RESUMEN
The basis for commitment to cell division in late G1 phase, called Start in yeast, is a critical but still poorly understood aspect of eukaryotic cell proliferation. Most dividing cells accumulate mass and grow to a critical cell size before traversing the cell cycle. This size threshold couples cell growth to division and thereby establishes long-term size homeostasis. At present, mechanisms involved in cell size homeostasis in fungal pathogens are not well described. Our previous survey of the size phenome in Candida albicans focused on 279 unique mutants enriched mainly in kinases and transcription factors (Sellam et al. PLoS Genet 15:e1008052, 2019). To uncover novel size regulators in C. albicans and highlight potential innovation within cell size control in pathogenic fungi, we expanded our genetic survey of cell size to include 1301 strains from the GRACE (Gene Replacement and Conditional Expression) collection. The current investigation uncovered both known and novel biological processes required for cell size homeostasis in C. albicans. We also confirmed the plasticity of the size control network as few C. albicans size genes overlapped with those of the budding yeast Saccharomyces cerevisiae. Many new size genes of C. albicans were associated with biological processes that were not previously linked to cell size control and offer an opportunity for future investigation. Additional work is needed to understand if mitochondrial activity is a critical element of the metric that dictates cell size in C. albicans and whether modulation of the onset of actomyosin ring constriction is an additional size checkpoint.
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Candida albicans , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Actomiosina/genética , Actomiosina/metabolismo , Citocinesis , Tamaño de la Célula , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión GénicaRESUMEN
Microbial natural products are specialized metabolites that are sources of many bioactive compounds including antibiotics, antifungals, antiparasitics, anticancer agents, and probes of biology. The assembly of libraries of producers of natural products has traditionally been the province of the pharmaceutical industry. This sector has gathered significant historical collections of bacteria and fungi to identify new drug leads with outstanding outcomes-upwards of 60% of drug scaffolds originate from such libraries. Despite this success, the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds in the drug industry. The advent of advanced mass spectrometry tools, along with rapid whole genome sequencing and in silico identification of biosynthetic gene clusters that encode the machinery necessary for the synthesis of specialized metabolites, offers the opportunity to revisit microbial natural product libraries with renewed vigor. Assembling a suitable library of microbes and extracts for screening requires the investment of resources and the development of methods that have customarily been the proprietary purview of large pharmaceutical companies. Here, we report a perspective on our efforts to assemble a library of natural product-producing microbes and the establishment of methods to extract and fractionate bioactive compounds using resources available to most academic labs. We validate the library and approach through a series of screens for antimicrobial and cytotoxic agents. This work serves as a blueprint for establishing libraries of microbial natural product producers and bioactive extract fractions suitable for screens of bioactive compounds. ONE-SENTENCE SUMMARY: Natural products are key to discovery of novel antimicrobial agents: Here, we describe our experience and lessons learned in constructing a microbial natural product and pre-fractionated extract library.
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Antineoplásicos , Productos Biológicos , Productos Biológicos/química , Biblioteca de Genes , Hongos/genética , Industria FarmacéuticaRESUMEN
Cell size is a complex trait that responds to developmental and environmental cues. Quantitative size analysis of mutant strain collections disrupted for protein kinases and transcriptional regulators in the pathogenic yeast Candida albicans uncovered 66 genes that altered cell size, few of which overlapped with known size genes in the budding yeast Saccharomyces cerevisiae. A potent size regulator specific to C. albicans was the conserved p38/HOG MAPK module that mediates the osmostress response. Basal HOG activity inhibited the SBF G1/S transcription factor complex in a stress-independent fashion to delay the G1/S transition. The HOG network also governed ribosome biogenesis through the master transcriptional regulator Sfp1. Hog1 bound to the promoters and cognate transcription factors for ribosome biogenesis regulons and interacted genetically with the SBF G1/S machinery, and thereby directly linked cell growth and division. These results illuminate the evolutionary plasticity of size control and identify the HOG module as a nexus of cell cycle and growth regulation.
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Candida albicans/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Candida albicans/metabolismo , Ciclo Celular , División Celular , Tamaño de la Célula , Regulación Fúngica de la Expresión Génica/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Factores de Transcripción , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward.
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Proteína BRCA1/genética , Proteínas Portadoras/genética , Ciclo Celular/genética , Reparación del ADN , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/genética , Proteínas de Unión a Telómeros/genética , Proteína BRCA1/metabolismo , Sitios de Unión , Proteínas Portadoras/metabolismo , Reparación del ADN por Unión de Extremidades/genética , Endodesoxirribonucleasas , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Fase S , Proteínas de Unión a Telómeros/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
The continued efficacy of glycopeptide antibiotics (GPAs) against Gram-positive bacteria is challenged by the emergence and spread of GPA-resistant pathogens, particularly vancomycin-resistant enterococci (VRE). The growing frequency of GPA resistance propels the need for innovative development of more effective antibiotics. Unlike canonical GPAs like vancomycin, Type V GPAs adopt a distinct mode of action by binding peptidoglycan and blocking the activity of autolysins essential for cell division, rendering them a promising class of antibiotics for further development. In this study, the Type V GPA, rimomycin A, was modified to generate 32 new analogues. Compound 17, derived from rimomycin A through N-terminal acylation and C-terminal amidation, exhibited improved anti-VRE activity and solubility. In a VRE-A neutropenic thigh infection mouse model, compound 17 significantly lowered the bacterial load by 3-4 orders of magnitude. This study sets the stage to develop next-generation GPAs in response to growing VRE infections.
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Infecciones por Bacterias Grampositivas , Enterococos Resistentes a la Vancomicina , Animales , Ratones , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antibacterianos/química , Glicopéptidos/farmacología , Glicopéptidos/uso terapéutico , Glicopéptidos/química , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Infecciones por Bacterias Grampositivas/microbiología , Pruebas de Sensibilidad Microbiana , Biología Sintética , Vancomicina/farmacología , Vancomicina/químicaRESUMEN
Antibiotics have transformed modern medicine. They are essential for treating infectious diseases and enable vital therapies and procedures. However, despite this success, their continued use in the 21st century is imperiled by two orthogonal challenges. The first is that the microbes targeted by these drugs evolve resistance to them over time. The second is that antibiotic discovery and development are no longer cost-effective using traditional reimbursement models. Consequently, there are a dwindling number of companies and laboratories dedicated to delivering new antibiotics, resulting in an anemic pipeline that threatens our control of infections. The future of antibiotics requires innovation in a field that has relied on highly traditional methods of discovery and development. This will require substantial changes in policy, quantitative understanding of the societal value of these drugs, and investment in alternatives to traditional antibiotics. These include narrow-spectrum drugs, bacteriophage, monoclonal antibodies, and vaccines, coupled with highly effective diagnostics. Addressing the antibiotic crisis to meet our future needs requires considerable investment in both research and development, along with ensuring a viable marketplace that encourages innovation. This review explores the past, present, and future of antimicrobial therapy.
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Antibacterianos , Vacunas , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Vacunas/uso terapéuticoRESUMEN
We identify a selective nanomolar inhibitor of blood-stage malarial proliferation from a screen of microbial natural product extracts. The responsible compound, PDE-I2, is a precursor of the anticancer duocarmycin family that preserves the class's sequence-specific DNA binding but lacks its signature DNA alkylating cyclopropyl warhead. While less active than duocarmycin, PDE-I2 retains comparable antimalarial potency to chloroquine. Importantly, PDE-I2 is >1,000-fold less toxic to human cell lines than duocarmycin, with mitigated impacts on eukaryotic chromosome stability. PDE-I2 treatment induces severe defects in parasite nuclear segregation leading to impaired daughter cell formation during schizogony. Time-of-addition studies implicate parasite DNA metabolism as the target of PDE-I2, with defects observed in DNA replication and chromosome integrity. We find the effect of duocarmycin and PDE-I2 on parasites is phenotypically indistinguishable, indicating that the DNA binding specificity of duocarmycins is sufficient and the genotoxic cyclopropyl warhead is dispensable for the parasite-specific selectivity of this compound class.
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Antimaláricos , Productos Biológicos , Antagonistas del Ácido Fólico , Malaria , Parásitos , Animales , Antimaláricos/farmacología , Productos Biológicos/farmacología , ADN/química , Duocarmicinas , HumanosRESUMEN
Stimulation of cardiac AMP-activated protein kinase (AMPK) has been demonstrated in both prohypertrophic and antihypertrophic settings, although the reasons for such discrepant results are not well understood. We determined how AMPK is regulated in response to phenylephrine-induced cardiomyocyte hypertrophy and assessed whether AMPK activity may be a factor underlying the antihypertrophic effect of adenosine receptor agonists. The role of AMPK in hypertrophic responses was determined by assessing the effect of the AMPK activator 5-aminoimidazole-4-carboxyamide ribonucleoside on three hypertrophic indexes, including protein synthesis, cell surface area, and fetal gene expression. The changes in phosphorylation of the catalytic alpha-subunit of AMPK at two different sites, Thr(172) and Ser(485/491), in response to phenylephrine and adenosine receptor agonists were also examined. 5-Aminoimidazole-4-carboxyamide ribonucleoside completely abolished phenylephrine-induced increases in protein synthesis, cell surface area, and fetal gene expression. AMPK phosphorylation time course studies revealed that phenylephrine induced a time-dependent activation at site Ser(485/491), in contrast to adenosine receptor agonists, which demonstrated rapid AMPK phosphorylation at Thr(172). Furthermore, the phosphorylation at Ser(485/491) by phenylephrine was not affected by the addition of adenosine receptor agonists, although, conversely, phosphorylation of AMPK at Thr(172) by adenosine receptor agonists was abrogated by the addition of phenylephrine. We propose from these results that cardiomyocyte hypertrophic and antihypertrophic responses, at least with respect to inhibition of phenylephrine-induced hypertrophy by adenosine receptor agonists, are mediated by multisite AMPK regulation. The latter are reflected by increased phosphorylation at Ser(485/491) and at Thr(172), associated with prohypertrophic and antihypertrophic responses, respectively.
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Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina/agonistas , Adenosina/farmacología , Animales Recién Nacidos/fisiología , Cardiomegalia/prevención & control , Cardiotónicos/farmacología , Fármacos Cardiovasculares/farmacología , Miocitos Cardíacos/efectos de los fármacos , Fenilefrina/farmacología , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Western Blotting , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Ventrículos Cardíacos/efectos de los fármacos , Leucina/metabolismo , Miocitos Cardíacos/fisiología , Miocitos Cardíacos/ultraestructura , Fosforilación , Ratas , Ratas Sprague-Dawley , Ribonucleótidos/farmacologíaRESUMEN
Adenosine has been shown to exert direct antihypertrophic effects on the heart, and plasma adenosine levels have been shown to be elevated in patients with heart failure. It has therefore been proposed that endogenously synthesized adenosine may function as a cardiac antihypertrophic factor. The present study was aimed to determine whether the adenosine system is altered in a potential adaptive manner following phenylephrine-induced hypertrophy in cultured neonatal rat ventricular myocytes. Phenylephrine produced significant hypertrophy as determined by cell size and atrial natriuretic peptide gene expression, which was accompanied by significantly increased gene and protein expression of adenosine A(1), A(2a), and A(3) receptors. These effects and the hypertrophic response were prevented by the alpha(1)-adrenoceptor antagonist prazosin as well as pharmacological agonists for all adenosine receptor subtypes. The upregulation of adenosine receptors by phenylephrine was also abrogated by adenosine 5'-(alpha,beta-methylene)diphosphate, an inhibitor of ectosolic 5'-nucleotidase. Moreover, phenylephrine significantly increased production of adenosine from myocytes in the presence of a nucleoside transport and adenosine deaminase inhibitor, the combination of which abrogated the hypertrophic effect of phenylephrine. The latter effect was reversed by adenosine receptor antagonists. Phenylephrine also produced a significant upregulation in expression levels of equilibrative nucleoside transporter 1 although expression levels of equilibrative nucleoside transporter 2 were unaffected. Taken together, our results suggest an adaptive upregulation of the adenosine system to phenylephrine-induced cardiomyocyte hypertrophy that serves to limit the hypertrophic effect of alpha(1-)adrenoceptor activation.
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Adenosina/metabolismo , Cardiomegalia/inducido químicamente , Cardiomegalia/patología , Ventrículos Cardíacos/patología , Miocitos Cardíacos/patología , Fenilefrina/efectos adversos , Regulación hacia Arriba/fisiología , Animales , Animales Recién Nacidos , Cardiomegalia/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Ventrículos Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas de Transporte de Nucleósidos/metabolismo , Fenilefrina/farmacología , Prazosina/farmacología , Antagonistas de Receptores Purinérgicos P1 , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P1/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Systematic genetic interaction profiles can reveal the mechanisms-of-action of bioactive compounds. The imipridone ONC201, which is currently in cancer clinical trials, has been ascribed a variety of different targets. To investigate the genetic dependencies of imipridone action, we screened a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) knockout library in the presence of either ONC201 or its more potent analog ONC212. Loss of the mitochondrial matrix protease CLPP or the mitochondrial intermediate peptidase MIPEP conferred strong resistance to both compounds. Biochemical and surrogate genetic assays showed that impridones directly activate CLPP and that MIPEP is necessary for proteolytic maturation of CLPP into a catalytically competent form. Quantitative proteomic analysis of cells treated with ONC212 revealed degradation of many mitochondrial as well as nonmitochondrial proteins. Prompted by the conservation of ClpP from bacteria to humans, we found that the imipridones also activate ClpP from Escherichia coli, Bacillus subtilis, and Staphylococcus aureus in biochemical and genetic assays. ONC212 and acyldepsipeptide-4 (ADEP4), a known activator of bacterial ClpP, caused similar proteome-wide degradation profiles in S. aureus ONC212 suppressed the proliferation of a number of Gram-positive (S. aureus, B. subtilis, and Enterococcus faecium) and Gram-negative species (E. coli and Neisseria gonorrhoeae). Moreover, ONC212 enhanced the ability of rifampin to eradicate antibiotic-tolerant S. aureus persister cells. These results reveal the genetic dependencies of imipridone action in human cells and identify the imipridone scaffold as a new entry point for antibiotic development.
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Antibacterianos/farmacología , Antineoplásicos/farmacología , Endopeptidasa Clp/metabolismo , Proteínas de Escherichia coli/agonistas , Imidazoles/farmacología , Proteolisis , Piridinas/farmacología , Pirimidinas/farmacología , Bacillus subtilis/efectos de los fármacos , Sitios de Unión , Secuencia Conservada , Depsipéptidos/metabolismo , Endopeptidasa Clp/química , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Metaloendopeptidasas/metabolismo , Unión Proteica , Rifampin/farmacología , Staphylococcus aureus/efectos de los fármacosRESUMEN
A switch in cellular identity in budding yeast requires the ubiquitin-dependent elimination of pre-existing master regulators encoded by the MAT locus. Failure to disassemble the prior state not only impairs the cell type transition but imparts a hybrid cellular fate. This theme will undoubtedly arise in many developmental and disease contexts.
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Diferenciación Celular , Regulación Fúngica de la Expresión Génica , Genes Fúngicos/genética , Saccharomycetales/genética , Transducción de Señal , Proteínas de Homeodominio/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción , Ubiquitina/metabolismoRESUMEN
One of the most critical but still poorly understood aspects of eukaryotic cell proliferation is the basis for commitment to cell division in late G1 phase, called Start in yeast and the Restriction Point in metazoans. In all species, a critical cell size threshold coordinates cell growth with cell division and thereby establishes a homeostatic cell size. While a comprehensive survey of cell size genetic determinism has been performed in the saprophytic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, very little is known in pathogenic fungi. As a number of critical Start regulators are haploinsufficient for cell size, we applied a quantitative analysis of the size phenome, using elutriation-barcode sequencing methodology, to 5639 barcoded heterozygous deletion strains of the opportunistic yeast Candida albicans Our screen identified conserved known regulators and biological processes required to maintain size homeostasis in the opportunistic yeast C. albicans We also identified novel C. albicans-specific size genes and provided a conceptual framework for future mechanistic studies. Interestingly, some of the size genes identified were required for fungal pathogenicity suggesting that cell size homeostasis may be elemental to C. albicans fitness or virulence inside the host.
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Candida albicans/genética , Proliferación Celular/genética , Tamaño de la Célula , Haploinsuficiencia/genética , Secuencia de Aminoácidos/genética , Candida albicans/patogenicidad , Ciclo Celular/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Genoma FúngicoRESUMEN
S1 domains occur in four of the major enzymes of mRNA decay in Escherichia coli: RNase E, PNPase, RNase II, and RNase G. Here, we report the structure of the S1 domain of RNase E, determined by both X-ray crystallography and NMR spectroscopy. The RNase E S1 domain adopts an OB-fold, very similar to that found with PNPase and the major cold shock proteins, in which flexible loops are appended to a well-ordered five-stranded beta-barrel core. Within the crystal lattice, the protein forms a dimer stabilized primarily by intermolecular hydrophobic packing. Consistent with this observation, light-scattering, chemical crosslinking, and NMR spectroscopic measurements confirm that the isolated RNase E S1 domain undergoes a specific monomer-dimer equilibrium in solution with a K(D) value in the millimolar range. The substitution of glycine 66 with serine dramatically destabilizes the folded structure of this domain, thereby providing an explanation for the temperature-sensitive phenotype associated with this mutation in full-length RNase E. Based on amide chemical shift perturbation mapping, the binding surface for a single-stranded DNA dodecamer (K(D)=160(+/-40)microM) was identified as a groove of positive electrostatic potential containing several exposed aromatic side-chains. This surface, which corresponds to the conserved ligand-binding cleft found in numerous OB-fold proteins, lies distal to the dimerization interface, such that two independent oligonucleotide-binding sites can exist in the dimeric form of the RNase E S1 domain. Based on these data, we propose that the S1 domain serves a dual role of dimerization to aid in the formation of the tetrameric quaternary structure of RNase E as described by Callaghan et al. in 2003 and of substrate binding to facilitate RNA hydrolysis by the adjacent catalytic domains within this multimeric enzyme.
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Endorribonucleasas/metabolismo , Oligonucleótidos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Endorribonucleasas/química , Endorribonucleasas/genética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mutación , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , TemperaturaRESUMEN
The ability of cells to respond to environmental changes and adapt their metabolism enables cell survival under stressful conditions. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) is particularly well adapted to the harsh conditions of anaerobic wine fermentation. However, S. cerevisiae gene function has not been previously systematically interrogated under conditions of industrial fermentation. We performed a genome-wide study of essential and nonessential S. cerevisiae gene requirements during grape juice fermentation to identify deletion strains that are either depleted or enriched within the viable fermentative population. Genes that function in autophagy and ubiquitin-proteasome degradation are required for optimal survival during fermentation, whereas genes that function in ribosome assembly and peroxisome biogenesis impair fitness during fermentation. We also uncover fermentation phenotypes for 139 uncharacterized genes with no previously known cellular function. We demonstrate that autophagy is induced early in wine fermentation in a nitrogen-replete environment, suggesting that autophagy may be triggered by other forms of stress that arise during fermentation. These results provide insights into the complex fermentation process and suggest possible means for improvement of industrial fermentation strains.
RESUMEN
BACKGROUND: Molecular barcode arrays provide a powerful means to analyze cellular phenotypes in parallel through detection of short (20-60 base) unique sequence tags, or "barcodes", associated with each strain or clone in a collection. However, costs of current methods for microarray construction, whether by in situ oligonucleotide synthesis or ex situ coupling of modified oligonucleotides to the slide surface are often prohibitive to large-scale analyses. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that unmodified 20mer oligonucleotide probes printed on conventional surfaces show comparable hybridization signals to covalently linked 5'-amino-modified probes. As a test case, we undertook systematic cell size analysis of the budding yeast Saccharomyces cerevisiae genome-wide deletion collection by size separation of the deletion pool followed by determination of strain abundance in size fractions by barcode arrays. We demonstrate that the properties of a 13K unique feature spotted 20 mer oligonucleotide barcode microarray compare favorably with an analogous covalently-linked oligonucleotide array. Further, cell size profiles obtained with the size selection/barcode array approach recapitulate previous cell size measurements of individual deletion strains. Finally, through atomic force microscopy (AFM), we characterize the mechanism of hybridization to unmodified barcode probes on the slide surface. CONCLUSIONS/SIGNIFICANCE: These studies push the lower limit of probe size in genome-scale unmodified oligonucleotide microarray construction and demonstrate a versatile, cost-effective and reliable method for molecular barcode analysis.
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Procesamiento Automatizado de Datos , Microscopía de Fuerza Atómica , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Sondas de ADN , Procesamiento Automatizado de Datos/economía , Genes Fúngicos , Genoma Fúngico , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos/economía , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Saccharomyces cerevisiaeRESUMEN
Recent evidence suggests that both adenosine receptor (AR) and K ATP channel activation exert antihypertrophic effects in cardiac myocytes. We studied the relative contributions of mitochondrial K ATP (mitoK ATP) and sarcolemmal K ATP (sarcK ATP) to the antihypertrophic effects of ARs in primary cultures of neonatal rat ventricular myocytes exposed for 24 h with the alpha1 adrenoceptor agonist phenylephrine (PE). The A1R agonist N6-cyclopentyladenosine (CPA), the A(2A)R agonist CGS21680 [2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine], and the A3R agonist N6-(3-iodobenzyl)adenosine-5'-methyluronamide (IB-MECA) all prevented PE-induced hypertrophy. Glibenclamide, a nonselective K(ATP) channel blocker reversed the antihypertrophic effect of all three AR agonists as determined by cell size and atrial natriuretic peptide expression and early c-fos up-regulation. In contrast, the mitoK(ATP) blocker 5-hydroxydecanoic acid selectively attenuated the effect of CGS21680 and IB-MECA, whereas HMR1098 [1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea, sodium salt], a specific blocker of sarcK(ATP), only abolished the antihypertrophic effect of CPA. Moreover, both CGS21680 and IB-MECA but not CPA decreased the mitochondrial membrane potential when PE was present, similarly to that seen with diazoxide, and both agents inhibited PE-stimulated elevation in mitochondrial Ca2+. All AR agonists diminished PE-induced phosphoserine/threonine kinase and protein kinase B up-regulation, which was unaffected by any K(ATP) blocker. Our data suggest that AR-mediated antihypertrophic effects are mediated by distinct K(ATP) channels, with sarcK(ATP) mediating the antihypertrophic effects of A1R activation, whereas mitoK(ATP) activation mediates the antihypertrophic effects of both A(2A)R and A3R agonists.
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Cardiomegalia/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Canales de Potasio/fisiología , Receptores Purinérgicos P1/fisiología , Animales , Animales Recién Nacidos , Calcio/metabolismo , Células Cultivadas , Ácidos Decanoicos/farmacología , Diazóxido/farmacología , Hidroxiácidos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
In Saccharomyces cerevisiae, DNA replication stress activates the replication checkpoint, which slows S-phase progression, stabilizes slowed or stalled replication forks, and relieves inhibition of the ribonucleotide reductase (RNR) complex. To identify novel genes that promote cellular viability after replication stress, the S. cerevisiae non-essential haploid gene deletion set (4812 strains) was screened for sensitivity to the RNR inhibitor hydroxyurea (HU). Strains bearing deletions in either CCR4 or CAF1/POP2, which encode components of the cytoplasmic mRNA deadenylase complex, were particularly sensitive to HU. We found that Ccr4 cooperated with the Dun1 branch of the replication checkpoint, such that ccr4Delta dun1Delta strains exhibited irreversible hypersensitivity to HU and persistent activation of Rad53. Moreover, because ccr4Delta and chk1Delta exhibited epistasis in several genetic contexts, we infer that Ccr4 and Chk1 act in the same pathway to overcome replication stress. A counterscreen for suppressors of ccr4Delta HU sensitivity uncovered mutations in CRT1, which encodes the transcriptional repressor of the DNA-damage-induced gene regulon. Whereas Dun1 is known to inhibit Crt1 repressor activity, we found that Ccr4 regulates CRT1 mRNA poly(A) tail length and may subtly influence Crt1 protein abundance. Simultaneous overexpression of RNR2, RNR3 and RNR4 partially rescued the HU hypersensitivity of a ccr4Delta dun1Delta strain, consistent with the notion that the RNR genes are key targets of Crt1. These results implicate the coordinated regulation of Crt1 via Ccr4 and Dun1 as a crucial nodal point in the response to DNA replication stress.
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Replicación del ADN/genética , Poli A/genética , ARN Mensajero/genética , Ribonucleasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Replicación del ADN/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Citometría de Flujo , Regulación Fúngica de la Expresión Génica , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Represoras , Ribonucleasas/genética , Ribonucleasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
Plasma adenosine levels are elevated in cardiovascular disease including hypertension and heart failure, and the nucleoside has been proposed to serve as an endogenous antimyocardial remodeling factor. We studied the modulation of phenylephrine-induced hypertrophy by adenosine receptor activation in isolated neonatal cultured ventricular myocytes. Phenylephrine (10 muM) increased cell size by 35% and significantly increased expression of atrial natriuretic peptide. These effects were reduced by the stable adenosine analog 2-chloroadenosine and were completely blocked by the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (1 microM), the A(2A) receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine (100 nM), and the A(3) receptor agonist N(6)-(3-iodobenzyl)adenosine-5'-methyluronamide (100 nM). The antihypertrophic effects of all three agonists were completely reversed by their respective antagonists. Phenylephrine significantly up-regulated expression of the immediate early gene c-fos especially within the first 30 min of phenylephrine treatment. These effects were almost completely inhibited by all adenosine receptor agonists. Although phenylephrine also induced early stimulation of both p38 mitogen-activated protein kinase and extracellular signal-regulated kinase, these responses were unaffected by adenosine agonists. The expression of the G-protein regulatory factors RGS2 and RGS4 were increased by nearly 3-fold by phenylephrine treatment although this was completely prevented by adenosine receptor agonists. These agents also blocked the ability of phenylephrine to up-regulate Na/H exchange isoform 1 (NHE1) expression in hypertrophied myocytes. Thus, our results demonstrate an antihypertrophic effect of adenosine acting via multiple receptor subtypes through a mechanism involving down-regulation of NHE1 expression. The ability to prevent regulators of G-protein signaling (RGS) up-regulation further suggests that adenosine receptor activation minimizes signaling which leads to hypertrophic responses.
Asunto(s)
Adenosina/análogos & derivados , Hipertrofia/inducido químicamente , Miocitos Cardíacos/efectos de los fármacos , Fenilefrina/efectos adversos , Receptor de Adenosina A1/fisiología , Receptor de Adenosina A2A/fisiología , Receptor de Adenosina A3/fisiología , Adenosina/farmacología , Agonistas del Receptor de Adenosina A1 , Agonistas del Receptor de Adenosina A2 , Agonistas del Receptor de Adenosina A3 , Agonistas alfa-Adrenérgicos/efectos adversos , Animales , Interacciones Farmacológicas , Fenetilaminas/farmacología , Fenilefrina/antagonistas & inhibidores , Ratas , Ratas Sprague-DawleyRESUMEN
The effect of the putative mitochondrial K(ATP) channel opener diazoxide (100 microM) was studied in terms of its ability to modulate the hypertrophic effect of 24 h treatment with the alpha(1) adrenoceptor agonist phenylephrine (PE; 10 microM) in cultured neonatal rat ventricular myocytes. PE on its own significantly increased cell size by 40%, (3)H leucine incorporation by 37% and produced more than a threefold elevation in both atrial natriuretic peptide and myosin light chain-2 expression. These effects were nearly completely prevented by diazoxide although the inhibitory effect of this agent was generally mitigated by the mitochondrial K(ATP) channel antagonists 5-hydroxydecanoic acid (100 microM) and glibenclamide (50 microM). Although PE produced an early threefold elevation in MAP kinase activation this was generally unaffected by diazoxide. PE also produced a greater than threefold increase in Na-H exchanger isoform 1 (NHE-1) expression which, was prevented by diazoxide treatment. Our study therefore, demonstrates a potential antihypertrophic influence of mitochondrial K(ATP) channel activation which, is related to diminished NHE-1 expression. Mitochondrial K(ATP) channel activation could represent an effective approach to minimize the myocardial hypertrophic process.