated (r = -.25, P = .005) with the aggregate range of hip motion, but had no relationship to the patient's Harris rating. In conclusion, the grading of heterotopic ossification is reliable. Although the severity of heterotopic ossification did not correlate with the Harris scale, the relationship between heterotopic ossification and range of motion indicates that the Brooker index is also a valid measurement.

Hip Joint↗
PubMed1994

Heterotopic ossification after primary cemented and noncemented total hip arthroplasty in patients with osteoarthritis and rheumatoid arthritis.

OBJECTIVE: To compare the frequency and severity of heterotopic ossification (HO) in patients with osteoarthritis or rheumatoid arthritis who undergo cemented or noncemented total hip arthroplasty. DESIGN: A prospective case study. SETTING: A university referral centre. PATIENTS: One hundred and sixty one patients underwent 184 total hip arthroplasties. The 184 hips were categorized as follows: cemented total hip arthroplasty--60 osteoarthritis hips, 26 hips affected by rheumatoid arthritis; noncemented total hip arthroplasty--67 osteoarthritic hips, 31 hips affected by rheumatoid arthritis. INTERVENTIONS: Total hip arthroplasty. A standard approach was used to implant either a cemented or noncemented prosthesis. Radiographs were obtained of each hip preoperatively, immediately postoperatively and 6 weeks, 3 months, 6 months and 1 year postoperatively. MAIN OUTCOME MEASURES: Radiographs were graded in blind fashion for HO, according to the criteria of Brooker. Modified Harris hip scores were calculated at 1 and 2 years postoperatively. RESULTS: HO (incidence of Brooker grades 2, 3 and 4) was greater after cemented (22%) than noncemented (9%) total hip arthroplasty in osteoarthritic hips (p < 0.05), but there was no significant difference between the two types of prosthesis in hips affected by rheumatoid arthritis. When both types of prosthesis were considered together there was no significant difference in the incidence of HO between osteoarthritic hips (15%) and hips affected by rheumatoid arthritis (12%). With respect to Harris hip score, the presence of grade 4 HO was associated with a significantly (p < 0.05) lower score (50.6) than grade 3 (89.4) and grade 2 (91) HO. CONCLUSIONS: Cemented total hip arthroplasty increases the frequency of HO only in osteoarthritic hips. Compared with Brooker's grades 2 and 3 HO, grade 4 HO significantly lowers the Harris hip score after total hip arthroplasty.

Adult↗
PubMed1993

Healing of cancellous bone osteotomy in rabbits--Part I: Regulation of bone volume and the regional acceleratory phenomenon in normal bone.

We performed a simple, undisplaced, incomplete osteotomy of the distal medial condyle of mature New Zealand White rabbits and permitted healing without fixation for 4 weeks. During the recovery period, the rabbits received the bone label calcein in their drinking water. From ground stained and unstained sections, we determined histomorphometric parameters in five zones: osteotomy gap, juxtaosteotomy, lateral condyle, metaphysis, and diaphysis. At 4 weeks, newly formed cancellous bone had obliterated the osteotomy gap. Osteotomy also stimulated new bone formation in cancellous bone of the medial condyle around the osteotomy. At that site, we found a fivefold increase in new bone without a change in bone volume (BV). Stimulation of bone formation was not observed in cancellous bone of the lateral condyle that had not been operated on or in cortical bone of the metaphysis or diaphysis. The maintenance of BV, despite increased new bone formation in the osteotomized medial femoral condyle, was probably due to a local acceleration of bone remodeling.

Animals↗
PubMed1993

Healing of cancellous bone osteotomy in rabbits--Part II: Local reversal of arthritis-induced osteopenia after osteotomy.

With use of the intravital bone label calcein, we previously identified, by histomorphometry, greatly increased new bone formation and juxtaarticular osteopenia (a high turnover state) in carrageenan-induced experimental inflammatory arthritis of the knee in rabbits. In Part I of this paper, we showed that a large increase in new bone formation after a stable incomplete distal femoral condylar osteotomy of normal bone in the rabbit does not increase bone volume (BV) in normal tissue adjacent to the osteotomy (zone 1). In this study, we performed an osteotomy of bone made osteopenic by experimental inflammatory arthritis. After 4 weeks of healing, we examined new bone formation and BV in five zones around the osteotomy. In zone 0 (the osteotomy gap), new bone formation filled the gap to normal BV, as we found for the normal/osteotomy group. In zone 1 (adjacent to but excluding the osteotomy gap), we found greatly increased new bone formation and a return to normal BV, increased from the osteopenic level induced by experimental inflammatory arthritis. In this model, osteotomy apparently reversed the bone-wasting process of inflammatory arthritis. This effect was not observed in other zones: the lateral condyle that had not been operated on, the metaphysis, or the diaphysis.

Animals↗
PubMed1993

DNA tumor virus transforming proteins and the cell cycle.

Studies using the transforming proteins of the small DNA tumor viruses point to the role of the E2F cellular transcription factor in regulating cell cycle specific gene expression. The evidence derived suggests that E2F is controlled by the retinoblastoma gene product and related proteins, including their associated cyclins. Studies using DNA tumor virus products further suggest the existence of additional pathways of cell-cycle activation.

Amino Acid Sequence↗
PubMed1993

Multiple drug resistance in the human ovarian carcinoma cell line OAW42-A.

A new multidrug-resistant variant (OAW42-A) of a human ovarian carcinoma line has been selected by exposure to increasing concentrations of doxorubicin. The variant is resistant to doxorubicin, vincristine (but surprisingly not to colchicine), etoposide, tenoposide and also to cisplatin (a drug not usually involved in classical multidrug resistance), but not to 5-fluorouracil. Overexpression of P-glycoprotein in the resistant line was demonstrated by immunofluorescence and western blotting. Direct evidence for P-glycoprotein as a determinant of resistance was provided by transfection with a specific antisense oligonucleotide. Reversal was incomplete and this, along with the pattern of cross-resistance observed, suggests that additional mechanisms of resistance may also be involved. Substantial clonal variation in resistance exists within the cell line.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Interaction of adenoviral proteins with pRB and p53.

The transforming gene products of the small DNA tumor viruses subvert host cell growth control mechanisms by binding to specific cell regulatory proteins. These include the retinoblastoma gene product (pRB) and p53. One indication of the pivotal roles played by these regulatory products is the observation that they are each targeted consistently by viruses of several groups, by adenoviruses, the human papillomaviruses, and the papovaviruses. In adenovirus, pRB and p53 are targeted by the E1A and E1B genes, respectively. The genetic probes made possible by manipulation of the virus genes in vitro have helped to illuminate the pathways in which pRB and p53 function. E1A studies have contributed to our current understanding that the retinoblastoma product is one of a family of related proteins, which with associated cyclins and kinases can modulate the activity of the cellular E2F transcription factor. E1B studies have helped explore models of p53 function, including the suggestion that p53, probably through aspects of its transcription regulating activity, can initiate a pathway in which programmed cell death can be invoked to stop unrestricted cell proliferation.

Adenovirus E1A Proteins↗
PubMed1993

Identification of specific adenovirus E1A N-terminal residues critical to the binding of cellular proteins and to the control of cell growth.

Adenovirus early region 1A (E1A) oncogene-encoded sequences essential for transformation- and cell growth-regulating activities are localized at the N terminus and in regions of highly conserved amino acid sequence designated conserved regions 1 and 2. These regions interact to form the binding sites for two classes of cellular proteins: those, such as the retinoblastoma gene product, whose association with the E1A products is specifically dependent on region 2, and another class which so far is known to include only a large cellular DNA-binding protein, p300, whose association with the E1A products is specifically dependent on the N-terminal region. Association between the E1A products and either class of cellular proteins can be disrupted by mutations in conserved region 1. While region 2 has been studied intensively, very little is known so far concerning the nature of the essential residues in the N-terminal region, or about the manner in which conserved region 1 participates in the binding of two distinct sets of cellular proteins. A combination of site-directed point mutagenesis and monoclonal antibody competition experiments reported here suggests that p300 binding is dependent on specific, conserved residues in the N terminus, including positively charged residues at positions 2 and 3 of the E1A proteins, and that p300 and pRB bind to distinct, nonoverlapping subregions within conserved region 1. The availability of precise point mutations disrupting p300 binding supports previous data linking p300 with cell cycle control and enhancer function.

Adenovirus E1A Proteins↗
PubMed1993

The E1A products of oncogenic adenovirus serotype 12 include amino-terminally modified forms able to bind the retinoblastoma protein but not p300.

The cell growth-regulating properties of the adenovirus type 5 (Ad5) E1A oncogene correlate closely with the binding of the E1A products to specific cellular proteins. These proteins include the products of the retinoblastoma tumor susceptibility gene and a 300-kDa product, p300. pRB binds to E1A sequences that are highly conserved among the E1A products of various serotypes, while p300 binding requires sequences in the E1A amino terminus, a region that is not highly conserved. To help evaluate the roles of the E1A-associated proteins in cell growth control, we have compared the p300-binding abilities of the E1A products of Ad5 and of the more oncogenic Ad12 serotype. We show here that despite encoding a sequence that varies somewhat from the p300-binding sequences of Ad5 E1A, the Ad12 E1A products associate with p300 with an affinity similar to that of the Ad5 E1A products. Both the 12S and 13S splice products of Ad12 E1A, like those of Ad5 E1A, encode proteins able to associate with p300. Interestingly, though, both also give rise to prominent forms that are amino terminally modified and unable to associate with p300. This modification, at least in the 13S product, does not appear to diminish the affinity of this product for the retinoblastoma protein.

Adenovirus E1A Proteins↗
PubMed1993

p300, and p300-associated proteins, are components of TATA-binding protein (TBP) complexes.

The transforming proteins encoded by the adenovirus E1A gene bind to a 300-kDa cellular product, p300, via the N-terminal E1A sequences. Residues important for p300 binding are required for the transformation function of E1A and for other E1A-mediated gene-regulating functions, including activation of cell cycle-regulated products and repression of tissue-specific enhancer activity. Recent evidence indicates that p300 is a DNA-binding protein with specific affinity for known enhancer motifs, suggesting that p300 may be a component of transcription factor complexes. The possibility that upstream element-binding factors might interact with basal transcription factors led us to investigate whether p300 interacts, directly or indirectly, with the TATA-binding protein (TBP). We report here that TBP-specific immunoprecipitations show a 300-kDa protein co-precipitating with TBP. This protein is lost from the precipitated material if the lysates are boiled in sodium dodecyl sulfate prior to immunoprecipitation, implying that its presence does not result from non-specific antibody cross-reactivity, but is dependent on specific association with TBP. The TBP-associated 300-kDa protein and p300 originally defined by E1A association show indistinguishable partial proteolytic digest patterns, indicating that these are identical or closely related species. Moreover, p300-specific complexes and TBP-specific complexes include at least two additional common polypeptide species, phosphoproteins of 64 and 59 kDa. These results suggest that p300 interacts with TBP, possibly through intermediate protein-protein associations. They thus provide additional biochemical evidence for postulated protein-protein interactions between upstream regulatory factors and the basal transcriptional machinery.

Antibodies↗
PubMed1992

Multiple drug-resistance in variant of a human non-small cell lung carcinoma cell line, DLKP-A.

A 300-fold adriamycin resistant variant (DLKP-A) of the human lung squamous cell carcinoma line DLKP was established by stepwise selection in increasing concentrations of adriamycin. Different levels of cross-resistance were observed towards VP-16, VM-26, colchicine, vincristine and, somewhat unexpectedly, cis-platin. Resistance was stable for at least 3 months in culture in the absence of drug. P-glycoprotein overexpression was detected by immunofluorescence and Western Blotting, and a direct causal role for P-glycoprotein overexpression in the resistant phenotype was established by transfection with an mdr1 specific antisense oligonucleotide. A modified cryopreservation procedure was necessary for the resistant variant line. The resistant population displays clonal heterogeneity with respect to resistance level. A higher frequency of double minute chromosomes was observed in DLKP-A when compared with the parental cell line.

Antineoplastic Agents↗
PubMed1992

Endothelin in the urinary bladder. II. Characterization of endothelin receptor subtypes.

Endothelins (ET-1, ET-2, ET-3) are a family of regulatory peptides with diverse biological functions, including modulation of smooth muscle tone. To evaluate the possible role of endothelins in the control of the detrusor smooth muscle of urinary bladder, we have investigated the responses of isolated strips of bladder dome and base to endothelins, in organ chambers, and characterized the receptors for these isopeptides in bladder membranes. ET-1, ET-2 and to a lesser extent ET-3 caused sustained concentration-dependent contraction of bladder dome and base. The contraction to endothelins, unlike the short-lived contraction produced by cholinergic agents, was long lasting and difficult to wash out. Equilibrium binding studies demonstrated that endothelin isoforms bound to bladder membrane receptors specifically, with high affinity (KD of 0.4-0.6 nM) and limited capacity (60-420 fmol/mg protein). Competition analysis showed two populations of receptors: one with high affinity for ET-1 and ET-2 and low affinity for ET-3 and another with high affinity for ET-1, ET-2 and ET-3. Chemical affinity labeling of endothelins to bladder membranes demonstrated that ET-1 and ET-2 were cross-linked to three proteins (75, 52 and 34 kDa, respectively), whereas ET-3 was cross-linked mainly to a 34 kDa protein. The data obtained from equilibrium binding studies, competition analysis and cross-linking experiments suggest that at least two endothelin receptor subtypes exist in bladder tissue. These observations further suggest that bladder smooth muscle tone may be modulated by endothelin or an endothelin-like substance via interaction with specific receptor sites.

Animals↗