Acanthamoeba polyphaga trophozoite binding of representative fungal single cell forms

Acanthamoeba polyphaga trophozoites bind yeast cells of Candida albicans isolates within a few hours, leaving few cells in suspension or still attached to trophozoite surfaces. The nature of yeast cell recognition, mediated by an acanthamoebal trophozoite mannose binding protein is confirmed by experiments utilizing concentration dependent mannose hapten blocking. Similarly, acapsulate cells of Cryptococcus neoformans are also bound within a relatively short timescale. However, even after protracted incubation many capsulate cells of Cryptococcus remain in suspension, suggesting that the capsulate cell form of this species is not predated by acanthamoebal trophozoites. Further aspects of the association of Acanthamoeba and fungi are apparent when studying their interaction with conidia of the biocontrol agent Coniothyrium minitans. Conidia which readily bind with increasing maturity of up to 42 days, were little endocytosed and even released. Cell and conidial surface mannose as determined by FITC-lectin binding, flow cytometry with associated ligand binding analysis and hapten blocking studies demonstrates the following phenomena. Candida isolates and acapsulate Cryptococcus expose most mannose, while capsulate Cryptococcus cells exhibit least exposure commensurate with yeast cellular binding or lack of trophozoites. Conidia of Coniothyrium, albeit in a localized fashion, also manifest surface mannose exposure but as shown by Bmax values, in decreasing amounts with increasing maturity. Contrastingly such conidia experience greater trophozoite binding with maturation, thereby questioning the primacy of a trophozoite mannose-binding-protein recognition model.

Design, synthesis and RAFT polymerisation of a quinoline-based monomer for use in metal-binding composite microfibres

Metal-binding polymer fibres have attracted major attention for diverse applications in membranes for metal sequestration from waste waters, non-woven wound dressings, matrices for photocatalysis, and many more. This paper reports the design and synthesis of an 8-hydroxyquinoline-based zinc-binding styrenic monomer, QuiBoc. Its subsequent polymerisation by reversible addition–fragmentation chain transfer (RAFT) yielded well-defined polymers, PQuiBoc, of controllable molar masses (6 and 12 kg mol−1) with low dispersities (Đ, Mw/Mn < 1.3). Protected (PQuiBoc) and deprotected (PQuiOH) derivatives of the polymer exhibited a high zinc-binding capacity, as determined by semi-quantitative SEM/EDXA analyses, allowing the electrospinning of microfibres from a PQuiBoc/polystyrene (PS) blend without the need for removal of the protecting group. Simple “dip-coating” of the fibrous mats into ZnO suspensions showed that PQuiBoc/PS microfibres with only 20% PQuiBoc content had almost three-fold higher loadings of ZnO (29%) in comparison to neat PS microfibres (11%).

EnDNA-Prot:identification of DNA-binding proteins by applying ensemble learning

DNA-binding proteins are crucial for various cellular processes, such as recognition of specific nucleotide, regulation of transcription, and regulation of gene expression. Developing an effective model for identifying DNA-binding proteins is an urgent research problem. Up to now, many methods have been proposed, but most of them focus on only one classifier and cannot make full use of the large number of negative samples to improve predicting performance. This study proposed a predictor called enDNA-Prot for DNA-binding protein identification by employing the ensemble learning technique. Experiential results showed that enDNA-Prot was comparable with DNA-Prot and outperformed DNAbinder and iDNA-Prot with performance improvement in the range of 3.97-9.52% in ACC and 0.08-0.19 in MCC. Furthermore, when the benchmark dataset was expanded with negative samples, the performance of enDNA-Prot outperformed the three existing methods by 2.83-16.63% in terms of ACC and 0.02-0.16 in terms of MCC. It indicated that enDNA-Prot is an effective method for DNA-binding protein identification and expanding training dataset with negative samples can improve its performance. For the convenience of the vast majority of experimental scientists, we developed a user-friendly web-server for enDNA-Prot which is freely accessible to the public. © 2014 Ruifeng Xu et al.

Identifying DNA-binding proteins by combining support vector machine and PSSM distance transformation

Background: DNA-binding proteins play a pivotal role in various intra- and extra-cellular activities ranging from DNA replication to gene expression control. Identification of DNA-binding proteins is one of the major challenges in the field of genome annotation. There have been several computational methods proposed in the literature to deal with the DNA-binding protein identification. However, most of them can't provide an invaluable knowledge base for our understanding of DNA-protein interactions. Results: We firstly presented a new protein sequence encoding method called PSSM Distance Transformation, and then constructed a DNA-binding protein identification method (SVM-PSSM-DT) by combining PSSM Distance Transformation with support vector machine (SVM). First, the PSSM profiles are generated by using the PSI-BLAST program to search the non-redundant (NR) database. Next, the PSSM profiles are transformed into uniform numeric representations appropriately by distance transformation scheme. Lastly, the resulting uniform numeric representations are inputted into a SVM classifier for prediction. Thus whether a sequence can bind to DNA or not can be determined. In benchmark test on 525 DNA-binding and 550 non DNA-binding proteins using jackknife validation, the present model achieved an ACC of 79.96%, MCC of 0.622 and AUC of 86.50%. This performance is considerably better than most of the existing state-of-the-art predictive methods. When tested on a recently constructed independent dataset PDB186, SVM-PSSM-DT also achieved the best performance with ACC of 80.00%, MCC of 0.647 and AUC of 87.40%, and outperformed some existing state-of-the-art methods. Conclusions: The experiment results demonstrate that PSSM Distance Transformation is an available protein sequence encoding method and SVM-PSSM-DT is a useful tool for identifying the DNA-binding proteins. A user-friendly web-server of SVM-PSSM-DT was constructed, which is freely accessible to the public at the web-site on http://bioinformatics.hitsz.edu.cn/PSSM-DT/.

Structural and Functional Analysis of the Caspase –dependent and –independent Domains of the X-linked Inhibitor of Apoptosis Protein in Inflammatory Breast Cancer Tumor Biology

Inflammatory breast cancer (IBC) is an extremely rare but highly aggressive form of breast cancer characterized by the rapid development of therapeutic resistance leading to particularly poor survival. Our previous work focused on the elucidation of factors that mediate therapeutic resistance in IBC and identified increased expression of the anti-apoptotic protein, X-linked inhibitor of apoptosis protein (XIAP), to correlate with the development of resistance to chemotherapeutics. Although XIAP is classically thought of as an inhibitor of caspase activation, multiple studies have revealed that XIAP can also function as a signaling intermediate in numerous pathways. Based on preliminary evidence revealing high expression of XIAP in pre-treatment IBC cells rather than only subsequent to the development of resistance, we hypothesized that XIAP could play an important signaling role in IBC pathobiology outside of its heavily published apoptotic inhibition function. Further, based on our discovery of inhibition of chemotherapeutic efficacy, we postulated that XIAP overexpression might also play a role in resistance to other forms of therapy, such as immunotherapy. Finally, we posited that targeting of specific redox adaptive mechanisms, which are observed to be a significant barrier to successful treatment of IBC, could overcome therapeutic resistance and enhance the efficacy of chemo-, radio-, and immuno- therapies. To address these hypotheses our objectives were: 1. to determine a role for XIAP in IBC pathobiology and to elucidate the upstream regulators and downstream effectors of XIAP; 2. to evaluate and describe a role for XIAP in the inhibition of immunotherapy; and 3. to develop and characterize novel redox modulatory strategies that target identified mechanisms to prevent or reverse therapeutic resistance.

Using various genomic and proteomic approaches, combined with analysis of cellular viability, proliferation, and growth parameters both in vitro and in vivo, we demonstrate that XIAP plays a central role in both IBC pathobiology in a manner mostly independent of its role as a caspase-binding protein. Modulation of XIAP expression in cells derived from patients prior to any therapeutic intervention significantly altered key aspects IBC biology including, but not limited to: IBC-specific gene signatures; the tumorigenic capacity of tumor cells; and the metastatic phenotype of IBC, all of which are revealed to functionally hinge on XIAP-mediated NFκB activation, a robust molecular determinant of IBC. Identification of the mechanism of XIAP-mediated NFκB activation led to the characterization of novel peptide-based antagonist which was further used to identify that increased NFκB activation was responsible for redox adaptation previously observed in therapy-resistant IBC cells. Lastly, we describe the targeting of this XIAP-NFκB-ROS axis using a novel redox modulatory strategy both in vitro and in vivo. Together, the data presented here characterize a novel and crucial role for XIAP both in therapeutic resistance and the pathobiology of IBC; these results confirm our previous work in acquired therapeutic resistance and establish the feasibility of targeting XIAP-NFκB and the redox adaptive phenotype of IBC as a means to enhance survival of patients.

Characterization of a Full-Length TTP Family Member Association with RNA Sequence Elements

Post-transcriptional regulation of cytoplasmic mRNAs is an efficient mechanism of regulating the amounts of active protein within a eukaryotic cell. RNA sequence elements located in the untranslated regions of mRNAs can influence transcript degradation or translation through associations with RNA-binding proteins. Tristetraprolin (TTP) is the best known member of a family of CCCH zinc finger proteins that targets adenosine-uridine rich element (ARE) binding sites in the 3’ untranslated regions (UTRs) of mRNAs, promoting transcript deadenylation through the recruitment of deadenylases. More specifically, TTP has been shown to bind AREs located in the 3’-UTRs of transcripts with known roles in the inflammatory response. The mRNA-binding region of the protein is the highly conserved CCCH tandem zinc finger (TZF) domain. The synthetic TTP TZF domain has been shown to bind with high affinity to the 13-mer sequence of UUUUAUUUAUUUU. However, the binding affinities of full-length TTP family members to the same sequence and its variants are unknown. Furthermore, the distance needed between two overlapping or neighboring UUAUUUAUU 9-mers for tandem binding events of a full-length TTP family member to a target transcript has not been explored. To address these questions, we recombinantly expressed and purified the full-length C. albicans TTP family member Zfs1. Using full-length Zfs1, tagged at the N-terminus with maltose binding protein (MBP), we determined the binding affinities of the protein to the optimal TTP binding sequence, UUAUUUAUU. Fluorescence anisotropy experiments determined that the binding affinities of MBP-Zfs1 to non-canonical AREs were influenced by ionic buffer strength, suggesting that transcript selectivity may be affected by intracellular conditions. Furthermore, electrophoretic mobility shift assays (EMSAs) revealed that separation of two core AUUUA sequences by two uridines is sufficient for tandem binding of MBP-Zfs1. Finally, we found evidence for tandem binding of MBP-Zfs1 to a 27-base RNA oligonucleotide containing only a single ARE-binding site, and showed that this was concentration and RNA length dependent; this phenomenon had not been seen previously. These data suggest that the association of the TTP TZF domain and the TZF domains of other species, to ARE-binding sites is highly conserved. Domains outside of the TZF domain may mediate transcript selectivity in changing cellular conditions, and promote protein-RNA interactions not associated with the ARE-binding TZF domain.

In summary, the evidence presented here suggests that Zfs1-mediated decay of mRNA targets may require additional interactions, in addition to ARE-TZF domain associations, to promote transcript destabilization and degradation. These studies further our understanding of post-transcriptional steps in gene regulation.

C-terminal residues of skeletal muscle calsequestrin are essential for calcium binding and for skeletal ryanodine receptor inhibition

AUXIN-BINDING-PROTEIN1 (ABP1) in phytochrome-B-controlled responses

The auxin receptor ABP1 directly regulates plasma membrane activities including the number of PIN-formed (PIN) proteins and auxin efflux transport. Red light (R) mediated by phytochromes regulates the steady-state level of ABP1 and auxin-inducible growth capacity in etiolated tissues but, until now, there has been no genetic proof that ABP1 and phytochrome regulation of elongation share a common mechanism for organ elongation. In far red (FR)-enriched light, hypocotyl lengths were larger in the abp1-5 and abp1/ABP1 mutants, but not in tir1-1, a null mutant of the TRANSPORT-INHIBITOR-RESPONSE1 auxin receptor. The polar auxin transport inhibitor naphthylphthalamic acid (NPA) decreased elongation in the low R: FR light-enriched white light (WL) condition more strongly than in the high red: FR light-enriched condition WL suggesting that auxin transport is an important condition for FR-induced elongation. The addition of NPA to hypocotyls grown in R-and FR-enriched light inhibited hypocotyl gravitropism to a greater extent in both abp1 mutants and in phyB-9 and phyA-211 than the wild-type hypocotyl, arguing for decreased phytochrome action in conjunction with auxin transport in abp1 mutants. Transcription of FR-enriched light-induced genes, including several genes regulated by auxin and shade, was reduced 3-5-fold in abp1-5 compared with Col and was very low in abp1/ABP1. In the phyB-9 mutant the expression of these reporter genes was 5-15-fold lower than in Col. In tir1-1 and the phyA-211 mutants shade-induced gene expression was greatly attenuated. Thus, ABP1 directly or indirectly participates in auxin and light signalling.

Protein quality and distribution determines anabolic signaling, cellular energetics, and body composition

Building and maintaining muscle is critical to the quality of life for adults and elderly. Physical activity and nutrition are important factors for long-term muscle health. In particular, dietary protein – including protein distribution and quality – are under-appreciated determinants of muscle health for adults. The most unequivocal evidence for the benefit of optimal dietary protein at individual meals is derived from studies of weight management. During the catabolic condition of weight loss, higher protein diets attenuate loss of lean tissue and partition weight loss to body fat when compared with commonly recommended high carbohydrate, low protein diets. Muscle protein turnover is a continuous process in which proteins are degraded, and replaced by newly synthesized proteins. Muscle growth occurs when protein synthesis exceeds protein degradation. Regulation of protein synthesis is complex, with multiple signals influencing this process. The mammalian target of rapamycin (mTORC1) pathway has been identified as a particularly important regulator of protein synthesis, via stimulation of translation initiation. Key regulatory points of translation initiation effected by mTORC1 include assembly of the eukaryotic initiation factor 4F (eIF4F) complex and phosphorylation of the 70 kilodalton ribosomal protein S6 kinase (S6K1). Assembly of the eIF4F initiation complex involves phosphorylation of the inhibitory eIF4E binding protein-1 (4E-BP1), which releases the initiation factor eIF4E and allows it to bind with eIF4G. Binding of eIF4E with eIF4G promotes preparation of the mRNA for binding to the 43S pre-initiation complex. Consumption of the amino acid leucine (Leu) is a key factor determining the anabolic response of muscle protein synthesis (MPS) and mTORC1 signaling to a meal. Research from this dissertation demonstrates that the peak activation of MPS following a complete meal is proportional to the Leu content of a meal and its ability to elevate plasma Leu. Leu has also been implicated as an inhibitor of muscle protein degradation (MPD). In particular, there is evidence suggesting that in muscle wasting conditions Leu supplementation attenuates expression of the ubiquitin-proteosome pathway, which is the primary mode of intracellular protein degradation. However, this is untested in healthy, physiological feeding models. Therefore, an experiment was performed to see if feeding isonitrogenous protein sources with different Leu contents to healthy adult rats would differentially impact ubiquitin-proteosome (protein degradation) outcomes; and if these outcomes are related to the meal responses of plasma Leu. Results showed that higher Leu diets were able to attenuate total proteasome content but had no effect on ubiquitin proteins. This research shows that dietary Leu determines postprandial muscle anabolism. In a parallel line of research, the effects of dietary Leu on changes in muscle mass overtime were investigated. Animals consuming higher Leu diets had larger gastrocnemius muscle weights; furthermore, gastrocnemius muscle weights were correlated with postprandial changes in MPS (r=0.471, P<0.01) and plasma Leu (r=0.400, P=0.01). These results show that the effect of Leu on ubiquitin-proteosome pathways is minimal for healthy adult rats consuming adequate diets. Thus, long-term changes in muscle mass observed in adult rats are likely due to the differences in MPS, rather than MPD. Factors determining the duration of Leu-stimulated MPS were further investigated. Despite continued elevations in plasma Leu and associated translation initiation factors (e.g., S6K1 and 4E-BP1), MPS returned to basal levels ~3 hours after a meal. However, administration of additional nutrients in the form of carbohydrate, Leu, or both ~2 hours after a meal was able to extend the elevation of MPS, in a time and dose dependent manner. This effect led to a novel discovery that decreases in translation elongation activity was associated with increases in activity of AMP kinase, a key cellular energy sensor. This research shows that the Leu density of dietary protein determines anabolic signaling, thereby affecting cellular energetics and body composition.

Biochemical studies of postsynaptic density signaling proteins with a focus on synaptic GTPase activating protein and PDZ domains

Memory storage in the brain involves adjustment of the strength of existing synapses and formation of new neural networks. A key process underlying memory formation is synaptic plasticity, the ability of excitatory synapses to strengthen or weaken their connections in response to patterns of activity between their connected neurons. Synaptic plasticity is governed by the precise pattern of Ca²⁺ influx through postsynaptic N-methyl-D-aspartate-type glutamate receptors (NMDARs), which can lead to the activation of the small GTPases Ras and Rap. Differential activation of Ras and Rap acts to modulate synaptic strength by promoting the insertion or removal of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptors (AMPARs) from the synapse. Synaptic GTPase activating protein (synGAP) regulates AMPAR levels by catalyzing the inactivation of GTP-bound (active) Ras or Rap. synGAP is positioned in close proximity to the cytoplasmic tail regions of the NMDAR through its association with the PDZ domains of PSD-95. SynGAP’s activity is regulated by the prominent postsynaptic protein kinase, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5), a known binding partner of CaMKII. Modulation of synGAP’s activity by phosphorylation may alter the ratio of active Ras to Rap in spines, thus pushing the spine towards the insertion or removal of AMPARs, subsequently strengthening or weakening the synapse. To date, all biochemical studies of the regulation of synGAP activity by protein kinases have utilized impure preparations of membrane bound synGAP. Here we have clarified the effects of phosphorylation of synGAP on its Ras and Rap GAP activities by preparing and utilizing purified, soluble recombinant synGAP, Ras, Rap, CaMKII, CDK5, PLK2, and CaM. Using mass spectrometry, we have confirmed the presence of previously identified CaMKII and CDK5 sites in synGAP, and have identified novel sites of phosphorylation by CaMKII, CDK5, and PLK2. We have shown that the net effect of phosphorylation of synGAP by CaMKII, CDK5, and PLK2 is an increase in its GAP activity toward HRas and Rap1. In contrast, there is no effect on its GAP activity toward Rap2. Additionally, by assaying the GAP activity of phosphomimetic synGAP mutants, we have been able to hypothesize the effects of CDK5 phosphorylation at specific sites in synGAP. In the course of this work, we also found, unexpectedly, that synGAP is itself a Ca²⁺/CaM binding protein. While Ca²⁺/CaM binding does not directly affect synGAP activity, it causes a conformational change in synGAP that increases the rate of its phosphorylation and exposes additional phosphorylation sites that are inaccessible in the absence of Ca²⁺/CaM.

The postsynaptic density (PSD) is an electron-dense region in excitatory postsynaptic neurons that contains a high concentration of glutamate receptors, cytoskeletal proteins, and associated signaling enzymes. Within the PSD, three major classes of scaffolding molecules function to organize signaling enzymes and glutamate receptors. PDZ domains present in the Shank and PSD-95 scaffolds families serve to physically link AMPARs and NMDARs to signaling molecules in the PSD. Because of the specificity and high affinity of PDZ domains for their ligands, I reasoned that these interacting pairs could provide the core components of an affinity chromatography system, including affinity resins, affinity tags, and elution agents. I show that affinity columns containing the PDZ domains of PSD-95 can be used to purify active PDZ domain-binding proteins to very high purity in a single step. Five heterologously expressed neuronal proteins containing endogenous PDZ domain ligands (NMDAR GluN2B subunit Tail, synGAP, neuronal nitric oxide synthase PDZ domain, cysteine rich interactor of PDZ three and cypin) were purified using PDZ domain resin, with synthetic peptides having the sequences of cognate PDZ domain ligands used as elution agents. I also show that conjugation of PDZ domain-related affinity tags to Proteins Of Interest (POIs) that do not contain endogenous PDZ domains or ligands does not alter protein activity and enables purification of the POIs on PDZ domain-related affinity resins.

The bacterial chromosome segregation protein Spo0J spreads along DNA from parS nucleation sites

Regulation of chromosome inheritance is essential to ensure proper transmission of genetic information. To accomplish accurate genome segregation, cells organize their chromosomes and actively separate them prior to cytokinesis. In Bacillus subtilis the Spo0J protein is required for accurate chromosome segregation and it regulates the developmental switch from vegetative growth to sporulation. Spo0J is a DNA-binding protein that recognizes at least eight identified parS sites located near the origin of replication. As judged by fluorescence microscopy, Spo0J forms discrete foci associated with the oriC region of the chromosome throughout the cell cycle. In an attempt to determine the mechanisms utilized by Spo0J to facilitate productive chromosome segregation, we have investigated the DNA binding activity of Spo0J. In vivo we find Spo0J associates with several kilobases of DNA flanking its specific binding sites (parS) through a parS-dependent nucleation event that promotes lateral spreading of Spo0J along the chromosome. Using purified components we find that Spo0J has the ability to coat non-specific DNA substrates. These 'Spo0J domains' provide large structures near oriC that could potentially demark, organize or localize the origin region of the chromosome.

Functional analysis of Arabidopsis thaliana lesion mimic mutant cfs1 in ESCRT complex-related protein trafficking

The Endosomal Sorting Complex Required for Transport (ESCRT)-complex is composed of four complexes, ESCRT-0-III. They sequentially act on a late endosome to sort mono-ubiquitinated transmembrane proteins into the intralumenal vesicle, forming of a multivesicular body(MVB) that is delivered to vacuole for degradation. In Arabidopsis thaliana, the loss of an ESCRT-I component, elch displays a cytokinesis defect; while a dominant negative expression of an ESCRT-III component results in cell death due to vacuolar loss. In this work, the function of a plant-specific ELCH-interactor, CELL DEATH RELATED FYVE/SYLF DOMAIN CONTAINING 1 (CFS1) and its influences on the ESCRT-complex function are investigated. CFS1 is a phosphatidylinositol-3-phosphate- and actin-binding protein. The cfs1 mutants mimic lesions in the first eldest leaf that propagate to the next eldest one. Genetic analyses have demonstrated that cell death in cfs1 does not require a functional ESCRT-I component; nevertheless, the loss of CFS1 alleviates elchcytokinesis defect, suggesting its influence on the ESCRT-I function. Further analyses reveal that cfs1 accumulates autophagosomes throughout its lifespan due to a decrease in autophagosome degradation, suggesting that as the plant ages, the cumulated autophagosomes falsely trigger effectors-triggered immunity that executes cell death in cfs1. As the ESCRT-complex has been demonstrated to be involved in the delivery of autophagosomes to vacuole and CFS1 homolog, CFS2 reportedly interacts with ATG8, it can be postulated from the results of this work that CFS1 alone or together with CFS2 function in sequestering mature autophagosomes onto MVBs. At the MVBs, the ESCRT-complex then mediates the fusion of autophagosome and MVB for subsequent delivery to vacuole.

Galectin-3 Up-regulation In Hypoxic And Nutrient Deprived Microenvironments Promotes Cell Survival.

Galectin-3 (gal-3) is a β-galactoside binding protein related to many tumoral aspects, e.g. angiogenesis, cell growth and motility and resistance to cell death. Evidence has shown its upregulation upon hypoxia, a common feature in solid tumors such as glioblastoma multiformes (GBM). This tumor presents a unique feature described as pseudopalisading cells, which accumulate large amounts of gal-3. Tumor cells far from hypoxic/nutrient deprived areas express little, if any gal-3. Here, we have shown that the hybrid glioma cell line, NG97ht, recapitulates GBM growth forming gal-3 positive pseudopalisades even when cells are grafted subcutaneously in nude mice. In vitro experiments were performed exposing these cells to conditions mimicking tumor areas that display oxygen and nutrient deprivation. Results indicated that gal-3 transcription under hypoxic conditions requires previous protein synthesis and is triggered in a HIF-1α and NF-κB dependent manner. In addition, a significant proportion of cells die only when exposed simultaneously to hypoxia and nutrient deprivation and demonstrate ROS induction. Inhibition of gal-3 expression using siRNA led to protein knockdown followed by a 1.7-2.2 fold increase in cell death. Similar results were also found in a human GBM cell line, T98G. In vivo, U87MG gal-3 knockdown cells inoculated subcutaneously in nude mice demonstrated decreased tumor growth and increased time for tumor engraftment. These results indicate that gal-3 protected cells from cell death under hypoxia and nutrient deprivation in vitro and that gal-3 is a key factor in tumor growth and engraftment in hypoxic and nutrient-deprived microenvironments. Overexpression of gal-3, thus, is part of an adaptive program leading to tumor cell survival under these stressing conditions.

Gene expression profile of the plant pathogen Fusarium graminearum under the antagonistic effect of Pantoea agglomerans

The pathogenic fungus Fusarium graminearum is an ongoing threat to agriculture, causing losses in grain yield and quality in diverse crops. Substantial progress has been made in the identification of genes involved in the suppression of phytopathogens by antagonistic microorganisms; however, limited information regarding responses of plant pathogens to these biocontrol agents is available. Gene expression analysis was used to identify differentially expressed transcripts of the fungal plant pathogen F. graminearum under antagonistic effect of the bacterium Pantoea agglomerans. A macroarray was constructed, using 1014 transcripts from an F. graminearum cDNA library. Probes consisted of the cDNA of F. graminearum grown in the presence and in the absence of P. agglomerans. Twenty-nine genes were either up (19) or down (10) regulated during interaction with the antagonist bacterium. Genes encoding proteins associated with fungal defense and/or virulence or with nutritional and oxidative stress responses were induced. The repressed genes coded for a zinc finger protein associated with cell division, proteins containing cellular signaling domains, respiratory chain proteins, and chaperone-type proteins. These data give molecular and biochemical evidence of response of F. graminearum to an antagonist and could help develop effective biocontrol procedures for pathogenic plant fungi.

Plasmodium vivax circumsporozoite genotypes: a limited variation or new subspecies with major biological consequences?

Background: Plasmodium vivax circumsporozoite variants have been identified in several geographical areas. The real implication of the genetic variation in this region of the P. vivax genome has been questioned for a long time. Although previous studies have observed significant association between VK210 and the Duffy blood group, we present here that evidences of this variation are limited to the CSP central portion. Methods: The phylogenetic analyses were accomplished starting from the amplification of conserved domains of 18 SSU RNAr and Cyt B. The antibodies responses against the CSP peptides, MSP-1, AMA-1 and DBP were detected by ELISA, in plasma samples of individuals infected with two P. vivax CS genotypes: VK210 and P. vivax-like. Results: These analyses of the two markers demonstrate high similarity among the P. vivax CS genotypes and surprisingly showed diversity equal to zero between VK210 and P. vivax-like, positioning these CS genotypes in the same clade. A high frequency IgG antibody against the N- and C-terminal regions of the P. vivax CSP was found as compared to the immune response to the R- and V-repetitive regions (p = 0.0005, Fisher's Exact test). This difference was more pronounced when the P. vivax-like variant was present in the infection (p = 0.003, Fisher's Exact test). A high frequency of antibody response against MSP-1 and AMA-1 peptides was observed for all P. vivax CS genotypes in comparison to the same frequency for DBP. Conclusions: This results target that the differences among the P. vivax CS variants are restrict to the central repeated region of the protein, mostly nucleotide variation with important serological consequences.