Interaction of chitosan and mucin in a biomembrane model environment

Chitosans have been widely exploited in biological applications, including drug delivery and tissue engineering, especially owing to their mucoadhesive properties, but the molecular-level mechanisms for the chitosan action are not known in detail. It is believed that chitosan could affect the mucus by interacting with the proteins mucins, in a process mediated by the cell membrane. In this study we used Langmuir monolayers of dimyristoylphosphatidic acid (DMPA) as simplified membrane models to investigate the interplay between the activity of mucins and chitosan. Surface pressure and surface potential measurements were performed with DMPA monolayers onto which chitosan and/or mucin was adsorbed. We found that the expanding effect from mucin was considerably reduced when chitosan was injected after mucin had been adsorbed on the DMPA monolayer. The results were consistent with the formation of complexes between mucin and chitosan, thus highlighting the importance of electrostatic interactions. Furthermore, chitosan could remove mucin that was co-deposited along with DMPA in Langmuir-Blodgett (LB) films, which could be ascribed to molecular-level interactions between chitosan and mucin inferred from the FTIR spectra of the LB films. In conclusion, the results with Langmuir and LB films suggest that electrostatic interactions are crucial for the mucoadhesive mechanism, which is affected by the complexation between chitosan and mucin. (C) 2012 Elsevier Inc. All rights reserved.

Effect of pH, mucin and bovine serum on rifampicin permeability through Caco-2 cells

Rifampicin, a poorly soluble drug, has great importance in therapeutics as it is the main drug used to treat tuberculosis. The characterization of its permeability and the factors that influence it represent an important tool for predicting its bioavailability. Caco-2 cell monolayers were used as models of the intestinal mucosa to assess the uptake and transport of rifampicin and the effects of various experimental conditions were investigated, in order to establish the influence of these variables on rifampicin permeability. Different pHs (5.8, 6.8 and 7.4) in the apical medium, the presence or absence of mucin (3.0% w/v) in the donor site and the presence or absence of bovine serum albumin (4.0% v/v) in the receptor chamber were the evaluated conditions. The quantification of rifampicin in the apical or basolateral chambers was performed by a validated HPLC-UV method. The change in the donor chamber pH showed that permeability values were greater at pH 6.8, although this increase does not result in an alteration of the qualitative classification of rifampicin, which has high permeability. Mucin and bovine serum showed no effects on the permeability of rifampicin at the concentration tested. Overall, the current study suggests that pH, artificial mucin and bovine serum proteins have no influence on rifampicin permeability. Copyright (c) 2012 John Wiley & Sons, Ltd.

The sialotranscriptome of Antricola delacruzi female ticks is compatible with non-hematophagous behavior and an alternative source of food

The hosts for Antricola delacruzi ticks are insectivorous, cave-dwelling bats on which only larvae are found. The mouthparts of nymphal and adult A. delacruzi are compatible with scavenging feeding because the hypostome is small and toothless. How a single blood meal of a larva provides energy for several molts as well as for oviposition by females is not known. Adults of A. delacruzi possibly feed upon an unknown food source in bat guano, a substrate on which nymphal and adult stages are always found. Guano produced by insectivorous bats contains twice the amount of protein and 60 times the amount of iron as beef. In addition, bacteria and chitin-rich fungi proliferate on guano. Comparative data on the transcriptome of the salivary glands of A. delacruzi is nonexistent and would help to understand the physiological adaptations of salivary glands that accompany different sources of food as well as the steps taken by the Acari toward haematophagy, believed to have evolved from scavenging dead animals. Annotation of the transcriptome of salivary glands from female instars of A. delacruzi collected on guano categorized 5.7% of the clusters of expressed genes as putative secreted proteins. They included abundantly expressed TIL-domain-containing proteins (possible anti-microbials), an abundantly expressed protein similar to a serum amyloid found in the sialotranscriptomes of Ornithodoros spp., a savignygrin, a family of mucin/peritrophin/cuticle-like proteins, anti-microbials and an HIV envelope-like glycoprotein also found in soft ticks. When comparing the transcriptome of A. delacruzi with those of blood-feeding female soft and hard ticks some notable differences were observed; they consisted of the following transcripts over- or under-represented or absent in the sialotranscriptome of A. delacruzi that may reflect its source of food: ferritin, mucins with chitin-binding domains and TIL-domain-containing proteins versus lipocalins, basic tail proteins, metalloproteases, glycine-rich proteins and Kunitz protease inhibitors, respectively. (C) 2012 Elsevier Ltd. All rights reserved.

MUC1 gene polymorphism in three Nelore lines selected for growth and its association with growth and carcass traits

The objective of this study was to describe the VNTR polymorphism of the mucin 1 gene (MUC1) in three Nelore lines selected for yearling weight to determine whether allele and genotype frequencies of this polymorphism were affected by selection for growth. In addition, the effects of the polymorphism on growth and carcass traits were evaluated. Birth, weaning and yearling weights, rump height, Longissimus muscle area, backfat thickness, and rump fat thickness, were analyzed. A total of 295 Nelore heifers from the Beef Cattle Research Center, Instituto de Zootecnia de Sertozinho, were used, including 41 of the control line, 102 of the selection line and 152 of the traditional. The selection and traditional lines comprise animals selected for higher yearling weight, whereas control line animals are selected for yearling weight close to the average. Five alleles were identified, with allele 1 being the most frequent in the three lines, especially in the lines selected for higher means for yearling weight. Heterozygosity was significantly higher in the control line. Association analyses showed significant effects of allele 1 on birth weight and weaning weight while the allele 3 exert significant effects on yearling weight and back fat thickness. Despite these findings, application of this marker to marker-assisted selection requires more consistent results based on the genotyping of a larger number of animals in order to increase the accuracy of the statistical analyses.

Involvement of TSSA (trypomastigote small surface antigen) in Trypanosoma cruzi invasion of mammalian cells

TSSA (trypomastigote small surface antigen) is a polymorphic mucin-like molecule displayed on the surface of Trypanosoma cruzi trypomastigote forms. To evaluate its functional properties, we undertook comparative biochemical and genetic approaches on isoforms present in parasite stocks from extant evolutionary lineages (CL Brener and Sylvio X-10). We show that CL Brener TSSA, but not the Sylvio X-10 counterpart, exhibits dose-dependent and saturable binding towards non-macrophagic cell lines. This binding triggers Ca2+-based signalling responses in the target cell while providing an anchor for the invading parasite. Accordingly, exogenous addition of either TSSA-derived peptides or specific antibodies significantly inhibits invasion of CL Brener, but not Sylvio X-10, trypomastigotes. Non-infective epimastigote forms, which do not express detectable levels of TSSA, were stably transfected with TSSA cDNA from either parasite stock. Although both transfectants produced a surface-associated mucin-like TSSA product, epimastigotes expressing CL Brener TSSA showed a similar to 2-fold increase in their attachment to mammalian cells. Overall, these findings indicate that CL Brener TSSA functions as a parasite adhesin, engaging surface receptor(s) and inducing signalling pathways on the host cell as a prerequisite for parasite internalization. More importantly, the contrasting functional features of TSSA isoforms provide one appealing mechanism underlying the differential infectivity of T. cruzi stocks.

Expression of non-TLR pattern recognition receptors in the spleen of BALB/c mice infected with Plasmodium yoelii and Plasmodium chabaudi chabaudi AS

The spleen plays a crucial role in the development of immunity to malaria, but the role of pattern recognition receptors (PRRs) in splenic effector cells during malaria infection is poorly understood. In the present study, we analysed the expression of selected PRRs in splenic effector cells from BALB/c mice infected with the lethal and non-lethal Plasmodium yoelii strains 17XL and 17X, respectively, and the non-lethal Plasmodium chabaudi chabaudi AS strain. The results of these experiments showed fewer significant changes in the expression of PRRs in AS-infected mice than in 17X and 17XL-infected mice. Mannose receptor C type 2 (MRC2) expression increased with parasitemia, whereas Toll-like receptors and sialoadhesin (Sn) decreased in mice infected with P. chabaudi AS. In contrast, MRC type 1 (MRC1), MRC2 and EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80) expression decreased with parasitemia in mice infected with 17X, whereas MRC1 an MRC2 increased and F4/80 decreased in mice infected with 17XL. Furthermore, macrophage receptor with collagenous structure and CD68 declined rapidly after initial parasitemia. SIGNR1 and Sn expression demonstrated minor variations in the spleens of mice infected with either strain. Notably, macrophage scavenger receptor (Msr1) and dendritic cell-associated C-type lectin 2 expression increased at both the transcript and protein levels in 17XL-infected mice with 50% parasitemia. Furthermore, the increased lethality of 17X infection in Msr1 -/- mice demonstrated a protective role for Msr1. Our results suggest a dual role for these receptors in parasite clearance and protection in 17X infection and lethality in 17XL infection.

Identification of protein expression signatures in gastric carcinomas using clustering analysis

Background and Aim: The identification of gastric carcinomas (GC) has traditionally been based on histomorphology. Recently, DNA microarrays have successfully been used to identify tumors through clustering of the expression profiles. Random forest clustering is widely used for tissue microarrays and other immunohistochemical data, because it handles highly-skewed tumor marker expressions well, and weighs the contribution of each marker according to its relatedness with other tumor markers. In the present study, we e identified biologically- and clinically-meaningful groups of GC by hierarchical clustering analysis of immunohistochemical protein expression. Methods: We selected 28 proteins (p16, p27, p21, cyclin D1, cyclin A, cyclin B1, pRb, p53, c-met, c-erbB-2, vascular endothelial growth factor, transforming growth factor [TGF]-beta I, TGF-beta II, MutS homolog-2, bcl-2, bax, bak, bcl-x, adenomatous polyposis coli, clathrin, E-cadherin, beta-catenin, mucin (MUC) 1, MUC2, MUC5AC, MUC6, matrix metalloproteinase [ MMP]-2, and MMP-9) to be investigated by immunohistochemistry in 482 GC. The analyses of the data were done using a random forest-clustering method. Results: Proteins related to cell cycle, growth factor, cell motility, cell adhesion, apoptosis, and matrix remodeling were highly expressed in GC. We identified protein expressions associated with poor survival in diffuse-type GC. Conclusions: Based on the expression analysis of 28 proteins, we identified two groups of GC that could not be explained by any clinicopathological variables, and a subgroup of long-surviving diffuse-type GC patients with a distinct molecular profile. These results provide not only a new molecular basis for understanding the biological properties of GC, but also better prediction of survival than the classic pathological grouping.