Expressão dos genes da via mTORC1 e seu envolvimento nas Neoplasias Mieloproliferativas

As Neoplasias Mieloproliferativas (NMPs) se caracterizam por apresentarem acúmulo de eritrócitos, leucócitos e plaquetas morfologicamente normais e seus precursores. Nos últimos anos vários estudos buscaram conhecer os mecanismos celulares e moleculares envolvidos na fisiopatologia e evolução dessas desordens, com o intuito de encontrar marcadores de diagnóstico, prognóstico e terapias eficazes. A mutação pontual no gene que codifica a enzima Janus Kinase 2 (JAK2 V617F), presente em aproximadamente 90% dos pacientes com PV e em 50% dos pacientes com TE e MF, foi o principal achado genético anormal associado a essas doenças. Essa mutação resulta na ativação constitutiva da enzima JAK2 e na desregulação da proliferação celular e resistência à apoptose. Nosso grupo de pesquisa descreveu em PV, TE e MF a expressão alterada de genes reguladores da apoptose e dados da literatura indicam que a desregulação do ciclo celular contribui para a fisiopatologia das NMPs. Nesse projeto o intuito foi investigar a associação da via de sinalização m-TOR com as alterações do ciclo celular e via JAK/STAT nas NMPs. A via de sinalização m-TOR participa dos processos celulares de sobrevivência e proliferação. A estratégia experimental foi avaliar a expressão de genes e proteínas, reguladores da via m-TOR, em leucócitos de pacientes com NPMC e linhagens celulares JAK2+ tratadas com inibidores de JAK2 e AKT. Para determinar a relação da via m-TOR nas NMPs foi escolhido o gene eIF4E, alterado nessas doenças, para observar sua modulação diante da inibição farmacológica nas linhagens celulares JAK2 positivas. Os resultados desse estudo contribuem para a descrição de novos alvos terapêuticos dependentes e indepentendes da atividade quinase JAK2 e para o melhor conhecimento da participação da via de sinalização m-TOR na fisiopatologia das NMPs.

Identification of differentially expressed genes in a Giardia lamblia WB C6 clone resistant to nitazoxanide and metronidazole.

OBJECTIVES The characterization of differential gene expression in Giardia lamblia WB C6 strain C4 resistant to metronidazole and nitazoxanide using microarray technology and quantitative real-time PCR. METHODS In a previous study, we created and characterized the G. lamblia WB C6 clone C4 resistant to nitazoxanide and metronidazole. In this study, using a microarray-based approach, we have identified open-reading frames (ORFs) that were differentially expressed in C4 when compared with its wild-type WB C6. Using quantitative real-time PCR, we have validated the expression patterns of some of those ORFs, focusing on chaperones such as heat-shock proteins in wild-type and C4 trophozoites. In order to induce an antigenic shift, trophozoites of both strains were subjected to a cycle of en- and excystation. Expression of selected genes and resistance to nitazoxanide and metronidazole were investigated after this cycle. RESULTS Forty of a total of 9115 ORFs were found to be up-regulated and 46 to be down-regulated in C4 when compared with wild-type. After a cycle of en- and excystation, resistance of C4 to nitazoxanide and metronidazole was lost. Resistance formation and en-/excystation were correlated with changes in expression of ORFs encoding for major surface antigens such as the variant surface protein TSA417 or AS7 ('antigenic shift'). Moreover, expression patterns of the cytosolic heat-shock protein HSP70 B2, HSP40, and of the previously identified nitazoxanide-binding proteins nitroreductase and protein disulphide isomerase PDI4 were correlated with resistance and loss of resistance after en-/excystation. C4 trophozoites had a higher thermotolerance level than wild-type trophozoites. After en-/excystation, this tolerance was lost. CONCLUSIONS These results suggest that resistance formation in Giardia to nitazoxanide and metronidazole is correlated with altered expression of genes involved in stress response such as heat-shock proteins.

Community-acquired methicillin-resistant Staphylococcus aureus carrying Panton-Valentine leukocidin genes: Worldwide emergence

Infections caused by community-acquired (CA)-methicillin-resistant Staphylococcus aureus (MRSA) have been reported worldwide. We assessed whether any common genetic markers existed among 117 CA-MRSA isolates from the United States, France, Switzerland, Australia, New Zealand, and Western Samoa by performing polymerase chain reaction for 24 virulence factors and the methicillin-resistance determinant. The genetic background of the strain was analyzed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The CA-MRSA strains shared a type IV SCCmec cassette and the Panton-Valentine leukocidin locus, whereas the distribution of the other toxin genes was quite specific to the strains from each continent. PFGE and MLST analysis indicated distinct genetic backgrounds associated with each geographic origin, although predominantly restricted to the agr3 background. Within each continent, the genetic background of CA-MRSA strains did not correspond to that of the hospital-acquired MRSA.

Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis

The plant hormones abscisic acid (ABA), jasmonic acid (JA), and ethylene are involved in diverse plant processes, including the regulation of gene expression during adaptive responses to abiotic and biotic stresses. Previously, ABA has been implicated in enhancing disease susceptibility in various plant species, but currently very little is known about the molecular mechanisms underlying this phenomenon. In this study, we obtained evidence that a complex interplay between ABA and JA-ethylene signaling pathways regulate plant defense gene expression and disease resistance. First, we showed that exogenous ABA suppressed both basal and JA-ethylene-activated transcription from defense genes. By contrast, ABA deficiency as conditioned by the mutations in the ABA1 and ABA2 genes, which encode enzymes involved in ABA biosynthesis, resulted in upregulation of basal and induced transcription from JA-ethylene responsive defense genes. Second, we found that disruption of AtMYC2 (allelic to JASMONATE INSENSITIVE1 [JIN1]), encoding a basic helix-loop-helix Leu zipper transcription factor, which is a positive regulator of ABA signaling, results in elevated levels of basal and activated transcription from JA-ethylene responsive defense genes. Furthermore, the jin1/myc2 and aba2-1 mutants showed increased resistance to the necrotrophic fungal pathogen Fusarium oxysporum. Finally, using ethylene and ABA signaling mutants, we showed that interaction between ABA and ethylene signaling is mutually antagonistic in vegetative tissues. Collectively, our results indicate that the antagonistic interactions between multiple components of ABA and the JA-ethylene signaling pathways modulate defense and stress responsive gene expression in response to biotic and abiotic stresses.

Factors that influence the prevalence of acaricide resistance and tick-borne diseases

This manuscript provides a summary of the results presented at a symposium organized to accumulate information on factors that influence the prevalence of acaricide resistance and tick-borne diseases. This symposium was part of the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP), held in New Orleans, LA, USA, during August 10-14, 2003. Populations of southern cattle ticks, Boophilus microplus, from Mexico have developed resistance to many classes of acaricide including chlorinated hydrocarbons (DDT), pyrethroids, organ ophosphates, and formamidines (amitraz). Target site mutations are the most common resistance mechanism observed, but there are examples of metabolic mechanisms. In many pyrethroid resistant strains, a single target site mutation on the Na+ channel confers very high resistance (resistance ratios: >1000x) to both DDT and all pyrethroid acaricides. Acetylcholine esterase affinity for OPs is changed in resistant tick populations. A second mechanism of OP resistance is linked to cytochrome P450 monooxygenase activity. A PCR-based assay to detect a specific sodium channel gene mutation that is associated with resistance to permethrin has been developed. This assay can be performed on individual ticks at any life stage with results available in a few hours. A number of Mexican strains of B. microplus with varying profiles of pesticide resistance have been genotyped using this test. Additionally, a specific metabolic esterase with permethrin-hydrolyzing activity, CzEst9, has been purified and its gene coding region cloned. This esterase has been associated with high resistance to permethrin in one Mexican tick population. Work is continuing to clone specific acetylcholinesterase (AChE) and carboxylesterase genes that appear to be involved in resistance to organophosphates. Our ultimate goal is the design of a battery of DNA- or ELISA-based assays capable of rapidly genotyping individual ticks to obtain a comprehensive profile of their susceptibility to various pesticides. More outbreaks of clinical bovine babesisois and anaplasmosis have been associated with the presence of synthetic pyrethroid (SP) resistance when compared to OP and amidine resistance. This may be the result of differences in the temporal and geographic patterns of resistance development to the different acaricides. If acaricide resistance develops slowly, herd immunity may not be affected. The use of pesticides for the control of pests of cattle other than ticks can affect the incidence of tick resistance and tick-borne diseases. Simple analytical models of tick- and tsetse-bome diseases suggest that reducing the abundance of ticks, by treating cattle with pyrethroids for example, can have a variety of effects on tick-bome diseases. In the worst-case scenario, the models suggest that treating cattle might not only have no impact on trypanosomosis but could increase the incidence of tick-bome disease. In the best-case, treatment could reduce the incidence of both trypanosomosis and tick-bome diseases Surveys of beef and dairy properties in Queensland for which tick resistance to amitraz was known were intended to provide a clear understanding of the economic and management consequences resistance had on their properties. Farmers continued to use amitraz as the major acaricide for tick control after the diagnosis of resistance, although it was supplemented with moxidectin (dairy farms) or fluazuron, macrocyclic lactones or cypermethrin/ chlorfenvinphos. (C) 2004 Published by Elsevier B.V.

Sulfadoxine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site

Sulfadoxine is predominantly used in combination with pyrimethamine, commonly known as Fansidar, for the treatment of Plasmodium falciparum. This combination is usually less effective against Plasmodium vivax, probably due to the innate refractoriness of parasites to the sulfadoxine component. To investigate this mechanism of resistance by P. vivax to sulfadoxine, we cloned and sequenced the P. vivax dhps (pvdhps) gene. The protein sequence was determined, and three-dimensional homology models of dihydropteroate synthase (DHPS) from P. vivax as well as P. falciparum were created. The docking of sulfadoxine to the two DHPS models allowed us to compare contact residues in the putative sulfadoxine-binding site in both species. The predicted sulfadoxine-binding sites between the species differ by one residue, V585 in P. vivax, equivalent to A613 in P. falciparum. V585 in P. vivax is predicted by energy minimization to cause a reduction in binding of sulfadoxine to DHPS in P. vivax compared to P. falciparum. Sequencing dhps genes from a limited set of geographically different P. vivax isolates revealed that V585 was present in all of the samples, suggesting that V585 may be responsible for innate resistance of P. vivax to sulfadoxine. Additionally, amino acid mutations were observed in some P. vivax isolates in positions known to cause resistance in P. falciparum, suggesting that, as in P. falciparum, these mutations are responsible for acquired increases in resistance of P. vivax to sulfadoxine.

Evolution of resistance to sulfadoxine-pyrimethamine in Plasmodium falciparum

The development of resistance to sulfadoxine-pyrimethamine by Plasmodium parasites is a major problem for the effective treatment of malaria, especially P. falciparum malaria. Although the molecular basis for parasite resistance is known, the factors promoting the development and transmission of these resistant parasites are less clear. This paper reports the results of a quantitative comparison of factors previously hypothesized as important for the development of drug resistance, drug dosage, time of treatment, and drug elimination half-life, with an in-host dynamics model of P. falciparum malaria in a malaria-naive host. The results indicate that the development of drug resistance can be categorized into three stages. The first is the selection of existing parasites with genetic mutations in the dihydrofolate reductase or dihydropteroate synthetase gene. This selection is driven by the long half-life of the sulfadoxine-pyrimethamine combination. The second stage involves the selection of parasites with allelic types of higher resistance within the host during an infection. The timing of treatment relative to initiation of a specific anti-P. falciparum EMP1 immune response is an important factor during this stage, as is the treatment dosage. During the third stage, clinical treatment failure becomes prevalent as the parasites develop sufficient resistance mutations to survive therapeutic doses of the drug combination. Therefore, the model output reaffirms the importance of correct treatment of confirmed malaria cases in slowing the development of parasite resistance to sulfadoxine-pyrimethamine.

Repressor- and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression

To identify transcription factors (TFs) involved in jasmonate (JA) signaling and plant defense, we screened 1,534 Arabidopsis (Arabidopsis thaliana) TFs by real-time quantitative reverse transcription-PCR for their altered transcript at 6 h following either methyl JA treatment or inoculation with the incompatible pathogen Alternaria brassicicola. We identified 134 TFs that showed a significant change in expression, including many APETALA2/ethylene response factor (AP2/ERF), MYB, WRKY, and NACTF genes with unknown functions. Twenty TF genes were induced by both the pathogen and methyl JA and these included 10 members of the AP2/ERF TF family, primarily from the B1a and B3 subclusters. Functional analysis of the B1a TF AtERF4 revealed that AtERF4 acts as a novel negative regulator of JA-responsive defense gene expression and resistance to the necrotrophic fungal pathogen Fusarium oxysporum and antagonizes JA inhibition of root elongation. In contrast, functional analysis of the B3 TF AtERF2 showed that AtERF2 is a positive regulator of JA-responsive defense genes and resistance to F. oxysporum and enhances JA inhibition of root elongation. Our results suggest that plants coordinately express multiple repressor-and activator-type AP2/ERFs during pathogen challenge to modulate defense gene expression and disease resistance.

Identification of three gene candidates for multicellular resistance in colon carcinoma

Solid tumours display elevated resistance to chemo- and radiotherapies compared to individual tumour derived cells. This so-called multicellular resistance (MCR) phenomenon can only be partly explained by reduced diffusion and altered cell cycle status; even fast growing cells on the surface of solid tumours display MCR. Multicellular spheroids (MCS) recapture this phenomenon ex vivo and here we compare gene expression in exponentially growing MCS with gene expression in monolayer culture. Using an 18,664 gene microarray, we identified 42 differentially expressed genes and three of these genes can be linked to potential mechanisms of MCR. A group of interferon response genes were also up-regulated in MCS, as were a number of genes that that are indicative of greater differentiation in three-dimensional cultures.

Heterotrimeric G proteins facilitate Arabidopsis resistance to necrotrophic pathogens and are involved in jasmonate signaling

Heterotrimeric G proteinshave been previously linked to plant defense; however a role for the G beta gamma dimer in defense signaling has not been described to date. Using available Arabidopsis (Arabidopsis thaliana) mutants lacking functional G alpha or G beta subunits, we show that defense against the necrotrophic pathogens Alternaria brassicicola and Fusarium oxysporum is impaired in G beta- deficient mutants while G alpha-deficient mutants show slightly increased resistance compared to wild-type Columbia ecotype plants. In contrast, responses to virulent (DC3000) and avirulent (JL1065) strains of Pseudomonas syringae appear to be independent of heterotrimeric G proteins. The induction of a number of defense-related genes in G beta-deficient mutants were severely reduced in response to A. brassicicola infection. In addition, G beta-deficient mutants exhibit decreased sensitivity to a number of methyl jasmonate- induced responses such as induction of the plant defensin gene PDF1.2, inhibition of root elongation, seed germination, and growth of plants in sublethal concentrations of methyl jasmonate. In all cases, the behavior of the G alpha- deficient mutants is coherent with the classic heterotrimeric mechanism of action, indicating that jasmonic acid signaling is influenced by the Gbg functional subunit but not by G alpha. We hypothesize that G beta gamma acts as a direct or indirect enhancer of the jasmonate signaling pathway in plants.

Salicylic acid mediates resistance to the vascular wilt pathogen Fusarium oxysporum in the model host Arabidopsis thaliana

Fusarium oxysporum is a soilborne fungal pathogen that causes major economic losses by inducing necrosis and wilting symptoms in many crop plants. In this study, the interaction between F. oxysporum and the model plant Arabidopsis thaliana has been investigated to better understand the nature of host defences that are effective against the Fusarium wilt pathogen. The expression of salicylate- and jasmonate-responsive defence genes in F. oxysporum-challenged roots of A. thaliana plants as well as in the roots of plants whose leaves were treated with salicylate or jasmonate was analysed. Unexpectedly, genes (e.g. PR1, PDF1.2, and CHIB) encoding proteins with defensive functions or transcription factors (e.g. ERF1, AtERF2, AtERF4 and AtMYC2) known to positively or negatively regulate defences against F. oxysporum were not activated in F. oxysporum-inoculated roots. In contrast, the jasmonate-responsive defence gene PDF1.2 was induced in the leaves of plants whose roots were challenged with F. oxysporum, but the salicylate- responsive PR1 gene was not induced in the leaves of inoculated plants. Exogenous salicylic acid treatment prior to inoculation, however, activated PR1 and BGL2 defence gene expression in leaves and provided increased F. oxysporum resistance as evidenced by reduced foliar necrosis and plant death. Exogenous salicylic acid treatment of the foliar tissue did not activate defence gene expression in the roots of plants. This suggests that salicylate- dependent defences may function in foliar tissue to reduce the development of pathogen-induced wilting and necrosis. Despite the induction of defence gene expression in the leaves by jasmonate, this treatment did not lead to increased resistance to F. oxysporum. Overall, the results presented here suggest that the genetic manipulation of plant defence signalling pathways is a useful strategy to provide increased Fusarium wilt resistance.

A DHA14 drug efflux gene from Xanthomonas albilineans confers high-level albicidin antibiotic resistance in Escherichia coli

Aims: Identification of a gene for self-protection from the antibiotic-producing plant pathogen Xanthomonas albilineans, and functional testing by heterologous expression. Methods and Results: Albicidin antibiotics and phytotoxins are potent inhibitors of prokaryote DNA replication. A resistance gene (albF) isolated by shotgun cloning from the X. albilineans albicidin-biosynthesis region encodes a protein with typical features of DHA14 drug efflux pumps. Low-level expression of albF in Escherichia coli increased the MIC of albicidin 3000-fold, without affecting tsx-mediated albicidin uptake into the periplasm or resistance to other tested antibiotics. Bioinformatic analysis indicates more similarity to proteins involved in self-protection in polyketide-antibiotic-producing actinomycetes than to multi-drug resistance pumps in other Gram-negative bacteria. A complex promoter region may co-regulate albF with genes for hydrolases likely to be involved in albicidin activation or self-protection. Conclusions: AlbF is the first apparent single-component antibiotic-specific efflux pump from a Gram-negative antibiotic producer. It shows extraordinary efficiency as measured by resistance level conferred upon heterologous expression. Significance and Impact of the Study: Development of the clinical potential of albicidins as potent bactericidial antibiotics against diverse bacteria has been limited because of low yields in culture. Expression of albF with recently described albicidin-biosynthesis genes may enable large-scale production. Because albicidins are X. albilineans pathogenicity factors, interference with AlbF function is also an opportunity for control of the associated plant disease.

Journey of net blotch: From pathotype diversity to useful resistance in barley

Studies on variation, occurrence and distribution of virulence in Pyrenophora teres f. teres are essential to identify effective sources of resistance for net type net blotch. Disease surveys suggested two different stains are prevalent in Western Australia and 13 in all around Australia. Sixty nine barley lines from different breeding groups in Australia and elsewhere were tested against most prevalent pathotypes. Majority of lines have partial to complete resistance while some have elite resistances to net type net blotch. Four lines out of 69 were chosen for further studies. These four lines: WA 4794 (103 IBON 91), Pompadour, CI 9214, and WPG 8412-9-2-1 were highly diverse and resistant to most of the isolates, and were crossed with Stirling-a highly adaptive but susceptible cultivar. Doubled haploids, F2s, and resistant x resistant crosses were studied against five prevalent isolates. Four genes from WA 4794 (all dominant), three (two dominant and one recessive) from Pompadour, five (two dominant and three recessive) from CI 9214, and two (one dominant and one recessive) from WPG 8412-9-2-1 were identified. In total, 11 different genes were operative against P. teres f. teres isolates. Molecular work is initiated to develop markers which would aid screening of the breeding populations for these resistances.