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Oxidation of PUFAs in LDLs trapped in the arterial intima plays a critical role in atherosclerosis. Though there have been many studies on the atherogenicity of oxidized derivatives of PUFA-esters of cholesterol, the effects of cholesteryl hemiesters (ChEs), the oxidation end products of these esters, have not been studied. Through lipidomics analyses, we identified and quantified two ChE types in the plasma of CVD patients and identified four ChE types in human endarterectomy specimens. Cholesteryl hemiazelate (ChA), the ChE of azelaic acid (n-nonane-1,9-dioic acid), was the most prevalent ChE identified in both cases. Importantly, human monocytes, monocyte-derived macrophages, and neutrophils exhibit inflammatory features when exposed to subtoxic concentrations of ChA in vitro. ChA increases the secretion of proinflammatory cytokines such as interleukin-1ß and interleukin-6 and modulates the surface-marker profile of monocytes and monocyte-derived macrophage. In vivo, when zebrafish larvae were fed with a ChA-enriched diet, they exhibited neutrophil and macrophage accumulation in the vasculature in a caspase 1- and cathepsin B-dependent manner. ChA also triggered lipid accumulation at the bifurcation sites of the vasculature of the zebrafish larvae and negatively impacted their life expectancy. We conclude that ChA behaves as an endogenous damage-associated molecular pattern with inflammatory and proatherogenic properties.
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Aterosclerosis , Pez Cebra , Animales , Humanos , Ésteres del Colesterol , Monocitos , Inflamación , ÉsteresRESUMEN
Neutrophils are prominent immune components of tumors, having either anti-tumor (N1) or pro-tumor activity (N2). Circulating neutrophils, divided into high density neutrophils (HDN) and low density neutrophils (LDN), functionally mirror those N1 and N2 cells, respectively. LDN are rare in non-pathological conditions, but frequent in cancer, exhibiting a pro-tumor phenotype. These findings have been mainly demonstrated in animal models, thus proper validation in humans is still imperative. Here, we observed that LDN were increased in the blood of breast cancer (BC) patients, particularly with metastatic disease. Within the population of non-metastatic patients, LDN were more prevalent in patients with poor response to neoadjuvant chemotherapy than patients with a good response. The higher incidence of LDN in BC patients with severe disease or resistance to treatment can be explained by their pro-tumor/immunosuppressive characteristics. Moreover, the percentage of LDN in BC patients' blood was negatively correlated with activated cytotoxic T lymphocytes and positively correlated with immunosuppressive regulatory T cells. The ability of LDN to spoil anti-tumor immune responses was further demonstrated ex vivo. Hence, this study reveals the potential of LDN as a biomarker of BC response to treatment and opens new avenues for developing new immunotherapies.
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The Notch-signaling ligand DLL1 has emerged as an important player and promising therapeutic target in breast cancer (BC). DLL1-induced Notch activation promotes tumor cell proliferation, survival, migration, angiogenesis and BC stem cell maintenance. In BC, DLL1 overexpression is associated with poor prognosis, particularly in estrogen receptor-positive (ER+) subtypes. Directed therapy in early and advanced BC has dramatically changed the natural course of ER+ BC; however, relapse is a major clinical issue, and new therapeutic strategies are needed. Here, we report the development and characterization of a novel monoclonal antibody specific to DLL1. Using phage display technology, we selected an anti-DLL1 antibody fragment, which was converted into a full human IgG1 (Dl1.72). The Dl1.72 antibody exhibited DLL1 specificity and affinity in the low nanomolar range and significantly impaired DLL1-Notch signaling and expression of Notch target genes in ER+ BC cells. Functionally, in vitro treatment with Dl1.72 reduced MCF-7 cell proliferation, migration, mammosphere formation and endothelial tube formation. In vivo, Dl1.72 significantly inhibited tumor growth, reducing both tumor cell proliferation and liver metastases in a xenograft mouse model, without apparent toxicity. These findings suggest that anti-DLL1 Dl1.72 could be an attractive agent against ER+ BC, warranting further preclinical investigation.
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Neoadjuvant chemotherapy (NACT) is common in breast cancer (BC) treatment, though more than half of the patients lack an effective response. Therefore, new predictive biomarkers and alternative therapies are crucial. Previously, we proposed HLA-DR-expressing cytotoxic T lymphocytes (CTLs) as a potential biomarker of the response to NACT. To validate this observation and further investigate these cells, 202 BC patients were enrolled. Flow cytometry analyses were performed in 61 biopsies and 41 blood samples pre-NACT and 100 non-NACT tumor samples. All the patients were followed up for 34 months. Blood-isolated immune cells were cultured with BC cell lines in a 3D system. We confirmed that HLA-DR level in CTLs is a highly sensitive, specific, and independent biomarker to predict response to NACT and developed a predictive probability model. This biomarker was also associated with progression-free survival, regardless of the treatment. The clinical observations are substantiated by the anti-tumor properties of HLA-DR-expressing CTLs. Intriguingly, HLA-DR level in CTLs can be modulated ex vivo, boosting their capacity to kill tumor cells synergistically with doxorubicin. Thus, HLA-DR expression in CTLs is a validated tool to select patients that will actually benefit from NACT, and its stimulation might be a novel therapeutic approach for BC.
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3D cell culture including different cell types, such as immune cells, is a representative platform that mimics the tumor microenvironment. Here we disclose an easy-to-handle 3D co-culture protocol using a scaffold-free technique with the breast cancer cell line MDA-MB-231 and breast cancer patient-derived immune cells from peripheral blood. The method presented is simple, less time-consuming and less expensive when compared to other 3D techniques. Additionally, this is an optimized protocol for the establishment of a 3D system for this cell line, which is normally seen as challenging to spontaneously form spheroids. The addition of patient-derived immune cells to the cancer cells' spheroid allows the study of the crosstalk between both cell types, as well as the assessment of individual therapeutic approaches to intensify the antitumor immune response. In fact, with this model, we observed that patients' immune cells exhibit a wide range of antitumor responses and we further demonstrated that it is possible to manipulate the less effective ones with a canonical stimulus, as a proof-of-concept, in order to improve their ability to lower the viability of tumor cells. Therefore, this platform could be applied for a personalized immune-based drug screening, with results after a maximum of 10 days of culture, in order to develop more tailored breast cancer treatments and ameliorate patients' survival rate.
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Seroprevalence studies are crucial both for estimating the prevalence of SARS-CoV-2 exposure and to provide a measure for the efficiency of the confinement measures. Portuguese universities were closed on March 16th 2020, when Portugal only registered 62 SARS-CoV-2 infection cases per million. We have validated a SARS-CoV-2 ELISA assay to a stabilized full-length spike protein using 216 pre-pandemic and 19 molecularly diagnosed SARS-CoV-2 positive individual's samples. At NOVA University of Lisbon, presential work was partially resumed on May 25th with staggered schedules. From June 15th to 30th, 3-4 weeks after the easing of confinement measures, we screened 1,636 collaborators of NOVA university of Lisbon for the presence of SARS-CoV-2 spike specific IgA and IgG antibodies. We found that spike-specific IgG in 50 of 1,636 participants (3.0%), none of which had anti-spike IgA antibodies. As participants self-reported as asymptomatic or paucisymptomatic, our study also provides a measurement of the prevalence of asymptomatic/paucisymptomatic SARS-CoV-2 infections. Our study suggests that essential workers have a 2-fold increase in viral exposure, when compared to non-essential workers that observed confinement. Additional serological surveys in different population subgroups will paint a broader picture of the effect of the confinement measures in the broader community.
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Prediction of breast cancer response to Neoadjuvant Chemotherapy (NACT) is an urgent need to promptly direct non-responder patients to alternative therapies. Infiltrating T lymphocytes, namely cytotoxic T lymphocytes (CTLs) have been appointed as predictors of response. However, cancer cells have the ability to dampen CTLs' activity and thus, the prognostic value of the CTLs, per se, is debatable. Here, we disclose that more than the occurrence of CTLs, it is their activation state, revealed by HLA-DR expression, that can accurately predict response to NACT. Flow cytometry analysis of breast cancer biopsies showed that the frequency of CTLs and other lymphocytes were similar regardless disease stage and between NACT responders and non-responders. However, only breast cancer patients without axillary lymph node metastasis and NACT responders have HLA-DRhi CTLs. Interestingly, HLA-DR levels in tumor CTLs is correlated with HLA-DR levels in systemic CTLs. These HLA-DR+ CTLs produce IFN-γ and Granzyme B, enlightening their effector and probable anti-tumor activity profile. Moreover, the level of HLA-DR in CTLs is negatively correlated with the level of HLA-DR in T regulatory lymphocytes and with immunosuppressive and pro-tumor molecules in the tumor microenvironment. Hence, HLA-DR levels in CTLs is a highly sensitive and specific potential predictive factor of NACT-response, which can be assessed in blood to guide therapeutic decisions.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Antígenos HLA-DR/inmunología , Linfocitos T Citotóxicos/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular , Femenino , Granzimas/farmacología , Humanos , Interferón gamma/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Metástasis Linfática/inmunología , Persona de Mediana Edad , Terapia Neoadyuvante/métodos , Pronóstico , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
Dendritic cells (DCs) play an essential role in immunity against bacteria by phagocytosis and by eliciting adaptive immune responses. Previously, we demonstrated that human monocyte-derived DCs (MDDCs) express a high content of cell surface α2,6-sialylated glycans. However, the relative role of these sialylated structures in phagocytosis of bacteria has not been reported. Here, we show that treatment with a sialidase significantly improved the capacity of both immature and mature MDDCs to phagocytose Escherichia coli. Desialylated MDDCs had a significantly more mature phenotype, with higher expression of MHC molecules and interleukin (IL)-12, tumour necrosis factor-α, IL-6 and IL-10 cytokines, and nuclear factor-κB activation. T lymphocytes primed by desialylated MDDCs expressed more interferon-γ when compared with priming by sialylated MDDCs. Improved phagocytosis required E. coli sialic acids, indicating a mechanism of host-pathogen interaction dependent on sialic acid moieties. The DCs harvested from mice deficient in the ST6Gal.1 sialyltransferase showed improved phagocytosis capacity, demonstrating that the observed sialidase effect was a result of the removal of α2,6-sialic acid. The phagocytosis of different pathogenic E. coli isolates was also enhanced by sialidase, which suggests that modifications on MDDC sialic acids may be considered in the development of MDDC-based antibacterial therapies. Physiologically, our findings shed new light on mechanisms that modulate the function of both immature and mature MDDCs, in the context of host-bacteria interaction. Hence, with particular relevance to DC-based therapies, the engineering of α2,6-sialic acid cell surface is a novel possibility to fine tune DC phagocytosis and immunological potency.
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Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ácido N-Acetilneuramínico/deficiencia , Fagocitosis/inmunología , Animales , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Escherichia coli/inmunología , Humanos , Ratones , Ratones Noqueados , Ácido N-Acetilneuramínico/metabolismo , Fagocitosis/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The limited efficacy of monocyte-derived dendritic cell (mo-DC)-based vaccines is primarily attributed to the reduced mo-DC migratory capacity. One undefined aspect is the initial binding of mo-DCs to endothelial cells and vascular selectins. In this study, we investigated the role and modulation of the selectin binding determinant sialyl Lewis(x) (sLe(x)) in selectin-dependent mo-DC binding. Our data reveal that sLe(x) is required for maximal binding of mo-DCs to tumor necrosis factor (TNF)-α-activated endothelial cells under static conditions, as evidenced by the use of sialidase. Sialidase treatment also abrogated mo-DC cell tethering to immobilized, purified P-, L-, or E-selectin under flow. The requirement of sLe(x)-dependent binding of mo-DC to selectins was further substantiated by using sLe(x) free sugar and anti-sLe(x) antibody, which significantly suppressed mo-DC-selectin binding. P-selectin glycoprotein ligand-1 is required for mo-DC binding to both P- and L-selectin, but it is dispensable for E-selectin recognition. Interestingly, the extent of mo-DC tethering was maximal on P-selectin, followed by E- and L- selectin. Accordingly, L-selectin mediated faster mo-DC rolling than E- or P-selectin. Interferon (IFN)-γ induces a significant increase in mo-DC surface sLe(x) expression, which is probably due to the enhanced synthesis of C2GnT-I. These findings may contribute to improving mo-DC-based vaccination protocols.
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Células Dendríticas/inmunología , Interferón gamma/inmunología , Oligosacáridos/inmunología , Selectinas/inmunología , Vacunas contra el Cáncer/inmunología , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Monocitos/inmunología , Antígeno Sialil Lewis XRESUMEN
INTRODUCTION: A functional vascular endothelial growth factor A (VEGF-A) autocrine loop is crucial for bladder cancer cell survival. We reasoned that treatment with the anti-VEGF antibody bevacizumab may result either in cell growth prevention or in the cell adaptation to compensate VEGF deprivation. METHODS: The cytotoxicity of different levels of bevacizumab and its effect on the gene expression was analyzed in human bladder cancer cell lines. RESULTS: Inhibition of bladder cancer cell proliferation was observed at >2.5 mg/ml of bevacizumab. Non-muscle-invasive bladder cancer cells expressed high concentrations of VEGF-A, and were less susceptible to bevacizumab inhibition. At 0.5 mg/ml (FDA approved concentration) of bevacizumab, cells increase their expression of VEGF-A, VEGF-A receptors and related growth factors. CONCLUSIONS: Bevacizumab cytotoxicity is only observed at high concentration, and it is inversely correlated with the basal VEGF-A expression of the bladder cancer cells. This is the first report showing that, at clinical bevacizumab concentrations, cancer cells compensate the VEGF-A blockade, by improving the expression of VEGF-A and related genes, highlighting the need to follow the patient's adaptation response to bevacizumab treatment.
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Anticuerpos Monoclonales/toxicidad , Comunicación Autocrina/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Bevacizumab , Línea Celular Tumoral , Humanos , Neoplasias de la Vejiga Urinaria/tratamiento farmacológicoRESUMEN
Human monocyte-derived dendritic cells (mo-DCs) express highly sialylated structures, with recognized but poorly understood function in maturation, immunogenicity and endocytosis capacity. We have previously shown that mo-DCs surface sialylation is changeable upon different stimuli, which led us to hypothesise the existence of cell surface (non-intracellular) sialyltransferases, rapidly restoring or altering mo-DC surface sialylation, thus modulating specific functions. Here, we demonstrate that, in the presence of exogenous CMP-Neu5Ac, mo-DCs incorporate considerable amounts of sialic acids into cell surface, predominantly when mo-DCs were previously desialylated or matured. This is a genuine sialyltransferase activity, confirmed by specific inhibition assays, which is not influenced by secreted enzymes. Functionally, the ecto-sialyltransferase activity causes a significant down-regulation of mo-DCs endocytic capacity, without affecting the maturation state. These findings suggest that ecto-sialyltransferases participate in a dynamic control of mo-DC sialylation, with functional repercussions. This activity is possibly related with specific physiological and pathological conditions, as inflammation and infection, contributing to protection and homeostasis regulation.
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Membrana Celular/enzimología , Células Dendríticas/enzimología , Sialiltransferasas/metabolismo , Células Dendríticas/citología , Células Dendríticas/ultraestructura , Endocitosis , Citometría de Flujo , Regulación de la Expresión Génica , Humanos , Microscopía Confocal , Monocitos/citología , Ácidos Siálicos/metabolismo , Sialiltransferasas/genéticaRESUMEN
Sialic acids are key structural determinants and contribute to the functionality of a number of immune cell receptors. Previously, we demonstrated that differentiation of human dendritic cells (DCs) is accompanied by an increased expression of sialylated cell surface structures, putatively through the activity of the ST3Gal.I and ST6Gal.I sialyltransferases. Furthermore, DC endocytosis was reduced upon removal of the cell surface sialic acid residues by neuraminidase. In the present work, we evaluate the contribution of the sialic acid modifications in DC maturation. We demonstrate that neuraminidase-treated human DCs have increased expression of major histocompatibility complex (MHC) and costimulatory molecules, increased gene expression of specific cytokines and induce a higher proliferative response of T lymphocytes. Together, the data suggest that clearance of cell surface sialic acids contributes to the development of a T helper type 1 proinflammatory response. This postulate is supported by mouse models, where elevated MHC class II and increased maturation of specific DC subsets were observed in DCs harvested from ST3Gal.I(-/-) and ST6Gal.I(-/-) mice. Moreover, important qualitative differences, particularly in the extent of reduced endocytosis and in the peripheral distribution of DC subsets, existed between the ST3Gal.I(-/-) and ST6Gal.I(-/-) strains. Together, the data strongly suggest not only a role of cell surface sialic acid modifications in maturation and functionality of DCs, but also that the sialic acid linkages created by different sialyltransferases are functionally distinct. Consequently, with particular relevance to DC-based therapies, cell surface sialylation, mediated by individual sialyltransferases, can influence the immunogenicity of DCs upon antigen loading.
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Células Dendríticas/inmunología , Ácidos Siálicos/inmunología , Sialiltransferasas/inmunología , Linfocitos T/inmunología , Animales , Antígeno B7-1/efectos de los fármacos , Antígeno B7-1/inmunología , Antígeno B7-2/efectos de los fármacos , Antígeno B7-2/inmunología , Células Cultivadas , Citocinas/efectos de los fármacos , Citocinas/inmunología , Células Dendríticas/efectos de los fármacos , Endocitosis/inmunología , Antígenos de Histocompatibilidad/efectos de los fármacos , Antígenos de Histocompatibilidad/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuraminidasa/farmacología , Sialiltransferasas/genética , Linfocitos T/efectos de los fármacos , beta-D-Galactósido alfa 2-6-Sialiltransferasa , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
The use of plants for production of recombinant proteins is becoming widely accepted. More recently, plant cell cultures have been proposed as valuable systems for producing a wide range of biologically active proteins. Such systems provide certain advantages over whole plants, but yields are still considered a limitation. In this study we established a Medicago truncatula cell suspension line expressing phytase from Aspergillus niger. Phytase is an N-glycosylated enzyme that breaks down indigestible phytate, resulting in an increased availability of phosphorus and other minerals in monogastric animals and reduced levels of phosphorus output in their manure. Various production systems have previously been used to express heterologous phytase, including several plant species. In this work, remarkable amounts of enzymatically active recombinant phytase were produced and secreted into the culture medium. Recombinant phytase accumulated to at least 25 mg/L and remained stable along the growth curve, and an enriched fraction with high enzymatic activity was easily obtained. We therefore propose M. truncatula cell suspension cultures as a potential system for the production of recombinant proteins. Most importantly, we have shown that, contrary to general belief, it is possible to achieve high levels of a functional recombinant protein in plant cell culture systems.
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6-Fitasa/biosíntesis , Medicago truncatula/enzimología , Medicago truncatula/genética , Plantas Modificadas Genéticamente , Proteínas Recombinantes/biosíntesis , 6-Fitasa/aislamiento & purificación , 6-Fitasa/metabolismo , Aspergillus niger/enzimología , Células Cultivadas , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/biosíntesis , Regulación de la Expresión Génica de las Plantas , Medicago truncatula/crecimiento & desarrollo , Medicago truncatula/metabolismo , Ácido Fítico/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Several glycoconjugates are involved in the immune response. Sialic acid is frequently the glycan terminal sugar and it may modulate immune interactions. Dendritic cells (DCs) are antigen-presenting cells with high endocytic capacity and a central role in immune regulation. On this basis, DCs derived from monocytes (mo-DC) are utilised in immunotherapy, though many features are ignored and their use is still limited. We analyzed the surface sialylated glycans expressed during human mo-DC generation. This was monitored by lectin binding and analysis of sialyltransferases (ST) at the mRNA level and by specific enzymatic assays. We showed that alpha 2-3-sialylated O-glycans and alpha 2-6- and alpha 2-3-sialylated N-glycans are present in monocytes and their expression increases during mo-DC differentiation. Three main ST genes are committed with this rearrangement: ST6Gal1 is specifically involved in the augmented alpha 2-6-sialylated N-glycans; ST3Gal1 contributes for the alpha2-3-sialylation of O-glycans, particularly T antigens; and ST3Gal4 may contribute for the increased alpha2-3-sialylated N-glycans. Upon mo-DC maturation, ST6Gal1 and ST3Gal4 are downregulated and ST3Gal1 is altered in a stimulus-dependent manner. We also observed that removing surface sialic acid of immature mo-DC by neuraminidase significantly decreased its endocytic capacity, while it increased in monocytes. Our results indicate the STs expression modulates the increased expression of surface sialylated structures during mo-DC generation, which is probably related with changes in cell mechanisms. The ST downregulation after mo-DC maturation probably results in a decreased sialylation or sialylated glycoconjugates involved in the endocytosis, contributing to the downregulation of one or more antigen-uptake mechanisms specific of mo-DC.
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Membrana Celular/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Endocitosis , Monocitos/citología , Monocitos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Diferenciación Celular , Células Cultivadas , Regulación Enzimológica de la Expresión Génica , Humanos , Sialiltransferasas/genética , Sialiltransferasas/metabolismoRESUMEN
Yeast cells adapted to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) exhibit a plasma membrane less susceptible to 2,4-D-induced disruption and are more tolerant than unadapted cells to lethal concentrations of the herbicide. These cells, adapted to grow in the presence of increasing concentrations of 2,4-D, were found to exhibit a dose-dependent increase of the saturation degree of membrane fatty acids, associated to the higher percentage of stearic (C(18:0)) and palmitic (C(16:0)) acids, and to the decreased percentage of palmitoleic (Delta9-cisC(16:1)) and oleic (Delta9-cisC(18:1)) acids. The decreased transcription of the OLE1 gene (encoding the Delta9 fatty acid desaturase that catalyses the conversion of palmitic and stearic acids to palmitoleic and oleic acids, respectively) registered in 2,4-D adapted cells suggests that yeast adaptation to the herbicide involves the enhancement of the ratio of saturated (C(16:0) and C(18:0)) to monounsaturated (C(16:1) and C(18:1)) membrane fatty acids through a reduced OLE1 expression.
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Ácido 2,4-Diclorofenoxiacético/farmacología , Adaptación Fisiológica/efectos de los fármacos , Ácido Graso Desaturasas/genética , Ácidos Grasos/metabolismo , Transcripción Genética/fisiología , Secuencia de Bases , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cartilla de ADN , Estearoil-CoA DesaturasaRESUMEN
We report results on the functional analysis of Saccharomyces cerevisiae ORF YNL065w, predicted to code for a protein belonging to the poorly characterized major facilitator superfamily (MFS) of transporters that are involved in multidrug resistance (MDR). YNL065w is important for a moderate increase of yeast tolerance to ketoconazole and to the cationic dye crystal violet; it protects the cell against short-chain monocarboxylic acids (C(2)-C(6)), but not against highly liposoluble acids such as octanoic acid or the phenoxyacetic-acid herbicides 2,4-D and MCPA; it is also a determinant of resistance to the antiarrhytmic and antimalarial drug quinidine. The encoding ORF was, thus, denominated the AQR1 gene. Results obtained using an AQR1-lacZ fusion indicate that gene expression is very low and it is not stimulated under weak acid stress. The encoded putative transporter was localized in the plasma membrane by fluorescence microscopy observation of the overproduced Aqr1-GFP fusion protein distribution.