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Biomedical subjects

M Downes

Publications and source records attributed to M Downes.

At least 19 recordsLinked to original sources
PubMed2005

Pancreas transplantation in Ireland.

BACKGROUND: We report on our experience of pancreatic transplants performed in Beaumont Hospital from 1992 to January 2003. During this period, 63 pancreatic transplants were carried out. Simultaneous pancreas and kidney transplantation is regarded as the treatment of choice for Type 1 diabetics with end stage renal failure (ESRF) by the American Diabetes Association. METHODS: A retrospective review of all the pancreas transplants between 1992 and January 2003 was carried out. All of the patients had insulin-dependent diabetes mellitus (IDDM) and, in addition, 57 of the recipients had concomitant end stage renal failure. For two of the patients, this was a second transplant, one of them having had a previous renal graft which thrombosed. The second patient underwent a simultaneous pancreas and kidney (SPK) transplant in 1998 with loss of the pancreatic graft shortly afterwards. She subsequently received a pancreas only transplant in 2002. RESULTS: The follow-up period ranges from one month to ten years and parameters used to assess graft function include Hb A1c and serum creatnine (SPK only). There have been 22 graft losses (six kidneys, nine pancreases and seven involving both the kidney and pancreas), to date. The one-year graft survival was 80.4% (pancreas) and 92.7% (kidney), with a one-year patient survival of 94.8%, which compares favourably with figures from other centres. At follow-up, the average Hb A1c at six months post-transplant was 5.7% with a serum creatnine of 121micmol/L being recorded at one month. CONCLUSION: Our experience of pancreas transplants at Beaumont Hospital has been very encouraging with graft and patient survival equivalent to other international centres.

Adult↗
PubMed2003

A pilot study of adjuvant intraperitoneal 5-fluorouracil using 4% icodextrin as a novel carrier solution.

AIM: This pilot study utilised the sustained intraperitoneal (i.p.) dwell properties of an iso-osmotic solution of 4% icodextrin to investigate the tolerability, toxicity and feasibility of home-based i.p. 5FU adjuvant chemotherapy following resective surgery for colorectal cancer. METHODS: Twenty eligible patients (Dukes' stage B and C with potentially curative resection) underwent perioperative Tenckhoff catheter placement. Ten (6 male, 4 female, aged 46-85; mean 67.5 years) received 5FU chemotherapy. After initial flushing and gradual increase in volumes of 4% icodextrin alone, patients received home-based i.p. 5FU (150-300 mg/m(2)/day given as equal doses at 12-hourly intervals) for 14 days, with a 14-day recovery period, for a maximum of 6 courses. Two incurable patients, treated on compassionate grounds, provided further safety data. RESULTS: Nine of the 10 patients became proficient in self-treatment with 5FU and two completed 6 courses. Frequent abdominal pain was the main dose-limiting toxicity of 5FU, causing withdrawal of three patients after a high (300 mg/m(2)/day) first course and one following a third course at lower doses. I.p. 5FU concentrations (mean>30000 ngml(-1)) were 1000 fold higher than systemic venous levels. Bacterial peritonitis led to two withdrawals but was not a frequent event (microbiologically confirmed incidence of 1 per 27 catheter-months). CONCLUSIONS: Home-based i.p. adjuvant chemotherapy is a feasible treatment option in patients with surgically resected colorectal carcinoma.

Adenocarcinoma↗
PubMed2001

Active repression of RAR signaling is required for head formation.

The retinoic acid receptors (RARs) recruit coactivator and corepressor proteins to activate or repress the transcription of target genes depending on the presence of retinoic acid (RA). Despite a detailed molecular understanding of how corepressor complexes function, there is no in vivo evidence to support a necessary function for RAR-mediated repression. Signaling through RARs is required for patterning along the anteroposterior (A-P) axis, particularly in the hindbrain and posterior, although the absence of RA is required for correct anterior patterning. Because RARs and corepressors are present in regions in which RA is absent, we hypothesized that repression mediated through unliganded RARs might be important for anterior patterning. To test this hypothesis, specific reagents were used that either reduce or augment RAR-mediated repression. Derepression of RAR signaling by expressing a dominant-negative corepressor resulted in embryos that exhibited phenotypes similar to those treated by RA. Anterior structures such as forebrain and cement gland were greatly reduced, as was the expression of molecular markers. Enhancement of target gene repression using an RAR inverse agonist resulted in up-regulation of anterior neural markers and expansion of anterior structures. Morpholino antisense oligonucleotide-mediated RARalpha loss-of-function phenocopied the effects of RA treatment and dominant-negative corepressor expression. Microinjection of wild-type or dominant-negative RARalpha rescued the morpholino phenotype, confirming that RAR is functioning anteriorly as a transcriptional repressor. Lastly, increasing RAR-mediated repression potentiated head-inducing activity of the growth factor inhibitor cerberus, whereas releasing RAR-mediated repression blocked cerberus from inducing ectopic heads. We conclude that RAR-mediated repression of target genes is critical for head formation. This requirement establishes an important biological role for active repression of target genes by nuclear hormone receptors and illustrates a novel function for RARs during vertebrate development.

Animals↗
PubMed2001

Sharp, an inducible cofactor that integrates nuclear receptor repression and activation.

A yeast two-hybrid screen using the conserved carboxyl terminus of the nuclear receptor corepressor SMRT as a bait led to the isolation of a novel human gene termed SHARP (SMRT/HDAC1 Associated Repressor Protein). SHARP is a potent transcriptional repressor whose repression domain (RD) interacts directly with SMRT and at least five members of the NuRD complex including HDAC1 and HDAC2. In addition, SHARP binds to the steroid receptor RNA coactivator SRA via an intrinsic RNA binding domain and suppresses SRA-potentiated steroid receptor transcription activity. Accordingly, SHARP has the capacity to modulate both liganded and nonliganded nuclear receptors. Surprisingly, the expression of SHARP is itself steroid inducible, suggesting a simple feedback mechanism for attenuation of the hormonal response.

Amino Acid Motifs↗
PubMed2001

A dynamic role for HDAC7 in MEF2-mediated muscle differentiation.

The overlapping expression profile of MEF2 and the class-II histone deacetylase, HDAC7, led us to investigate the functional interaction and relationship between these regulatory proteins. HDAC7 expression inhibits the activity of MEF2 (-A, -C, and -D), and in contrast MyoD and Myogenin activities are not affected. Glutathione S-transferase pulldown and immunoprecipitation demonstrate that the repression mechanism involves direct interactions between MEF2 proteins and HDAC7 and is associated with the ability of MEF2 to interact with the N-terminal 121 amino acids of HDAC7 that encode repression domain 1. The MADS domain of MEF2 mediates the direct interaction of MEF2 with HDAC7. MEF2 inhibition by HDAC7 is dependent on the N-terminal repression domain and surprisingly does not involve the C-terminal deacetylase domain. HDAC7 interacts with CtBP and other class-I and -II HDACs suggesting that silencing of MEF2 activity involves corepressor recruitment. Furthermore, we show that induction of muscle differentiation by serum withdrawal leads to the translocation of HDAC7 from the nucleus into the cytoplasm. This work demonstrates that HDAC7 regulates the function of MEF2 proteins and suggests that this class-II HDAC regulates this important transcriptional (and pathophysiological) target in heart and muscle tissue. The nucleocytoplasmic trafficking of HDAC7 and other class-II HDACs during myogenesis provides an ideal mechanism for the regulation of HDAC targets during mammalian development and differentiation.

Animals↗
PubMed2001

SOX18 and the transcriptional regulation of blood vessel development.

SOX18 is a transcription factor that is transiently expressed in nascent endothelial cells during embryonic development and adult neovascularization. This protein belongs to the SOX family of transcription factors, which are proving to be some of the key regulators of cell-type specification in the vertebrate embryo. Natural mutations in the Sox18 gene have been shown to result in cardiovascular dysfunction, in some cases leading to death. Available evidence thus implicates Sox18 as an important regulator of vascular development, most likely playing a key role in endothelial cell specification. However, the genetic knockout of Sox18 in mice has produced a confounding result that complicates our understanding of the molecular mode of action of the SOX18 protein. We speculate that Sox18 may act in a redundant fashion with closely related genes such as Sox7 and/or Sox17.

Animals↗
PubMed2001

Virus-directed, enzyme prodrug therapy with nitroimidazole reductase: a phase I and pharmacokinetic study of its prodrug, CB1954.

CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] is converted by the bacterial enzyme nitroimidazole reductase (NTR) into a potent cytotoxic bifunctional alkylating agent, which can be delivered to tumors in adenoviral vectors as virus-directed, enzyme prodrug therapy. This report summarizes a Phase I and pharmacokinetic study of the prodrug, CB1954. Thirty patients, ages 23-78 years (median 62 years), with predominantly gastrointestinal malignancies were treated. CB1954 was administered by i.v. injection every 3 weeks or i.p. followed by 3-weekly i.v. injections, toward a maximum of six cycles. The dose was escalated from 3 to 37.5 mg/m2. No significant toxicity was seen until 24 mg/m2 (recommended i.v. dose). Dose-limiting toxicities (DLT) were diarrhea and hepatic toxicity, seen at 37.5 mg/m2. DLT has not been observed at the current i.p. dose of 24 mg/m2. There was no alopecia, marrow suppression, or nephrotoxicity. Clearance data suggest hepatic metabolism, and <5% of CB1954 was renally excreted. There was a nonlinear relationship between i.v. dose and area under the curve (AUC). At the recommended i.v. dose of 24 mg/m2, the AUC was 5.8 microM/h. Intraperitoneal administration (24 mg/m2) achieved an AUC of 387 microM/h, giving a considerable regional advantage. In vitro, the AUC required to achieve the IC50 for CB1954, in NTR-expressing cancer cells, ranges from 10-50 microM/h. Thus, CB1954 is well tolerated at a dose of 24 mg/m2, and sufficient serum/peritoneal levels are achieved for an enzyme-prodrug approach to be feasible. We are now conducting a Phase I trial combining adenovirus-mediated NTR and i.v. CB1954 (24 mg/m2) in patients with primary and secondary liver tumors.

Abdominal Pain↗
PubMed2000

Identification of a nuclear domain with deacetylase activity.

Here, we describe the identification and characterization of a nuclear body (matrix-associated deacetylase body) whose formation and integrity depend on deacetylase activity. Typically, there are 20-40 0.5-microM bodies per nucleus, although the size and number can vary substantially. The structure appears to contain both class I and the recently described class II histone deacetylases (HDAC)5 and 7 along with the nuclear receptor corepressors SMRT (silencing mediator for retinoid and thyroid receptor) and N-CoR (nuclear receptor corepressor). Addition of the deacetylase inhibitors trichostatin A and sodium butyrate completely disrupt these nuclear bodies, providing a demonstration that the integrity of a nuclear body is enzyme dependent. We demonstrate that HDAC5 and 7 can associate with at least 12 distinct proteins, including several members of the NuRD and Sin3A repression complexes, and appear to define a new but related complex.

Amino Acid Sequence↗
PubMed2000

Humanized xenobiotic response in mice expressing nuclear receptor SXR.

The cytochrome CYP3A gene products, expressed in mammalian liver, are essential for the metabolism of lipophilic substrates, including endogenous steroid hormones and prescription drugs. CYP3A enzymes are extremely versatile and are inducible by many of their natural and xenobiotic substrates. Consequently, they form the molecular basis for many clinical drug-drug interactions. The induction of CYP3A enzymes is species-specific, and we have postulated that it involves one or more cellular factors, or receptor-like xeno-sensors. Here we identify one such factor unequivocally as the nuclear receptor pregnenolone X receptor (PXR) and its human homologue, steroid and xenobiotic receptor (SXR). We show that targeted disruption of the mouse PXR gene abolishes induction of CYP3A by prototypic inducers such as dexamethasone or pregnenolone-16alpha-carbonitrile. In transgenic mice, an activated form of SXR causes constitutive upregulation of CYP3A gene expression and enhanced protection against toxic xenobiotic compounds. Furthermore, we show that the species origin of the receptor, rather than the promoter structure of CYP3A genes, dictates the species-specific pattern of CYP3A inducibility. Thus, we can generate 'humanized' transgenic mice that are responsive to human-specific inducers such as the antibiotic rifampicin. We conclude that SXR/PXR genes encode the primary species-specific xeno-sensors that mediate the adaptive hepatic response, and may represent the critical biochemical mechanism of human xenoprotection.

Animals↗
PubMed2000

The genome sequence of Drosophila melanogaster.

The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.

Animals↗
PubMed2000

Isolation of a novel histone deacetylase reveals that class I and class II deacetylases promote SMRT-mediated repression.

The transcriptional corepressor SMRT functions by mediating the repressive effect of transcription factors involved in diverse signaling pathways. The mechanism by which SMRT represses basal transcription has been proposed to involve the indirect recruitment of histone deacetylase HDAC1 via the adaptor mSin3A. In contrast to this model, a two-hybrid screen on SMRT-interacting proteins resulted in the isolation of the recently described HDAC5 and a new family member termed HDAC7. Molecular and biochemical results indicate that this interaction is direct and in vivo evidence colocalizes SMRT, mHDAC5, and mHDAC7 to a distinct nuclear compartment. Surprisingly, HDAC7 can interact with mSin3A in yeast and in mammalian cells, suggesting association of multiple repression complexes. Taken together, our results provide the first evidence that SMRT-mediated repression is promoted by class I and class II histone deacetylases and that SMRT can recruit class II histone deacetylases in a mSin3A-independent fashion.

Amino Acid Sequence↗
PubMed2000

Structure-function analysis of the Rev-erbA and RVR ligand-binding domains reveals a large hydrophobic surface that mediates corepressor binding and a ligand cavity occupied by side chains.

Rev-erbA/RVR are closely related orphan nuclear receptors (NRs) functioning as dominant transcriptional silencers through an association with the nuclear receptor corepressor N-CoR. In contrast with ligand-regulated NRs, Rev-erbA/RVR lack the ligand-binding domain (LBD) C-terminal activation helix, H12. In the case of retinoid acid receptor and thyroid hormone receptor, ligand binding is thought to reposition H12, causing corepressor dissociation and coactivator recruitment, thus leading to transcriptional activation. Here we present homology models of the Rev-erbA/RVR LBDs, which show that the putative ligand cavity is occupied by side chains, suggesting the absence of endogenous ligands. Modeling also revealed a very hydrophobic surface due to the absence of H12, exposing residues from H3, loop 3-4, H4, and H11. Mutation of specific residues from this surface severely impaired the in vitro and in vivo interaction of the Rev-erbA/RVR LBD with the receptor-interacting domain of the corepressors N-CoR or its splice variant RIP13delta1, reinforcing the view of the physical association of N-CoR with a LBD surface encompassing H3-H4 and H11. Furthermore, mutations in the LBD surface significantly reduced the ability of Rev-erbA and RVR to function as repressors of transcription. Interestingly, a hydrophobic surface comprised of H3-H4 and H12 in liganded NRs mediates the interaction with coactivators. Hence, it appears that corepressors and coactivators bind to overlapping surfaces of NR LBDs, the conformational change associated with H12 upon ligand binding resulting in a switch from a corepressor- to a coactivator-binding surface.

Amino Acid Sequence↗
PubMed1999

Unique forms of human and mouse nuclear receptor corepressor SMRT.

Nuclear hormone receptors have been shown to repress transcription in the absence of ligand. This repression is mediated by a corepressor complex that contains the Sin3A protein and histone deacetylases (HDAC1 and 2). Studies by several groups demonstrate that this complex is recruited to nuclear receptors through the highly related corepressors SMRT (silencing mediator of retinoid acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor). We describe here the cloning, characterization, and chromosomal mapping of forms of human and mouse SMRT that includes a 1,000-aa extension, which reveals striking homology to the amino terminus of N-CoR. Structure and function studies of wild-type and natural splicing variants suggest the presence of 3-4 amino terminal domains that repress in a cooperative as well as mechanistically distinct fashion.

Amino Acid Sequence↗
PubMed1999

A patient preference study comparing raltitrexed ('Tomudex') and bolus or infusional 5-fluorouracil regimens in advanced colorectal cancer: influence of side-effects and administration attributes.

Current chemotherapy regimens used in advanced colorectal cancer (ACRC) are similar in terms of efficacy, but differ importantly in terms of side-effects and administration profiles. These differences may impact significantly on patients' lives. We have evaluated patient preferences between raltitrexed ('Tomudex') and 5-fluorouracil-based chemotherapy regimens, with regard to side-effect attributes (raltitrexed and Mayo regimens) and administration attributes (raltitrexed, Mayo, De Gramont and Lokich regimens) in a study based on 82 patients with ACRC. Patients completed a series of rating tasks on how 'upsetting' these attributes were to them using a visual analogue scale (VAS) in a structured interview conducted by a research nurse. Mucositis and asthenia were the most and least upsetting side-effects, respectively. The side-effect profile of raltitrexed was clearly preferred by 78% of patients versus 14% for Mayo (P < 0.001). When side-effects and administration attributes were combined into an overall profile, 91% of patients selected raltitrexed as their clearly preferred regimen, versus 6% for Mayo (P < 0.001). The administration regimen of raltitrexed was ranked most acceptable (mean rank score 1.5 compared with Mayo 2.6, De Gramont 2.7, and Lokich 3.3). Given similar palliative effects, patients with ACRC exhibit preferences for raltitrexed over other regimens, based on administration and/or side-effect attributes. Such preferences should constitute an important part of decisions relating to the choice of chemotherapy regimen in ACRC.

Adult↗
PubMed1999

Partitioning effects during terminal carbon and electron flow in sediments of a low-salinity meltwater pond near Bratina Island, McMurdo Ice Shelf, Antarctica.

A study of anaerobic sediments below cyanobacterial mats of a low-salinity meltwater pond called Orange Pond on the McMurdo Ice Shelf at temperatures simulating those in the summer season (<5 degrees C) revealed that both sulfate reduction and methane production were important terminal anaerobic processes. Addition of [2-(14)C]acetate to sediment samples resulted in the passage of label mainly to CO(2). Acetate addition (0 to 27 mM) had little effect on methanogenesis (a 1.1-fold increase), and while the rate of acetate dissimilation was greater than the rate of methane production (6.4 nmol cm(-3) h(-1) compared to 2.5 to 6 nmol cm(-3) h(-1)), the portion of methane production attributed to acetate cleavage was <2%. Substantial increases in the methane production rate were observed with H(2) (2.4-fold), and H(2) uptake was totally accounted for by methane production under physiological conditions. Formate also stimulated methane production (twofold), presumably through H(2) release mediated through hydrogen lyase. Addition of sulfate up to 50-fold the natural levels in the sediment (interstitial concentration, approximately 0.3 mM) did not substantially inhibit methanogenesis, but the process was inhibited by 50-fold chloride (36 mM). No net rate of methane oxidation was observed when sediments were incubated anaerobically, and denitrification rates were substantially lower than rates for sulfate reduction and methanogenesis. The results indicate that carbon flow from acetate is coupled mainly to sulfate reduction and that methane is largely generated from H(2) and CO(2) where chloride, but not sulfate, has a modulating role. Rates of methanogenesis at in situ temperatures were four- to fivefold less than maximal rates found at 20 degrees C.

Acetates↗
PubMed1999

The nuclear receptor corepressor N-CoR regulates differentiation: N-CoR directly interacts with MyoD.

Classical ligand-activated nuclear receptors (e.g. thyroid hormone receptor, retinoic acid receptor), orphan nuclear receptors (e.g. Rev-erbAalpha/beta), Mad/Max bHLH (basic helix loop helix)-LZ proteins, and oncoproteins, PLZF and LAZ3/BCL6, bind DNA and silence transcription by recruiting a repressor complex that contains N-CoR (nuclear receptor corepressor)/SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), Sin3A/B, and HDAc-1/-2 proteins. The function of the corepressor, N-CoR, in the process of cellular differentiation and coupled phenotypic acquisition, has not been investigated. We examined the functional role of N-CoR in myogenesis (muscle differentiation), an ideal paradigm for the analysis of the determinative events that govern the cell's decision to divide or differentiate. We observed that the mRNA encoding N-CoR was suppressed as proliferating myoblasts exited the cell cycle, and formed morphologically and biochemically differentiated myotubes. Exogenous expression of N-CoR (but not RIP13) in myogenic cells ablated 1) myogenic differentiation, 2) the expression of the myoD gene family that encode the myogenic specific bHLH proteins, and 3) the crucial cell cycle regulator, p21Waf-1/Cip-1 mRNA. Furthermore, N-CoR expression efficiently inhibits the myoD-mediated myogenic conversion of pluripotential C3H10T1/2 cells. We demonstrate that MyoD-mediated transactivation and activity are repressed by N-CoR. The mechanism involves direct interactions between MyoD and N-CoR; moreover, the interaction was dependent on the amino-terminal repression domain (RD1) of N-CoR and the bHLH region of MyoD. Trichostatin A treatment significantly stimulated the activity of MyoD by approximately 10-fold and inhibited the ability of N-CoR to repress MyoD-mediated transactivation, consistent with the involvement of the corepressor and the recruitment of a histone deacteylase activity in the process. This work demonstrates that the corepressor N-CoR is a key regulator of MyoD activity and mammalian differentiation, and that N-CoR has a multifaceted role in myogenesis.

Animals↗