A Paracoccidioides brasiliensis glycan shares serologic and functional properties with cryptococcal glucuronoxylomannan

The cell wall of the yeast form of the dimorphic fungus Paracoccidioides brasiliensis is enriched with α1,3-glucans. In Cryptococcus neoformans, α1,3-glucans interact with glucuronoxylomannan (GXM), a heteropolysaccharide that is essential for fungal virulence. In this study, we investigated the occurrence of P. brasiliensis glycans sharing properties with cryptococcal GXM. Protein database searches in P. brasiliensis revealed the presence of sequences homologous to those coding for enzymes involved in the synthesis of GXM and capsular architecture in C. neoformans. In addition, monoclonal antibodies (mAbs) raised to cryptococcal GXM bound to P. brasiliensis cells. Using protocols that were previously established for extraction and analysis of C. neoformans GXM, we recovered a P. brasiliensis glycan fraction composed of mannose and galactose, in addition to small amounts of glucose, xylose and rhamnose. In comparison with the C. neoformans GXM, the P. brasiliensis glycan fraction components had smaller molecular dimensions. The P. brasiliensis components, nevertheless, reacted with different GXM-binding mAbs. Extracellular vesicle fractions of P. brasiliensis also reacted with a GXM-binding mAb, suggesting that the polysaccharide-like molecule is exported to the extracellular space in secretory vesicles. An acapsular mutant of C. neoformans incorporated molecules from the P. brasiliensis extract onto the cell wall, resulting in the formation of surface networks that resembled the cryptococcal capsule. Coating the C. neoformans acapsular mutant with the P. brasiliensis glycan fraction resulted in protection against phagocytosis by murine macrophages. These results suggest that P. brasiliensis and C. neoformans share metabolic pathways required for the synthesis of similar polysaccharides and that P. brasiliensis yeast cell walls have molecules that mimic certain aspects of C. neoformans GXM. These findings are important because they provide additional evidence for the sharing of antigenically similar components across phylogenetically distant fungal species. Since GXM has been shown to be important for the pathogenesis of C. neoformans and to elicit protective antibodies, the finding of similar molecules in P. brasiliensis raises the possibility that these glycans play similar functions in paracoccidiomycosis.     

N-glycosylation of the mammalian dipeptidyl aminopeptidase-like protein 10 (DPP10) regulates trafficking and interaction with Kv4 channels

The dipeptidyl aminopeptidase-like protein 10 (DPP10) is a type II transmembrane protein homologue to the serine protease DPPIV/CD26 but enzymatically inactive. In the mammalian brain, DPP10 forms a complex with voltage-gated potassium channels of the Kv4 family, regulating their cell surface expression and biophysical properties. DPP10 is a glycoprotein containing eight predicted N-glycosylation sites in the extracellular domain. In this study we investigated the role of N-glycosylation on DPP10 trafficking and functional activity. Using site-directed mutagenesis (N to Q) we showed that N-glycosylation occured at six positions. Glycosylation at these specific residues was necessary for DPP10 trafficking to the plasma membrane as observed by flow cytometry. The surface expression levels of the substitutions N90Q, N119Q, N257Q and N342Q were reduced by more than 60%. Hence the interaction with the Kv4.3/KChIP2a channel complex was disrupted preventing the hastening effect of wild type DPP10 on current kinetics. Interestingly, N257 was crucial for this function and its substitution to glutamine completely blocked DPP10 sorting to the cell surface and prevented DPP10 dimerization. In summary, we demonstrated that glycosylation was necessary for both DPP10 trafficking to the cell surface and functional interaction with Kv4 channels.     

High fat-induced obesity associated with insulin-resistance increases FGF-2 content and causes stromal hyperplasia in rat ventral prostate

Obesity affects sex hormone secretion, which can negatively influence prostatic structure, homeostasis, and disease. This investigation aimed to evaluate the repercussions of obesity induced by a high-fat diet on the rat prostate, with or without treatment with the aromatase inhibitor, Letrozole. Adult Wistar rats were fed a high-fat diet (20% saturated fat, O) for 15?weeks to induce obesity or received a balanced diet (4% fat, C). Then, a group of C and O rats were daily treated with Letrozole (1?mg/kg b.w. per day) for 2?weeks (CL and OL, respectively). Subsequently, ventral prostate was processed for analysis by transmission electron microscopy, immunohistochemistry, and Western blotting. Obesity decreased 70% of the testosterone plasma level. The prostate showed epithelial atrophy and dilated acini in the intermediate portion and epithelial wrinkling in the distal tips. The relative frequency of smooth muscle α-actin in the O group increased by 67%. Ultrastructurally, epithelial cells in obese animals presented altered secretory organelles, lipid droplets, and thicker subjacent fibromuscular layer. Letrozole treatment caused a partial restoration of the prostatic changes caused by obesity. Obesity increased the prostatic content of fibroblast growth factor-2 (FGF-2) by 150%, and Letrozole treatment increased this protein even more in the control and obese groups. This investigation shows that obesity provokes structural and ultrastructural changes in the epithelium of rat prostate; these changes might affect gland homeostasis and physiology. The epithelial and smooth muscle cell hyperplasia and increased FGF-2 expression observed in this experimental model of obesity/insulin-resistance might explain the high frequency of benign prostatic hyperplasia in insulin-resistant men.     

Ion channels and transporters in cancer. 6. Vascularizing the tumor: TRP channels as molecular targets

Tumor vascularization is a critical process that determines tumor growth and metastasis. In the last decade new experimental evidence obtained from in vitro and in vivo studies have challenged the classical angiogenesis model forcing us to consider new scenarios for tumor neovascularization. In particular, the genetic stability of tumor-derived endothelial cells (TECs) has been recently questioned in several studies, which show that TECs, as well as pericytes, differ significantly from their normal counterparts at genetic and functional levels. In addition to such an epigenetic action of tumor microenvironment on endothelial cells (ECs) commitment, the distinct characteristics of TECs could be due to differences in their origin compared with preexisting differentiated ECs. Intracellular Ca(2+) signals are involved at different critical phases in the regulation of the complex process of angiogenesis and tumor progression. These signals are generated by a wide variety of intrinsic and extrinsic factors. Several key components of Ca(2+) signaling including Ca(2+) channels in the plasma membrane, endoplasmic reticulum, calcium pumps, and mitochondria contribute to the generation, amplitude, and frequency of these Ca(2+) change. In particular, several members of the transient receptor potential (TRP) family of calcium-permeable channels have profound effects on the function of ECs. Because of its multifaceted role in the control of cell function, proliferation, and motility, TRP channels have been suggested as a potential molecular target for control of tumor neovascularization. Since plasma membrane Ca(2+) channels are easily and directly accessible via the bloodstream, they are potential targets for a number of pharmacological and antibody-targeted therapeutic strategies, with specificity being the main limitation. In this review we discuss recent advances in understanding the role of Ca(2+) channels, with specific reference to TRP channels, in tumor vascularization process.     

Amino acids and protein profile in floral nectar: Much more than a simple reward

Nectar is considered a primary alimentary reward for a large variety of pollinators. Recent studies demonstrated that nectar may have other functions in addition to attracting pollinators. Mainly other two functions have been recognized: defense against microbial invasion and post-secretion modification of sugar profile. The floral nectar of Cucurbita pepo L. has been analyzed to confirm the presence of defense proteins, namely xylosidases that were identified by means of a proteomic approach in a previous study. An enzyme assay was also performed that revealed low invertase activity for which the optimal pH was determined. This invertase activity is not able to modify the sugar profile significantly during the short period of anthesis (6 h). The amino acids complement of the nectar of both sexes was also determined. Sixteen of the 20 protein amino acids have been detected. Proline comprises more than 30% of total amino acid content in male and female flowers. Three non-protein amino acids (taurine, β-alanine, and GABA) represent almost one third of the total amino acid content, and of these, GABA is the most abundant (16%). Several biological activities were attributed to these amino acids and further studies are needed to assess their presumed involvement in managing the foraging behavior of insects. More recent studies, including our own, demonstrate that the various functions of nectar are much more complex than previously thought.     

Coxiella burnetii, the agent of Q fever in Brazil: its hidden role in seronegative arthritis and the importance of molecular diagnosis based on the repetitive element IS1111 associated with the transposase gene

Coxiella burnetii is the agent of Q fever , an emergent worldwide zoonosis of wide clinical spectrum. Although C. burnetii infection is typically associated with acute infection, atypical pneumonia and flu-like symptoms, endocarditis, osteoarticular manifestations and severe disease are possible, especially when the patient has a suppressed immune system; however, these severe complications are typically neglected. This study reports the sequencing of the repetitive element IS1111 of the transposase gene of C. burnetii from blood and bronchoalveolar lavage (BAL) samples from a patient with severe pneumonia following methotrexate therapy, resulting in the molecular diagnosis of Q fever in a patient who had been diagnosed with active seronegative polyarthritis two years earlier. To the best of our knowledge, this represents the first documented case of the isolation of C. burnetii DNA from a BAL sample.     

Cell processing for haplo-identical hematopoietic stem cell transplantation: automated washing and immunomagnetic-positive selection

Background aimsImmunomagnetic cell selection (ICS) of CD34⁺ cells is being used increasingly in allogeneic transplantation in order to reduce T-cell quantity. The aim of this study was to evaluate an automated washing protocol before immunomagnetic selection.MethodsThe automated method was compared with a conventional washing procedure. In the study group the cell processing using the automated procedure, both before and after antibody incubation, was performed with a Sepax S-100 device. The efficacy of the automated procedure was compared with the control group, where washing were performed using a standard method.ResultsThe results obtained after pre-incubation washing performed using the automated system showed a total nucleated cell (NC) and CD34⁺ cell recovery of 84.87% (71.80–105, SD 8.62; range, standard deviation) and 83.45% (47–109, SD 16.12), respectively. The NC and CD34⁺ cell recovery after the pre-incubation washing cycle was performed using the standard method was 75.54% (38.36–97.76, SD 22.5) and 61.51% (30.87–81.79, SD 19.3), respectively. The CD34⁺ cell recovery after ICS was 51.27% (13.77–98.82, SD 24.97) and 48.89% (15.57–88.24, SD 25.91) for group 1 and group 2, respectively. The average purity in group 1 was 86.46% (67.4–96.10, SD 13.07) and in group 2 84.97% (58.1–97.8, SD 15.58). Conclusions. The efficacy of the ICS led to an optimal purity without affecting cell recovery, which was higher in group 1. Overall, our data suggest that the automated method is suitable for washing hematopoietic progenitor cell apheresis (HPC-A) concentrates before immunomagnetic cell selection in daily clinical routines.