980 resultados para Cloning, Organism
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Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES)
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This project was developed to fully assess the indoor air quality in archives and libraries from a fungal flora point of view. It uses classical methodologies such as traditional culture media for the viable fungi and modern molecular biology protocols, especially relevant to assess the non-viable fraction of the biological contaminants. Denaturing high-performance liquid chromatography (DHPLC) has emerged as an alternative to denaturing gradient gel electrophoresis (DGGE) and has already been applied to the study of a few bacterial communities. We propose the application of DHPLC to the study of fungal colonization on paper-based archive materials. This technology allows for the identification of each component of a mixture of fungi based on their genetic variation. In a highly complex mixture of microbial DNA this method can be used simply to study the population dynamics, and it also allows for sample fraction collection, which can, in many cases, be immediately sequenced, circumventing the need for cloning. Some examples of the methodological application are shown. Also applied is fragment length analysis for the study of mixed Candida samples. Both of these methods can later be applied in various fields, such as clinical and sand sample analysis. So far, the environmental analyses have been extremely useful to determine potentially pathogenic/toxinogenic fungi such as Stachybotrys sp., Aspergillus niger, Aspergillus fumigatus, and Fusarium sp. This work will hopefully lead to more accurate evaluation of environmental conditions for both human health and the preservation of documents.
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Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a'_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.
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A 5-unit polyubiquitin gene, TTU3, was isolated from a T. thermophila genomic library and sequenced. This gene presents an extra triplet coding for Phe, a AGAGA motif and a putative HSE element in its 5'-non-coding region. The ubiquitin gene expression in this ciliate was investigated by Northern blot hybridization in conjugating cells or cells under stress conditions. Exponentially growing cells express two ubiquitin mRNAs of 0.75 and 1.8 kb and a new species of 1.4 kb is induced under hyperthermic stress. During sexual reproduction of the cells (conjugation) the 1.8-kb mRNA is still transcribed whereas the steady-state population of the 0.75 mRNA transcripts is strongly diminished. Southern blot analysis suggests that ubiquitin in T. thermophila constitutes a large family of about ten members.
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We report here the cloning and the characterization of the T. pyriformis CCT eta gene (TpCCT eta) and also a partial sequence of the corresponding T. thermophila gene (TtCCT eta). The TpCCt eta gene encodes a protein sharing a 60.3% identity with the mouse CCT eta. We have studied the expression of these genes in Tetrahymena exponentially growing cells, cells regenerating their cilia for different periods and during different stages of the cell sexual reproduction. These genes have similar patterns of expression to those of the previously identified TpCCt gamma gene. Indeed, the Tetrahymena CCT eta and CCT gamma genes are up-regulated at 60-120 min of cilia recovery, and in conjugation when vegetative growth was resumed and cell division took place. Our results seem to indicate that both CCT subunits play an important role in the biogenesis of the newly synthesized cilia of Tetrahymena and during its cell division.
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Ochratoxin A (OTA) is a mycotoxin produced by a variety of fungi, such as Penicillium verrucosum and Aspergillium spp., which has been found to have a wide number of potentially deadly toxic effects, and can enter the human organism through a variety of means. It then finds its way into the bloodstream and, after a lengthy process, is eventually excreted through the urine. It can thus be detected in its original form not only in blood samples but also in this biological medium. As such, and in an attempt to evaluate the exposure of the Portuguese population to this mycotoxin, morning urine samples were collected during the Winter of 2007, from each of five geographically distinct Portuguese locations Bragana, Porto, Coimbra, Alentejo, and Algarve and subjected to extraction by immunoaffinity columns and to OTA quantification through liquid chromatography coupled with fluorescence detection. Prevalent incidence was higher than 95% with Coimbra being the exception (incidence of 73.3%). In nearly all locations, the OTA content of most samples was found to be above the limit of quantification (LOQ) of 0.008 ng/ml. Indeed, excluding Coimbra, with an OTA content level of 0.014 ng/ml, all regions featured content values over 0.021 ng/ml.
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The purpose of the present work is to determine the antioxidant capacity (AC) of 27 commercial beers. The AC indicates the degree of protection of a certain organism against oxidative damage provoked by reactive oxygen and nitrogen species. Assays were carried out by the following methods: (i) total radical trapping antioxidant parameter (TRAP); (ii) trolox equivalent antioxidant capacity (TEAC); (iii) trolox equivalent antioxidant capacity (DPPH); (iv) ferric-ion reducing antioxidant parameter (FRAP); (v) cupric reducing antioxidant capacity (CUPRAC); (vi) oxygen radical absorbance capacity (ORAC). Ascorbic acid (AA), gallic acid (GA) and trolox (TR) were used as standards. All beers showed antioxidant power, but a wide range of ACs was observed. The effect of several factors upon these differences was studied. Statistical differences were found between ACs of beers of different colours. ORAC method provided always higher experimental ACs, of significant statistical differences to other assays.
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Eur. J. Biochem. 271, 23612369 (2004)
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Thesis submitted to the Faculdade de Cincias e Tecnologia to obtain the Masters degree in Environmental Engineering, profile in Ecological Engineering
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Campylobacter jejuni was looked for in the feaces of 262 children, beeing 189 with acute diarrhoea, 31 with chronic diarrhoea and 42 controls. The organism was isolated from 14 (7.4%), 2 (6.5%) and 6 (14.3%) of the children, and each group, respectively.
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Resumo Uma estratgia de avaliao e preveno de riscos na exposio a agentes qumicos deve ter sempre em conta que a vigilncia do ambiente de trabalho e a da sade dos trabalhadores so aspectos complementares de uma mesma realidade os riscos resultantes da interaco entre um agente qumico e os trabalhadores a ele expostos. Se Vigilncia Ambiental compete apreciar o risco, pela caracterizao do agente no ambiente de trabalho, a Vigilncia Biolgica pronuncia-se sobre a interaco entre o txico e o organismo, avaliando a resposta agresso qumica e a evoluo das reaces de adaptao ou de desajuste face absoro do txico. Os Indicadores Biolgicos, deste modo, assumem um estatuto de instrumento privilegiado na vigilncia da sade dos trabalhadores expostos, na medida em que medem a quantidade de txico que efectivamente penetrou e foi absorvido, ou o resultado (efeito) determinado por essa mesma dose. O presente estudo procura contribuir para a definio de um quadro metodolgico de utilizao dos Indicadores Biolgicos na avaliao/gesto da exposio profissional ao chumbo, designadamente apreciando a variao da protoporfirina-zinco (PPZ), indicador at ao presente ainda no utilizado em Portugal. O chumbo um metal de ocorrncia natural, cujos nveis nos diversos ecossistemas resultam, principalmente, das actividades antropognicas de natureza domstica e industrial. A sua capacidade poluente assinalvel, representando uma fonte de exposio permanente para o homem, demonstrvel pela sua constante presena no organismo apesar de no desempenhar qualquer tipo de funo fisiolgica. So actualmente inmeras as suas aplicaes, tornando a exposio profissional ao chumbo uma realidade vasta: indstrias de acumuladores elctricos, de vidros, de plsticos e de munies, construo civil, manuteno e reparao automvel e de navios, fabrico de tintas, indstrias electrnicas, fundies e actividades de soldadura so, entre outras, situaes onde uma realidade a ter em conta. A penetrao dos compostos inorgnicos de chumbo no organismo efectua-se principalmente por via respiratria, no sendo, no entanto, desprezvel, a sua penetrao por via digestiva. As partculas absorvidas so transportadas pelo sangue principalmente ligadas aos eritrocitos (95%), distribuem-se pelos tecidos moles e depositam-se essencialmente no tecido sseo, onde representam mais de 90% da carga corporal do total absorvido e tendo a um elevado tempo de semi-vida (mais de 20 anos). No metabolizado no organismo e a sua eliminao efectua-se essencialmente por via renal,sendo igualmente excretado, em menor escala, atravs das fezes, do suor, da saliva, das faneras e do leite materno. O conhecimento cientfico evidencia que concentraes sanguneas de chumbo entre 20 e 50 mg/dL so susceptveis de determinar efeitos adversos no homem, podendo ser afectados o sistema hematopoitico, o sistema nervoso, o sistema cardiovascular, o sistema reprodutor e o sistema imunitrio. Contudo, ainda muito h a clarificar no mbito da toxicidade do chumbo. Os nveis de exposio a que correspondem as alteraes nos diversos rgos e sistemas continuam a ser motivo de alguma controvrsia. As caractersticas carcinognicas e mutagnicas do chumbo so, ainda, um campo de vasta exigncia de investigao. A intoxicao por chumbo e seus sais (Saturnismo) de origem ocupacional reconhecida em Portugal como doena profissional (grupo 1 - Doenas Provocadas por Agentes Qumicos, da Lista das Doenas Profissionais). uma intoxicao do tipo crnico, fruto da absoro contnua de doses relativamente pequenas durante longo perodo, evidenciando-se no seu incio por sinais e sintomas vagos e difusos de grande inespecificidade, que podem incluir, nomeadamente, perda de apetite, sabor metlico na boca, palidez, mal-estar e fadiga, cefaleias, mialgias e artralgias, irritabilidade, tremores finos, obstipao, clicas abdominais, insnias, dficit da memria de curto prazo e da capacidade de concentrao. Um importante conjunto de indicadores biolgicos pode ser utilizado na vigilncia peridica da sade de trabalhadores nestas condies de exposio. Tais indicadores (de dose ou de efeito), encerram diferentes significados e comportam distintas exigncias, competindo ao Mdico do Trabalho, no mbito dos programas de preveno dos efeitos adversos relacionados com a exposio profissional a chumbo, seleccionar a sua utilizao e interpretar a sua informao, de modo a avaliar a interaco do txico com o organismo numa fase de reversibilidade. O presente estudo envolveu 180 trabalhadores dos quais 110 apresentavam plumbmias (Pb-S) iguais ou superiores a 40 mg/dL. Alm da Pb-S, a todos foi doseada a protoporfirina-zinco (PPZ) e efectuado o Hemograma e a cerca de 25% foi determinada a concentrao do cido d-aminolevulnico urinrio (ALA-U). Os doseamentos da PPZ efectuados em amostra de sangue capilar atravs de um hematofluormetro porttil revelaram-se de total fiabilidade, dando significado a uma tcnica de fcil execuo e baixo custo. A avaliao do tipo de colheita urinria para doseamento do ALA-U concluiu pela necessidade de recurso a urinas de 24 horas.Os resultados do estudo evidenciaram uma elevada associao entre a PPZ e a Pb-S, com uma maior magnitude e de incio mais precoce do que o que registado na associao da Pb-S com o ALA-U. Revelaram, ainda, fracos nveis de associao da hemoglobina (e outros parmetros hematolgicos) com a Pb-S. E demonstraram para um cut-off de 100 mg/ dL de PPZ, taxa de falsos negativos e falsos positivos, para plumbmias a partir de 70 mg/dL, inferiores a 20%. Assim, concluiu-se que, nos protocolos de vigilncia de sade de trabalhadores expostos a chumbo, o doseamento da PPZ por hematofluormetro, em sangue de colheita capilar, adequado, fivel e de realizao preferencial em relao ao do ALA-U. Concluiu-se, tambm, que a realizao do hemograma apenas se justifica em situaes individuais que clinicamente o tornem aconselhvel. E que estes protocolos devem incluir a realizao da Pb-S e da PPZ, podendo, em situaes de controlo rigoroso (ambiental, biolgico e clnico), basear-se apenas na determinao da PPZ reservando os outros indicadores para aprofundar a investigao mdica nos casos de taxas elevadas desta ou de situaes limitantes. Rsume Une stratgie dvaluation et de prvention des risques dexposition aux agents chimiques doit toujours tenir en considration que la vigilance du lieu de travail et de la sant des travailleurs sont des aspects complmentaires dune mme ralit les risques rsultant dune interaction entre lagent chimique et les travailleurs exposs. Si cest la Vigilance Ambiantale de juger le risque, par la caractrisation de lagent dans le lieu de travail, la Vigilance Biologique, elle, se prononce sur linteraction entre le toxique et lorganisme, valuant la rponse lagression chimique et lvolution des ractions dadaptation ou de rupture face labsorption du toxique. Les Indicateurs Biologiques assument ainsi un statut dinstrument privilgi de vigilance de la sant des travailleurs exposs, dans la mesure o ils dterminent la quantit de toxique qui a effectivement t pntr et absorb, ou le rsultat (effet) dtermin par cette dose. Cette tude-ci cherche contribuer la dfinition dun cadre mthodologique dutilisation des Indicateurs Biologiques dans lvaluation/ gestion de lexposition professionnelle au plomb inorganique, valuant spcialement le comportement de la protoporphirine-zinc (PPZ), indicateur pas encore utilis au Portugal.Le plomb est un mtal doccurrence naturelle dont les niveaux dans les diffrents cosystmes en rsultent, principalement, des activits anthropogniques de nature domestique et industrielle. Sa capacit polluante peut tre signale, reprsentant une source dexposition permanente pour lhomme, celle-ci dmontrable par sa prsence continue dans lorganisme, mme si elle ny accomplit aucune fonction physiologique. Actuellement ses applications sont innombrables, faisant de lexposition professionnelle au plomb une ralit de grande ampleur : industries daccumulateurs lectriques, de verre, de plastique et de munitions, btiments, manutention et rparation automobile et navale, fabrication dencres, industries lectroniques, fontes et activits de soudure sont, entre autres, des situations relles a en tenir compte. La pntration du plomb inorganique dans lorganisme se fait principalement par voie respiratoire, pouvant se faire galement par voie digestive. Les particules absorbes sont transportes par le sang, surtout lies aux rythrocytes (95%), se repartent travers les tissus mous et se dposent essentiellement dans le tissu osseux, o elles reprsentent plus de 90% de la charge corporelle de ce qui a t absorb et ont un temps de demi-vie lev (plus de 20 ans). Le plomb nest pas mtabolis dans lorganisme et son limination se fait essentiellement par voie rnale, pouvant tout de mme, une moindre chelle, tre excrt dans les fces, de la sueur, de la salive, des ongles, des cheveux et du lait maternel. La connaissance scientifique met en vidence que des concentrations sanguines de plomb entre 20 et 50 mg/dL sont susceptibles de dterminer des effets adverses dans lhomme, pouvant les systmes hmatopotique, nerveux, cardiovasculaire, reproducteur et immunitaire en tre affects. Cependant, il en reste beaucoup claircir dans le domaine de la toxicit du plomb. Les niveaux dexposition auxquels correspondent les modifications des divers organes et systmes, demeurent toujours sujet de quelque controverse. Les caractristiques carcinogniques et mutagniques du plomb restent toujours un champ dinvestigation dune grande exigence. Lintoxication par le plomb et ses sels (Saturnisme) dorigine occupationnelle est reconnue, au Portugal, comme une maladie professionnelle (groupe 1- Maladies Provoques par des Agents Chimiques, de la Liste des Maladies Professionnelles). Cest une intoxication du tipe chronique, due labsorption continue de doses relativement petites pendant une longue priode, mise en vidence travers des signes et des symptmes vagues et diffus sans grande spcificit, lesquels peuvent inclure, particulirement, le manque dapptit, got mtallique dans la bouche, pleur, malaise et fatigue, cphales, myalgies et arthralgies, irritabilit, tremblements fins, constipation, coliques abdominales, insomnies, dficit de la mmoire court terme et de la capacit de concentration.Un ensemble important dindicateurs biologiques peut tre employ dans la vigilance priodique de la sant des travailleurs dans ces conditions dexposition. Ces indicateurs (de dose ou deffet) renferment diffrentes significations et comportent diverses exigences, devant le Mdecin de Travail, dans le domaine des programmes de prvention des effets adverses qui sont en relation avec lexposition professionnelle au plomb, slectionner son utilisation et interprter son information de faon valuer linteraction de llment toxique avec lorganisme un stade de rversibilit. Ltude ci-prsent engloba 180 travailleurs desquels 110 prsentaient des plombmies (Pb-S) gales ou suprieures 40 mg/dL. part la Pb-S, la protoporphyrine-zinc (PPZ) leur a t prise en dosage et un Hmogramme fut effectu et fut dtermin lacide d- aminolvulinique urinaire (ALA-U) sur environ 25% des travailleurs. Le dosage de la PPZ efectu en chantillon de sang capillaire par un fluorimtre portable, sest accomplit dune fiabilit total, donnant du sgnificat une tchnique de facile execution et bas prix. Lvaluation de la prise urinaire par dosage du ALA-U conclut au besoin dun recours aux urines de 24 heures Les rsultats de ltude ont mis en vidence une association leve entre la PPZ et la Pb- S, avec une intensit majeure et de dbut plus prcoce par rapport celui qui fut registr lors de lassociation de la Pb-S avec la ALA-U. Ces rsultats ont galement montr de faibles niveaux dassociation entre lhmoglobine (et autres paramtres hmatologiques) et la Pb-S. Ils ont dmontr aussi, une valeur de cut-off de 100 mg/dL de PPZ, des taux de faux ngatifs et faux positifs, pour des plombmies de 70 mg/dL, infrieurs 20%. On peut donc conclure que dans les protocoles de vigilance de la sant des travailleurs exposs au plomb, le dosage de la PPZ par fluorimetrie dans le sang capillaire est adquat, fiable et de ralisation prfrentielle par rapport celui du ALA-U. On peut galement conclure que la ralisation de lhmogramme ne se justifie que dans les cas individuels o, cliniquement, celui-ci est conseill. De plus, ces protocoles doivent inclure la ralisation de la Pb-S et de la PPZ, pouvant, en cas de contrle rigoureux (ambiantal, biologique et clinique), sappuyer que dans la dtermination de la PPZ rservant les autres indicateurs pour approfondir linvestigation mdicale dans les cas o les taux de celle-ci sont levs ou dans les cas de situations limitantes. Summary Any strategy to evaluate and prevent the risks of chemical agents exposure must always regard the work environment and workers health as complementary aspects of one reality - the resulting risks from the interaction between the chemical agent and the exposed workers. It is the responsibility of Environmental Monitoring to evaluate the risks of exposure by the characterization of the chemical agent in the work environment. Biological Monitoring, on the other hand, pronounces itself over the toxin and body interaction, evaluating human response to the chemical aggression and the body adaptations to the toxic absorption. Biological Exposure Indices (BEI) assume, therefore, a privileged status among exposed workers' health monitoring instruments, as they measure the actual penetrated and absorbed toxic quantity and the effect it produces. This research study aims to contribute to the definition of a methodological strategy on the utilization of BEIs in evaluating inorganic lead's occupational exposure, more specifically appreciating the zinc protoporphyrin (ZPP) variation, an index that has never been taken under consideration in Portugal until now. Lead is a natural metal whose ecosystems levels are mainly due to domestic and industrial anthropogenic activities. Its pollutant capacity is notable, representing a permanent exposure risk shown by its constant presence in the human body, although it has no physiologic function. Nowadays, lead's applications are countless, turning its professional exposure a huge reality: storage batteries industries, glass industries, plasterers and munitions industries, building construction, ships and motor car maintenance and repairing, ink manufacture, electronics industries, foundries and other soldering activities are, among so many other, realities to attend to. Respiration is the main cause of human body's inorganic lead absorption, although digestive pathway must not to be ignored. The absorbed particles are transported by blood, essentially bounded to erythrocytes (95%). It is distributed by soft tissues and settled mainly on bone tissues, where it represents approximately 90% of the total body charge and has a high half-life time (more than 20 years). It is not metabolized by the organism, its elimination being effectuated by renal activity and, in smaller scale, through lees, sweat, saliva, nails, hair and maternal milk.Scientific knowledge shows that concentrations of lead in blood between 20 e 50 mg/dL are susceptible to determine adverse effects in man and able to affect the hematopoietic system, the nervous system, the cardiovascular system, the reproductive system and the immunological system. Nevertheless, there's still much to be learned and clarified about lead's toxicity. The correlation between exposure levels and human's systems and organs alteration levels continues to be a centre of controversies. Still, lead's carcinogenic and mutagenic characteristics continue to be a high demanding research field. Intoxication by lead and its compounds (saturnism), from occupational origin, is recognized in Portugal as an occupational disease, included in Group 1 - Chemical Agents Caused Diseases, on the Occupational Diseases List. It is a chronic intoxication caused by a continuous absorption of small doses, throughout a long period of time. Its signs and symptoms are diffuse and imprecise, of great unspecificity, such as loss of appetite, metallic flavor in the mouth, paleness, ailment and fatigue, headaches, myalgia and arthralgia, irritability, thin tremors, constipation, abdominal pain, insomnias, short memory loses and inability to concentrate. A considered number of BEIs can be used in Periodic Health Monitoring of workers in such exposure conditions. Such BEI (dose indices or effect indices) provide different meanings and imply different procedures, being Occupational Doctors responsibility, in the context of lead related adverse effects preventive programmes, to select and interpret its information, in order to evaluate the interaction between toxic and organism in a reversible phase of the toxic action. The present research study involved 180 workers, 110 of which presented blood lead levels (PbB) above or equal to 40 mg/dL. Besides PbB, all workers has been evaluated for zinc protoporphyrin levels (PPZ) and submitted to a haemogram. About 25% of the workers were selected for d-aminolevulinic urinary acid (ALA-U) determination. The evaluation of PPZ, by a portable hematofluorometer using capillary blood samples, turned out to be an easy procedure with low costs and total warrantability. As in regard for ALA- U procedure, it was concluded the necessity of 24 hours urine samples. This research results underlined a strong connection between ZPP and PbB, which was found to to be stringer and to begin earlier than it was registered for PbB and ALA-U association. The same study also revealed a low association level between PbB and hemoglobin or other hematological indices. It was also verified less than 20% of false negatives and false positives cases when admitted a ZPP 100 mg/dL cut off value for PbB 70 mg/dL. As in result it was concluded that in Health Monit
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Applied and Environmental Microbiology, Vol. 73, No.4
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A radiometric assay system has been used to study oxidation patterns of (1-14C) fatty acids by drug-susceptible and drug-resistant organisms of the genus Mycobacterium. Two strains of M. tuberculosis susceptible to all drugs, H37Rv and Erdman, were used. Drug-resistant organisms included in this investigation were M. tuberculosis H37Rv resistant to 5 ug/ml isoniazid, M. bovis, M. avium, M. intracellular, M. kansasii and M. chelonei. The organisms were inoculated in sterile reaction vials containing liquid 7H9 medium, 10% ADC enrichment and 1.0 uCi of one of the (1-14C) fatty acids (butyric, hexnoic, octanoic, decanoic, lauric, myristic, palmitic, stearic, oleic, linoleic, linolenic). Vials were incubated at 37C and the 14CO2 envolved was measured daily for 3 days with a Bactec R-301 instrument. Although each individual organism displayed a different pattern of fatty oxidation, these patterns were not distinctive enough for identification of the organism. No combination of fatty acids nor preferential oxidation of long chain or of short chain fatty acids were able to separate susceptible from resistant organisms. Further investigation with a larger number of drug susceptible mycobacteria including assimilation studies and oxidation of other substrates may be required to achieve a distinction between drug-susceptible and drug-resistant mycobacteria.
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A radiometric assay system has been used to study oxidation patterns of (U-14C) L-amino acids by drug-susceptible and drug-resistant mycobacteria. Drug-susceptible M. tuberculosis (H37Rv TMC 102 and Erdman) along with the drug-resistant organism M. tuberculosis (H37 Rv TMC 303), M. bovis, M. avium, M. intracellulare, M. kansasii and M. chelonei were used. The organisms were inoculated into a sterile reaction system with liquid 7H9 medium and one of the (U-14C) L-amino acids. Each organism displayed a different pattern of amino acid oxidation, but these patterns were not distinctive enough for identification of the organism. Complex amino acids such as proline, phenylalanine and tyrosine were of no use in identification of mycobacteria, since virtually all organisms failed to oxidize them. There was no combination of substrates able to separate susceptible from resistant organisms.
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The yeast Saccharomyces cerevisiae is a useful model organism for studying lead (Pb) toxicity. Yeast cells of a laboratory S. cerevisiae strain (WT strain) were incubated with Pb concentrations up to 1,000 mol/l for 3 h. Cells exposed to Pb lost proliferation capacity without damage to the cell membrane, and they accumulated intracellular superoxide anion (O2 .) and hydrogen peroxide (H2O2). The involvement of the mitochondrial electron transport chain (ETC) in the generation of reactive oxygen species (ROS) induced by Pb was evaluated. For this purpose, an isogenic derivative 0 strain, lacking mitochondrial DNA, was used. The 0 strain, without respiratory competence, displayed a lower intracellular ROS accumulation and a higher resistance to Pb compared to the WT strain. The kinetic study of ROS generation in yeast cells exposed to Pb showed that the production of O2 . precedes the accumulation of H2O2, which is compatible with the leakage of electrons from the mitochondrial ETC. Yeast cells exposed to Pb displayed mutations at the mitochondrial DNA level. This is most likely a consequence of oxidative stress. In conclusion, mitochondria are an important source of Pb-induced ROS and, simultaneously, one of the targets of its toxicity.
