RESUMEN
The application of a One Health approach recognizes that human health, animal health, plant health and ecosystem health are intrinsically connected. Tackling complex challenges associated with foodborne zoonoses, antimicrobial resistance, and emerging threats is imperative. Therefore, the One Health European Joint Programme was established within the European Union research programme Horizon 2020. The One Health European Joint Programme activities were based on the development and harmonization of a One Health science-based framework in the European Union (EU) and involved public health, animal health and food safety institutes from almost all EU Member States, the UK and Norway, thus strengthening the cooperation between public, medical and veterinary organizations in Europe. Activities including 24 joint research projects, 6 joint integrative projects and 17 PhD projects, and a multicountry simulation exercise facilitated harmonization of laboratory methods and surveillance, and improved tools for risk assessment. The provision of sustainable solutions is integral to a One Health approach. To ensure the legacy of the work of the One Health European Joint Programme, focus was on strategic communication and dissemination of the outputs and engagement of stakeholders at the national, European and international levels.
Asunto(s)
Unión Europea , Salud Única , Humanos , Animales , Salud Pública , Europa (Continente) , Zoonosis/prevención & control , Comunicación , Inocuidad de los AlimentosRESUMEN
The COVID-19 pandemic exemplifies a One Health issue at the intersection of human, animal, and environmental health that requires collaboration across sectors to manage it successfully. The global One Health community includes professionals working in many different fields including human medicine, veterinary medicine, public health, ecosystem health, and, increasingly, social sciences. The aims of this cross-sectional study were to describe the involvement of the global One Health community in COVID-19 pandemic response activities. One Health networks (OHNs) have formed globally to serve professionals with common interests in collaborative approaches. We assessed the potential association between being part of an OHN and involvement in COVID-19 response activities. Data were collected in July-August 2020 using an online questionnaire that addressed work characteristics, perceived connection to OHNs, involvement in COVID-19 pandemic response activities, and barriers and facilitators to the involvement. The sample included 1,050 respondents from 94 countries across a range of organizations and work sectors including, but not restricted to, those typically associated with a One Health approach. Sixty-four percent of survey respondents indicated involvement in pandemic response activities. Being part of an OHN was positively associated with being involved in the COVID-19 response (odds ratio: 1.8, 95% confidence interval: 1.3-2.4). Lack of opportunities was a commonly reported barrier to involvement globally, with lack of funding the largest barrier in the WHO African region. This insight into diverse workforce involvement in the pandemic helps fill a gap in the global health workforce and public health education literature. An expanded understanding of the perceived roles and value of OHNs can inform targeted interventions to improve public health education and workforce capacity to prepare for and respond to public health emergencies.
Asunto(s)
COVID-19 , Salud Única , COVID-19/epidemiología , Estudios Transversales , Ecosistema , Humanos , PandemiasRESUMEN
Helicobacter pylori is a major chronic health problem, infecting more than half of the population worldwide. H. pylori infection is linked with various clinical complications ranging from gastritis to gastric cancer. The resolution of gastritis and peptic ulcer appears to be linked with the eradication of H. pylori. However, resistance to antibiotics and eradication failure rates are reaching alarmingly high levels. This calls for urgent action in finding alternate methods for H. pylori eradication. Here, we discuss the recently identified mechanism of H. pylori known as cholesterol glucosylation, mediated by the enzyme cholesterol-α-glucosyltransferase, encoded by the gene cgt. Cholesterol glucosylation serves several functions that include promoting immune evasion, enhancing antibiotic resistance, maintaining the native helical morphology, and supporting functions of prominent virulence factors such as CagA and VacA. Consequently, strategies aiming at inhibition of the cholesterol glucosylation process have the potential to attenuate the potency of H. pylori infection and abrogate H. pylori immune evasion capabilities. Knockout of H. pylori cgt results in unsuccessful colonization and elimination by the host immune responses. Moreover, blocking cholesterol glucosylation can reverse antibiotic susceptibility in H. pylori. In this work, we review the main roles of cholesterol glucosylation in H. pylori and evaluate whether this mechanism can be targeted for the development of alternate methods for eradication of H. pylori infection.
Asunto(s)
Gastritis , Infecciones por Helicobacter , Helicobacter pylori , Colesterol , Glucosiltransferasas , HumanosRESUMEN
Carcinoma of the gallbladder (GBC) is the most frequent tumor of the biliary tract. Despite epidemiological studies showing a correlation between chronic infection with Salmonella enterica Typhi/Paratyphi A and GBC, the underlying molecular mechanisms of this fatal connection are still uncertain. The murine serovar Salmonella Typhimurium has been shown to promote transformation of genetically predisposed cells by driving mitogenic signaling. However, insights from this strain remain limited as it lacks the typhoid toxin produced by the human serovars Typhi and Paratyphi A. In particular, the CdtB subunit of the typhoid toxin directly induces DNA breaks in host cells, likely promoting transformation. To assess the underlying principles of transformation, we used gallbladder organoids as an infection model for Salmonella Paratyphi A. In this model, bacteria can invade epithelial cells, and we observed host cell DNA damage. The induction of DNA double-strand breaks after infection depended on the typhoid toxin CdtB subunit and extended to neighboring, non-infected cells. By cultivating the organoid derived cells into polarized monolayers in air-liquid interphase, we could extend the duration of the infection, and we observed an initial arrest of the cell cycle that does not depend on the typhoid toxin. Non-infected intoxicated cells instead continued to proliferate despite the DNA damage. Our study highlights the importance of the typhoid toxin in causing genomic instability and corroborates the epidemiological link between Salmonella infection and GBC.IMPORTANCE Bacterial infections are increasingly being recognized as risk factors for the development of adenocarcinomas. The strong epidemiological evidence linking Helicobacter pylori infection to stomach cancer has paved the way to the demonstration that bacterial infections cause DNA damage in the host cells, initiating transformation. In this regard, the role of bacterial genotoxins has become more relevant. Salmonella enterica serovars Typhi and Paratyphi A have been clinically associated with gallbladder cancer. By harnessing the stem cell potential of cells from healthy human gallbladder explant, we regenerated and propagated the epithelium of this organ in vitro and used these cultures to model S. Paratyphi A infection. This study demonstrates the importance of the typhoid toxin, encoded only by these specific serovars, in causing genomic instability in healthy gallbladder cells, posing intoxicated cells at risk of malignant transformation.
Asunto(s)
Daño del ADN , Células Epiteliales/microbiología , Células Epiteliales/patología , Vesícula Biliar/citología , Salmonella paratyphi A/patogenicidad , Adulto , Anciano , Animales , Células Cultivadas , Femenino , Vesícula Biliar/microbiología , Interacciones Huésped-Patógeno , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Serogrupo , Virulencia/genéticaRESUMEN
Infection of the human stomach caused by Helicobacter pylori is very common, as the pathogen colonizes more than half of the world's population. It is associated with varied outcomes of infection, such as peptic ulcer disease, gastric ulcers, and mucosa-associated lymphoid tissue lymphoma, and is generally considered a risk factor for the development of gastric adenocarcinoma. Cholesteryl glucosides (CGs) constitute a vital component of the cell wall of H. pylori and contribute to its pathogenicity and virulence. The hp0421 gene, which encodes cholesteryl-α-glucoside transferase (CGT), appears critical for the enzymatic function of integrating unique CGs into the cell wall of H. pylori, and deletion of this gene leads to depletion of CGs and their variants. Herein, we report that the deletion of hp0421 and consequent deficiency of cholesterol alter the morphology, shape, and cell wall composition of H. pylori cells, as demonstrated by high-resolution confocal microscopy and flow cytometry analyses of two different type strains of H. pylori, their isogenic knockouts as well as a reconstituted strain. Moreover, measurement of ethidium bromide (EtBr) influx by flow cytometry showed that lack of CGs increased cell wall permeability. Antimicrobial susceptibility testing revealed that the hp0421 isogenic knockout strains (Hp26695Δ421 and Hp76Δ421) were sensitive to antibiotics, such as fosfomycin, polymyxin B, colistin, tetracycline, and ciprofloxacin, in contrast to the wild-type strains that were resistant to the above antibiotics and tended to form denser biofilms. Lipid profile analysis of both Hp76 and Hp76Δ421 strains showed an aberrant profile of lipopolysaccharides (LPS) in the Hp76Δ421 strain. Taken together, we herein provide a set of mechanistic evidences to demonstrate that CGs play critical roles in the maintenance of the typical spiral morphology of H. pylori and its cell wall integrity, and any alteration in CG content affects the characteristic morphological features and renders the H. pylori susceptible to various antibiotics.IMPORTANCEHelicobacter pylori is an important cause of chronic gastritis leading to peptic ulcer and is a major risk factor for gastric malignancies. Failure in the eradication of H. pylori infection and increasing antibiotic resistance are two major problems in preventing H. pylori colonization. Hence, a deeper understanding of the bacterial survival strategies is needed to tackle the increasing burden of H. pylori infection by an appropriate intervention. Our study demonstrated that the lack of cholesteryl glucosides (CGs) remarkably altered the morphology of H. pylori and increased permeability of the bacterial cell wall. Further, this study highlighted the substantial role of CGs in maintaining the typical H. pylori morphology that is essential for retaining its pathogenic potential. We also demonstrated that the loss of CGs in H. pylori renders the bacterium susceptible to different antibiotics.
Asunto(s)
Pared Celular/metabolismo , Colesterol/análogos & derivados , Glucosiltransferasas/metabolismo , Helicobacter pylori/citología , Helicobacter pylori/enzimología , Antibacterianos/farmacología , Biopelículas/crecimiento & desarrollo , Colesterol/metabolismo , Citometría de Flujo , Eliminación de Gen , Prueba de Complementación Genética , Glucosiltransferasas/genética , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Pruebas de Sensibilidad Microbiana , Microscopía Confocal , PermeabilidadRESUMEN
In the adult mammalian brain, newborn granule cells are continuously integrated into hippocampal circuits, and the fine-tuning of this process is important for hippocampal function. Thus, the identification of factors that control adult neural stem cells (NSCs) maintenance, differentiation and integration is essential. Here we show that the deletion of the iron trafficking protein lipocalin-2 (LCN2) induces deficits in NSCs proliferation and commitment, with impact on the hippocampal-dependent contextual fear discriminative task. Mice deficient in LCN2 present an increase in the NSCs population, as a consequence of a G0/G1 cell cycle arrest induced by increased endogenous oxidative stress. Of notice, supplementation with the iron-chelating agent deferoxamine rescues NSCs oxidative stress, promotes cell cycle progression and improves contextual fear conditioning. LCN2 is, therefore, a novel key modulator of neurogenesis that, through iron, controls NSCs cell cycle progression and death, self-renewal, proliferation and differentiation and, ultimately, hippocampal function.
Asunto(s)
Discriminación en Psicología/fisiología , Lipocalina 2/metabolismo , Neurogénesis/fisiología , Animales , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Giro Dentado/metabolismo , Miedo/fisiología , Hipocampo/citología , Hipocampo/metabolismo , Lipocalina 2/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/fisiología , Neurogénesis/genética , Neuronas/citología , Neuronas/metabolismoRESUMEN
Non-Hodgkin lymphoma (NHL) has been associated with immunological defects, chronic inflammatory and autoimmune conditions. Given the link between immune dysfunction and NHL, genetic variants in toll-like receptors (TLRs) have been regarded as potential predictive factors of susceptibility to NHL. Adequate anti-tumoral responses are known to depend on TLR9 function, such that the use of its synthetic ligand is being targeted as a therapeutic strategy. We investigated the association between the functional rs5743836 polymorphism in the TLR9 promoter and risk for B-cell NHL and its major subtypes in three independent case-control association studies from Portugal (1160 controls, 797 patients), Italy (468 controls, 494 patients) and the US (972 controls, 868 patients). We found that the rs5743836 polymorphism was significantly overtransmitted in both Portuguese (odds ratio (OR), 1.85; P=7.3E-9) and Italian (OR, 1.84; P=6.0E-5) and not in the US cohort of NHL patients. Moreover, the increased transcriptional activity of TLR9 in mononuclear cells from patients harboring rs5743836 further supports a functional effect of this polymorphism on NHL susceptibility in a population-dependent manner.
Asunto(s)
Linfoma no Hodgkin/genética , Polimorfismo Genético , Receptor Toll-Like 9/genética , Femenino , Genética de Población , Humanos , Linfoma no Hodgkin/epidemiología , Masculino , Persona de Mediana Edad , Factores de RiesgoRESUMEN
Yeast metacaspase (Yca1p) is required for the execution of apoptosis upon a wide range of stimuli. However, the specific degradome of this yeast protease has not been unraveled so far. By combining different methodologies described as requisites for a protein to be considered a protease substrate, such as digestome analysis, cleavage of recombinant GAPDH by metacaspase and evaluation of protein levels in vivo, we show that upon H(2)O(2)-induced apoptosis, the metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a specific target of metacaspase. Nitric oxide (NO) signaling, which mediates H(2)O(2)-induced apoptosis, is required for metacaspase specific GAPDH cleavage. In conclusion, in this work we identified GAPDH as the first direct yeast metacaspase substrate described so far. Although mammalian caspases and yeast metacaspase apparently have distinct target cleavage sites, GAPDH arises as a common substrate for these proteases.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Peróxido de Hidrógeno/farmacología , Óxido Nítrico/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Immunoblotting , Datos de Secuencia Molecular , Oxidantes/farmacología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The ease by which yeast can be manipulated in conjunction with their similarities to cells of more complex metazoans makes many yeast species, particularly Saccharomyces cerevisae, very attractive models for the study of conserved evolutionary processes that occur in eukaryotes. The ability to functionally express heterologous genes in these cells has allowed the development of countless new and elegant approaches leading to detailed structure-function analysis of numerous mammalian genes. Of these, the most informative have been the studies involving the analysis of regulators that have no direct or obvious sequence orthologue in yeast, including members of the Bcl-2 family of proteins, caspases and tumour suppressors. Here we review the field and provide evidence that these studies have served to further understand mammalian apoptosis.
Asunto(s)
Apoptosis/fisiología , Mamíferos/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Animales , Humanos , Modelos Biológicos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Paracoccidioides brasiliensis is characterized by a multiple budding phenotype and a polymorphic cell growth, leading to the formation of cells with extreme variations in shape and size. Since Cdc42 is a pivotal molecule in establishing and maintaining polarized growth for diverse cell types, as well as during pathogenesis of certain fungi, we evaluated its role during cell growth and virulence of the yeast-form of P. brasiliensis. We used antisense technology to knock-down PbCDC42's expression in P. brasiliensis yeast cells, promoting a decrease in cell size and more homogenous cell growth, altering the typical polymorphism of wild-type cells. Reduced expression levels also lead to increased phagocytosis and decreased virulence in a mouse model of infection. We provide genetic evidences underlying Pbcdc42p as an important protein during host-pathogen interaction and the relevance of the polymorphic nature and cell size in the pathogenesis of P. brasiliensis.
Asunto(s)
Proteínas Fúngicas/metabolismo , Paracoccidioides/citología , Paracoccidioides/patogenicidad , Paracoccidioidomicosis/microbiología , Proteína de Unión al GTP cdc42/metabolismo , Animales , Células Cultivadas , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Interacciones Huésped-Patógeno , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Paracoccidioides/genética , Paracoccidioides/fisiología , Fagocitosis , ARN sin Sentido , Virulencia , Proteína de Unión al GTP cdc42/genéticaRESUMEN
In order to alter the impact of diseases on human society, drug development has been one of the most invested research fields. Nowadays, cancer and infectious diseases are leading targets for the design of effective drugs, in which the primary mechanism of action relies on the modulation of programmed cell death (PCD). Due to the high degree of conservation of basic cellular processes between yeast and higher eukaryotes, and to the existence of an ancestral PCD machinery in yeast, yeasts are an attractive tool for the study of affected pathways that give insights into the mode of action of both antitumour and antifungal drugs. Therefore, we covered some of the leading reports on drug-induced apoptosis in yeast, revealing that in common with mammalian cells, antitumour drugs induce apoptosis through reactive oxygen species (ROS) generation and altered mitochondrial functions. The evidence presented suggests that yeasts may be a powerful model for the screening/development of PCD-directed drugs, overcoming the problem of cellular specificity in the design of antitumour drugs, but also enabling the design of efficient antifungal drugs, targeted to fungal-specific apoptotic regulators that do not have major consequences for human cells.
Asunto(s)
Antifúngicos/farmacología , Antineoplásicos/farmacología , Apoptosis/fisiología , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Levaduras/efectos de los fármacos , Animales , Humanos , Mitocondrias/efectos de los fármacos , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/fisiología , Levaduras/citología , Levaduras/fisiologíaRESUMEN
Invasive fungal infections, specifically candidemia, constitute major public health problems with high mortality rates. Therefore, in the last few years, the development of novel diagnostic methods has been considered a critical issue. Herein we describe a multiplex PCR strategy allowing the identification of 8 clinically relevant yeasts of the Candida genus, namely C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. guilliermondii, C. lusitaniae and C. dubliniensis. This method is based on the amplification of two fragments from the ITS1 and ITS2 regions by the combination of 2 yeast-specific and 8 species-specific primers in a single PCR reaction. Results from the identification of 231 clinical isolates are presented pointing to the high specificity of this procedure. Furthermore, several Candida isolates were identified directly from clinical specimens which also attests to the method's direct laboratory application. The results from the multiplex reactions with other microorganisms that usually co-infect patients also confirmed its high specificity in the identification of Candida species. Moreover, this method is simple and presents a sensitivity of approximately 2 cells per ml within 5 hours. Furthermore, it allows discrimination of individual Candida species within polyfungal samples. This novel method may therefore provide a clinical diagnostic procedure with direct applicability.
Asunto(s)
Candida/aislamiento & purificación , Candidiasis/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Candida/clasificación , Candida/genética , Candidiasis/microbiología , Cartilla de ADN , ADN Espaciador Ribosómico , Humanos , Sensibilidad y EspecificidadRESUMEN
We herein report the development of a molecular toolbox for the dimorphic fungus Paracoccidioides brasiliensis, specifically a more efficient transformation and a gene expression system. We evaluated several parameters that influence Agrobacterium tumefaciens-mediated transformation (ATMT), such as co-cultivation conditions and host cell susceptibility. Our results show that cellular recovery and air drying of A. tumefaciens:P. brasiliensis mixtures are essential for ATMT. Overall, our data indicate a transformation efficiency of 78+/-9 transformants/co-cultivation (5+/-1 transformants/10(6) target cells). P. brasiliensis GFP-expressing isolates were also constructed by insertion of the GFP gene under the control of several fungal promoters. RT-PCR, epifluorescence microscopy and flow cytometry analysis revealed Gfp visualization for all studied promoters but without significant differences in fluorescence and gene expression levels. Moreover, we present evidence for the occurrence of random single gene copy integration per haploid nuclei and the generation of homokaryon progeny, relevant for the future use in targeted mutagenesis and linking mutations to phenotypes.
Asunto(s)
Biología Molecular/métodos , Paracoccidioides/genética , Agrobacterium tumefaciens/genética , Azaserina/farmacología , Southern Blotting , Dermoscopía , Citometría de Flujo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Isoxazoles/farmacología , Paracoccidioides/efectos de los fármacos , Paracoccidioides/crecimiento & desarrollo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleósidos/farmacología , Transformación GenéticaRESUMEN
The aim of this study was to evaluate genome size and ploidy of the dimorphic pathogenic fungus Paracoccidioides brasiliensis. The cell cycle analysis of 10 P. brasiliensis isolates by flow cytometry (FCM) revealed a genome size ranging from 26.3+/-0.1Mb (26.9+/-0.1fg) to 35.5+/-0.2Mb (36.3+/-0.2fg) per uninucleated yeast cell. The DNA content of conidia from P. brasiliensis ATCC 60855-30.2+/-0.8Mb (30.9+/-0.8fg) -showed no significant differences with the yeast form, possibly excluding the occurrence of ploidy shift during morphogenesis. The ploidy of several P. brasiliensis isolates was assessed by comparing genome sizing by FCM with the previously described average haploid size obtained from electrophoretic karyotyping. The analysis of intra-individual variability of a highly polymorphic P. brasiliensis gene, GP43, indicated that only one allele seems to be present. Overall, the results showed that all analysed isolates presented a haploid, or at least aneuploid, DNA content and no association was detected between genome size/ploidy and the clinical-epidemiological features of the studied isolates. This work provides new knowledge on P. brasiliensis genetics/genomics, important for future research in basic cellular/molecular mechanisms and for the development/design of molecular techniques in this fungus.
Asunto(s)
Antígenos Fúngicos/genética , ADN de Hongos/análisis , Proteínas Fúngicas/genética , Genoma Fúngico , Glicoproteínas/genética , Haploidia , Paracoccidioides/genética , Cromosomas Fúngicos , ADN de Hongos/química , Citometría de Flujo , Cariotipificación , Paracoccidioides/clasificación , Paracoccidioides/crecimiento & desarrollo , Ploidias , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Modifications in social habits together with the increase of emigration have contributed not only to increased dermatophytoses but also to an altered etiology. During the last few years, Braga has suffered a radical change from a rural to a cosmopolitan life-style. METHODS: A statistical study of dermatophytoses and the etiology of their causative agents was performed by a retrospective survey carried out among patients of Hospital de São Marcos, Braga, Portugal, from 1983-2002. In this study, a total of 10,003 patients were analyzed. RESULTS: Over this period the frequency of dermatophytoses, as defined by the recovery of a dermatophyte in culture, was found to be 23.6%, whereas nondermatophytic infections accounted for 7.0%. Analysis of the clinical forms and the isolated fungi supports that the dermatophyte species have a predilection for certain body areas (P Asunto(s)
Dermatomicosis/epidemiología
, Adolescente
, Adulto
, Distribución por Edad
, Anciano
, Anciano de 80 o más Años
, Niño
, Preescolar
, Dermatomicosis/diagnóstico
, Dermatomicosis/microbiología
, Femenino
, Encuestas Epidemiológicas
, Humanos
, Lactante
, Masculino
, Persona de Mediana Edad
, Portugal/epidemiología
, Estudios Retrospectivos
, Distribución por Sexo
RESUMEN
Yeasts as eukaryotic microorganisms with simple, well known and tractable genetics, have long been powerful model systems for studying complex biological phenomena such as the cell cycle or vesicle fusion. Until recently, yeast has been assumed as a cellular 'clean room' to study the interactions and the mechanisms of action of mammalian apoptotic regulators. However, the finding of an endogenous programmed cell death (PCD) process in yeast with an apoptotic phenotype has turned yeast into an 'unclean' but even more powerful model for apoptosis research. Yeast cells appear to possess an endogenous apoptotic machinery including its own regulators and pathway(s). Such machinery may not exactly recapitulate that of mammalian systems but it represents a simple and valuable model which will assist in the future understanding of the complex connections between apoptotic and non-apoptotic mammalian PCD pathways. Following this line of thought and in order to validate and make the most of this promising cell death model, researchers must undoubtedly address the following issues: what are the crucial yeast PCD regulators? How do they play together? What are the cell death pathways shared by yeast and mammalian PCD? Solving these questions is currently the most pressing challenge for yeast cell death researchers.
Asunto(s)
Apoptosis/fisiología , Modelos Biológicos , Transducción de Señal/fisiología , Levaduras/fisiología , Factor Inductor de la Apoptosis , Citocromos c/metabolismo , Flavoproteínas/metabolismo , Serina Peptidasa A2 que Requiere Temperaturas Altas , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales , Especies Reactivas de Oxígeno/metabolismo , Serina Endopeptidasas/metabolismoRESUMEN
In yeast the use of rhodamine 123 (Rh123) has been restricted to the evaluation of mitochondrial respiratory function including the discrimination between respiratory-competent and -deficient cells. This study describes the optimization and validation of a low-concentration Rh123 staining protocol for the flow-cytometric assessment of mitochondrial membrane potential (Delta Psi m) changes in whole yeast cells. The optimized protocol was validated by the use of compounds that specifically affect mitochondrial energetics. Epifluorescence microscopy was used to monitor Rh123 distribution within the cell. Incubation of yeast cell suspensions with Rh123 (50 nM, 10 min) gave minimal non-specific binding and cytotoxicity of the dye. The ratio (R) between the green fluorescence and forward scatter (both measured as log values) was used to measure Delta Psi m with only little dependence on cell 'volume' and mitochondrial concentration. Cells treated with mitochondrial membrane de- or hyper-polarizing agents displayed a decrease and an increase of R values respectively, indicating that changes of the Rh123 distribution in cells indicate variations in the Delta Psi m. Live and dead cells also displayed significantly different R values. The method described here allows assessment of Delta Psi m changes in whole yeast cells in response to a given drug. Moreover, the relationship between drug effects and disorders of mitochondrial energetics might be addressed.