Protein-mediated surface structuring in biomembranes

The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein), integral (Folch-Lees proteolipid protein) and amphitropic (c-Fos and c-Jun) proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase), in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

Restoration of the rabbit corneal surface after total epithelial debridement and complete limbal excision

How is the corneal epithelium restored when all of it plus the limbus have been eliminated? This investigation explored the possibility that this may be achieved through the conjunctival epithelium. The corneal epithelium of the right eye of 12 rabbits (Oryctolagus cuniculus) was totally scraped followed by surgical excision of the limbus plus 1.0-1.5 mm of the adjacent conjunctiva. Antibiotics and corticosteroids were applied for 1 week after surgery. Histological and immunohistochemical techniques were used to monitor the events taking place on the eye surface 2 weeks and 1, 3 and 6 months thereafter. Initially, the corneal surface was covered by conjunctival-like epithelium. After 1 month and more prominently at 3 and 6 months an epithelium displaying the morphological features of the cornea and reacting with the AE5 antibody was covering the central region. It is likely that the corneal epithelium originated from undifferentiated cells of the conjunctiva interacting with the corneal stroma.

Imbalanced expression of functional surface molecules in regulatory and effector T cells in systemic lupus erythematosus

Regulatory T (TREG) cells play an important role in maintaining immune tolerance and avoiding autoimmunity. We analyzed the expression of membrane molecules in TREG and effector T cells in systemic lupus erythematosus (SLE). TREG and effector T cells were analyzed for the expression of CTLA-4, PD1, CD28, CD95, GITR, HLA-DR, OX40, CD40L, and CD45RO in 26 patients with active disease, 31 with inactive disease, and 26 healthy controls. TREG cells were defined as CD25+/highCD127Ø/lowFoxP3+, and effector T cells were defined as CD25+CD127+FoxP3Ø. The ratio of TREG to effector T cells expressing GITR, PD1, HLA-DR, OX40, CD40L, and CD45RO was determined in the three groups. The frequency of TREG cells was similar in patients with SLE and controls. However, SLE patients had a decreased frequency of CTLA-4+TREG and CD28+TREG cells and an increased frequency of CD40L+TREG cells. There was a decrease in the TREG/effector-T ratio for GITR+, HLA-DR+, OX40+, and CD45RO+ cells, and an increased ratio of TREG/effector-T CD40L+ cells in patients with SLE. In addition, CD40L+TREG cell frequency correlated with the SLE disease activity index (P=0.0163). In conclusion, our findings showed several abnormalities in the expression of functionally critical surface molecules in TREG and effector T cells in SLE that may be relevant to the pathogenesis of this disease.

Influence of carbon source and the fermentation process on levan production by Zymomonas mobilis analyzed by the surface response method

The aim of this study is to assess sugar cane juice and sucrose as substrates, the batch and fed batch processes and their interaction in the levan production using a complete factorial design. Zymomonas mobilis was cultivated in different sugar cane juice and sucrose concentrations in two fermentation processes at 25 °C for 20 h. A complete factorial design (2³) was used to analyze the effects of the type and concentration of the substrate, as well as the batch and fed batch processes. A complete second factorial design (2²) was used to observe the importance of sugar cane juice. The results indicated that the batch process improved the levan production reaching 40.14 g/L. The addition of sugar cane juice was not statistically significant for levan formation, however sugar cane juice stimulated biomass, sorbitol and ethanol production. The best medium for levan production was 150 g/L sucrose in batch.

Hygienic-sanitary quality of vegetables and evaluation of treatments for the elimination of indigenous E. coli and E. coli O157:H7 from the surface of leaves of lettuce (Lactuca sativa L.)

The purpose of this study is to evaluate the hygienic-sanitary quality of vegetables and irrigation water and assess the effectiveness of lemon juice and vinegar in reducing E. coli strains inoculated on lettuce. One hundred and forty samples of vegetables and 45 samples of irrigation water were investigated for thermotolerant coliforms and Salmonella spp. In order to verify the effectiveness of natural household sanitizers in reducing E. coli in inoculated lettuce, four treatment solutions were tested: fresh lemon juice, alcohol vinegar, lemon juice-vinegar mixture, and lemon juice-vinegar-water mixture. The microbiological analysis revealed high rates of contamination by thermotolerant coliforms and identified the presence of E. coli in 32% of the tested vegetable samples and 56% of the water samples. While no significant statistical difference (p < 0, 05) was identified in the tested solutions, the treatment with a combination of lemon juice and vinegar resulted in the highest Decimal Reductions (DR) of E. coli O157: H7 while the treatment with vinegar alone was the most effective against the indigenous E. coli strain

Instrumental color and sensory acceptance of soy-based emulsions: a response surface approach

Response Surface Methodology (RSM) was applied to evaluate the chromatic features and sensory acceptance of emulsions that combine Soy Protein (SP) and red Guava Juice (GJ). The parameters analyzed were: instrumental color based on the coordinates a* (redness), b* (yellowness), L* (lightness), C* (chromaticity), h* (hue angle), visual color, acceptance, and appearance. The analyses of the results showed that GJ was responsible for the high measured values of red color, hue angle, chromaticity, acceptance, and visual color, whereas SP was the variable that increased the yellowness intensity of the assays. The redness (R²adj = 74.86%, p < 0.01) and hue angle (R²adj = 80.96%, p < 0.01) were related to the independent variables by linear models, while the sensory data (color and acceptance) could not be modeled due to a high variability. The models of yellowness, lightness, and chromaticity did not present lack of fit but presented adjusted determination coefficients bellow 70%. Notwithstanding, the linear correlations between sensory and instrumental data were not significant (p > 0.05) and low Pearson coefficients were obtained. The results showed that RSM is a useful tool to develop soy-based emulsions and model some chromatic features of guava-based emulsions through RSM.