RESUMO
AIM: Percutaneous coronary intervention (PCI) is the gold standard for the treatment of acute myocardial infarction (AMI), with the main limitation of in-stent restenosis for BMS and late stent thrombosis (ST) for both BMS and DES. Endothelial progenitor cells (EPC) CD34+ capture stents, promoting vascular healing, may be advantageous in preventing ST. Aim of the study is to evaluate the outcomes of AMI patients treated with EPC CD34+ capture stent and describe the mobilization kinetics of CD34+ and their clinical correlation. METHODS: Fifty AMI patients underwent primary PCI with EPC CD34+ capture stent. Serial assays of CD34+ were performed by flow-cytometric analysis. RESULTS: Procedural success rate was 100%. At six-months follow-up cardiac death, myocardial infarction, target lesion revascularization (TLR) and target vessel revascularization (TVR) occurred respectively in 2%, 4%, 10% and 12% of patients. No case of ST was observed. The MACE-free survival was 81,2%. The mean peak value of plasmatic CD34+ was 4.69±3.76 cells/µL. A positive correlation was found between CD34+ concentration, age and infarct area. No correlation was detected between CD34+ concentration and occurrence of TVR, TLR and MACE. CONCLUSION: EPC capture stent implantation seems to be safe and effective in the clinical setting of AMI, representing a possible alternative to BMS and DES. CD34+ cells plasmatic concentration seems not to correlate to coronary restenosis and atheromasic disease progression.
Assuntos
Mobilização de Células-Tronco Hematopoéticas , Infarto do Miocárdio/terapia , Intervenção Coronária Percutânea/instrumentação , Stents , Idoso , Antígenos CD34/análise , Contagem de Células Sanguíneas , Comorbidade , Reestenose Coronária/epidemiologia , Reestenose Coronária/prevenção & controle , Reestenose Coronária/cirurgia , Trombose Coronária/epidemiologia , Trombose Coronária/prevenção & controle , Intervalo Livre de Doença , Endotélio Vascular/fisiologia , Feminino , Seguimentos , Humanos , Tempo de Internação/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/mortalidade , Infarto do Miocárdio/cirurgia , Intervenção Coronária Percutânea/métodos , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/prevenção & controle , Estudos Prospectivos , Regeneração , Sistema de Registros , Fatores de Risco , Stents/efeitos adversos , Resultado do TratamentoRESUMO
The Turin Metropolitan Transplant Centre (CIC 305) includes four flow-cytometry laboratories assessing quality control on hematopoietic stem cells (HSC) with different instruments and operators. Therefore, the CD34+ enumeration assay should be validated on a regular basis. We describe here the validation plan to test the inter-laboratory reproducibility of CD34+ enumeration assay, based on the risk analysis. Stabilized blood samples were analysed using Stem-Kit reagent according to manufacturer's instructions and acquired using the Beckman Coulter Navios at Regina Margherita Children's' Hospital (305-1), Beckman Coulter FC500 at Candiolo Cancer Institute FPO-IRCCS (305-2), BD Biosciences FACSLyric™ at S. Luigi Hospital (305-3), and Beckman Coulter Navios EX at Mauriziano Hospital (305-4). The ISHAGE guidelines were followed for estimating % and absolute number of CD34+ cells in single-platform method. For each sample repeatability limit (r), reproducibility error, uncertainty of reproducibility error and coefficient of variation (CV) were reported. The repeated measurements from each laboratory or instrument have a variability, expressed as reproducibility error, lower than the repeatability limit for that single parameter. The corrected reproducibility error is always lower than the repeatability limit except for the percentage value of the "low" count. The analysis of inter-laboratory variance is within the maximum acceptable variance value, and the CV of all measurements for each parameter is less than 8%, indicating low measurement variability among laboratories. Evaluating the overall data, we can conclude that the four laboratories are perfectly aligned and the results are reproducible.
RESUMO
A particular feature of gammadelta T cell biology is that cells expressing T cell receptor (TCR) using specific Vgamma/Vdelta segments are localized in distinct epithelial sites, e.g., in mouse epidermis nearly all gammadelta T cells express Vgamma3/Vdelta1. These cells, referred to as dendritic epidermal T cells (DETC) originate from fetal Vgamma3+ thymocytes. The role of gammadelta TCR specificity in DETC's migration/localization to the skin has remained controversial. To address this issue we have generated transgenic (Tg) mice expressing a TCR delta chain (Vdelta6.3-Ddelta1-Ddelta2-Jdelta1-Cdelta), which can pair with Vgamma3 in fetal thymocytes but is not normally expressed by DETC. In wild-type (wt) Vdelta6.3Tg mice DETC were present and virtually all of them express Vdelta6.3. However, DETC were absent in TCR-delta(-/-) Vdelta6.3Tg mice, despite the fact that Vdelta6.3Tg gammadelta T cells were present in normal numbers in other lymphoid and nonlymphoid tissues. In wt Vdelta6.3Tg mice, a high proportion of in-frame Vdelta1 transcripts were found in DETC, suggesting that the expression of an endogenous TCR-delta (most probably Vdelta1) was required for the development of Vdelta6.3+ epidermal gammadelta T cells. Collectively our data demonstrate that TCR specificity is essential for the development of gammadelta T cells in the epidermis. Moreover, they show that the TCR-delta locus is not allelically excluded.
Assuntos
Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Pele/imunologia , Linfócitos T/fisiologia , Animais , Movimento Celular , Células Dendríticas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T gama-delta/genética , Células-Tronco/fisiologia , Timo/citologiaRESUMO
Thymic dendritic cells (DCs) form a discrete subset of bone marrow (BM)-derived cells, the function of which is to mediate negative selection of autoreactive thymocytes. The developmental origin of thymic DCs remains controversial. Although cell transfer studies support a model in which T cells and thymic DCs develop from the same intrathymic pluripotential precursor, it remains possible that these two types of cells develop from independent intrathymic precursors. Notch proteins are cell surface receptors involved in the regulation of cell fate specification. We have recently reported that T cell development in inducible Notch1-deficient mice is severely impaired at an early stage, before the expression of T cell lineage markers. To investigate whether development of thymic DCs also depends on Notch1, we have constructed mixed BM chimeric mice. We report here that thymic DC development from Notch1(-/)- BM precursors is absolutely normal (in terms of absolute number and phenotype) in this competitive situation, despite the absence of Notch1(-/)- T cells. Furthermore, we find that peripheral DCs and Langerhans cells are also not affected by Notch1 deficiency. Our results demonstrate that the development of DCs is totally independent of Notch1 function, and strongly suggest a dissociation between intrathymic T cell and DC precursors.
Assuntos
Células Dendríticas/citologia , Proteínas de Membrana/fisiologia , Receptores de Superfície Celular , Linfócitos T/citologia , Timo/citologia , Fatores de Transcrição , Animais , Linfócitos B/citologia , Células da Medula Óssea/citologia , Diferenciação Celular , Granulócitos/citologia , Células-Tronco Hematopoéticas/citologia , Células Matadoras Naturais/citologia , Macrófagos/citologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptor Notch1 , Projetos de Pesquisa , Linfócitos T/imunologiaRESUMO
Mitochondrial DNA depletion syndromes (MDSs) form a group of autosomal recessive disorders characterized by profoundly decreased mitochondrial DNA copy numbers in affected tissues. Three main clinical presentations are known: myopathic, encephalomyopathic and hepatocerebral. The first is associated with mutations in thymidine kinase 2 (TK2) and p53-induced ribonucleotide reductase B subunit (RRM2B); the second with mutations in succinate synthase A (SUCLA2) and B (SUCLG1); the third with mutations in Twinkle (PEO1), pol-gammaA (POLG1), deoxyguanosine kinase (DGUOK) and MPV17 (MPV17). In this work, we review the MDS-associated phenotypes and present our own experience of 32 MDS patients, with the aim of defining the mutation frequency of the known genes, the clinical spectrum of the diseases, and the genotype-phenotype correlations. Five of our patients carried previously unreported mutations in one of the eight MDS genes.
Assuntos
DNA Mitocondrial/genética , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Acidose Láctica/etiologia , Idade de Início , Encéfalo/patologia , Criança , Pré-Escolar , Estudos de Coortes , Eletroencefalografia , Eletromiografia , Feminino , Humanos , Lactente , Recém-Nascido , Fígado/patologia , Imageamento por Ressonância Magnética , Masculino , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/patologia , Miopatias Mitocondriais/genética , Miopatias Mitocondriais/patologia , Músculo Esquelético/patologia , Mutação/fisiologia , Timidina Quinase/genéticaRESUMO
BACKGROUND AND AIMS: Mesenchymal stem cells from bone marrow (MSCs) may have the potential to differentiate in vitro and in vivo into hepatocytes. We investigated whether transplanted human MSCs (hMSCs) may engraft the liver of non-obese diabetic severe combined immuno-deficient (NOD/SCID) mice and differentiate into cells of hepatic lineage. METHODS: Ex vivo expanded, highly purified and functionally active hMSCs from bone marrow were transplanted (caudal vein) in sublethally irradiated NOD/SCID mice that were either exposed or not to acute liver injury or submitted to a protocol of chronic injury (single or chronic intraperitoneal injection of CCl(4), respectively). Chimeric livers were analysed for expression of human transcripts and antigens. RESULTS: Liver engraftment of cells of human origin was very low in normal and acutely injured NOD/SCID mice with significantly higher numbers found in chronically injured livers. However, hepatocellular differentiation was relatively rare, limited to a low number of cells (ranging from less than 0.1% to 0.23%) as confirmed by very low or not detectable levels of human transcripts for alpha-fetoprotein, CK18, CK19 and albumin in either normal or injured livers. Finally, a significant number of cells of human origin exhibited a myofibroblast-like morphology. CONCLUSIONS: Transplanted hMSCs have the potential to migrate into normal and injured liver parenchyma, particularly under conditions of chronic injury, but differentiation into hepatocyte-like cells is a rare event and pro-fibrogenic potential of hMSC transplant should be not under-evaluated.
Assuntos
Regeneração Hepática/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Medicina Regenerativa/métodos , Animais , Células da Medula Óssea , Tetracloreto de Carbono , Expressão Gênica , Sobrevivência de Enxerto/fisiologia , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Amyotrophic lateral sclerosis (ALS) is a lethal disease affecting motoneurons. In familial ALS, patients bear mutations in the superoxide dismutase gene (SOD1). We transplanted human bone marrow mesenchymal stem cells (hMSCs) into the lumbar spinal cord of asymptomatic SOD1(G93A) mice, an experimental model of ALS. hMSCs were found in the spinal cord 10 weeks after, sometimes close to motoneurons and were rarely GFAP- or MAP2-positive. In females, where progression is slower than in males, astrogliosis and microglial activation were reduced and motoneuron counts with the optical fractionator were higher following transplantation. Motor tests (Rotarod, Paw Grip Endurance, neurological examination) were significantly improved in transplanted males. Therefore hMSCs are a good candidate for ALS cell therapy: they can survive and migrate after transplantation in the lumbar spinal cord, where they prevent astrogliosis and microglial activation and delay ALS-related decrease in the number of motoneurons, thus resulting in amelioration of the motor performance.
Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Esclerose Lateral Amiotrófica/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Mielite/terapia , Medula Espinal/fisiopatologia , Medula Espinal/cirurgia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Feminino , Gliose/metabolismo , Gliose/fisiopatologia , Gliose/cirurgia , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Microglia/citologia , Microglia/metabolismo , Neurônios Motores/patologia , Transtornos dos Movimentos/etiologia , Transtornos dos Movimentos/fisiopatologia , Transtornos dos Movimentos/cirurgia , Mutação/genética , Mielite/fisiopatologia , Degeneração Neural/fisiopatologia , Degeneração Neural/prevenção & controle , Degeneração Neural/cirurgia , Recuperação de Função Fisiológica/fisiologia , Caracteres Sexuais , Medula Espinal/patologia , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Taxa de Sobrevida , Resultado do TratamentoRESUMO
Although their role in the cardiovascular system is still largely unknown, mast cells are present in the myocardium of both experimental animals and humans. Interestingly, cathecolaminergic nerve fibres and mast cells are often described in close morphological and functional interactions in various organs. In the present study we investigated the effects of chronic interference with beta-adrenergic receptors (via either sympathectomy or beta-blockade) on cardiac mast cell morphology/activation and on interstitial collagen deposition. In rats subjected to chemical sympathectomizy with the neurotoxin 6-hydroxydopamine (6-OHDA) we observed a significant increase of mast cell density, and in particular of degranulating mast cells, suggesting a close relationship between the cardiac catecholaminergic system and mast cell activation. In parallel, chronic 6-OHDA treatment was associated with increased collagen deposition. The influence of the beta-adrenergic receptor component was investigated in rats subjected to chronic propranolol administration, that caused a further significant increase in mast cell activation associated with a lower extent of collagen deposition when compared to chemical sympathectomy. These data are the first demonstration of a close relationship between rat cardiac mast cell activation and the catecholaminergic system, with a complex interplay with cardiac collagen deposition. Specifically, abrogation of the cardiac sympathetic efferent drive by chemical sympathectomy causes mast cell activation and interstitial fibrosis, possibly due to the local effects of the neurotoxin 6-hydroxydopamine. In contrast, beta-adrenergic blockade is associated with enhanced mast cell degranulation and a lower extent of collagen deposition in the normal myocardium. In conclusion, cardiac mast cell activation is influenced by beta-adrenergic influences.
Assuntos
Degranulação Celular/efeitos dos fármacos , Colágeno/química , Mastócitos/citologia , Mastócitos/fisiologia , Miocárdio/citologia , Simpatolíticos/farmacologia , Animais , Contagem de Células , Ventrículos do Coração/anatomia & histologia , Ventrículos do Coração/citologia , Imuno-Histoquímica , Masculino , Mastócitos/efeitos dos fármacos , Oxidopamina/farmacologia , Pericárdio/anatomia & histologia , Pericárdio/citologia , Propranolol/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
The rag4 mutant of Kluyveromyces lactis was previously isolated as a fermentation-deficient mutant, in which transcription of the major glucose transporter gene RAG1 was affected. The wild-type RAG4 was cloned by complementation of the rag4 mutation and found to encode a protein homologous to Snf3 and Rgt2 of Saccharomyces cerevisiae. These two proteins are thought to be sensors of low and high concentrations of glucose, respectively. Rag4, like Snf3 and Rgt2, is predicted to have the transmembrane structure of sugar transporter family proteins as well as a long C-terminal cytoplasmic tail possessing a characteristic 25-amino-acid sequence. Rag4 may therefore be expected to have a glucose-sensing function. However, the rag4 mutation was fully complemented by one copy of either SNF3 or RGT2. Since K. lactis appears to have no other genes of the SNF3/RGT2 type, we suggest that Rag4 of K. lactis may have a dual function of signaling high and low concentrations of glucose. In rag4 mutants, glucose repression of several inducible enzymes is abolished.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Glucose/metabolismo , Kluyveromyces/genética , Kluyveromyces/metabolismo , Proteínas de Saccharomyces cerevisiae , Transporte Biológico , Northern Blotting , Membrana Celular/metabolismo , Sistema Livre de Células , Clonagem Molecular , Proteínas Fúngicas , Deleção de Genes , Glucose/farmacocinética , Proteínas de Membrana/genética , Modelos Genéticos , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Fenótipo , Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA , Transdução de Sinais , Transcrição GênicaRESUMO
Transforming growth factor (TGF)-beta is a protein family which affects multiple cellular functions including survival, proliferation, differentiation and adhesion. Among the three known isoforms, TGF-beta1 is commonly overexpressed in solid malignancies. Recent studies in knock-out mice demonstrated non-redundant roles of different TGF-beta isoforms in development. The present study was performed to assess tumour-associated expression of the three TGF-beta isoforms in colon carcinoma. We report that colon carcinoma progression is associated with gradual and significant increases in expression of TGF-beta1 and TGF-beta2 mRNA and proteins. By contrast, TGF-beta3 expression was detected in normal colonic mucosa and, at slightly higher levels, in tumour tissues. In addition, plasma levels of both TGF-beta1 and TGF-beta2 were significantly higher in cancer patients when compared with unaffected individuals. Taken together, our results indicate distinct expression patterns of the three TGF-beta isoforms in colon carcinoma cells and possible systemic effects of TGF-beta1 and TGF-beta2 in tumour patients.
Assuntos
Carcinoma in Situ/diagnóstico , Neoplasias do Colo/diagnóstico , Proteínas de Neoplasias/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Metástase Linfática , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1 , Fator de Crescimento Transformador beta2 , Fator de Crescimento Transformador beta3RESUMO
Mouse thymic dendritic cells (DC) have been isolated after collagenase digestion, selection of the low-density cell fraction, then depletion of T-lineage cells and other non-DC by treatment with specific monoclonal antibodies (mAb) and removal with anti-Ig-coated magnetic beads. The resulting DC preparation represented 0.1-0.2% of total thymic cells and contained 70-80% DC. Flow cytometry analysis of MHC class II (MHC II) expression by DC showed that 40% of DC expressed intermediate levels of MHC II, and 60% expressed high levels of this marker. Moreover, immunofluorescent 2-colour staining allowed the characterization of two clearly distinguishable DC subpopulations: MHC IIinter DC were CD45hi, CD44hi, HSAhi, whereas MHC IIhi DC were CD45lo, CD44lo, HSAlo. These results are discussed with regard to the functional significance of MHC IIinter and MHC IIhi DC subpopulations in the mouse thymus.
Assuntos
Células Dendríticas , Timo/citologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD/análise , Biomarcadores , Separação Celular , Células Dendríticas/química , Células Dendríticas/ultraestrutura , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/análise , Separação Imunomagnética , Imunofenotipagem , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de InterferênciaRESUMO
Rat thymic dendritic cells (DC) have been analyzed by flow cytometry in order to study the variations on the cell surface marker expression upon culture at 37 degrees C. Our results demonstrate that whereas expression of major histocompatibility complex (MHC) molecules, CD45, Mac-1, LFA-1, B-cell markers, macrophage markers and some T-cell markers (as CD2, CD4 and CD8) did not undergo changes in culture, the level of expression of the adhesion molecules VLA-4 and ICAM-1, and the T-cell markers CD5, CD25 and Thy-1 increased after 14 h incubation at 37 degrees C. VLA-4, ICAM-1 and Thy-1 expression was up-regulated from intermediate to high levels, the percentage of CD5+ cells increased from 20% to 50%, and the interleukin-2 (IL-2) receptor alpha chain (CD25) was induced in 50% of DC after the culture period. These results are discussed with regard to the functional significance of DC phenotypic variations, and their implications concerning the development of in vitro systems designed for T-cell differentiation studies involving purified DC.
Assuntos
Antígenos de Superfície/imunologia , Células Dendríticas/imunologia , Timo/imunologia , Animais , Anticorpos Monoclonais , Biomarcadores , Moléculas de Adesão Celular/imunologia , Células Cultivadas , Citometria de Fluxo , Ativação Linfocitária , Glicoproteínas de Membrana/imunologia , Ratos , Ratos Wistar , Receptores de Interleucina-2/biossíntese , Linfócitos T/imunologia , Antígenos Thy-1 , Timo/citologia , Regulação para CimaRESUMO
Programmed cell death, also known as apoptosis, is a normal physiologic process which occurs during embryonic development as well as in maintenance of tissue homeostasis. Increasing evidence suggests that alterations in cell death contribute to the pathogenesis of a number of human diseases, including cancer, viral infections, autoimmune diseases and acquired immunodeficiency syndrome (AIDS). The extraordinary research activity of the past few years has resulted in the characterization of the principal proteins involved in the apoptosis machinery. An area of particular interest has been the induction of apoptosis by two death receptor/ligand pairs, Fas/Fas Ligand and DR4-DR5/TRAIL. The identification of these molecules with the recruited signaling pathways could clarify their physiopathological implications, having a significant impact upon potential therapeutic interventions in diseases associated with cell survival alterations.
Assuntos
Apoptose , Glicoproteínas de Membrana/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Animais , Proteína Ligante Fas , Humanos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Transdução de SinaisRESUMO
Amyotrophic Lateral Sclerosis (ALS) is a devastating incurable disease. Stem-cell-based therapies represent a new possible strategy for ALS clinical research. The objectives of this Phase 1 clinical study were to assess the feasibility and toxicity of mesenchymal stem cell transplantation and to test the impact of a cell therapy in ALS patients. The trial was approved and monitored by the National Institute of Health and by the Ethics Committees of all participating Institutions. Autologous MSCs were isolated from bone marrow, expanded in vitro and analyzed according to GMP conditions. Expanded MSCs were suspended in the autologous cerebrospinal fluid (CSF) and directly transplanted into the spinal cord at a high thoracic level with a surgical procedure. Ten ALS patients were enrolled and regularly monitored before and after transplantation by clinical, psychological, neuroradiological and neurophysiological assessments. There was no immediate or delayed transplant-related toxicity. Clinical, laboratory, and radiographic evaluations of the patients showed no serious transplant-related adverse events. Magnetic resonance images (MRI) showed no structural changes (including tumor formation) in either the brain or the spinal cord. However the lack of post mortem material prevents any definitive conclusion about the vitality of the MSCs after transplantation. In conclusion, this study confirms that MSC transplantation into the spinal cord of ALS patients is safe and that MSCs might have a clinical use for future ALS cell based clinical trials.
Assuntos
Esclerose Lateral Amiotrófica/cirurgia , Transplante de Células-Tronco Mesenquimais/métodos , Adulto , Idoso , Esclerose Lateral Amiotrófica/fisiopatologia , Antígenos CD/metabolismo , Células da Medula Óssea/fisiologia , Estudos de Coortes , Imagem de Difusão por Ressonância Magnética/métodos , Estimulação Elétrica/métodos , Potenciais Somatossensoriais Evocados/fisiologia , Feminino , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Medula Espinal/patologia , Fatores de Tempo , Adulto JovemAssuntos
Antígenos de Superfície/análise , Células Dendríticas/imunologia , Citometria de Fluxo , Imunofenotipagem , Timo/citologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD/análise , Biomarcadores/análise , Glicoproteínas de Membrana/análise , Camundongos , Ratos , Ratos Wistar/imunologia , Antígenos Thy-1RESUMO
The aim of our research project was to consolidate a multiplex RT-PCR protocol to detect aflatoxigenic strains of Aspergillus flavus. Several independent A. flavus strains were isolated from corn and flour samples from the North of Italy and from three European countries. Aflatoxin producing/not producing phenotype was assessed by qualitative and quantitative assays at day five of growth in aflatoxin inducing conditions. Expression of 16 genes belonging to the aflatoxin cluster was assayed by multiplex or monomeric RT-PCR. There is a good correlation between gene expression and aflatoxin production. Strains that apparently transcribed all the relevant genes but did not release aflatoxin in the medium ("false positives") were re-assessed for mycotoxin production after extended growth in inducing condition. All the "false positive" strains in actual fact were positive when aflatoxin determination was performed after 10 days of growth. These strains should then be re-classified as "slow aflatoxin accumulators". To optimise the diagnostic procedure, a quintuplex RT-PCR procedure was designed consisting of a primer set directed against four informative aflatoxin cluster genes and the beta-tubulin gene as an internal amplification control. In conclusion we have provided evidence for the robustness and reliability of our RT-PCR protocol in discriminating mycotoxin producer from non-producer strains of A. flavus. and the molecular procedure we devised is a promising tool with which to screen and control the endemic population of A. flavus colonising different areas of the World.
Assuntos
Aspergillus flavus/metabolismo , Micotoxinas/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Aspergillus flavus/classificação , Aspergillus flavus/genética , Reações Falso-Positivas , Regulação Fúngica da Expressão Gênica/fisiologia , Genes Fúngicos , RNA FúngicoRESUMO
AIMS: To develop a multiplex reverse transciption-polymerase chain reaction (RT-PCR) protocol to discriminate aflatoxin-producing from aflatoxin-nonproducing strains of Aspergillus flavus. METHODS AND RESULTS: The protocol was first optimized on a set of strains obtained from laboratory collections and then validated on A. flavus strains isolated from corn grains collected in the fields of the Po Valley (Italy). Five genes of the aflatoxin gene cluster of A. flavus, two regulatory (aflR and aflS) and three structural (aflD, aflO and aflQ), were targeted with specific primers to highlight their expression in mycelia cultivated under inducing conditions for aflatoxins production. 48-h-old cultures expressed the complete set of the genes analysed here whereas 24-h-old ones did not. Genomic PCR (quadruplex PCR) was also performed in parallel using chromosomal DNA extracted from the same set of strains to correlate the integrity of the genes with their expression. CONCLUSIONS: We show that a good correlation exists between gene expression of the aflatoxin genes, here analysed by multipex RT-PCR, and aflatoxin production, except for one strain that apparently transcribed all the relevant genes but did not produce aflatoxin in the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first example of the application of a combination of multiplex PCR and RT-PCR approaches to screen a population of A. flavus for the presence of aflatoxigenic and nonaflatoxigenic strains. The proposed protocol will be helpful in evaluating the risk posed by A. flavus in natural environments and might also be a useful tool to monitor its presence during the processing steps of food and feed commodities.
Assuntos
Aflatoxinas/biossíntese , Aspergillus flavus/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ração Animal/microbiologia , Aspergilose/genética , Aspergilose/metabolismo , Aspergillus flavus/genética , Aspergillus flavus/isolamento & purificação , Meios de Cultura , DNA Fúngico/genética , Microbiologia de Alimentos , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos/genética , Micélio/genética , Micélio/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Venenos/metabolismo , Transcrição Gênica/genética , Zea mays/microbiologiaRESUMO
In Saccharomyces cerevisiae the utilization of lactate occurs via specific oxidation of L- and D-lactate to pyruvate catalysed by L-lactate ferricytochrome c oxidoreductase (L-LCR) (EC 1.1.2.3) encoded by the CYB2 gene, and D-lactate ferricytochrome c oxidoreductase (D-LCR) (EC 1.1.2.4), respectively. We selected several lactate- pyruvate+ mutants in a cyb2 genetic background. Two of them were devoid of D-LCR activity (dld mutants, belonging to the same complementation group). The mutation mapped in the structural gene. This was demonstrated by a gene dosage effect and by the thermosensitivity of the enzyme activity of thermosensitive revertants. The DLD gene was cloned by complementation for growth on D-, L-lactate in the strain WWF18-3D, carrying both a CYB2 disruption and the dld mutation. The minimal complete complementing sequence was localized by subcloning experiments. From the sequence analysis an open reading frame (ORF) was identified that could encode a polypeptide of 576 amino-acids, corresponding to a calculated molecular weight of 64000 Da. The deduced protein sequence showed significant homology with the previously described microsomal flavoprotein L-gulono-gamma-lactone oxidase isolated from Rattus norvegicus, which catalyses the terminal step of L-ascorbic acid biosynthesis. These results are discussed together with the role of L-LCR and D-LCR in lactate metabolism of S. cerevisiae.
Assuntos
Genes Fúngicos , L-Lactato Desidrogenase/genética , Lactato Desidrogenases , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Citocromos/biossíntese , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Mutação , Plasmídeos , Ratos , Saccharomyces cerevisiae/genéticaRESUMO
The codon bias index (CBI) of several genes of Kluyveromyces lactis was calculated and compared with corresponding data from Saccharomyces cerevisiae. Genes encoding cytoplasmic as well as mitochondrial proteins were analyzed. The CBI of K. lactis and S. cerevisiae genes are similar for the majority of the cases considered with the exception of genes encoding mitochondrial proteins which display higher CBI values in K. lactis, indicating a higher level of gene expression. This could be related to the key role played by mitochondria in this yeast.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Kluyveromyces/genética , Mitocôndrias/química , Saccharomyces cerevisiae/genética , Códon/genética , Homologia de Sequência , Especificidade da EspécieRESUMO
Catabolite repression by galactose was investigated in several strains of Saccharomyces cerevisiae grown on different carbon sources. Galactose repressed as much as glucose; raffinose was less effective. Full derepression was achieved with lactate. The functions tested were L-lactate ferricytochrome c oxidoreductase, NAD-glutamate dehydrogenase, and respiration. Galactose repression was observed only in the GAL4 but not in the gal4 strain. The presence of multiple copies of the GAL4 gene enhanced the repression by galactose. Different alleles of the GAL4 gene and the copy number did not affect glucose repression.