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Biomedical subjects

M Braddock

Publications and source records attributed to M Braddock.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of a selective and potent antagonist of human P2X(7) receptors, AZ11645373.

BACKGROUND AND PURPOSE: The ATP-gated P2X(7) receptor has been shown to play a role in several inflammatory processes, making it an attractive target for anti-inflammatory drug discovery. We have recently identified a novel set of cyclic imide compounds that inhibited P2X(7) receptor-mediated dye uptake in human macrophage THP-1 cells. In this study the actions and selectivity of one of these compounds, AZ11645373, were characterized. EXPERIMENTAL APPROACH: We measured membrane currents, calcium influx, and YOPRO-1 uptake from HEK cells expressing individual P2X receptors, and YOPRO1 uptake and interleukin-1beta release from THP-1 cells in response to ATP and the ATP analogue benzoylbenzoyl ATP (BzATP). KEY RESULTS: AZ11645373 up to 10 microM, had no agonist or antagonist actions on membrane currents due to P2X receptor activation at human P2X(1), rat P2X(2), human P2X(3), rat P2X(2/3), human P2X(4), or human P2X(5) receptors expressed in HEK cells. AZ11645373 inhibited human P2X(7) receptor responses in HEK cells in a non-surmountable manner with K (B) values ranging from 5 - 20 nM, with mean values not significantly different between assays. K (B) values were not altered by removing extracellular calcium and magnesium. ATP-evoked IL-1beta release from lipopolysaccharide-activated THP-1 cells was inhibited by AZ11645373, IC(50) = 90 nM. AZ11645373 was > 500-fold less effective at inhibiting rat P2X(7) receptor-mediated currents with less than 50% inhibition occurring at 10 microM. CONCLUSIONS AND IMPLICATIONS: AZ11645373 is a highly selective and potent antagonist at human but not rat P2X(7) receptors and will have much practical value in studies of human cells.

Adenosine Triphosphate↗
PubMed2003

Novel P2X7 receptor antagonists.

The synthesis and pharmacological evaluation of a new series of potent P2X(7) receptor antagonists is disclosed. The compounds inhibit BzATP-mediated pore formation in THP-1 cells. The distribution of the P2X(7) receptor in inflammatory cells, most notably the macrophage, mast cell and lymphocyte, suggests that P2X(7) antagonists have a significant role to play in the treatment of inflammatory disease.

Adenosine Triphosphate↗
PubMed2001

The transcriptional corepressor NAB2 blocks Egr-1-mediated growth factor activation and angiogenesis.

Effective tissue repair results from a rapid, temporally orchestrated series of events. At the site of local tissue injury, the production of many growth factors and cytokines is, in part, stimulated by the early growth response transcription factors such as Egr-1. Egr-1 protein binds to a family of corepressor proteins called NAB which function to block or limit Egr-1 trans-activation of cognate target genes. NAB2 blocks Egr-1 activation of the tissue factor (TF) promoter, Egr-1 stimulated production of PDGF-AB, HGF, TGFbeta(1), and VEGF and the endogenous expression of PDGF-AB and TGFbeta(1). Expression of a wild-type NAB2 but not a dominant negative NAB2 mutant abrogates Egr-1 driven TF promoter activity and tubule formation in an in vitro model of angiogenesis. These findings may have importance in any tissue that is subject to scarring after acute or chronic injury.

Cells, Cultured↗
PubMed2001

Homing markers for atherosclerosis: applications for drug delivery, gene delivery and vascular imaging.

Endothelial dysfunction plays a major role in the pathogenesis of atherosclerosis. Pro-inflammatory cytokines such as interleukin-1 beta and tumour necrosis factor alpha activate endothelial cells changing their resting phenotype to become pro-adhesive, pro-thrombotic and pro-atherogenic. Phage display in vivo biopanning has been used to identify peptide sequences that home to diseased regions of the vessel wall in low density lipoprotein receptor (LDLr) knockout mice. In LDLr knockout mice, peptide sequence determinants exhibiting organ specificity have been isolated. These sequences have applications for gene delivery, drug delivery and for improving contrast agents for vascular imaging.

Animals↗
PubMed2001

Born again bone: tissue engineering for bone repair.

Destruction of bone tissue due to disease and inefficient bone healing after traumatic injury may be addressed by tissue engineering techniques. Growth factor, cytokine protein, and gene therapies will be developed, which, in conjunction with suitable carriers, will regenerate missing bone or help in cases of defective healing.

Animals↗
PubMed2001

The transcription factor Egr-1: a potential drug in wound healing and tissue repair.

In the United States, between 40 and 90 million hospital days are lost per year as a result of trauma and surgical procedures which result in the loss of functional tissue. This is estimated to cost the economy and healthcare providers in excess of US$ 500 billion, a figure that is increasing because of extending population lifespan. Tissue engineering and gene therapies are radical new treatments that are aimed at tissue regeneration ranging from dermal, osteal and occular repair to the replacement of failing tissue with entire biosynthetic organs. Over the last decade, numerous proteins have been identified that are able to direct the synthesis of new tissue. Such proteins include growth factors, cytokines and, more recently, transcription factors.

Arthritis, Rheumatoid↗
PubMed2000

Tissue repair with a therapeutic transcription factor.

The healing of tissue involves a wide range of molecular, cellular, and physiological events that are coordinated in a temporally specific manner. The cellular transcription factor early growth response factor 1 (Egr-1) is expressed minutes after acute injury and serves to stimulate the production of a class of growth factors whose role is to promote tissue repair. We have studied the effects of Egr-1 expression at the site of dermal wounding in rodents. We find that Egr-1 promotes angiogenesis in vitro and in vivo, increases collagen production, and accelerates wound closure. These results show that Egr-1 gene therapy accelerates the normal healing process and raises the potential use of this therapeutic transcription factor for any aspect of tissue repair.

Animals↗
PubMed2000

Full promoter sequence of human early growth response factor-1 (Egr-1): demonstration of a fifth functional serum response element.

The early growth response factor-1 (Egr-1) gene encodes a zinc finger transcription factor which is critical for cell proliferation and differentiation. The human Egr-1 promoter comprises regulatory elements including two Sp1 sites, an AP1 site, two cAMP response elements and an Egr-1 binding site. In addition to these transcription factor binding sites, the promoter harbours five serum response elements (SREs) and associated binding sites for the Ets transcription factor family, previously identified from partial sequence data (Sakamoto et al, Oncogene 6; 867-871, 1991). We now report the full sequence of the human Egr-1 promoter and confirm the presence of a fifth serum response element. This element is functionally active in a minimal promoter vector in response to the MAP kinase kinase MEK1.

Base Sequence↗
PubMed2000

Calculating the costs of gun injuries. A new methodology.

A new method for assessing the costs of gun injuries to a health system examines data on paid amounts, comprehensive medical expenses, and expenses over time. The authors extracted claims using injury diagnosis codes from billing forms and medical charts. The study demonstrates that a claims database can be used to accurately measure health care costs associated with gun injuries. The study is the first to include gun-related injuries treated in ambulatory care settings and to track actual payments over time.

Adolescent↗
PubMed1999

Delivery and expression of fluid shear stress-inducible promoters to the vessel wall: applications for cardiovascular gene therapy.

In atherosclerosis, endothelial cells at sites of stenosis experience elevated levels of shear stress. We have constructed a series of shear stress-inducible transcription units (SITUs) expressing the luciferase reporter gene and determined their activation by fluid shear stress in transfected endothelial cells. Chimeric promoters were constructed that comprised basal transcription factor-binding sites coupled to a shear stress response element (SSRE). We have used consensus binding sites for transcription factors NF-kappaB, Ap1, Sp1, Oct1, and Egr-1/Sp1 in either the presence or absence of the previously defined "GAGACC" SSRE. The response of the promoters to shear stress was determined after transfection into human umbilical vein endothelial cells (HUVECs). After transient transfection into HUVECs, fluid shear stress activated the promoters by between two- and eightfold. The most responsive SITUs comprised an overlapping Sp1/Egr-1-binding site linked to a TATA box with (SP5) or without (SP7) the GAGACC SSRE. Instillation of SP5 DNA in vivo into the left carotid artery of rabbit and subsequent generation of a stenosis using a mechanical wire occluder caused a 10-fold upregulation of luciferase reporter gene expression at the site of vessel occlusion. These vectors show promise for therapeutic gene expression at sites of occlusive vascular disease.

Animals↗
PubMed1999

Fluid shear stress induction of the tissue factor promoter in vitro and in vivo is mediated by Egr-1.

Hemodynamic forces such as fluid shear stress have been shown to modulate the activity of an expanding family of genes involved in vessel wall homeostasis and the pathogenesis of vascular disease. We have investigated the effect of shear stress on tissue factor (TF) gene expression in human endothelial cells (ECs) and in a rat arterial model of occlusion. As measured by reverse transcriptase polymerase chain reaction, exposure of ECs to 1.5 N/m2 shear stress resulted in a time-dependent induction of endogenous TF transcripts of over 5-fold. Transient transfection of TF promoter mutants into cultured ECs suggests the involvement of the transcription factor Egr-1 in mediating the response of the TF promoter to shear stress. To address the importance of flow induction of Egr-1 in vivo, we have established a flow-restricted rat arterial model and determined the level of expressed Egr-1 and TF at the site of restricted flow using immunohistochemistry. We report an increase in the level of Egr-1 and TF protein in ECs expressed at the site of restricted flow. Elevated expression of Egr-1 and TF is restricted to a highly localized area, as evidenced by the fact that no significant increase in level can be detected at arterial sites distal to the site of occlusion. These findings suggest a direct role for Egr-1 in flow-mediated induction of TF and further substantiate the importance of shear stress as a modulator of vascular endothelial gene function in vivo.

Animals↗
PubMed1998

Fluid shear stress activation of egr-1 transcription in cultured human endothelial and epithelial cells is mediated via the extracellular signal-related kinase 1/2 mitogen-activated protein kinase pathway.

The primary response transcription factor, early growth response-1 (Egr-1), is rapidly activated by a variety of extracellular stimuli. Egr-1 binds to a sequence found in the promoters of genes involved in vascular injury, such as PDGF-A and tissue factor, and trans-activates their expression in endothelial cells in response to fluid shear stress. Here we show that egr-1 mRNA is increased after 30 min of flow in human aortic endothelial cell and HeLa cell cultures. Transient transfection of HeLa cells with reporter gene constructs driven by the murine or human egr-1 5' flanking sequence revealed a five- and ninefold induction, respectively, in transcriptional activity after exposure to a shear stress of 5 dynes/cm2 for 3 h. Deletion of sequences in the murine promoter containing two AP1 sites and an inhibitory Egr-1 binding sequence, did not reduce shear stress inducibility. However, progressive deletion of five serum response elements, reduced both the basal promoter activity and its capacity to be activated by shear stress. Further examination indicated that the three upstream serum response elements are predominantly responsible for shear stress activation of the egr-1 promoter. Treatment of cells with PD98059, a specific inhibitor of mitogen-activated protein kinase-1 inhibited shear stress activation of egr-1. We suggest that egr-1 activation by shear stress involves activation of Elk-1 but not c-jun activity. These data, which are consistent with previous findings for shear mediated signaling via the mitogen-activated protein kinase cascade, now implicate shear modulation of the Egr-1 transcription factor in this pathway.

Animals↗
PubMed1995

Promoter control of translation in Xenopus oocytes.

The HIV-1 promoter directs the high level production of transcripts in Xenopus oocytes. However, despite being exported to the cytoplasm, the transcripts are not translated [M. Braddock, A. M. Thorburn, A. Chambers, G. D. Elliott, G. J. Anderson, A. J. Kingsman and S. M. Kingsman (1990) Cell, 62, 1123-1133]. We have shown previously that this is a function of promoter sequences and is independent of the TAR RNA element that is normally located at the 5' end of all HIV mRNAs. We now show that a three nucleotide substitution at position -340, upstream of the RNA start site, reverses the translation inhibition. This site coincides with a sequence that can bind the haematopoietic transcription factor GATA. The inhibition of translation can also be reversed by treatment with inhibitors of casein kinase II or by injection into the nucleus of antibodies specific for the FRGY2 family of RNP proteins. We suggest that the -340 site influences the quality of the transcription complex such that transcripts are diverted to a nucleus-dependent translation inhibition pathway.

Animals↗
PubMed1995

Requirement for HIV-1 TAR sequences for Tat activation in rodent cells.

HIV-1 gene expression is activated via an interaction between the virally encoded Tat protein and a target RNA, TAR. TAR is located at the immediate 5' end of all viral mRNAs and comprises a partially base-paired stem with a tripyrimidine bulge in the upper stem and a hexanucleotide loop. In vitro, Tat binds specifically to the bulge and upper stem region with no requirement for the loop. In contrast, when Tat activation is analyzed in primate cells, mutations in the loop abolish activation, suggesting a critical role for loop binding cellular factors. However, in rodent cells the reverse is true. Messages with a mutation in the TAR loop are activated whereas messages harboring a wild-type TAR sequence are not activated. By testing the effect of mutations in the bulge and stem in the context of mutation in the loop we now show that this loop-independent activation by Tat in rodent cells requires the critical bulge-stem sequences needed for Tat binding in vitro. These data suggest that in rodent cells, as in vitro, Tat does not require a loop binding cofactor. In rodent cells containing human chromosome 12 (CHO12), however, Tat activation is both bulge and loop dependent. It appears that rodent cells, but not CHO12 cells, are refractory to the normal Tat/TAR activation pathway not by virtue of lacking a loop binding cofactor, but rather by the presence of a loop binding inhibitor of either Tat binding or the activation process.

Animals↗