Analysis of Immune Response Markers in Jorge Lobo's Disease Lesions Suggests the Occurrence of Mixed T Helper Responses with the Dominance of Regulatory T Cell Activity

Jorge Lobo’s disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-β and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-β1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-β1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-β1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis.     

Comparative study of the inactivation kinetics of pectinmethylesterase in tomato juice and purified form

Pectinmethylesterase (PME) extracted from tomato fruit was purified by affinity chromatography. A single peak of PME activity was observed, presenting a molar mass of 33.6 kDa, an isoelectric point higher than 9.3, and an optimal temperature and pH for activity of 55 degrees C and 8.0, respectively. The processing stability of purified tomato PME in buffer solution was compared to PME stability in tomato juice. In both media, thermal inactivation of PME presented first-order inactivation kinetics, PME in tomato juice being more heat-labile than purified PME. Regarding high-pressure treatment, tomato PME showed to be very pressure-resistant, revealing an outspoken antagonistic effect of temperature and pressure. To avoid cloud loss in tomato juice, a time-temperature treatment of 1 min at 76.5 degrees C was calculated in order to have a residual PME activity of 1 x 10(-)(4) U/mL.     

Construction and partial characterization of a Corynebacterium pseudotuberculosis bacterial artificial chromosome library through genomic survey sequencing

Corynebacterium pseudotuberculosis is a gram-positive bacterium that causes caseous lymphadenitis in sheep and goats. However, despite the economic losses caused by caseous lymphadenitis, there is little information about the molecular mechanisms of pathogenesis of this bacterium. Genomic libraries constructed in bacterial artificial chromosome (BAC) vectors have become the method of choice for clone development in high-throughput genomic-sequencing projects. Large-insert DNA libraries are useful for isolation and characterization of important genomic regions and genes. In order to identify targets that might be useful for genome sequencing, we constructed a C. pseudotuberculosis BAC library in the vector pBeloBAC11. This library contains about 18,000 BAC clones, with inserts ranging in size from 25 to 120 kb, theoretically representing a 390-fold coverage of the C. pseudotuberculosis genome (estimated to be 2.5-3.1 Mb). Many genomic survey sequences (GSSs) with homology to C. diphtheriae, C. glutamicum, C. efficiens, and C. jeikeium proteins were observed within a sample of 215 sequenced clones, confirming their close phylogenetic relationship. Computer analyses of GSSs did not detect chimeric, deleted, or rearranged BAC clones, showing that this library has low redundancy. This GSSs collection is now available for further genetic and physical analysis of the C. pseudotuberculosis genome. The GSS strategy that we used to develop our library proved to be efficient for the identification of genes and will be an important tool for mapping, assembly, comparative, and functional genomic studies in a C. pseudotuberculosis genome sequencing project that will begin this year.     

Conformational stability of the epidermal growth factor (EGF) receptor as influenced by glycosylation,dimerization and EGF hormone binding

The epidermal growth factor receptor (EGFR) is an important transmembrane glycoprotein kinase involved the initiation or perpetuation of signal transduction cascades within cells. These processes occur after EGFR binds to a ligand [epidermal growth factor (EGF)], thus inducing its dimerization and tyrosine autophosphorylation. Previous publications have highlighted the importance of glycosylation and dimerization for promoting proper function of the receptor and conformation in membranes; however, the effects of these associations on the protein conformational stability have not yet been described. Molecular dynamics simulations were performed to characterize the conformational preferences of the monomeric and dimeric forms of the EGFR extracellular domain upon binding to EGF in the presence and absence of N‐glycan moieties. Structural stability analyses revealed that EGF provides the most conformational stability to EGFR, followed by glycosylation and dimerization, respectively. The findings also support that EGF–EGFR binding takes place through a large‐scale induced‐fitting mechanism. Proteins 2017; 85:561–570. © 2016 Wiley Periodicals, Inc.     

Interactions Between Chronic Stress and Chronic Consumption of Caffeine on the Enzymatic Antioxidant System

We studied the effect of chronic caffeine on parameters related to oxidative stress in different brain regions of stressed and non-stressed rats. Wistar rats were divided into three groups: control (receiving water), caffeine 0.3 g/L and caffeine 1.0 g/L (in the drinking water). These groups were subdivided into non-stressed and stressed (repeated restraint stress during 40 days). Lipid peroxide levels and the total radical-trapping potential were assessed, as well as antioxidant enzyme activities superoxide dismutase, gluthatione peroxidase, and catalase in hippocampus, striatum and cerebral cortex. Results showed interactions between stress and caffeine, especially in the cerebral cortex, since caffeine increased the activity of some antioxidant enzymes, but not in stressed animals. We concluded that chronic administration of caffeine led, in some cases, to increased activity of antioxidant enzymes. However, these effects were not observed in the stressed animals.     

Occurrence of <Emphasis Type="Italic">TRGV-BJ</Emphasis> hybrid gene in SV40-transformed fibroblast cell lines

Illegitimate V(D)J-recombination in lymphoid malignancies involves rearrangements in immunoglobulin or T-cell receptor genes, and these rearrangements may play a role in oncogenic events. High frequencies of TRGV-BJ hybrid gene (rearrangement between the TRB and TRG loci at 7q35 and 7p14-15, respectively) have been detected in lymphocytes from patients with ataxia telangiectasia (AT), and also in patients with lymphoid malignancies. Although the TRGV-BJ gene has been described only in T-lymphocytes, we previously detected the presence of TRGV-BJ hybrid gene in the genomic DNA extracted from SV40-transformed AT5BIVA fibroblasts from an AT patient. Aiming to determine whether the AT phenotype or the SV40 transformation could be responsible for the production of the hybrid gene by illegitimate V(D)J-recombination, DNA samples were extracted from primary and SV40-transformed (normal and AT) cell lines, following Nested-PCR with TRGV- and TRBJ-specific primers. The hybrid gene was only detected in SV40-transformed fibroblasts (AT-5BIVA and MRC-5). Sequence alignment of the cloned PCR products using the BLAST program confirmed that the fragments corresponded to the TRGV-BJ hybrid gene. The present results indicate that the rearrangement can be produced in nonlymphoid cells, probably as a consequence of the genomic instability caused by the SV40-transformation, and independently of ATM gene mutation.     

Erratum to: Interactions Between Chronic Stress and Chronic Consumption of Caffeine on the Enzymatic Antioxidant System

Neurochemical Research -     

Influence of beta-subunit on thermal and high-pressure process stability of tomato polygalacturonase

Polygalacturonase (PG; E.C. 3.2.1.15) was extracted from tomato fruit and purified by cation-exchange chromatography. Two peaks containing PG activity were detected: the first denotes a thermolabile PG fraction (PG2) and the second a thermostable fraction (PG1). PG1 is a dimer of PG2 and a heat-stable protein called the beta-subunit. In contrast to its resistance to heat, PG is easily inactivated at elevated pressure. Although the thermal stability of purified tomato PG1 and PG2 is distinctly different, they show an identical pressure stability. To gain further insight into the thermal and pressure stability of both PG isoenzymes, the in vitro recombination of PG2 and beta-subunit was studied. After severe heat (up to 140 degrees C for 5 min) and pressure (up to 800 MPa for 15 min) treatments, the residual fractions containing the beta-subunit were able to convert PG2 into the heat-stable PG1, showing the extreme thermal and pressure stability of the beta-subunit. PG1 was detected in heat-treated tomato juice and, to a lesser extent, in tomato pieces. In contrast, as was the case for purified PG, no pressure-stable fraction was observed when tomato juice and pieces were treated under pressure. These data clearly show the differing behavior of the PG1-PG2-beta-subunit system under thermal and high-pressure treatments and offer the possibility of inactivating tomato PG using high pressure without the need for high temperatures.