RESUMO
Access to deep-sea sponges brings with it the potential to discover novel antimicrobial candidates, as well as novel cold- and pressure-adapted bacteria with further potential clinical or industrial applications. In this study, we implemented a combination of different growth media, increased pressure and high-throughput techniques to optimize recovery of isolates from two deep-sea hexactinellid sponges, Pheronema carpenteri and Hertwigia sp., in the first culture-based microbial analysis of these two sponges. Using 16S rRNA gene sequencing for isolate identification, we found a similar number of cultivable taxa from each sponge species, as well as improved recovery of morphotypes from P. carpenteri at 22-25 °C compared to other temperatures, which allows a greater potential for screening for novel antimicrobial compounds. Bacteria recovered under conditions of increased pressure were from the phyla Proteobacteria, Actinobacteria and Firmicutes, except at 4â%O2/5 bar, when the phylum Firmicutes was not observed. Cultured isolates from both sponge species displayed antimicrobial activity against Micrococcus luteus, Staphylococcus aureus and Escherichia coli.
Assuntos
Actinobacteria , Poríferos , Actinobacteria/genética , Animais , Antibacterianos/farmacologia , Bactérias , Filogenia , Poríferos/genética , RNA Ribossômico 16S/genéticaRESUMO
OBJECTIVES: To measure the variability in carbapenem susceptibility conferred by different OxaAb variants, characterize the molecular evolution of oxaAb and elucidate the contribution of OxaAb and other possible carbapenem resistance factors in the clinical isolates using WGS and LC-MS/MS. METHODS: Antimicrobial susceptibility tests were performed on 10 clinical Acinetobacter baumannii isolates. Carbapenem MICs were evaluated for all oxaAb variants cloned into A. baumannii CIP70.10 and BM4547, with and without their natural promoters. Molecular evolution analysis of the oxaAb variants was performed using FastTree and SplitsTree4. Resistance determinants were studied in the clinical isolates using WGS and LC-MS/MS. RESULTS: Only the OxaAb variants with I129L and L167V substitutions, OxaAb(82), OxaAb(83), OxaAb(107) and OxaAb(110) increased carbapenem MICs when expressed in susceptible A. baumannii backgrounds without an upstream IS element. Carbapenem resistance was conferred with the addition of their natural upstream ISAba1 promoter. LC-MS/MS analysis on the original clinical isolates confirmed overexpression of the four I129L and L167V variants. No other differences in expression levels of proteins commonly associated with carbapenem resistance were detected. CONCLUSIONS: Elevated carbapenem MICs were observed by expression of OxaAb variants carrying clinically prevalent substitutions I129L and L167V. To drive carbapenem resistance, these variants required overexpression by their upstream ISAba1 promoter. This study clearly demonstrates that a combination of IS-driven overexpression of oxaAb and the presence of particular amino acid substitutions in the active site to improve carbapenem capture is key in conferring carbapenem resistance in A. baumannii and other mechanisms are not required.
Assuntos
Acinetobacter baumannii , Proteínas de Bactérias , beta-Lactamases , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Cromatografia Líquida , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Espectrometria de Massas em Tandem , beta-Lactamases/genéticaRESUMO
First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.
Assuntos
Bactérias/efeitos dos fármacos , Bactérias/genética , Proteínas de Bactérias/genética , Genes Bacterianos , Proteínas Recombinantes/genética , Resistência a Tetraciclina , Animais , Proteínas de Bactérias/metabolismo , Humanos , Proteínas Recombinantes/metabolismoRESUMO
Type 2 diabetes (T2DM) promotes premature atherosclerosis and inferior prognosis after arterial reconstruction. Vascular smooth muscle cells (SMC) respond to patho/physiological stimuli, switching between quiescent contractile and activated synthetic phenotypes under the control of microRNAs (miRs) that regulate multiple genes critical to SMC plasticity. The importance of miRs to SMC function specifically in T2DM is unknown. This study was performed to evaluate phenotype and function in SMC cultured from non-diabetic and T2DM patients, to explore any aberrancies and investigate underlying mechanisms. Saphenous vein SMC cultured from T2DM patients (T2DM-SMC) exhibited increased spread cell area, disorganised cytoskeleton and impaired proliferation relative to cells from non-diabetic patients (ND-SMC), accompanied by a persistent, selective up-regulation of miR-143 and miR-145. Transfection of premiR-143/145 into ND-SMC induced morphological and functional characteristics similar to native T2DM-SMC; modulating miR-143/145 targets Kruppel-like factor 4, alpha smooth muscle actin and myosin VI. Conversely, transfection of antimiR-143/145 into T2DM-SMC conferred characteristics of the ND phenotype. Exposure of ND-SMC to transforming growth factor beta (TGFß) induced a diabetes-like phenotype; elevated miR-143/145, increased cell area and reduced proliferation. Furthermore, these effects were dependent on miR-143/145. In conclusion, aberrant expression of miR-143/145 induces a distinct saphenous vein SMC phenotype that may contribute to vascular complications in patients with T2DM, and is potentially amenable to therapeutic manipulation.
Assuntos
Diabetes Mellitus Tipo 2/genética , MicroRNAs/genética , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Veia Safena/metabolismo , Actinas/genética , Actinas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Feminino , Regulação da Expressão Gênica , Glucose/metabolismo , Glucose/farmacologia , Humanos , Hipoglicemiantes/uso terapêutico , Interleucina-1alfa/farmacologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Fenótipo , Cultura Primária de Células , Veia Safena/efeitos dos fármacos , Veia Safena/patologia , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Since its inception in the 1980s, advancements in PCR technology using improved thermal cyclers, engineered DNA polymerases and commercial master mixes, have led to increased PCR productivity. Despite these advancements, PCR cycling protocols have largely remained unchanged over the same period. This study aimed to systemically evaluate the effect of reduced PCR cycling parameters on amplicon production. The 1466bp fragment from the 16S rRNA gene present in low-, medium- and high-CG bacteria was amplified using three commercially available PCR master mixes. The shortest cycling parameters required to successfully amplify the 16S fragment from all bacteria and master mixes comprised 30-cycles of 5 s denaturation, 25 s annealing, and 25 s extension. While all produced an amplicon with sufficient yield to enable downstream sequence analysis, the PCRBIO Ultra Mix in conjunction with the shortened parameters was found to achieve the highest amplicon yield across low-, medium- and high CG bacteria. Comparing the run times to that of a typical 16S PCR protocol, the shortened cycling parameters reduced the program duration by 46 % and consumed 50 % less electricity, translating into increased productivity and helping to improve laboratory environmental sustainability.
Assuntos
Bactérias , Reação em Cadeia da Polimerase , RNA Ribossômico 16S , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase/métodos , Bactérias/genética , DNA Bacteriano/genéticaRESUMO
Tumor cell survival and proliferation is attributable in part to suppression of apoptotic pathways, yet the mechanisms by which cancer cells resist apoptosis are not fully understood. Many cancer cells constitutively express heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). These breakdown products may play a role in the ability of cancer cells to suppress apoptotic signals. K(+) channels also play a crucial role in apoptosis, permitting K(+) efflux which is required to initiate caspase activation. Here, we demonstrate that HO-1 is constitutively expressed in human medulloblastoma tissue, and can be induced in the medulloblastoma cell line DAOY either chemically or by hypoxia. Induction of HO-1 markedly increases the resistance of DAOY cells to oxidant-induced apoptosis. This effect was mimicked by exogenous application of the heme degradation product CO. Furthermore we demonstrate the presence of the pro-apoptotic K(+) channel, Kv2.1, in both human medulloblastoma tissue and DAOY cells. CO inhibited the voltage-gated K(+) currents in DAOY cells, and largely reversed the oxidant-induced increase in K(+) channel activity. p38 MAPK inhibition prevented the oxidant-induced increase of K(+) channel activity in DAOY cells, and enhanced their resistance to apoptosis. Our findings suggest that CO-mediated inhibition of K(+) channels represents an important mechanism by which HO-1 can increase the resistance to apoptosis of medulloblastoma cells, and support the idea that HO-1 inhibition may enhance the effectiveness of current chemo- and radiotherapies.
Assuntos
Monóxido de Carbono/farmacologia , Heme Oxigenase-1/metabolismo , Meduloblastoma/metabolismo , Canais de Potássio Shab/metabolismo , Apoptose , Western Blotting , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Diamida/farmacologia , Eletrofisiologia , Citometria de Fluxo , Heme Oxigenase-1/genética , Humanos , Imidazóis/farmacologia , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Canais de Potássio Shab/genéticaRESUMO
OBJECTIVES: To identify the genes responsible for tetracycline resistance in a strain of Streptococcus australis isolated from pooled saliva from healthy volunteers in France. S. australis is a viridans Streptococcus, originally isolated from the oral cavity of children in Australia, and subsequently reported in the lungs of cystic fibrosis patients and as a cause of invasive disease in an elderly patient. METHODS: Agar containing 2 mg/L tetracycline was used for the isolation of tetracycline-resistant organisms. A genomic library in Escherichia coli was used to isolate the tetracycline resistance determinant. In-frame deletions and chromosomal repair were used to confirm function. Antibiotic susceptibility was determined by agar dilution and disc diffusion assay. RESULTS: The tetracycline resistance determinant from S. australis FRStet12 was isolated from a genomic library in E. coli and DNA sequencing showed two open reading frames predicted to encode proteins with similarity to multidrug resistance-type ABC transporters. Both genes were required for tetracycline resistance (to both the naturally occurring and semi-synthetic tetracyclines) and they were designated tetAB(46). CONCLUSIONS: This is the first report of a predicted ABC transporter conferring tetracycline resistance in a member of the oral microbiota.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antiporters/genética , Proteínas de Bactérias/genética , Boca/microbiologia , Multimerização Proteica/genética , Streptococcus/genética , Resistência a Tetraciclina/genética , Transportadores de Cassetes de Ligação de ATP/química , Adulto , Antiporters/química , Proteínas de Bactérias/química , Humanos , Testes de Sensibilidade Microbiana/métodos , Valor Preditivo dos Testes , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/genética , Streptococcus/isolamento & purificaçãoRESUMO
Global antimicrobial resistance is a health crisis that can change the face of modern medicine. Exploring diverse natural habitats for bacterially-derived novel antimicrobial compounds has historically been a successful strategy. The deep-sea presents an exciting opportunity for the cultivation of taxonomically novel organisms and exploring potentially chemically novel spaces. In this study, the draft genomes of 12 bacteria previously isolated from the deep-sea sponges Phenomena carpenteri and Hertwigia sp. are investigated for the diversity of specialized secondary metabolites. In addition, early data support the production of antibacterial inhibitory substances produced from a number of these strains, including activity against clinically relevant pathogens Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. Draft whole-genomes are presented of 12 deep-sea isolates, which include four potentially novel strains: Psychrobacter sp. PP-21, Streptomyces sp. DK15, Dietzia sp. PP-33, and Micrococcus sp. M4NT. Across the 12 draft genomes, 138 biosynthetic gene clusters were detected, of which over half displayed less than 50% similarity to known BGCs, suggesting that these genomes present an exciting opportunity to elucidate novel secondary metabolites. Exploring bacterial isolates belonging to the phylum Actinomycetota, Pseudomonadota, and Bacillota from understudied deep-sea sponges provided opportunities to search for new chemical diversity of interest to those working in antibiotic discovery.
RESUMO
The OXA ß-lactamases are responsible for hydrolysing ß-lactam antibiotics and contribute to the multidrug-resistant phenotype of several major human pathogens. The OXAAb enzymes are intrinsic to Acinetobacter baumannii and can confer resistance to carbapenem antibiotics. Here we determined the structure of the most prevalent OXAAb enzyme, OXA-66. The structure of OXA-66 was solved at a resolution of 2.1 Å and found to be very similar to the structure of OXA-51, the only other OXAAb enzyme that has had its structure solved. Our data contained one molecule per asymmetric unit, and analysis of positions responsible for dimer formation in other OXA enzymes suggest that OXA-66 likely exists as a monomer.
RESUMO
Reactive oxygen species (ROS) have for some time been implicated in the onset and progression of medical conditions including cancer, ageing, heart disease and Alzheimer's disease. Recently, it has been postulated that ROS play a much more subtle role in intracellular signalling mechanisms as second messengers. Given the importance of these species in influencing cellular processes, it is surprising that tools for studying intracellular levels of ROS are extremely limited and devices for studying the cells' response to internally generated ROS are virtually non-existent. In order to study the response of cells to intracellular ROS we have designed a nano-scale device that can both generate ROS and simultaneously monitor the cells' reaction as a function of changes in the important signalling ion, calcium. Here we report the synthesis, characterisation, and calibration of a new ROS nano-probe and demonstrate its ability to detect cellular response to elevated levels of intracellular ROS.
Assuntos
Nanopartículas/química , Porfirinas/química , Espécies Reativas de Oxigênio/química , Resinas Acrílicas/química , Técnicas Biossensoriais , Cálcio/química , Linhagem Celular , Humanos , Espécies Reativas de Oxigênio/metabolismo , Sistemas do Segundo Mensageiro , Transdução de SinaisRESUMO
Individuals with Type 2 diabetes mellitus (T2DM) are at increased risk of saphenous vein (SV) graft stenosis following coronary artery bypass. Graft stenosis is caused by intimal hyperplasia, a pathology characterized by smooth muscle cell (SMC) proliferation and migration. We hypothesized that SV-SMC from T2DM patients were intrinsically more proliferative and migratory than those from nondiabetic individuals. SV-SMC were cultured from nondiabetic and T2DM patients. Cell morphology (light microscopy, immunocytochemistry), S100A4 expression (real-time RT-PCR, immunoblotting), proliferation (cell counting), migration (Boyden chamber assay), and cell signaling (immunoblotting with phosphorylation state-specific antibodies) were studied. SV-SMC from T2DM patients were morphologically distinct from nondiabetic patients and exhibited a predominantly rhomboid phenotype, accompanied by disrupted F-actin cytoskeleton, disorganized alpha-smooth muscle actin network, and increased focal adhesion formation. However, no differences were observed in expression of the calcium-binding protein S100A4, a marker of rhomboid SMC phenotype, between the two cell populations. T2DM cells were less proliferative in response to fetal calf serum than nondiabetic cells, but both populations had similar proliferative responses to insulin plus PDGF. Under high glucose concentration conditions in the presence of insulin, migration of diabetic SV-SMC was greater than nondiabetic cells. Glucose concentration did not affect SV-SMC proliferation. No differences in insulin or PDGF-induced phosphorylation of ERK-1/2 or components of the Akt pathway (Akt-Ser473, Akt-Thr308, and GSK-3beta) were apparent between the two populations. In conclusion, SV-SMC from T2DM patients differ from nondiabetic SV-SMC in that they exhibit a rhomboid phenotype and are more migratory, but less proliferative, in response to serum.
Assuntos
Movimento Celular , Proliferação de Células , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Adulto , Idoso , Western Blotting , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/biossíntese , Veia Safena/metabolismo , Veia Safena/patologia , Transdução de Sinais/fisiologiaRESUMO
tet(32) Was identified in three bacterial isolates and in metagenomic DNA from the human oral cavity. The regions immediately flanking the gene were found to have similarities to the mobile elements TnB1230 from Butyrivibrio fibrisolvens, ATE-3 from Arcanobacterium pyogenes, and CTn5 from Clostridium difficile.
Assuntos
Boca/microbiologia , Proteínas Repressoras/genética , Arcanobacterium/efeitos dos fármacos , Arcanobacterium/genética , Butyrivibrio/efeitos dos fármacos , Butyrivibrio/genética , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/genética , Elementos de DNA Transponíveis/genética , Elementos de DNA Transponíveis/fisiologia , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Proteínas Repressoras/classificaçãoRESUMO
Cardiac myofibroblasts (CMF) play a key role in infarct repair and scar formation following myocardial infarction (MI) and are also an important source of proinflammatory cytokines. We postulated that interleukin-1alpha (IL-1alpha), a potential early trigger of acute inflammation post-MI, could stimulate human CMF to express additional proinflammatory cytokines. Furthermore, we hypothesized that these effects may be modulated by the anti-inflammatory cytokine interleukin-10 (IL-10). Human CMF were cultured from atrial biopsies from multiple patients. Interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and cardiotrophin-1 (CT-1) mRNA expression and secretion were measured using quantitative real-time RT-PCR and enzyme-linked immunosorbent assay. IL-1alpha (0.001-10 ng/ml, 0-6 h) stimulated IL-1beta, TNF-alpha, and IL-6 mRNA expression with distinct temporal and concentration profiles, resulting in increased cytokine secretion. The response to IL-1alpha was much greater than with TNF-alpha. Neither IL-1alpha nor TNF-alpha treatment modulated CT-1 mRNA expression. Immunoblotting with phosphospecific antibodies revealed that IL-1alpha stimulated the extracellular signal-regulated kinase (ERK)-1/2, p38 mitogen-activated protein kinase (MAPK), c-Jun NH(2)-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt), and nuclear factor (NF)-kappaB signaling pathways. Pharmacological inhibitor studies indicated roles for PI 3-kinase/Akt and NF-kappaB pathways in mediating IL-1beta expression, and for NF-kappaB and p38 MAPK pathways in mediating TNF-alpha expression. IL-1alpha-induced IL-6 mRNA expression was reduced by p38 MAPK inhibition, but increased by ERK and JNK pathway inhibitors. IL-10 produced a consistent but modest reduction in IL-1alpha-induced IL-6 mRNA levels (not IL-1beta or TNF-alpha), but this was not reflected by reduced IL-6 protein secretion. In conclusion, IL-1alpha stimulates human CMF to express IL-1beta, TNF-alpha, and IL-6 via specific signaling pathways, responses that are unaffected by IL-10 exposure.
Assuntos
Fibroblastos/imunologia , Interleucina-1alfa/imunologia , Miocardite/imunologia , Miocárdio/imunologia , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Interleucina-10/imunologia , Interleucina-10/farmacologia , Interleucina-1alfa/metabolismo , Interleucina-1alfa/farmacologia , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/imunologia , Miocárdio/citologia , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
1. Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor (PPAR) gamma agonists that are used to lower insulin resistance in Type 2 diabetic patients. Although TZDs exhibit beneficial effects on the vasculature, their effects on the heart are less clear and are the subject of current clinical debate. Thiazolidinediones have been reported to reduce adverse myocardial remodelling, a pathology in which cardiac myofibroblasts (CMF) are pivotal. 2. The aim of the present study was to investigate whether TZDs modulate specific human CMF functions of importance to the myocardial remodelling process and to determine whether any of these effects were mediated via PPARgamma activation. 3. Immunoblotting of cultured human CMF homogenates revealed strong expression of PPARgamma (approximately 50 kDa). Three different TZDs (ciglitazone, rosiglitazone and troglitazone) and the endogenous PPARgamma ligand 15-deoxy-delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) inhibited CMF proliferation (cell number and expression of proliferating cell nuclear antigen) in a concentration-dependent manner (range 0.1-10 micromol/L) with similar potencies. This antiproliferative effect of TZDs was not reversed by the PPARgamma antagonists GW9662 or T0070907 (10-25 micromol/L). None of the TZDs or 15d-PGJ(2) affected cell migration or invasion (Boyden chamber assays without or with Matrigel barrier), matrix metalloproteinase-2 or -9 secretion (gelatin zymography) or the actin cytoskeleton (rhodamine/phalloidin fluorescent confocal microscopy). 4. In conclusion, TZDs reduce human CMF proliferation via a PPARgamma-independent mechanism. Although TZDs do not inhibit CMF invasion, their antiproliferative activity may contribute to the ability of this class of drugs to modulate adverse myocardial remodelling.
Assuntos
Miócitos Cardíacos/efeitos dos fármacos , PPAR gama/fisiologia , Tiazolidinedionas/farmacologia , Células 3T3-L1 , Animais , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Hipoglicemiantes/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , PPAR gama/agonistas , PPAR gama/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: In addition to direct effects on myocardial cell function, tumor necrosis factor alpha (TNFalpha) contributes to adverse cardiac remodeling by increasing production of other pro-inflammatory cytokines [e.g. interleukin (IL)-1 and IL-6]. Both statins and thiazolidinediones (TZDs) have beneficial effects on cardiac remodeling, possibly due to their anti-inflammatory properties. The present study examined the mechanisms by which TNFalpha stimulates expression of pro-inflammatory cytokines in cultured human cardiac fibroblasts and determined the effects of statin or TZD treatment. METHODS: Human cardiac fibroblasts were cultured from biopsies of right atrial appendages. Cytokine mRNA expression and secretion was measured using quantitative real-time RT-PCR and ELISA. Activation of signaling pathways was determined by immunoblotting with phospho-specific antibodies. RESULTS: TNFalpha (0.1-10 ng/ml) stimulated IL-6, IL-1alpha and IL-1beta mRNA expression in cardiac fibroblasts in a concentration-dependent manner. Pharmacological inhibitors and receptor-neutralizing antibodies established that both TNFalpha-induced IL-6 and IL-1beta expression was mediated via the TNFRI receptor and p38 mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/Akt and nuclear factor (NF)-kappaB pathways. In contrast, TNFalpha-induced IL-1alpha expression required both TNFRI and TNFRII subtypes and p38 MAPK and PI3K/Akt pathways, but was negatively regulated by the NF-kappaB pathway. Neither statins (simvastatin, fluvastatin) nor TZDs (ciglitazone, rosiglitazone, troglitazone) had inhibitory effects on TNFalpha-induced IL-6 secretion or IL-1alpha/beta mRNA expression; indeed, cytokine expression was increased in response to TZDs. CONCLUSIONS: Our data provide important insights into the regulation of pro-inflammatory cytokine expression in human cardiac fibroblasts and suggest that the myocardial anti-inflammatory effects of statins and TZDs are not due to inhibition of TNFalpha-induced IL-1 or IL-6 expression by cardiac fibroblasts.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Interleucinas/biossíntese , Miócitos Cardíacos/imunologia , Transdução de Sinais/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Análise de Variância , Anticorpos Monoclonais/farmacologia , Células Cultivadas , Cromanos/farmacologia , Doença das Coronárias/imunologia , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática/métodos , Ácidos Graxos Monoinsaturados/farmacologia , Fluvastatina , Humanos , Indóis/farmacologia , Interleucina-1alfa/análise , Interleucina-1alfa/biossíntese , Interleucina-1alfa/genética , Interleucina-1beta/análise , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Interleucina-6/análise , Interleucina-6/biossíntese , Interleucina-6/genética , Miócitos Cardíacos/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rosiglitazona , Sinvastatina/farmacologia , Estimulação Química , TroglitazonaRESUMO
It is currently unclear whether activation of the AT1 receptor by agonists involves conformational selection or induction. We evaluated the pharmacological properties of wild type and N111G CAM human AT1 receptors stably expressed in HEK293 cells. Although [Sar1]-Ang II and Ang IV were full agonists at both receptors, the potency of Ang IV was 280-fold lower at the wild type receptor. [Sar1, Ile8]-Ang II was only a full agonist at the N111G CAM AT1 receptor. [Sar1]-Ang II and [Sar1, Ile8]-Ang II displayed similar high affinity binding to both receptors. In contrast, Ang IV displayed low affinity binding to the wild type and high affinity binding to the N111G CAM AT1 receptor. Based on these observations we provide strong evidence that conformational induction is the key process for activation of the AT1 receptor. Only by the creation of CAMs can conformational selection be envisaged to take place.
Assuntos
Conformação Proteica , Receptor Tipo 1 de Angiotensina/metabolismo , Angiotensina II/análogos & derivados , Angiotensina II/farmacologia , Ligação Competitiva , Linhagem Celular , Humanos , Fosfatos de Inositol/metabolismo , Modelos Biológicos , Receptor Tipo 1 de Angiotensina/agonistas , Receptor Tipo 1 de Angiotensina/genética , Fatores de TempoRESUMO
Screening of a bacterial artificial chromosome (BAC) library containing metagenomic DNA from human plaque and saliva allowed the isolation of four clones producing antimicrobial activity. Three of these were pigmented and encoded homologues of glutamyl-tRNA reductase (GluTR), an enzyme involved in the C5 pathway leading to tetrapyrole synthesis, and one clone had antibacterial activity with no pigmentation. The latter contained a BAC with an insert of 15.6 kb. Initial attempts to localize the gene(s) responsible for antimicrobial activity by subcloning into pUC-based vectors failed. A new plasmid for toxic gene expression (pTGEX) was designed enabling localization of the antibacterial activity to a 4.7-kb HindIII fragment. Transposon mutagenesis localized the gene to an open reading frame of 483 bp designated antibacterial protein1 (abp1). Abp1 was 94% identical to a hypothetical protein of Neisseria subflava (accession number WP_004519448.1). An Escherichia coli clone expressing Abp1 exhibited antibacterial activity against Bacillus subtilis BS78H, Staphylococcus epidermidis NCTC 11964 and B4268, and S. aureus NCTC 12493,ATCC 35696 and NCTC 11561. However, no antibacterial activity was observed against Pseudomonas aeruginosa ATCC 9027, N. subflava ATCC A1078, E. coli K12 JM109 and BL21(DE3) Fusobacterium nucleatum ATCC 25586 and NCTC 11326, Prevotella intermedia ATCC 25611, Veillonella parvula ATCC 10790 or Lactobacillus casei NCTC 6375.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Placa Dentária/microbiologia , Metagenoma , Neisseria/química , Saliva/microbiologia , Aldeído Oxirredutases/genética , Sequência de Aminoácidos , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/isolamento & purificação , Cromossomos Artificiais Bacterianos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli K12/efeitos dos fármacos , Vetores Genéticos , Biblioteca Genômica , Humanos , Lacticaseibacillus casei/efeitos dos fármacos , Dados de Sequência Molecular , Mutagênese , Plasmídeos , Pseudomonas aeruginosa/efeitos dos fármacos , Análise de Sequência de DNA , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacosRESUMO
Type 2 diabetes (T2DM) confers increased risk of endothelial dysfunction, coronary heart disease, and vulnerability to vein graft failure after bypass grafting, despite glycaemic control. This study explored the concept that endothelial cells (EC) cultured from T2DM and nondiabetic (ND) patients are phenotypically and functionally distinct. Cultured human saphenous vein- (SV-) EC were compared between T2DM and ND patients in parallel. Proliferation, migration, and in vitro angiogenesis assays were performed; western blotting was used to quantify phosphorylation of Akt, ERK, and eNOS. The ability of diabetic stimuli (hyperglycaemia, TNF-α, and palmitate) to modulate angiogenic potential of ND-EC was also explored. T2DM-EC displayed reduced migration (~30%) and angiogenesis (~40%) compared with ND-EC and a modest, nonsignificant trend to reduced proliferation. Significant inhibition of Akt and eNOS, but not ERK phosphorylation, was observed in T2DM cells. Hyperglycaemia did not modify ND-EC function, but TNF-α and palmitate significantly reduced angiogenic capacity (by 27% and 43%, resp.), effects mimicked by Akt inhibition. Aberrancies of EC function may help to explain the increased risk of SV graft failure in T2DM patients. This study highlights the importance of other potentially contributing factors in addition to hyperglycaemia that may inflict injury and long-term dysfunction to the homeostatic capacity of the endothelium.
Assuntos
Diabetes Mellitus Tipo 2/patologia , Células Endoteliais/patologia , Endotélio Vascular/patologia , Veia Safena/patologia , Idoso , Idoso de 80 Anos ou mais , Movimento Celular , Proliferação de Células , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Feminino , Rejeição de Enxerto , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo III/metabolismo , Fenótipo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Veia Safena/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The aim of this study was to screen for the presence of antimicrobial resistance genes within the saliva and faecal microbiomes of healthy adult human volunteers from five European countries. Two non-culture based approaches were employed to obviate potential bias associated with difficult to culture members of the microbiota. In a gene target-based approach, a microarray was employed to screen for the presence of over 70 clinically important resistance genes in the saliva and faecal microbiomes. A total of 14 different resistance genes were detected encoding resistances to six antibiotic classes (aminoglycosides, ß-lactams, macrolides, sulphonamides, tetracyclines and trimethoprim). The most commonly detected genes were erm(B), blaTEM, and sul2. In a functional-based approach, DNA prepared from pooled saliva samples was cloned into Escherichia coli and screened for expression of resistance to ampicillin or sulphonamide, two of the most common resistances found by array. The functional ampicillin resistance screen recovered genes encoding components of a predicted AcrRAB efflux pump. In the functional sulphonamide resistance screen, folP genes were recovered encoding mutant dihydropteroate synthase, the target of sulphonamide action. The genes recovered from the functional screens were from the chromosomes of commensal species that are opportunistically pathogenic and capable of exchanging DNA with related pathogenic species. Genes identified by microarray were not recovered in the activity-based screen, indicating that these two methods can be complementary in facilitating the identification of a range of resistance mechanisms present within the human microbiome. It also provides further evidence of the diverse reservoir of resistance mechanisms present in bacterial populations in the human gut and saliva. In future the methods described in this study can be used to monitor changes in the resistome in response to antibiotic therapy.