Acetic acid induces a programmed cell death process in the food spoilage yeast Zygosaccharomyces bailii

Here we show that 320-800 mM acetic acid induces in Zygosaccharomyces bailii a programmed cell death (PCD) process that is inhibited by cycloheximide, is accompanied by structural and biochemical alterations typical of apoptosis, and occurs in cells with preserved mitochondrial and plasma membrane integrity (as revealed by rhodamine 123 (Rh123) and propidium iodide (PI) staining, respectively). Mitochondrial ultrastructural changes, namely decrease of the cristae number, formation of myelinic bodies and swelling were also seen. Exposure to acetic acid above 800 mM resulted in killing by necrosis. The occurrence of an acetic acid-induced active cell death process in Z. bailii reinforces the concept of a physiological role of the PCD in the normal yeast life cycle.Fundação para a Ciência e a Tecnologia (FCT) - PRAXIS XX

Rapid detection of efflux pumps and their relation with drug resistance in yeast cells

Cell drug resistance can be due to the presence of active efflux pumps (AEP). Identification of yeast cells with a resistance phenotype is important either from a clinical, agricultural or biotechnological point of view. Rapid and reliable methods to detect AEP can be therefore very useful. Some yeast cells change their staining by cal-cein-AM, BCECF-AM, rhodamine 123 and DiOC5,when pretreated with verapamil, CCCP or ATP depletion, or when pretreated with specific antimicrobial agents. This fact may be interpreted as an indication of the presence/absence of AEP. Six yeast species were tested with a flowcytometric method (FCM) and an epifluorescence micro-scopic method (EFM), and ten other species were evalu-ated only by EFM. The minimum inhibitory concentration(MIC) of penconazol, benomyl and cycloheximide for Saccharomyces cerevisiae and Kluyveromyces marxia-nus, were determined by growth inhibition on solid me-dium and were compared to the staining changes de-tected by FCM. The FCM and the EFM allowed the detection of AEP in all the yeast species tested. High MIC values for adrug were related with the presence of at least one AEP indicated by the cytometric data. The FCM revealed to be a robust assay where as the EFM can be used as a preliminary test. It is possible to identify resistance/sensitivity patterns in yeast cells through cytometric detection methods of different efflux pumping systems.info:eu-repo/semantics/publishedVersio

Antifungal activity of local anesthetics against Candida species.

OBJECTIVE: To evaluate the activity of benzydamine, lidocaine, and bupivacaine, three drugs with local anesthetic activity, against Candida albicans and non-albicans strains and to clarify their mechanism of activity. METHODS: The minimal inhibitory concentration (MIC) was determined for 20 Candida strains (18 clinical isolates and two American Type Culture Collection strains). The fungistatic activity was studied with the fluorescent probe FUN-1 and observation under epifluorescence microscopy and flow cytometry. The fungicidal activity of the three drugs was assayed by viability counts. Membrane alterations induced in the yeast cells were evaluated by staining with propidium iodide, by quantitation of intracellular K+ leakage and by transmission electron microscopy of intact yeast cells and prepared spheroplasts. RESULTS: The MIC ranged from 12.5-50.0 microg/mL, 5.0-40.0 mg/mL, and 2.5-10.0 mg/mL for benzydamine, lidocaine, and bupivacaine, respectively. The inhibitory activity of these concentrations could be detected with the fluorescent probe FUN-1 after incubation for 60 minutes. A very fast fungicidal activity was shown by 0.2, 50, and 30 mg/mL of benzydamine, lidocaine, and bupivacaine, respectively. CONCLUSIONS: At lower concentrations, the tested drugs have a fungistatic activity, due to yeast metabolic impairment, while at higher concentrations they are fungicidal, due to direct damage to the cytoplasmic membrane

Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli

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Nitrogen and carbon source balance determined longevity, independently of fermentative or respiratory metabolism in the yeast Saccharomyces cerevisiae

Dietary regimens have proven to delay aging and age-associated diseases in several eukaryotic model organisms but the input of nutritional balance to longevity regulation is still poorly understood. Here, we present data on the role of single carbon and nitrogen sources and their interplay in yeast longevity. Data demonstrate that ammonium, a rich nitrogen source, decreases chronological life span (CLS) of the prototrophic Saccharomyces cerevisiae strain PYCC 4072 in a concentration-dependent manner and, accordingly, that CLS can be extended through ammonium restriction, even in conditions of initial glucose abundance. We further show that CLS extension depends on initial ammonium and glucose concentrations in the growth medium, as long as other nutrients are not limiting. Glutamine, another rich nitrogen source, induced CLS shortening similarly to ammonium, but this effect was not observed with the poor nitrogen source urea. Ammonium decreased yeast CLS independently of the metabolic process activated during aging, either respiration or fermentation, and induced replication stress inhibiting a proper cell cycle arrest in G0/G1 phase. The present results shade new light on the nutritional equilibrium as a key factor on cell longevity and may contribute for the definition of interventions to promote life span and healthy aging.The research leading to these results received funding from the Fundação para a Ciência e Tecnologia (FCT), cofunded by Programa Operacional Regional do Norte (ON.2—O Novo Norte); from the Quadro de Referência Estratégico Nacional (QREN) through the Fundo Europeu de Desenvolvimento Regional (FEDER) and from the Projeto Estratégico – LA 26 – 2013–2014 (PEst-C/SAU/LA0026/2013). Júlia Santos holds a PostDoc fellowship (SFRH/BPD/112108/2015) funded by FCT

Ammonium is a key determinant on the dietary restriction of yeast chronological aging in culture medium

New evidences have recently emerged from studies in yeast and in higher eukaryotes showing the importance of nutrient balance in dietary regimes and its effects on longevity regulation.We have previously shown that manipulation ofammoniumconcentration in the culture and/or aging medium can drastically affect chronological lifespan (CLS)of Saccharomyces cerevisiae, especially in amino acid restricted cells. Here we describe that the CLS shortening under amino acid restriction can be completely reverted by removing ammonium from the culture medium. Furthermore, the absence of ammonium, and of any rich nitrogen source, was so effective in extending CLS that no beneficial effect could be observed by further imposing calorie restriction conditions. When present in the culture medium,ammoniumimpaired the consumption of theauxotrophy-complementing amino acidsand caused in an improper cell cycle arrest of the culture.TOR1deletion reverted ammonium effects both in amino acid restricted and non-restricted cultures, whereas, Ras2p and Sch9p seem to have only a milder effect in the mediation ofammonium toxicity under amino acid restriction and no effect on non-restricted cultures.Our studies highlight ammonium as a key effector in the nutritional equilibrium between rich and essential nitrogen sources and glucose required for longevity promotion.Julia Santos holds a Post-Doc fellowship (UMINHO/ BPD / 39/ 2013) funded by QREN-FEDER

Cancro Avançado do Pulmão doença previsível?

RESUMO: O carcinoma do pulmão continua a ser a maior causa de mortalidade por cancro em grande parte dos países. O hábito de fumar, a incapacidade do rastreio em reduzir a taxa de mortalidade, o diagnóstico tardio e a inexistência de tratamento eficaz para as situações avançadas, são as razões mais relevantes para esta situação.O objectivo foi determinar o contributo, da ploidia do ADN e da percentagem de células em fase de síntese do ciclo celular(fase S), associadas a características clínicas bem conhecidas, na avaliação prognóstica dos doentes com carcinoma avançado do pulmão e identificar entre os parâmetros estudados alguns que pudessem servir de marcadores do comportamento da doença.Estudaram-se prospectivamente 93 doentes com carcinoma do pulmão diagnosticados e tratados no Serviço de Pneumologia do H.S. João.Os doentes eram predominantemente do sexo masculino,70 (75%) e 23 (25%) do sexo feminino com 64 anos de idade mediana. O estado geral da maioria dos doentes era bom, só 12 doentes estavam gravemente limitados. Setenta e quatro doentes (80%) apresentavam doença avançada, sendo em 57 (61,3%) metastática. Dezanove casos (20%) eram carcinomas de células pequenas,41 eram adenocarcinomas e 25 eram carcinomas epidermóides.A análise da sobrevida permitiu identificar como principais factores: o estado geral (Zubrod), o estádio da doença, as contagens, leucocitária e linfocitária, a fosfatase alcalina, a ploidia do ADN e a% de células em fase S. Assinale-se, que a presença de aneuploidia e% de células em fase Sâ¥15%, se associaram a bom prognóstico, ao contrário do referido na maioria dos estudos publicados.Na análise do comportamento da doença identificaram-se três parâmetros: o estado geral, a% de células em fase S e o valor das proteínas séricas totais. Utilizando estes parâmetros foi possível prever a evolução tumoral na grande maioria dos doentes.Concluímos referindo o importante contributo adicional, dos dois parâmetros tumorais de índole biológica estudados quando associados aos de natureza clínica, tanto na avaliação do prognóstico como na determinação do comportamento da doença.REV PORT PNEUMOL 2001; VII (3): SUMMARY: Lung Cancer continues to be a major cause of death from cancer in most countries mainly due to continued smoking, late diagnosis and the lack of an effective treatment.The objective was to evaluate the prognostic significance of clinical evaluation in association with two biological markers: DNA ploidy and S phase fraction determined by flow cytometry.We studied 93 patients with cancer of the lung diagnosed and treated at the São João Hospital Department of Pneumology. They were predominantly male 70 (75%) and mean age was 64 years the majority with good performance status; only 12 patients were severely disabled. Seventy-four patients (80%) presented with advanced disease, and this was metastatic on diagnosis in 57 (61.3%). Nineteen cases (20%) were small cell cancers; 41 were adenocarcinomas and 25 were scamous cell carcinomas. Forty seven (50,5%) were aneuploid and 22 had âhighâ S phase fraction.The survival analysis enabled us to identify the following as the principle independent prognostic factors: PS, stage of the disease, leukocyte and lymphocyte counts, alkaline phosphatase, DNA ploidy and the S phase fraction. We noted that the presence of aneuploidy and a S phase fractionâ¥15%, was associated with a good prognosis.The principle indicators of the disease progression were: PS, S phase fraction and the value of total serum proteins.We conclude by referring the importance of using clinical evaluation and biological markers in association, on prognostic evaluation and as indicators of the progression of the disease.REV PORT PNEUMOL 2001; VII (3): Palavras-chave: Carcinoma do pulmão, citometria de fluxo, factores de prognóstico, marcadores do comportamento da doença, Key-words: lung cancer, flow cytometry, prognostic factors, disease progression indicator