ESS - CQB - Artigos
Permanent URI for this collection
Browse
Browsing ESS - CQB - Artigos by Issue Date
Now showing 1 - 10 of 152
Results Per Page
Sort Options
- Contributos da citologia analítica para estudos de biologia de levedurasPublication . Côrte-Real, Manuela; Sansonetty, Filipe; Ludovico, Paula; Fortuna, Margarida; Sousa, Maria João; Silva, Manuel Teixeira da; Leão, Cecília; Prudêncio, Cristina; Rodrigues, FernandoNeste artigo começamos por apresentar uma breve introdução à citologia analítica descrevendo alguns conceitos importantes desta disciplina bem como um dos instrumentos que a torna possível - o citómetro de fluxo. Referimos alguns dos aspectos relevantes desta instrumentação, em particular a possibilidade de medir a estrutura e o funcionamento de células vivas e de separar sub-populações celulares para posterior obtenção de culturas puras. Discutimos ainda os fundamentos de algumas aplicações e apresentamos os resultados obtidos nos nossos laboratórios com os seguintes estudos: i) efeitos de fungicidas no ciclo celular de leveduras; ii) relação entre a presença de proteínas de efluxo activas e a menor susceptibilidade de leveduras a antifúngicos; iii) avaliação de alterações estruturais e funcionais induzidas pelo ácido acético e sua relação com perda de viabilidade celular em populações de Saccharomyces cerevisiae e Zygosaccharomyces bailii; iv) determinação do potencial de membrana mitocondrial e avaliação da função mitocondrial de leveduras; v) determinação da ploidia de DNA em leveduras; vi) monitorização in vivo de processos celulares em S. cerevisiae e vii) morte celular programada induzida pelo ácido acético em S. cerevisiae. No decurso destes trabalhos, optimizámos diferentes protocolos de marcação celular recorrendo aos seguintes fl uorocromos: SYBR® Green I para a marcação de DNA; calceína-M, BCECF-AM, Rh123 e DiOC5 para a detecção de proteínas de efl uxo activo; DAF, FUN-1®, IP e Rh123 para a avaliação da integridade estrutural e funcional da célula; Rh123 para a avaliação de potencial de membrana mitocondrial, anexina V-FITC para a detecção de fosfatidilserina no folheto externo da membrana citoplasmática e FITC conjugado com dUTP (reacção de TUNEL) para a detecção de fragmentação internucleosomal de DNA.
- Rapid detection of efflux pumps and their relation with drug resistance in yeast cellsPublication . Prudêncio, Cristina; Sansonetty, Filipe; Sousa, Maria João; Côrte-Real, Manuela; Leão, Cecı́liaCell 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.
- Human salivary α-amylase (EC.3.2.1.1) activity and periodic acid and schiff reactive (PAS) staining: a useful tool to study polysaccharides at an undergraduate levelPublication . Fernandes, Ruben; Correia, Rossana; Fonte, Rosália; Prudêncio, CristinaHealth science education is presently in discussion throughout Europe due to the Bologna Declaration. Teaching basic sciences such as biochemistry in a health sciences context, namely in allied heath education, can be a challenging task since the students of preclinical health sciences are not often convinced that basic sciences are clinically valuable (J. R. Rudland, S. C. Rennie ( 2003) The determination of the relevance of basic sciences learning objectives to clinical practice using a questionnaire survey, Med. Educ. (Oxf.) 37, 962-965; E. C. Wragg ( 2003) How can we determine the relevance of basic sciences learning objectives to clinical practice?, Med. Educ. ( Oxf.) 37, 948-949). Thus, nowadays teachers are compelled to use their imagination to be able to elaborate laboratory sessions aiming for the understanding of theoretical concepts that are also clinically related: in other words, basic concepts and skills that underlie the competencies demanded of the future health professional. In the present work, we describe a set of laboratory sessions implemented in the discipline of biochemistry, belonging to the first year of several courses of allied health professionals, which can also be implemented in other health sciences courses. These sessions focus on the characteristics and properties of carbohydrates. The exercises we propose include two different laboratory practical sessions based on a histopathological routine technique known as periodic acid and Schiff reactive that is currently used to detect sugar metabolic and tumor diseases ( J. M. T. Rivera, C. T. Lopez, B. C. Segui ( 2001) Bioquimica Estructural: Conceptos y Tests, Tebar Flores, Madrid). The methodology described enables the demonstration of some biochemical properties of polysaccharides, namely animal and vegetable, and the catalytic activity of the human salivary alpha-amylase (EC.3.2.1.1) enzyme. A further comparison between alpha-amylase activity in vitro and in situ is also possible by the proposed methodology. Additionally, to this extent, a comparison between the results of the learning improvement that occurred after the implementation of this tool is presented.
- Unanticipated stereoselectivity in the reaction of primaquine α-aminoamides with substituted benzaldehydes: A computational and experimental study†Publication . Ferraz, Ricardo; Gomes, José R. B.; Oliveira, Eliandre de; Moreira, Rui; Gomes, PaulaImidazolidin-4-ones are commonly employed as skeletal modifications in bioactive oligopeptides, either as proline surrogates or for protection of the N-terminal amino acid against aminopeptidase- and endopeptidase-catalyzed hydrolysis. Imidazolidin-4-one synthesis usually involves the reaction of an α-aminoamide moiety with a ketone or an aldehyde to yield an imine, followed by intramolecular cyclization. We have unexpectedly found that imidazolidin-4-one formation is stereoselective when benzaldehydes containing o-carboxyl or o-methoxycarbonyl substituents are reacted with α-aminoamide derivatives of the antimalarial drug primaquine. A systematic computational and experimental study on the stereoselectivity of imidazolidin-4-one formation from primaquine α-aminoamides and various substituted benzaldehydes has been carried out, and they have allowed us to conclude that intramolecular hydrogen-bonds involving the CO oxygen of the o-substituent play a crucial role.
- Anti-Pneumocystis carinii and antiplasmodial activities of primaquine-derived imidazolidin-4-onesPublication . Vale, Nuno; Collins, Margaret S.; Gut, Jiri; Ferraz, Ricardo; Rosenthal, Philip J.; Cushion, Melanie T.; Moreira, Rui; Gomes, PaulaA series of primaquine-derived imidazolidin-4-ones were screened for their in vitro activity against Pneumocystis carinii and Plasmodium falciparum W2 strain. Most compounds were active against P. carinii above 10 lg/mL and displayed slight to marked activity. The imidazolidin-4-ones most active against P. carinii were also those most active antiplasmodial agents, in the lM range. One of the tested imidazolidin-4-ones was slightly more active than the parent primaquine and may represent a lead com pound for the development of novel anti-P. carinii 8-aminoquinolines with increased stability and resistance to metabolic inactivation.
- Photodynamic inactivation of recombinant bioluminescent Escherichia coli by cationic porphyrins under artificial and solar irradiationPublication . Alves, Eliana; Carvalho, Carla M. B.; Tomé, João P. C.; Faustino, Maria A. F.; Neves, Maria G. P. M. S.; Tomé, Augusto C.; Cavaleiro, João A. S.; Cunha, Ângela; Mendo, Sónia; Almeida, AdelaideA faster and simpler method to monitor the photoinactivation process of Escherichia coli involving the use of recombinant bioluminescent bacteria is described here. Escherichia coli cells were transformed with luxCDABE genes from the marine bioluminescent bacterium Vibrio fischeri and the recombinant bioluminescent indicator strain was used to assess, in real time, the effect of three cationic meso-substituted porphyrin derivatives on their metabolic activity, under artificial (40 W m−2) and solar irradiation (≈620 W m−2). The photoinactivation of bioluminescent E. coli is effective (>4 log bioluminescence decrease) with the three porphyrins used, the tricationic porphyrin Tri-Py+-Me-PF being the most efficient compound. The photoinactivation process is efficient both with solar and artificial light, for the three porphyrins tested. The results show that bioluminescence analysis is an efficient and sensitive approach being, in addition, more affordable, faster, cheaper and much less laborious than conventional methods. This approach can be used as a screening method for bacterial photoinactivation studies in vitro and also for the monitoring of the efficiency of novel photosensitizer molecules. As far as we know, this is the first study involving the use of bioluminescent bacteria to monitor the antibacterial activity of porphyrins under environmental conditions.
- High resistance to fourth-generation cephalosporins among clinical isolates of Enterobacteriaceae producing extended-spectrum ß-lactamases isolated in PortugalPublication . Fernandes, Rúben; Gestoso, Álvaro; Freitas, José Mota; Santos, Perpétua; Prudêncio, CristinaCarta ao editor da revista International Journal of Antimicrobial Agents. Here we report the molecular and antimicrobial susceptibility profile of extended-spectrum β-lactamase (ESBL)-producing strains found in the Portuguese northern occidental coast region (Minho).
- Resistance to β-lactams in bacteria isolated from different types of Portuguese cheesePublication . Amador, Paula; Fernandes, Rúben; Prudêncio, Cristina; Brito, LuísaThe purpose of this study was to investigate the presence of beta-lactam-resistant bacteria in six different types of Portuguese cheese. The numbers of ampicillin resistant (AMP(r)) bacteria varied from 4.7 x 10(2) to 1.5 x 10(7) CFU/g. Within 172 randomly selected beta-lactam-resistant bacteria, 44 resistant phenotypes were found and 31.4% were multidrug resistant. The majority (85%) of the isolates identified belonged to the Enterobacteriaceae family. The presence of the bla(TEM) gene was detected in 80.9% of the tested isolates. The results suggest that without thermal processing of the milk and good hygienic practices, cheese may act as a vehicle of transfer of beta-lactam-resistant bacteria to the gastrointestinal tract of consumers.
- ß-lactamases in the biochemistry and molecular biology laboratoryPublication . Amador, Paula; Prudêncio, Cristina; Vieira, Mónica; Ferraz, Ricardo; Fonte, Rosália; Silva, Nuno; Coelho, Pedro; Fernandes, Rúbenβ-lactamases are hydrolytic enzymes that inactivate the β-lactam ring of antibiotics such as penicillins and cephalosporins. The major diversity of studies carried out until now have mainly focused on the characterization of β-lactamases recovered among clinical isolates of Gram-positive staphylococci and Gram-negative enterobacteria, amongst others. However, only some studies refer to the detection and development of β-lactamases carriers in healthy humans, sick animals, or even in strains isolated from environmental stocks such as food, water, or soils. Considering this, we proposed a 10-week laboratory programme for the Biochemistry and Molecular Biology laboratory for majors in the health, environmental, and agronomical sciences. During those weeks, students would be dealing with some basic techniques such as DNA extraction, bacterial transformation, polymerase chain reaction (PCR), gel electrophoresis, and the use of several bioinformatics tools. These laboratory exercises would be conducted as a mini research project in which all the classes would be connected with the previous ones. This curriculum was compared in an experiment involving two groups of students from two different majors. The new curriculum, with classes linked together as a mini research project, was taught to a major in Pharmacy and an old curriculum was taught to students from environmental health. The results showed that students who were enrolled in the new curriculum obtained better results in the final exam than the students who were enrolled in the former curriculum. Likewise, these students were found to be more enthusiastic during the laboratory classes than those from the former curriculum.
- High resistance to fourth-generation cephalosporins among clinical isolates of Enterobacteriaceae producing extended-spectrum β-lactamases isolated in PortugalPublication . Prudêncio, Cristina; Gestoso, Álvaro; Freitas, José Mota; Santos, Perpétua; Prudêncio, Cristina; Fernandes, Ruben; Fernandes, RúbenHere we report the molecular and antimicrobial susceptibility profile of extended-spectrum β-lactamase (ESBL)-producing strains found in the Portuguese northern occidental coast region (Minho). For this purpose, bacteria isolated from clinical hospitalised and non-hospitalised patients over a period of 2 years were identified and minimal inhibitory concentrations (MICs) were determined by microdilution methods according to the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) guidelines on Enterobacteriaceae. Additionally, ESBL phenotypic identification was confirmed by the Etest (AB BIODISK, Solna, Sweden). Various methods of molecular identification of the β-lactamase (bla) genes, involving polymerase chain reaction (PCR) and sequencing strategies, were used in this study. The ESBL-producing strains (n = 193) were isolated from urine (n = 127), sputum (n = 42), bronchoalveolar lavage (n = 14), blood (n = 7) and ascitic fluid (n = 3). The most frequent ESBL-producing organism isolated in the present study was Escherichia coli (67.9%; n = 131), followed by Klebsiella pneumoniae (30.6%; n = 59), Klebsiella oxytoca (0.5%; n = 1), Enterobacter aerogenes (0.5%; n = 1) and Citrobacter freundii (0.5%; n = 1). The ESBL detected in the present studywere the TEM type (40.4%), CTX-M type (36.8%) and SHV type (22.8%). TEM-52 and TEM-24 were the most frequent TEM types (20.2% and 12.9%, respectively). Members of TEM-10 (4.1%) and TEM-116 (2.1%) were also detected. Within the CTX-M family, CTX-M-9 group was represented by CTX-M-9 (13.5%) and CTX-M-14 (8.4%). In the CTX-M-1 group, CTXM-15 was the most frequent type (12.4 %), followed by CTX-M-1 (2.1%), CTX-M-3 (0.5%) and CTX-M-32 (0.5%). Regarding CTX-M types, it appears that CTX-M-14 is widespread among the northwestern Iberian Peninsula [1]. Klebsiella pneumoniae harbouring a CTX-M-15 enzyme was described for the first time in Portugal in 2005 [2] in the Lisbon area, but CTX-M-15 enzyme has also recently been found by us in the north of Portugal in another Enterobacteriaceae member, isolated from bloodstream infections [3] among seven patients in two different hospitals. Other ESBL-producing species (not E. coli or K. pneumoniae)were also found. This is the first time that C. freundii has been described as a producer of CTX-M-32 in this country. The SHV enzymes occurred only in 23.3% of all ESBL-producing organisms. Within this type, the most frequent type was SHV-12 (12.4%), followed by SHV-5 (8.8%) and finally SHV-2 (2.1%). Some isolates co-produced more than one ESBL type: TEM-52/CTX-M-14 (0.5%); TEM-116/CTX-M-14 (0.5%); and TEM-116/CTX-M-15 (0.5%). MIC testing showed that isolates producing ESBLs were mostly susceptible to carbapenems (100%) and amikacin (99.5%). In contrast, ESBL-producing strains presented low susceptibility rates to cefepime and quinolones. Indeed, 98.9% of the ESBL-producing strains were cefepime-resistant and 85.4% were resistant to quinolones (ciprofloxacin and norfloxacin). In the generality, these high levels of resistance to quinolones were more conspicuous in members of the CTX-M family (98.1%) than TEM and SHV types (80.8% and 72.1%, respectively). In this study, cefepime presented a surprisingly low activity against ESBL-producing microorganisms. Recent literature refers to the inoculum effect exhibited by cefepime [4]. Nevertheless, we believe that this should not be pointed out as a single explanation once MIC determination is performed using inoculum concentrations of 0.5 McFarland standard. In our sample, only two K. pneumoniae harbouring SHV-2 ESBL were susceptible to cefepime. All the other clinical isolates (98.9%) expressing the ESBL phenotype were resistant to cefepime. It seems interesting that a recent study showed that cefepimewas successfully administered to three patients (two females and one male) aged between 47 years and 87 years carrying a Gram-negative ESBL-positive strain [5]. Nevertheless, other studies worldwide have begun to describe the emergence of high resistance to cefepime among Gram-negative ESBL-producers [6]. The present work showed a high diversity of ESBL enzymes occurring in the north of Portugal. In this country, the most prevalent type is still the TEM type, but CTX-M is growing rapidly [7]. The emergence of ESBL-producers resistant to cefepime in Portugal is a matter of concern. We believe that the uncontrolled use of cephalosporins may have an important role in the acquisition of resistance mechanisms, particularly the production of ESBL enzymes. Establishment of policies to monitor drug delivery in hospital and ambulatory pharmacies as well as implementation of public health defence strategies towards health promotion and drug resistance prevention appear to be urgent.