Crystal structure of Archaeoglobus fulgidus CTP:inositol-1-phosphate cytidylyltransferase, a key enzyme for di-myo-inositol-phosphate synthesis in (hyper)thermophiles

Many Archaea and Bacteria isolated from hot, marine environments accumulate di-myo-inositol-phosphate (DIP), primarily in response to heat stress. The biosynthesis of this compatible solute involves the activation of inositol to CDP-inositol via the action of a recently discovered CTP:inositol-1-phosphate cytidylyltransferase (IPCT) activity. In most cases, IPCT is part of a bifunctional enzyme comprising two domains: a cytoplasmic domain with IPCT activity and a membrane domain catalyzing the synthesis of di-myo-inositol-1,3′-phosphate-1′-phosphate from CDP-inositol and l-myo-inositol phosphate. Herein, we describe the first X-ray structure of the IPCT domain of the bifunctional enzyme from the hyperthermophilic archaeon Archaeoglobus fulgidus DSMZ 7324. The structure of the enzyme in the apo form was solved to a 1.9-Å resolution. The enzyme exhibited apparent K_m values of 0.9 and 0.6 mM for inositol-1-phosphate and CTP, respectively. The optimal temperature for catalysis was in the range 90 to 95°C, and the V_max determined at 90°C was 62.9 μmol · min⁻¹ · mg of protein⁻¹. The structure of IPCT is composed of a central seven-stranded mixed β-sheet, of which six β-strands are parallel, surrounded by six α-helices, a fold reminiscent of the dinucleotide-binding Rossmann fold. The enzyme shares structural homology with other pyrophosphorylases showing the canonical motif G-X-G-T-(R/S)-X₄-P-K. CTP, l-myo-inositol-1-phosphate, and CDP-inositol were docked into the catalytic site, which provided insights into the binding mode and high specificity of the enzyme for CTP. This work is an important step toward the final goal of understanding the full catalytic route for DIP synthesis in the native, bifunctional enzyme.     

Phylogenetic Groups Among <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> Isolates from Brazil: Relationship with Antimicrobial Resistance and Origin

The objectives of this study were to determine the distribution of phylogenetic groups among Klebsiella pneumoniae isolates from Recife, Brazil and to assess the relationship between the groups and the isolation sites and resistance profile. Ninety four isolates of K. pneumoniae from hospital or community infections and from normal microbiota were analyzed by gyrA PCR–RFLP, antibiotic susceptibility, and adonitol fermentation. The results revealed the distinction of three phylogenetic groups, as it has also been reported in Europe, showing that these clusters are highly conserved within K. pneumoniae. Group KpI was dominantly represented by hospital and community isolates while groups KpII and KpIII displayed mainly normal microbiota isolates. The resistance to third generation cephalosporins, aztreonam, imipenem, amoxicillin/clavulanic acid, and streptomycin was only observed in KpI. The percentage of resistance was higher in KpI, followed by KpII and KpIII. The differences in the distribution of K. pneumoniae phylogenetic groups observed in this study suggest distinctive clinical and epidemiological characteristics among the three groups, which is important to understand the epidemiology of infections caused by this organism. This is the first study in Brazil on K. pneumoniae isolates from normal microbiota and community infections regarding the distribution of phylogenetic groups based on the gyrA gene.     

CD4<Superscript>+</Superscript> T cell response against a non-tumor antigen is unaffected in melanoma-bearing mice

The tumor microenvironment is complex and creates an immunosuppressive network to tolerize tumor-specific immune responses; however, little information is available regarding the response against non-tumor antigens in tumor-bearing individuals. The goal of the present study was to evaluate if tumor burden could influence a CD4⁺ T cell response against a soluble protein, not expressed by the tumor, in the absence of in vitro stimulation. Using an experimental system in which we can compare CD4⁺ T cell responses to the Ea antigen when it is either expressed by B16F10 melanoma cells (B16EaRFP cells) or is an exogenous, non-tumor antigen (soluble EaRFP protein), in immunizations of B16F10 tumor-bearing mice, we observed that the tumor can modulate the CD4⁺ T cell-specific response to the antigen when it is expressed by the tumor cells. TEa cells proliferated poorly and produced less IFN-γ in mice bearing B16F10 melanoma expressing Ea peptide, and tumor growth was impervious to this response. However, in mice bearing 7 days B16F10 tumors, not expressing the Ea antigen, priming of TEa cells was similar to that observed in tumor-free mice, based on the total number of cells recovered and proliferation assessed by CFSE dilution after EaRFP immunization. We also investigated if tumor burden could influence recall responses of already differentiated effector cells. We immunized mice with EaRFP antigen and after a few days injected B16F10 cells. After 10 days of tumor growth, we challenged the mice with the non-tumor antigen. We found that the number of TEa cells producing IFN-γ in tumor-bearing mice was not different compared to tumor-free mice. No differences in antigen presentation, assessed by YAe antibody staining, were verified in the draining lymph node of these two groups. Collectively, our data indicate that tumor burden does not affect immune responses to non-tumor antigens. These results have important implications in the design of anti-cancer therapy.     

Cytotoxicity and genotoxicity of chitooligosaccharides upon lymphocytes

Two COS mixtures and a low molecular weight chitosan (LMWC) were tested for potential cytotoxicity and genotoxicity upon human lymphocytes. Genotoxicity was evaluated in vitro by cytokinesis-blocked micronucleus and alkaline comet assays, while cytotoxicity was assessed by flow cytometry analysis. Our results suggest that COS do not exhibit any genotoxicity upon human lymphocytes, independently of MW or concentration. However, above 0.07 mg/mL COS induced strong cytotoxic effects. According to the concentration used, such cytotoxicity will induce cell death, essentially by necrosis (>0.10 mg/mL) and/or apoptosis (<0.10 mg/mL). The level of necrosis/apoptosis induced by high COS concentrations, suggests a promising use as apoptosis inducers in specific cancer situations.     

Glioblastoma cells: a heterogeneous and fatal tumor interacting with the parenchyma

Glioblastomas (GBMs) are considered to be one of the deadliest human cancers, characterized by a high proliferative rate, aggressive invasiveness and insensitivity to radio- and chemotherapy, as well as a short patient survival period. Moreover, GBMs are among the most vascularized and invasive cancers in humans. Angiogenesis in GBMs is correlated with the grade of malignancy and is inversely correlated with patient survival. One of the first steps in tumor invasions is migration. GBM cells have the ability to infiltrate and disrupt physical barriers such as basement membranes, extracellular matrix and cell junctions. The invasion process includes the overexpression of several members of a super-family of zinc-based proteinases, the Metzincin, in particular a sub-group, metalloproteinases. Another interesting aspect is that, inside the GBM tissue, there are up to 30% of microglia or macrophages. However, little is known about the immune performance and interactions of the microglia with GBMs. These singular properties of GBMs will be described here. A sub-population of cells with stem-like properties may be the source of tumors since, apparently, GBM stem cells (GSCs) are highly resistant to current cancer treatments. These cancer therapies, while killing the majority of tumor cells, ultimately fail in GBM treatment because they do not eliminate GSCs, which survive to regenerate new tumors. Finally, GBM patient prognostic has shown little improvement in decades. In this context, we will discuss how the membrane-acting toxins called cytolysins can be a potential new tool for GBM treatment.     

Phage-displayed peptides as capture antigens in an innovative assay for Taenia saginata-infected cattle

Bovine cysticercosis is detected during the routine post mortem examination of carcasses by visual inspection (knife and eye method). However, the sensitivity of this procedure is several times lower than immunoassays, even when it is performed by qualified professionals. In the present study, a new generation capture antigens were screened from a phage display peptide library using antibodies from Taenia saginata-infected animals. Eight phage clones were selected, and one, Tsag 3 (VHTSIRPRCQPRAITPR), produced similar results to the T. saginata metacestode crude antigen (TsCa) when used as a capture antigen in an ELISA. The phage-displayed peptides competed with TsCa for binding sites, reducing the reactivity by approximately 30 %. Alanine scanning indicated that proline, arginine, and serine are important residues for antibody binding. Tsag 1 (HFYQITWLPNTFPAR), the most frequent affinity-selected clone, and Tsag 6 (YRWPSTPSASRQATL) shared similarity with highly conserved proteins from the Taeniidae family with known immunogenicity. Due to their epitopic or mimotopic properties, these affinity-selected phages could contribute to the rational design of an ante mortem immunodiagnosis method for bovine cysticercosis, as well as an epitope-based vaccine to interrupt the taeniosis/cysticercosis complex.     

Defense Response of Native and Alien Mealybugs (Hemiptera: Pseudococcidae) Against the Solitary Parasitoid Anagyrus sp. nr. pseudococci (Girault) (Hymenoptera: Encyrtidae)

The host behavioral and immune (encapsulation) defenses against the parasitoid Anagyrus sp. nr. pseudococci were compared for five mealybug species with different phylogenetic relationships and geographical origins: i) a Mediterranean native mealybug species, Planococcus ficus, with a long co-evolutionary history with the parasitoid; ii) three alien mealybugs species, Planococcus citri, Pseudococcus calceolariae and Pseudococcus viburni, with a more recent co-evolutionary history; and iii) a fourth alien mealybug species, Phenacoccus peruvianus, with no previous common history with the parasitoid. Three host defense behaviors were registered: abdominal flipping, reflex bleeding and walking away. The native host Pl. ficus and its congener Pl. citri exhibited the lowest probability of defense behavior (0.11?±?0.01 and 0.09?±?0.01 respectively), whereas the highest value was observed in P. viburni (0.31?±?0.02). Intermediate levels of defense behavior were registered for Ps. calceolariae, and Ph. peruvianus. The probability of parasitoid encapsulation was lowest and highest for two alien host species, Ph. peruvianus (0.20?±?0.07) and Ps. viburni (0.86?±?0.05), respectively. The native host Pl. ficus, its congener Pl. citri and Ps. calceolariae showed intermediate values (0.43?±?0.07, 0.52?±?0.06, and 0.45?±?0.09, respectively). The results are relevant with respect to biological control and to understand possible evolutionary processes involved in host range of A. sp. nr. pseudococci.     

Field evaluation of (E)-2-hexenal efficacy for behavioral manipulation of egg parasitoids in soybean

We studied the influence of synthetic (E)-2-hexenal on the abundance of Telenominae aiming to attract parasitoids and enhance stink bug egg parasitism in treated areas. We conducted experiments in 2006, 2007 and 2008 soybean seasons with two short-term (one week) and one long-term (seven weeks) experiment, respectively. We evaluated the abundance of parasitoids with yellow sticky traps and estimated the incidence and intensity of parasitism with sentinel eggs of Euschistus heros Fabricius (Hemiptera: Pentatomidae) or with tulle bags, enclosing laboratory pregnant E. heros as an egg source. In short-term experiments, there was increased abundance of Trissolcus spp. (Hymenoptera: Platygastridae: Telenominae) in treated areas, associated with greater intensity of parasitism. In long-term experiments, treatment with (E)-2-hexenal did not influence the abundance of Telenominae or parasitism levels, but increased egg predation. Applications of (E)-2-hexenal can be used to increase recruitment of Trissolcus spp. in the early flowering stages of soybean and also to attract other natural enemies, but do not increase egg parasitism.