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1.
Sci Rep ; 12(1): 8485, 2022 05 19.
Article in English | MEDLINE | ID: mdl-35590028

ABSTRACT

Enterococcus faecalis is a natural inhabitant of the human gastrointestinal tract. This bacterial species is subdominant in a healthy physiological state of the gut microbiota (eubiosis) in adults, but can become dominant and cause infections when the intestinal homeostasis is disrupted (dysbiosis). The relatively high concentrations of bile acids deoxycholate (DCA) and taurocholate (TCA) hallmark eubiosis and dysbiosis, respectively. This study aimed to better understand how E. faecalis adapts to DCA and TCA. We showed that DCA impairs E. faecalis growth and possibly imposes a continuous adjustment in the expression of many essential genes, including a majority of ribosomal proteins. This may account for slow growth and low levels of E. faecalis in the gut. In contrast, TCA had no detectable growth effect. The evolving transcriptome upon TCA adaptation showed the early activation of an oligopeptide permease system (opp2) followed by the adjustment of amino acid and nucleotide metabolisms. We provide evidence that TCA favors the exploitation of oligopeptide resources to fuel amino acid needs in limiting oligopeptide conditions. Altogether, our data suggest that the combined effects of decreased DCA and increased TCA concentrations can contribute to the rise of E. faecalis population during dysbiosis.


Subject(s)
Bile Acids and Salts , Enterococcus faecalis , Amino Acids/metabolism , Bile Acids and Salts/metabolism , Deoxycholic Acid/metabolism , Deoxycholic Acid/pharmacology , Dysbiosis , Enterococcus faecalis/genetics , Humans , Taurocholic Acid/metabolism , Taurocholic Acid/pharmacology
2.
Oncogenesis ; 5(10): e266, 2016 Oct 24.
Article in English | MEDLINE | ID: mdl-27775700

ABSTRACT

Acquired resistance to conventional and targeted therapies is becoming a major hindrance in cancer management. It is increasingly clear that cancer cells are able to evolve and rewire canonical signalling pathways to their advantage, thus evading cell death and promoting cell invasion. The Axl receptor tyrosine kinase (RTK) has been shown to modulate acquired resistance to EGFR-targeted therapies in both breast and lung cancers. Glioblastoma multiforme (GBM) is a highly infiltrative and invasive form of brain tumour with little response to therapy. Both Axl and EGFR have been identified as major players in gliomagenesis and invasiveness. However, the mechanisms underlying a potential signalling crosstalk between EGFR and Axl RTKs are unknown. The purpose of this study was to investigate this novel and unconventional interaction among RTKs of different families in human GBM cells. With the use of western blotting, in vitro kinase activity, co-immunoprecipitation and bimolecular fluorescence complementation assays, we show that EGF stimulates activation of Axl kinase and that there is a hetero-interaction between the two RTKs. Through small interfering RNA knockdown and quantitative PCR screening, we identified distinct gene expression patterns in GBM cells that were specifically regulated by signalling from EGFR-EGFR, Axl-Axl and EGFR-Axl RTK parings. These included genes that promote invasion, which were activated only via the EGFR-Axl axis (MMP9), while EGFR-EGFR distinctly regulated the cell cycle and Axl-Axl regulated invasion. Our findings provide critical insights into the role of EGFR-Axl hetero-dimerisation in cancer cells and reveal regulation of cell invasion via Axl as a novel function of EGFR signalling.

3.
Theriogenology ; 58(5): 947-61, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12212894

ABSTRACT

Our objective was to perform a retrospective analysis of breeding soundness evaluations (BSEs) as classified by the 1993 Society for Theriogenology (SFT) guidelines [Chenoweth et al., Guidelines for using the bull breeding soundness evaluation form, in: Theriogenology Handbook, 1993, pp. B-10]. Data included BSE information obtained from five performance-testing stations in South Carolina (SC1, SC2, SC3) and Tennessee (TN1, TN2) from 1986 through 1999 on 3648 Angus, Brangus, Charolais, Chianina, Gelbvieh, Limousin, Polled Hereford, Santa Gertrudis, Simbrah, and Simmental bulls. Analyses were simplified by classifying all bulls as either satisfactory or unsatisfactory potential breeders. Of the 3648 bulls evaluated, 76.2% were classified as satisfactory potential breeders. Of all bulls evaluated, 4.0% were unsatisfactory due to inadequate spermatozoal motility, 7.0% due to inadequate spermatozoal morphology and 2.6% due to a combination of inadequate motility and morphology. Unsatisfactory classifications due to non-spermatozoal parameters out of all bulls were 10.2%, with 7.1% for inadequate scrotal circumference and 3.1% for physical abnormalities. For satisfactory and unsatisfactory bulls, respectively, means and standard deviations were 35.8 +/- 2.7 and 33.0 +/- 4.1 cm (P < 0.001) for scrotal circumference, 63 +/- 18 and 35 +/- 24% (P < 0.001) for percent motility, and 86 +/- 7 and 63 +/- 21% (P < 0.001) for percent normal morphology.


Subject(s)
Breeding/methods , Cattle , Animals , Cattle/classification , Male , Scrotum/anatomy & histology , Sperm Motility , Spermatozoa/abnormalities
4.
Genome Res ; 11(10): 1641-50, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11591641

ABSTRACT

The genome of the halophilic archaeon Halobacterium sp. NRC-1 and predicted proteome have been analyzed by computational methods and reveal characteristics relevant to life in an extreme environment distinguished by hypersalinity and high solar radiation: (1) The proteome is highly acidic, with a median pI of 4.9 and mostly lacking basic proteins. This characteristic correlates with high surface negative charge, determined through homology modeling, as the major adaptive mechanism of halophilic proteins to function in nearly saturating salinity. (2) Codon usage displays the expected GC bias in the wobble position and is consistent with a highly acidic proteome. (3) Distinct genomic domains of NRC-1 with bacterial character are apparent by whole proteome BLAST analysis, including two gene clusters coding for a bacterial-type aerobic respiratory chain. This result indicates that the capacity of halophiles for aerobic respiration may have been acquired through lateral gene transfer. (4) Two regions of the large chromosome were found with relatively lower GC composition and overrepresentation of IS elements, similar to the minichromosomes. These IS-element-rich regions of the genome may serve to exchange DNA between the three replicons and promote genome evolution. (5) GC-skew analysis showed evidence for the existence of two replication origins in the large chromosome. This finding and the occurrence of multiple chromosomes indicate a dynamic genome organization with eukaryotic character.


Subject(s)
Adaptation, Biological/genetics , Computational Biology/methods , Genome, Bacterial , Halobacterium/genetics , Base Composition , Chromosome Mapping , Codon/genetics , Halobacterium/growth & development , Isoelectric Point , Multigene Family , Protein Structure, Tertiary/genetics , Proteome/genetics , Sequence Homology, Amino Acid
5.
Phys Rev Lett ; 86(18): 4148-51, 2001 Apr 30.
Article in English | MEDLINE | ID: mdl-11328117

ABSTRACT

We have observed that resonant Rayleigh scattering dominates the emission from poly(p-phenylene vinylene) excited with photons at energies below the threshold at which excitonic migration is reduced. The intensity of the resonant emission decays exponentially with a lifetime of up to 450 fs after pulsed excitation. The coherent nature of the emission was confirmed by angular variations in the far-field emission intensity-bright and dark speckles. Persistence of a coherent polarization was demonstrated by coherent control using phase-locked pulses.

6.
Bioorg Med Chem Lett ; 11(6): 803-7, 2001 Mar 26.
Article in English | MEDLINE | ID: mdl-11277524

ABSTRACT

Zoniporide (CP-597,396) is a potent and selective inhibitor of NHE-1, which exhibits high aqueous solubility and acceptable pharmacokinetics for intravenous administration. The discovery, synthesis, activities, and rat and dog pharmacokinetics of this compound are presented. The potency and selectivity of zoniporide may be due to the conformation that the molecule adopts due to the presence of a cyclopropyl and a 5-quinolinyl substituent on the central pyrazole ring of the molecule.


Subject(s)
Guanidines/pharmacology , Pyrazoles/pharmacology , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Animals , Dogs , Guanidines/chemistry , Guanidines/pharmacokinetics , Injections, Intravenous , Molecular Conformation , Protective Agents/pharmacokinetics , Protective Agents/pharmacology , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Rats , Sodium-Hydrogen Exchangers/metabolism , Solubility , Water/chemistry
7.
Proc Natl Acad Sci U S A ; 98(5): 2521-5, 2001 Feb 27.
Article in English | MEDLINE | ID: mdl-11226271

ABSTRACT

The extremely halophilic archaeon Halobacterium sp. NRC-1 can grow phototrophically by means of light-driven proton pumping by bacteriorhodopsin in the purple membrane. Here, we show by genetic analysis of the wild type, and insertion and double-frame shift mutants of Bat that this transcriptional regulator coordinates synthesis of a structural protein and a chromophore for purple membrane biogenesis in response to both light and oxygen. Analysis of the complete Halobacterium sp. NRC-1 genome sequence showed that the regulatory site, upstream activator sequence (UAS), the putative binding site for Bat upstream of the bacterio-opsin gene (bop), is also present upstream to the other Bat-regulated genes. The transcription regulator Bat contains a photoresponsive cGMP-binding (GAF) domain, and a bacterial AraC type helix-turn-helix DNA binding motif. We also provide evidence for involvement of the PAS/PAC domain of Bat in redox-sensing activity by genetic analysis of a purple membrane overproducer. Five additional Bat-like putative regulatory genes were found, which together are likely to be responsible for orchestrating the complex response of this archaeon to light and oxygen. Similarities of the bop-like UAS and transcription factors in diverse organisms, including a plant and a gamma-proteobacterium, suggest an ancient origin for this regulon capable of coordinating light and oxygen responses in the three major branches of the evolutionary tree of life. Finally, sensitivity of four of five regulon genes to DNA supercoiling is demonstrated and correlated to presence of alternating purine-pyrimidine sequences (RY boxes) near the regulated promoters.


Subject(s)
Archaea/genetics , Regulon , Amino Acid Sequence , Base Sequence , DNA, Archaeal , Genes, Archaeal , Molecular Sequence Data , Mutation , Sequence Homology, Amino Acid
8.
J Biol Chem ; 276(8): 5739-44, 2001 Feb 23.
Article in English | MEDLINE | ID: mdl-11092896

ABSTRACT

Bacteriorhodopsin, the light-driven proton pump of Halobacterium salinarum, consists of the membrane apoprotein bacterioopsin and a covalently bound retinal cofactor. The mechanism by which retinal is synthesized and bound to bacterioopsin in vivo is unknown. As a step toward identifying cellular factors involved in this process, we constructed an in-frame deletion of brp, a gene implicated in bacteriorhodopsin biogenesis. In the Deltabrp strain, bacteriorhodopsin levels are decreased approximately 4.0-fold compared with wild type, whereas bacterioopsin levels are normal. The probable precursor of retinal, beta-carotene, is increased approximately 3.8-fold, whereas retinal is decreased by approximately 3.7-fold. These results suggest that brp is involved in retinal synthesis. Additional cellular factors may substitute for brp function in the Deltabrp strain because retinal production is not abolished. The in-frame deletion of blh, a brp paralog identified by analysis of the Halobacterium sp. NRC-1 genome, reduced bacteriorhodopsin accumulation on solid medium but not in liquid. However, deletion of both brp and blh abolished bacteriorhodopsin and retinal production in liquid medium, again without affecting bacterioopsin accumulation. The level of beta-carotene increased approximately 5.3-fold. The simplest interpretation of these results is that brp and blh encode similar proteins that catalyze or regulate the conversion of beta-carotene to retinal.


Subject(s)
Bacteriorhodopsins/biosynthesis , Genes, Bacterial , Halobacterium salinarum/genetics , Retinaldehyde/biosynthesis , Gene Deletion , Mutagenesis, Insertional , beta Carotene/metabolism
9.
Photosynth Res ; 70(1): 3-17, 2001.
Article in English | MEDLINE | ID: mdl-16228359

ABSTRACT

Halobacterium species display a variety of responses to light, including phototrophic growth, phototactic behavior, and photoprotective mechanisms. The complete genome sequence of Halobacterium species NRC-1 (Proc Natl Acad Sci USA 97: 12176-12181, 2000), coupled with the availability of a battery of methods for its analysis makes this an ideal model system for studying photobiology among the archaea. Here, we review: (1) the structure of the 2.57 Mbp Halobacterium NRC-1 genome, including a large chromosome, two minichromosomes, and 91 transposable IS elements; (2) the purple membrane regulon, which programs the accumulation of large quantities of the light-driven proton pump, bacteriorhodopsin, and allows for a period of phototrophic growth; (3) components of the sophisticated pathways for color-sensitive phototaxis; (4) the gas vesicle gene cluster, which codes for cell buoyancy organelles; (5) pathways for the production of carotenoid pigments and retinal, (6) processes for the repair of DNA damage; and (7) putative homologs of circadian rhythm regulators. We conclude with a discussion of the power of systems biology for comprehensive understanding of Halobacterium NRC-1 photobiology.

10.
Circulation ; 102(18): 2185-9, 2000 Oct 31.
Article in English | MEDLINE | ID: mdl-11056090

ABSTRACT

BACKGROUND: Unstable atherosclerotic lesions typically have an abundant inflammatory cell infiltrate, including activated T cells, macrophages, and mast cells, which may decrease plaque stability. The pathophysiology of inflammatory cell recruitment and activation in the human atheroma is incompletely described. METHODS AND RESULTS: We hypothesized that differential gene expression with DNA microarray technology would identify new genes that may participate in vascular inflammation. RNA isolated from cultured human aortic smooth muscle cells treated with tumor necrosis factor-alpha (TNF-alpha) was examined with a DNA microarray with 8600 genes. This experiment and subsequent Northern analyses demonstrated marked increases in steady-state eotaxin mRNA (>20 fold), a chemokine initially described as a chemotactic factor for eosinophils. Because eosinophils are rarely present in human atherosclerosis, we then studied tissue samples from 7 normal and 14 atherosclerotic arteries. Immunohistochemical analysis demonstrated overexpression of eotaxin protein and its receptor, CCR3, in the human atheroma, with negligible expression in normal vessels. Eotaxin was predominantly located in smooth muscle cells. The CCR3 receptor was localized primarily to macrophage-rich regions as defined by immunopositivity for CD 68; a minority of mast cells also demonstrated immunopositivity for the CCR3 receptor. CONCLUSIONS: Eotaxin and its receptor, CCR3, are overexpressed in human atherosclerosis, suggesting that eotaxin participates in vascular inflammation. These data demonstrate how genomic differential expression technology can identify novel genes that may participate in the stability of atherosclerotic lesions.


Subject(s)
Arteriosclerosis/metabolism , Chemokines, CC , Cytokines/biosynthesis , Muscle, Smooth, Vascular/metabolism , Receptors, Chemokine/biosynthesis , Vasculitis/metabolism , Actins/metabolism , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Aorta, Thoracic/metabolism , Aorta, Thoracic/pathology , Arteriosclerosis/genetics , Arteriosclerosis/pathology , Blotting, Northern , Carotid Arteries/metabolism , Carotid Arteries/pathology , Cells, Cultured , Chemokine CCL11 , Cytokines/genetics , Gene Expression , Humans , Immunohistochemistry , Mast Cells/metabolism , Mast Cells/pathology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/pathology , Oligonucleotide Array Sequence Analysis , RNA, Messenger/metabolism , Receptors, CCR3 , Receptors, Chemokine/genetics , Tumor Necrosis Factor-alpha/pharmacology , Vasculitis/genetics , Vasculitis/pathology
11.
Proc Natl Acad Sci U S A ; 97(22): 12176-81, 2000 Oct 24.
Article in English | MEDLINE | ID: mdl-11016950

ABSTRACT

We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes. The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported. Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodium-proton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms. Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria. The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea.


Subject(s)
Genome, Bacterial , Halobacterium/genetics , Biological Evolution , Cell Membrane/metabolism , DNA Repair , DNA Replication , Energy Metabolism , Halobacterium/metabolism , Lipid Bilayers , Molecular Sequence Data , Protein Biosynthesis , Recombination, Genetic , Signal Transduction , Transcription, Genetic
12.
J Biol Chem ; 275(14): 10443-52, 2000 Apr 07.
Article in English | MEDLINE | ID: mdl-10744734

ABSTRACT

Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by G protein-coupled receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated in vitro by free Gbetagamma subunits and negatively charged membrane phospholipids through their pleckstrin homology (PH) domains. However, the molecular binding determinants and physiological role for these ligands remain unclear. To address these issues, we generated an array of site-directed mutants within the GRK2 PH domain and characterized their interaction with Gbetagamma and phospholipids in vitro. Mutation of several residues in the loop 1 region of the PH domain, including Lys-567, Trp-576, Arg-578, and Arg-579, resulted in a loss of receptor phosphorylation, likely via disruption of phospholipid binding, that was reversed by Gbetagamma. Alternatively, mutation of residues distal to the C-terminal amphipathic alpha-helix, including Lys-663, Lys-665, Lys-667, and Arg-669, resulted in decreased responsiveness to Gbetagamma. Interestingly, mutation of Arg-587 in beta-sheet 3, a region not previously thought to interact with Gbetagamma, resulted in a specific and profound loss of Gbetagamma responsiveness. To further characterize these effects, two mutants (GRK2(K567E/R578E) and GRK2(R587Q)) were expressed in Sf9 cells and purified. Analysis of these mutants revealed that GRK2(K567E/R578E) was refractory to stimulation by negatively charged phospholipids but bound Gbetagamma similar to wild-type GRK2. In contrast, GRK2(R587Q) was stimulated by acidic phospholipids but failed to bind Gbetagamma. In order to examine the role of phospholipid and Gbetagamma interaction in cells, wild-type and mutant GRK2s were expressed with a beta(2)-adrenergic receptor (beta(2)AR) mutant that is responsive to GRK2 phosphorylation (beta(2)AR(Y326A)). In these cells, GRK2(K567E/R578E) and GRK2(R587Q) were largely defective in promoting agonist-dependent phosphorylation and internalization of beta(2)AR(Y326A). Similarly, wild-type GRK2 but not GRK2(K567E/R578E) or GRK2(R587Q) promoted morphinedependent phosphorylation of the mu-opioid receptor in cells. Thus, we have (i) identified several specific GRK2 binding determinants for Gbetagamma and phospholipids, and (ii) demonstrated that Gbetagamma binding is the limiting step for GRK2-dependent receptor phosphorylation in cells.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/metabolism , GTP-Binding Protein beta Subunits , GTP-Binding Protein gamma Subunits , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , Heterotrimeric GTP-Binding Proteins , Phospholipids/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites , COS Cells , Cell Line , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Phospholipids/chemistry , Phosphorylation , Protein Structure, Secondary , Spodoptera , Transfection , beta-Adrenergic Receptor Kinases
13.
Circ Res ; 85(12): 1118-23, 1999.
Article in English | MEDLINE | ID: mdl-10590237

ABSTRACT

Vascular smooth muscle cells must monitor and respond to their mechanical environment; however, the molecular response of these cells to mechanical stimuli remains incompletely defined. By applying a highly uniform biaxial cyclic strain to cultured cells, we used DNA microarray technology to describe the transcriptional profile of mechanically induced genes in human aortic smooth muscle cells. We first identified vascular endothelial growth factor (VEGF) as a mechanically induced gene in these cells; VEGF served as a positive control for these experiments. We then used a DNA microarray with 5000 genes with putative functions to identify additional mechanically induced genes. Surprisingly, relatively few genes are mechanically induced in human aortic smooth muscle cells. Only 3 transcripts of 5000 were induced >2.5-fold: cyclooxygenase-1, tenascin-C, and plasminogen activator inhibitor-1. Downregulated transcripts included matrix metalloproteinase-1 and thrombomodulin. The transcriptional profile of mechanically induced genes in human aortic smooth muscle cells suggests a response of defense against excessive deformation. These data also demonstrate that in addition to identifying large clusters of genes that respond to a given stimulus, DNA microarray technology may be used to identify a small subset of genes that comprise a highly specific molecular response.


Subject(s)
Gene Expression Regulation/physiology , Muscle, Smooth, Vascular/physiology , Cells, Cultured , Humans , Muscle, Smooth, Vascular/pathology , Stress, Mechanical , Transcription, Genetic
14.
J Biol Chem ; 274(31): 21840-6, 1999 Jul 30.
Article in English | MEDLINE | ID: mdl-10419501

ABSTRACT

Mechanical overload may change cardiac structure through angiotensin II-dependent and angiotensin II-independent mechanisms. We investigated the effects of mechanical strain on the gene expression of tenascin-C, a prominent extracellular molecule in actively remodeling tissues, in neonatal rat cardiac myocytes. Mechanical strain induced tenascin-C mRNA (3.9 +/- 0.5-fold, p < 0.01, n = 13) and tenascin-C protein in an amplitude-dependent manner but did not induce secreted protein acidic and rich in cysteine nor fibronectin. RNase protection assay demonstrated that mechanical strain induced all three alternatively spliced isoforms of tenascin-C. An angiotensin II receptor type 1 antagonist inhibited mechanical induction of brain natriuretic peptide but not tenascin-C. Antioxidants such as N-acetyl-L-cysteine, catalase, and 1, 2-dihydroxy-benzene-3,5-disulfonate significantly inhibited induction of tenascin-C. Truncated tenascin-C promoter-reporter assays using dominant negative mutants of IkappaBalpha and IkappaB kinase beta and electrophoretic mobility shift assays indicated that mechanical strain increases tenascin-C gene transcription by activating nuclear factor-kappaB through reactive oxygen species. Our findings demonstrate that mechanical strain induces tenascin-C in cardiac myocytes through a nuclear factor-kappaB-dependent and angiotensin II-independent mechanism. These data also suggest that reactive oxygen species may participate in mechanically induced left ventricular remodeling.


Subject(s)
Antioxidants/pharmacology , Gene Expression Regulation , I-kappa B Proteins , Myocardium/cytology , Myocardium/metabolism , Reactive Oxygen Species/metabolism , Tenascin/genetics , Transcription, Genetic , 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt/pharmacology , Acetylcysteine/pharmacology , Alternative Splicing , Angiotensin Receptor Antagonists , Animals , Animals, Newborn , Catalase/pharmacology , Cells, Cultured , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fibronectins/genetics , Gene Expression Regulation/drug effects , Genes, Reporter , Heart Ventricles , I-kappa B Kinase , Indazoles/pharmacology , NF-KappaB Inhibitor alpha , Osteonectin/genetics , Promoter Regions, Genetic , Protein Isoforms/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Receptor, Angiotensin, Type 1 , Receptor, Angiotensin, Type 2 , Stress, Mechanical , Tenascin/biosynthesis , Transcription, Genetic/drug effects , Transfection
15.
J Mol Cell Cardiol ; 30(5): 1019-25, 1998 May.
Article in English | MEDLINE | ID: mdl-9618242

ABSTRACT

Myocardial tissue has been demonstrated to exhibit, in response to brief periods of ischemia, both an immediate period of cytoprotection [i.e. early or "first window" preconditioning response (EPR)], and a later period of cytoprotection [i.e. delayed or "second window" preconditioning response (DPR)], when exposed to a subsequent prolonged hypoxic insult. EPR has been documented in vitro in isolated cardiac myocytes, as well as in situ in intact hearts or trabeculae, for a number of vertebrate species, including humans. However, there are no reports to date of DPR in human cardiac myocytes. To address this question, human ventricular myocytes (HVM) primary isolates were prepared from fetal ventricular muscle, grown to confluency, and studied in primary culture in serum-free medium (> 90%) ventricular myocytes as determined by immunohistochemical analysis with an anti-myosin chain antibody). Using cell viability as determined by trypan blue exclusion, an EPR response could readily be detected following 15, 30, or 60 min of simulated ischemia (SI) in a hypoxic (< 1 tau pO2) buffer containing 11 mmol/l 2-deoxyglucose, followed by a prolonged (c. 17 h) SI challenge. In addition, HVM exposed to 60 min of SI, followed after 24 h by a period of SI, also exhibited a "second window" DPR (80 +/- 10% compared to 71 +/- 11% survival, in preconditioned and non-preconditioned cultures; P < 0.05; n = 18 independent experiments). Thus, in response to short periods of SI, human ventricular myocytes in vitro exhibit both "first window" and "second window" cytoprotective responses to subsequent, prolonged ischemic stress.


Subject(s)
Cell Hypoxia , Heart Ventricles/pathology , Ischemic Preconditioning, Myocardial , Myocardial Ischemia/pathology , Analysis of Variance , Cells, Cultured , Cytoprotection , Humans , Time Factors
16.
Biochem Biophys Res Commun ; 244(3): 832-7, 1998 Mar 27.
Article in English | MEDLINE | ID: mdl-9535752

ABSTRACT

There has been considerable difficulty in defining distinct adrenomedullin (AM) binding sites and function in vivo. However, a rat adrenomedullin receptor (rAMR) and a putative human adrenomedullin receptor (hAMR) have recently been reported. We attempted to confirm and extend the pharmacological characterization of these cloned receptors. COS-7 cells transfected with rAMR or epitope tagged rAMR display abundant rAMR mRNA expression and cell-surface receptor localization. Specific 125I-AM binding is detected in transfected cells; however, similar levels of binding are also detected in cells transfected with vector DNA alone. This AM binding site fails to mediate any changes in cAMP in response to AM. In contrast, Swiss 3T3 cells, expressing specific endogenous AM receptors, display AM binding and functional cAMP responses. Transfection studies performed with the putative hAMR yield similar results. These data suggest that the proposed rAMR and hAMR do not represent authentic adrenomedullin receptors.


Subject(s)
Membrane Proteins/biosynthesis , Peptides/metabolism , Receptors, Peptide , 3T3 Cells , Adrenomedullin , Animals , Artifacts , Binding, Competitive , COS Cells , Cloning, Molecular , Cyclic AMP/analysis , Humans , Membrane Proteins/genetics , Mice , Rats , Receptors, Adrenomedullin , Recombinant Proteins/biosynthesis , Transfection
17.
Cardiovasc Res ; 33(2): 410-5, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9074706

ABSTRACT

OBJECTIVE: The aim of this study was to determine whether selective activation of the adenosine A3 receptor reduces infarct size in a Langendorff model of myocardial ischemia-reperfusion injury. METHODS: Buffer-perfused rabbit hearts were exposed to 30 min regional ischemia and 120 min of reperfusion. Infarct size was measured by tetrazolium staining and normalized for area-at-risk (IA/AAR). RESULTS: Preconditioning by 5 min global ischemia and 10 min reperfusion reduced infarct size (IA/AAR) to 19 +/- 4% (controls: 67 +/- 5%). Replacing global ischemia with 5 min perfusion of the rabbit A3-selective agonist, IB-MECA (A3 Ki: 2 nM; A1 Ki: 30 nM) elicited a concentration-dependent reduction in infarct size; 50 nM IB-MECA reduced IA/AAR to 24 +/- 4%. The A1-selective agonist, R-PIA (25 nM) reduced IA/AAR to a similar extent (21 +/- 6%). However, while the cardioprotective effect of R-PIA was significantly inhibited (54 +/- 7% IA/AAR) by the rabbit A1-selective antagonist, BWA1433 (50 nM), the IB-MECA-dependent cardioprotection was unaffected (28 +/- 6% IA/AAR). A non-selective (A1 vs. A3) concentration of BWA1433 (5 microM) significantly attenuated the IB-MECA-dependent cardioprotection (61 +/- 7% IA/AAR). CONCLUSIONS: These data clearly demonstrate that selective A3 receptor activation provides cardioprotection from ischemia-reperfusion injury in the rabbit heart. Furthermore, the degree of A3-dependent cardioprotection is similar to that provided by A1 receptor stimulation or ischemic preconditioning.


Subject(s)
Adenosine/analogs & derivatives , Myocardial Ischemia/prevention & control , Phenylisopropyladenosine/therapeutic use , Receptors, Purinergic/drug effects , Adenosine/therapeutic use , Animals , Disease Models, Animal , Male , Myocardial Reperfusion Injury/prevention & control , Rabbits , Stimulation, Chemical
18.
J Pharmacol Exp Ther ; 280(1): 122-8, 1997 Jan.
Article in English | MEDLINE | ID: mdl-8996189

ABSTRACT

The role of adenosine A1 and A3 receptors in mediating cardioprotection has been studied predominantly in rabbits, yet the pharmacological characteristics of rabbit adenosine A1 and A3 receptor subtypes are unknown. Thus, the rabbit adenosine A3 receptor was cloned and expressed, and its pharmacology was compared with that of cloned adenosine A1 receptors. Stable transfection of rabbit A1 or A3 cDNAs in Chinese hamster ovary-K1 cells resulted in high levels of expression of each of the receptors, as demonstrated by high-affinity binding of the A1/A3 adenosine receptor agonist N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA). For both receptors, binding of 125I-ABA was inhibited by the GTP analog 5'-guanylimidodiphosphate, and forskolin-stimulated cyclic AMP accumulation was inhibited by the adenosine receptor agonist (R)-phenylisopropyladenosine. The rank orders of potency of adenosine receptor agonists for inhibition of 125I-ABA binding were as follows: rabbit A1, N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N-ethylcarboxamidoadenosine > or = I-ABA > or = N6-2-(4-aminophenyl) ethyladenosine > > N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > N6-(4-amino-3-benzyl)adenosine; rabbit A3, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > or = I-ABA > > N-ethylcarboxamidoadenosine > N6-2-(4-aminophenyl) ethyladenosine = N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N6-(4-amino-3-benzyl)adenosine. The adenosine receptor antagonist rank orders were as follow: rabbit A1, 8-cyclopentyl-1,3-dipropylxanthine > 1,3- dipropyl-8-(4-acrylate)phenylxanthine > or = xanthine amine congener > > 8-(p-sulfophenyl)theophylline; rabbit A3, xanthine amine congener > 1,3-dipropyl-8-(4-acrylate)phenylxanthine > or = 8-cyclopentyl-1,3-dipropylxanthine > > 8-(p-sulfophenyl)theophylline. These observations confirm the identity of the expressed proteins as A1 and A3 receptors. The results will facilitate further in-depth studies of the roles of A1 and A3 receptors in adenosine-mediated cardioprotection in rabbits, which can now be based on the appropriate recombinant rabbit A1 and A3 receptor pharmacology.


Subject(s)
Receptors, Purinergic P1/genetics , Adenosine/metabolism , Amino Acid Sequence , Animals , Base Sequence , CHO Cells , Cloning, Molecular , Cricetinae , Cyclic AMP/biosynthesis , Iodine Radioisotopes , Molecular Sequence Data , Rabbits , Receptors, Purinergic P1/biosynthesis , Receptors, Purinergic P1/physiology
19.
Cardiovasc Res ; 36(1): 52-9, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9415272

ABSTRACT

OBJECTIVE: Adenosine receptor activation has been implicated in the mechanism of ischaemic preconditioning protection. Evidence suggests adenosine A1 receptor involvement, and possibly A3 receptor involvement in the rabbit. This study investigated the roles of these receptors in human preconditioning. Human A1- and A3-selective compounds were chosen based on Ki values for inhibition of N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA) binding to stably expressed recombinant human A1 and A3 receptors. Cyclopentyladenosine (CPA), a 194-fold selective A1 agonist, and iodobenzylmethylcarboxamidoadenosine (IBMECA), a 10-fold selective A3 agonist were used alone and in combination with dipropylcyclopentylxanthine (DPCPX) a 62-fold selective A1 antagonist. METHODS: Human atrial trabeculae were superfused with oxygenated Tyrode's solution. After stabilisation, muscles underwent one of 8 protocols (n = 6 per group), followed by 90 min of simulated ischaemia and 120 min of reoxygenation. The experimental endpoint was recovery of contractile function, presented as percentage baseline function. RESULTS: 5 nM CPA (52.2 +/- 3.1%), 30 nM IBMECA (49.7 +/- 3.8%) and preconditioning (55.3 +/- 2.5%) produced similar functional recoveries at 120 min of reoxygenation; significantly different to controls (27.7 +/- 1.0%; P < 0.05, ANOVA). When DPCPX (200 nM) was added prior to 5 nM CPA, protection was lost (31.8 +/- 0.9%), but when added prior to 30 nM IBMECA, muscles continued to be significantly protected (41.5 +/- 2.3%). CONCLUSIONS: In human atrium both A1 and A3 receptor stimulation appears to mimic ischaemic preconditioning. This may represent the first evidence for A3 receptor involvement in 'pharmacological' preconditioning of human myocardium.


Subject(s)
Heart Atria/metabolism , Myocardial Ischemia/prevention & control , Receptors, Purinergic P1/physiology , Adenosine/analogs & derivatives , Adenosine/pharmacology , Dinucleoside Phosphates/pharmacology , Heart Atria/drug effects , Humans , In Vitro Techniques , Ischemic Preconditioning, Myocardial , Models, Biological , Myocardial Contraction/drug effects , Myocardial Ischemia/metabolism , Myocardial Reperfusion , Purinergic P1 Receptor Agonists , Purinergic P1 Receptor Antagonists , Receptor, Adenosine A3 , Xanthines/pharmacology
20.
J Biol Chem ; 269(46): 29212-9, 1994 Nov 18.
Article in English | MEDLINE | ID: mdl-7961888

ABSTRACT

Central to spectrin's function is its association with the plasma membrane. The linking proteins ankyrin and protein 4.1 partly mediate this association, and their interactions with spectrin are well understood. Both beta I (erythrocyte) and beta II (fodrin, beta G) spectrin also associate with unknown protein receptors in crude membrane preparations by ankyrin and protein 4.1 independent mechanisms. As a first step to understanding this interaction, kinetic and equilibrium assays have been used to monitor which regions of beta I and beta II spectrin inhibit the binding of purified 125I-labeled bovine brain spectrin to demyelinated and NaOH-stripped bovine brain membranes. A series of 19 recombinant proteins spanning the entire sequence of beta II spectrin, including an alternatively spliced NH2-terminal isoform (beta II epsilon 2 spectrin), were prepared as glutathione S-transferase fusion proteins. Also prepared were peptides representing the alternatively spliced COOH-terminal domain found in beta I epsilon 2 spectrin ("muscle spectrin"). Two distinct sequence motifs inhibited the binding of native brain spectrin. Membrane association domain 1 (MAD1) was represented in all fusion peptides that included spectrin repeat 1. These peptides slowed the kinetics of brain spectrin binding and inhibited up to 46% of the maximal binding under the conditions of these assays (apparent Ki < or = 0.2 microM). Peptides representative of repeats 2-17 of beta II spectrin were devoid of inhibitory activity. The second membrane association domain (MAD2) was identified in penultimate COOH-terminal sequences (domain III) of both beta II and beta I epsilon 2 spectrin. These sequences were absent in beta I epsilon 1 (erythrocyte) spectrin. MAD2 competitively inhibited over 80% of brain spectrin binding in these assays, with an apparent Ki < or = 0.1 microM. Direct binding studies confirmed that both MAD1 and MAD2 peptides associated with membranes with affinities comparable to their inhibition constants. Sequence comparisons suggest that MAD1 is created by the insertion of two non-homologous sequence motifs into repeat 1, extending it from 106 to 122 amino acids. Similarly, MAD2 encompasses a putative site of beta gamma-heterotrimeric G-protein binding called the pleckstrin homology domain, and MAD2 may in fact be the pleckstrin homology domain although this has not been rigorously proven. Collectively these studies identify two novel functional motifs in spectrin that mediate ankyrin independent association with membranes. We hypothesize that these motifs and their still to be discovered ligands play a primary role in the nascent assembly and stabilization of an ordered and polarized spectrin skeleton.


Subject(s)
Carrier Proteins/chemistry , Microfilament Proteins/chemistry , Muscle, Skeletal/chemistry , Spectrin/chemistry , Alternative Splicing , Amino Acid Sequence , Base Sequence , Carrier Proteins/genetics , Cell Membrane/chemistry , Humans , Microfilament Proteins/genetics , Molecular Sequence Data , Peptides/chemistry , RNA, Messenger/genetics , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid , Spectrin/genetics
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