Analysis of the mechanism of resistance of 7 novel MDR variants.
Explore the source record for details and available documents.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Biomedical subjects
E Moran
Publications and source records attributed to E Moran.
Preliminary studies of a monoclonal antibody raised to the over expressed plasma membrane associated glycoprotein of a multidrug resistant cell line.
Explore the source record for details and available documents.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Monoclonal antibodies to human endothelin-1: characterization and utilization in radioimmunoassay and immunocytochemistry.
A host of monoclonal antibodies directed against human endothelin-1 (ET-1) has been developed and characterized. The antibodies reacted with ET-1 specifically and with high affinity, as determined by competition analysis and sucrose density gradients. The antibodies did not cross-react with neuropeptide YY, beta-endorphin, calcitonin gene-related peptide, secretin or somatostatin. The antibodies cross-reacted with big endothelin (B-ET), endothelin-2 (ET-2), vasointestinal constrictor peptide (VIC), and endothelin-3 (ET-3) albeit with varying affinity but did not cross-react with sarafotoxin (SRTX-6b). None of the antibodies reacted with the C-terminal hexapeptide (HXPT) of ET-1, indicating that the epitopes are not located within this region of ET-1. The monoclonal antibodies exhibited binding activity in dilutions ranging from 1:1000, to 1:10(6). The isotypes of the monoclonal antibodies were determined by competition binding assay. Six of the monoclonal antibodies were of the IgG gamma 1, two were IgM and one of the IgG gamma 2a subclass. The antibodies detected immunoreactive ETs by radioimmunoassay and in immunocytochemical localization, suggesting the potential use of these antibodies as tools to determine the concentration of ETs in biological fluids and in immunocytochemical localization of ETs in specific cell types in various tissues.
Amino Acid Sequence↗
Transforming growth factor beta 1 (TGF beta 1) reduces cellular levels of p34cdc2, and this effect is abrogated by adenovirus independently of the E1A-associated pRB binding activity.
We have used E1A probes to study the roles of the p34cdc2 kinase and the retinoblastoma tumor susceptibility gene product (pRB) in transforming growth factor beta 1 (TGF beta 1)-mediated growth suppression in mink lung epithelial (Mv1Lu) cells. In agreement with previous reports, we see a decline in p34cdc2 kinase activity and a loss of pRB phosphorylation after TGF beta 1 treatment. We report here that TGF beta 1 induces not only a change in p34cdc2 kinase activity but a strong repression of p34cdc2 synthesis. Loss of p34cdc2 kinase activity is not seen until the steady-state level of p34cdc2 declines, suggesting that the intra-cellular signals induced by TGF beta 1 affect p34cdc2 at the level of expression, rather than by altering the posttranslational modifications of p34cdc2 that regulate its kinase activity. Infection with adenovirus expressing either wild-type E1A or a mutant E1A (pm928) defective for pRB binding alleviated TGF beta 1-mediated suppression of DNA synthesis, indicating that E1A does not need to bind pRB physically to keep cell growth-suppressing functions from being activated by TGF beta 1. The E1A.928 mutant virus is able to maintain p34cdc2 expression and kinase activity, as well as pRB phosphorylation in the presence of TGF beta 1, which may account for its ability to maintain cell cycle activity without directly sequestering pRB. Overall our results suggest that TGF beta 1 acts by signaling changes at the level of control of G1 gene expression, not at the level of posttranslational modification of p34cdc2 or its substrates.
Adenoviridae↗
Promoter-specific trans-activation by the adenovirus E1A12S product involves separate E1A domains.
Recent studies have shown that the adenovirus E1A12S product can trans-activate transcription by activating the transcription factor E2F. However, E2F cannot be the only target for the E1A12S product, since several cellular promoters have been found to be activated by the E1A12S protein even though they lack E2F sites. Indeed, we now show that activation of the hsp70 promoter by the E1A12S product requires the TATAA sequence. Moreover, activation of the hsp70 promoter requires the N-terminal domain of the E1A protein and does not require the conserved region 2 sequences which are required for the E2F-dependent activation of transcription. We conclude that the targeting of distinct transcription factors, leading to trans-activation of transcription of multiple promoters, involves distinct domains of the E1A proteins that are also required for oncogenic activity.
Adenovirus E1A Proteins↗
DNA-binding properties of the E1A-associated 300-kilodalton protein.
One of the major E1A-associated cellular proteins is a 300-kDa product (p300) that binds to the N-terminal region of the E1A products. The p300 binding site is distinct from sequences involved in binding the retinoblastoma product and other E1A-associated cellular products such as p60-cyclin A and p107. p300 binding to E1A is linked genetically to the enhancer repression function of E1A and the other E1A-mediated gene-regulating functions as well as to the transforming functions of E1A. However, the biochemical properties of p300 have not yet been characterized. We report here that p300 has an intrinsic DNA-binding activity and shows a preferential affinity for specific DNA sequences. The sequences selectively bound by p300 are related to those of a series of enhancer elements that are recognized by NF-kappa B. The direct physical interaction of p300 with enhancer elements provides a biochemical basis for the genetic evidence linking the E1A-mediated enhancer repression function with the p300-binding activity of E1A.
Adenovirus Early Proteins↗
A critical analysis of the use of sialic acid determination in the diagnosis of malignancy.
The measurement of serum sialic acid in sera from individuals with leukemia, breast, gastrointestinal, lung and bladder cancers, brucellosis and manic depression is described. The serum sialic acid levels were also examined in mice bearing Landschutz ascites tumours. The presence of sialic acid-enriched glycopeptides in serum was shown. The results indicate that while sialic acid may be significantly elevated in sera of some tumour-bearing individuals, it is not solely tumour-derived. Its application as a tumour marker is limited and requires critical analysis.
Animals↗
Meningococcal vaccines--present and future.
Over 20 years after the development of the meningococcal A and C vaccines, an effective vaccine against Neisseria meningitidis group B is still lacking. Major obstacles in the development of a B vaccine have been the remarkable capacity of the organism to evade the immune defences of the host and the lack of a predictive animal model. Three group B vaccines based on outer membrane proteins have been, or are currently being, evaluated in field trials. Nevertheless, a number of important questions remain such as the identity of the active components, the degree of efficacy against heterologous group B subtypes, and the duration of protection. In addition, work on a variety of alternative approaches to a group B vaccine is rapidly progressing. Among these are use of chemically modified group B polysaccharide, synthetic or natural lipopolysaccharide epitopes, synthetic peptides corresponding to bactericidal epitopes on the class 1 outer membrane protein, and iron binding proteins. Although each of these approaches has some problems associated with it, the prospects remain good for an effective solution to the group B problem.
Antigens, Bacterial↗
Domains of the adenovirus E1A protein required for oncogenic activity are also required for dissociation of E2F transcription factor complexes.
Recent experiments have shown that the cellular E2F transcription factor is found in complexes with cellular proteins and that one such complex contains the cyclin-A protein. Isolation of a cellular activity, which we term E2F-BF, can reconstitute the E2F-cyclin-A complex and has permitted a more detailed analysis of the mechanism of E1A dissociation. Through the analysis of a series of E1A mutants, we find that sequences in conserved region 1 (CR1) and conserved region 2 (CR2) are important for dissociation of the E2F complex, whereas amino-terminal sequences are not required. In contrast to the requirements for dissociation, only the CR1 sequences are required to block formation of the complex if E1A is added when the components are combined. We have also identified an activity, termed E2F-I, that inhibits E2F binding to DNA, again apparently through the formation of a complex with E2F. This inhibitory activity is also blocked by E1A, dependent on the same elements of the E1A protein that disrupt the interaction with E2F-BF. Because the E1A sequences that are important for releasing E2F from these interactions are also sequences necessary for oncogenesis, we suggest that this activity may be a critical component of the transforming activity of E1A.
Adenoviridae↗
Analysis with specific polyclonal antiserum indicates that the E1A-associated 300-kDa product is a stable nuclear phosphoprotein that undergoes cell cycle phase-specific modification.
Binding of a 300-kDa host cell protein (p300) is tightly correlated with the ability of the adenovirus E1A products to induce quiescent baby rat kidney cells to proliferate. We have generated rabbit polyclonal antibodies against p300 to characterize this protein further. We have found p300 to be a nuclear phosphoprotein that is actively synthesized in both quiescent and proliferating baby rat kidney cells. In partially purified mitotic cell populations, we observe a form of p300 with decreased electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels that shares a nearly identical partial proteolytic digest pattern with p300. The slower-migrating form of p300 is greatly reduced by treating immune complexes with potato acid phosphatase. The relative stability and presence of p300 even in resting cells suggests that p300 has a basal cell function, but the appearance of differentially modified forms during the cell cycle suggests the possibility that p300 function is modulated specifically in growing cells.
Adenovirus Early Proteins↗
Simian virus 40 large-T antigen expresses a biological activity complementary to the p300-associated transforming function of the adenovirus E1A gene products.
In this report we present evidence that simian virus 40 T antigen encodes a biological activity that is functionally equivalent to the transforming activity lost by deletion of the E1A p300-binding region. T-antigen constructs from which the pRb-binding region has been deleted are virtually unable to induce foci of transformed cells in a ras cooperation assay in primary baby rat kidney cells. Nevertheless, such a construct can cooperate with an E1A N-terminal deletion mutant, itself devoid of transforming activity, to induce foci in this assay. The heterologous trans-cooperating activity observed between E1A and T-antigen deletion products is as efficient as trans cooperation between mutants expressing individual E1A domains. The cooperating function can be impaired by a deletion near the N terminus of T antigen. Such a deletion impairs neither the p53-binding function nor the activity of the pRb-binding region.
Adenovirus Early Proteins↗
E1A induces phosphorylation of the retinoblastoma protein independently of direct physical association between the E1A and retinoblastoma products.
We have studied the initial effects of adenovirus E1A expression on the retinoblastoma (RB) gene product in normal quiescent cells. Although binding of the E1A products to pRB could, in theory, make pRB phosphorylation unnecessary for cell cycle progression, we have found that the 12S wild-type E1A product is capable of inducing phosphorylation of pRB in normal quiescent cells. The induction of pRB phosphorylation correlates with E1A-mediated induction of p34cdc2 expression and kinase activity, consistent with the possibility that p34cdc2 is a pRB kinase. Expression of simian virus 40 T antigen induces similar effects. Induction of pRB phosphorylation is independent of the pRB binding activity of the E1A products; E1A domain 2 mutants do not bind detectable levels of pRB but remain competent to induce pRB phosphorylation and to activate cdc2 protein kinase expression and activity. Although the kinetics of induction are slower, domain 2 mutants induce wild-type levels of pRB phosphorylation and host cell DNA synthesis and yet fail to induce cell proliferation. These results imply that direct physical interaction between the RB and E1A products does not play a required role in the early stages of E1A-mediated cell cycle induction and that pRB phosphorylation is not, of itself, sufficient to allow quiescent cells to divide. These results suggest that the E1A products do not need to bind pRB in order to stimulate resting cells to enter the cell cycle. Indeed, a more important role of the RB binding activity of the E1A products may be to prevent dividing cells from returning to G0.
Adenoviridae↗
Solubilization and sedimentation analysis of muscarinic acetylcholine receptors.
To investigate if G-protein-receptor interactions can be characterized using sucrose density gradients (SDG) we have determined the experimental conditions for muscarinic acetylcholine receptor (mAChR) solubilization and analysis on SDG. Solubilization of 65-80% of [3H]QNB bound mAChR was accomplished with 1% of detergent. Analysis of solubilized receptors on SDG containing 0.4 M KCl and 0.1% detergent demonstrated that the physical properties of the receptor-detergent complexes are influenced by the solubilizing detergent as well as detergents included in the SDG. Neither GTP gamma S nor NaF and AlCl3 altered the sedimentation properties of mAChR, suggesting that the solubilized mAChR is no longer associated with G-protein under these conditions. Receptors bound to [3H]oxotremorine and [3H]QNB had similar sedimentation properties, suggesting that, once solubilized, mAChRs do not remain associated with G-proteins. Covalent labeling with [3H]PrBCM followed by solubilization and analysis on SDS-gel electrophoresis demonstrated the presence of intact receptor molecule. These observations suggest that the changes in the sedimentation properties of detergent-receptor complexes are independent of G-protein interactions and are influenced by the nature of the detergent associated with the mAChR during analysis.
Animals↗
Meningococcal serogroup B vaccine protection trial and follow-up studies in Chile. The Chilean National Committee for Meningococcal Disease.
Explore the source record for details and available documents.
Adolescent↗
Physicochemical characterization and solubilization of endothelin receptors.
[125I]Endothelin-1 (ET-1) bound to specific endothelin receptors (ET-R) with high affinity (Kd = 0.4-1 nM) and dissociated very slowly from ET-R at 37 degrees C (K-1 = 2.4 x 10(-3)/h). The binding of ET-1 was reduced in acidic (pH = 4) and alkaline (pH > 8.5) medium but was stable at near neutral pH (pH 6.5-7.5). Covalent affinity labeling of ET-R with [125I] endothelins (ET-1,2,3) demonstrated that ET-1 and ET-2 bound specifically to three proteins with approximate mol masses of 75, 52, and 34 kDa, whereas [125I]ET-3 bound mainly to a protein with a mol mass of 34 kDa. The binding of [125I]ET-1 to the three mol-mass species was effectively displaced with ET-1 and ET-2. However, ET-3 displaced the binding to the 34-kDa band, only. ET-R were solubilized, in active endothelin-binding forms, with 1% digitonin and 0.1% cholate. Solubilized ET-R sedimented on sucrose density gradients (SDG) containing 0.1% CHAPS, 10% glycerol, and 0.4 M KCl, as 7-8S complexes. Binding of ET-1, ET-3, vasoactive intestinal constrictor (VIC) or sarafotoxin (SRTX-6b) to the solubilized ET-R, and subsequent analysis on SDG, demonstrated similar sedimentation characteristics, suggesting that these peptides are bound to similar receptors.
Affinity Labels↗
TGF-beta 1 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming proteins with pRB binding domains.
TGF-beta 1 is demonstrated to inhibit skin keratinocyte proliferation when added during the G1 phase of the cell cycle. Human foreskin keratinocytes transformed with either HPV-16 or -18 or SV40, however, were resistant to the growth inhibitory effects of TGF-beta 1. Since TGF-beta 1 appears to inhibit keratinocyte growth through down-regulation of c-myc, it was hypothesized that these DNA tumor viruses might be modulating the response to TGF-beta 1 via this pathway. Transient expression of proteins HPV-16 E7, adenovirus type 5 E1A, and SV40 large T antigen is demonstrated to block TGF-beta 1 suppression of c-myc transcription. This effect was not observed with DNA tumor virus transforming proteins mutated in their pRB binding domain. These observations indicate that pRB or another protein that interacts with this binding domain mediates TGF-beta 1 regulation of c-myc gene expression and growth inhibition.
Adenovirus Early Proteins↗
Methylprednisolone acetate does not cause inflammatory changes in the epidural space.
Few studies have examined the possible adverse effects that epidural injection of depot corticosteroid preparations may have on meningeal membranes and nervous tissue. Thirty-six healthy adult white rabbits received 0.3 ml/kg epidural injections of either lactated Ringer's solution (negative control group), 1% lidocaine containing methylprednisolone acetate (study group), or normal saline containing talc (positive control group). Animals were killed either 4 or 10 days after injection and stained sections of the spinal cord and meningeal membranes were examined by light microscopy. In all animals that received either lactated Ringer's solution or lidocaine with methylprednisolone acetate, microscopic examination of specimens taken from the L5-L6 interspace revealed no white cell infiltrates and no fibroblastic activity. All animals that received epidural injections of normal saline containing talc had marked infiltration of tissue macrophages in the epidural space. There was no thickening of the meningeal membranes or nerve roots in any animal. The complete lack of inflammatory changes and meningeal thickening demonstrated in this pilot study helps to confirm the safety of methylprednisolone acetate when injected into the epidural space.
Animals↗