Obtaining and selection of hexokinases-less strains of Saccharomyces cerevisiae for production of ethanol and fructose from sucrose

Saccharomyces cerevisiae hexokinase-less strains were produced to study the production of ethanol and fructose from sucrose. These strains do not have the hexokinases A and B. Twenty-three double-mutant strains were produced, and then, three were selected for presenting a smaller growth in yeast extract-peptone-fructose. In fermentations with a medium containing sucrose (180.3 g L-1) and with cell recycles, simulating industrial conditions, the capacity of these mutant yeasts in inverting sucrose and fermenting only glucose was well characterized. Besides that, we could also see their great tolerance to the stresses of fermentative recycles, where fructose production (until 90 g L-1) and ethanol production (until 42.3 g L-1) occurred in cycles of 12 h, in which hexokinase-less yeasts performed high growth (51.2% of wet biomass) and viability rates (77% of viable cells) after nine consecutive cycles.

Amphetamine modulates cellular recruitment and airway reactivity in a rat model of allergic lung inflammation

Asthma is characterized by pulmonary cellular infiltration, vascular exudation and airway hyperresponsiveness. Several drugs that modify central nervous system (CNS) activity can modulate the course of asthma. Amphetamine (AMPH) is a highly abused drug that presents potent stimulating effects on the CNS and has been shown to induce behavioral, biochemical and immunological effects. The purpose of this study was to investigate the effects of AMPH on pulmonary cellular influx, vascular permeability and airway reactivity. AMPH effects on adhesion molecule expression, IL-10 and IL-4 release and mast cell degranulation were also studied. Male Wistar rats were sensitized with ovalbumin (OVA) plus alum via subcutaneous injection. One week later, the rats received another injection of OVA-alum (booster). Two weeks after this booster, the rats were subjected to AMPH treatment 12 h prior to the OVA airway challenge. In rats treated with AMPH, the OVA challenge reduced cell recruitment into the lung, the vascular permeability and the cellular expression of ICAM-1 and Mac-1. Additionally, elevated levels of IL-10 and IL-4 were found in samples of lung explants from allergic rats. AMPH treatment, in comparison, increased IL-10 levels but reduced those of IL-4 in the lung explants. Moreover, the tracheal responsiveness to methacholine (MCh), as well as to an in vitro OVA challenge, was reduced by AMPH treatment, and levels of PCA titers were not modified by the drug. Our findings suggest that single AMPH treatment down-regulates several parameters of lung inflammation, such as cellular migration, vascular permeability and tracheal responsiveness. These results also indicate that AMPH actions on allergic lung inflammation include endothelium-leukocyte interaction mechanisms, cytokine release and mast cell degranulation. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Differential effects of female sex hormones on cellular recruitment and tracheal reactivity after formaldehyde exposure

Female sex hormones (FSHs) exert profound regulatory effects on the course of lung inflammation due to allergic and non-allergic immune responses. As pollution is one of the pivotal factors to induce lung dysfunction, in this study we investigated the modulatory role of FSHs on lung inflammation after a formaldehyde (FA) exposure. For this purpose, lung and systemic inflammatory responses were evaluated in terms of leukocytes countings in bronchoalveolar lavage (BAL), peripheral blood and bone marrow lavage from 7-day ovariectomized (OVx) and Sham-OVx rats subjected to FA inhalation for 3 consecutive days. The hypothesized link between effects of FSHs on expression of adhesion molecules and mast cells degranulation was also studied. Once exposed to FA, Sham-OVx rats increased the number of total cells recovered in BAL and of leukocytes in peripheral blood, and decreased the counts in bone marrow. By contrast, in OVx rats upon FA exposure there was a reduction of the total cells counts in BAL and of blood leukocytes: lung expressions of ICAM-1 and Mac-1 were depressed, but the number of bone marrow cells did not vary. Estradiol treatment of OVx rats increased the total cells in BAL and decreased the number of blood leukocytes, whereas the number of bone marrow cell remained unaltered. Progesterone treatment, in turn increased the total cells in BAL and blood leukocytes, but decreased the number of bone marrow cells. OVx rats exposed to FA developed tracheal hyperresponsiveness to methacholine (MCh). A similarly altered response was found between the tracheal segments of Sham-OVx rats after FA exposure and that found in tracheae of naive rats. Estradiol treatment prevented FA-induced tracheal hyperresponsiveness to MCh whereas progesterone was ineffective in this regard. In addition, OVx rats upon FA exposure significantly increased both, the ability of mast cell degranulation and serum corticosterone levels. In conclusion, it was found that FSHs act by distinct control mechanisms on FA-induced lung inflammation and tracheal hyperresponsiveness, since at low circulating levels of FSHs (such as those after OVx) there is some resistance to the development of a lung inflammatory response, but the cholinergic tracheal responsiveness is exacerbated. Our data also help to understand the involvement of FSHs on mast cells activity after pollutants exposure and add information regarding the role of FSHs on the mechanisms related to endothelium-leukocyte interactions. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

The pattern recognition receptors Nod1 and Nod2 account for neutrophil recruitment to the lungs of mice infected with Legionella pneumophila

The intracellular bacterium Legionella pneumophila induces a severe form of pneumonia called Legionnaires diseases, which is characterized by a strong neutrophil (NE) infiltrate to the lungs of infected individuals. Although the participation of pattern recognition receptors, such as Toll-like receptors, was recently demonstrated, there is no information on the role of nod-like receptors (NLRs) for bacterial recognition in vivo and for NE recruitment to the lungs. Here, we employed a murine model of Legionnaires disease to evaluate host and bacterial factors involved in NE recruitment to the mice lungs. We found that L. pneumophila type four secretion system, known as Dot/Icm, was required for NE recruitment as dot/icm mutants fail to trigger NE recruitment in a process independent of bacterial multiplication. By using mice deficient for Nod1, Nod2, and Rip2, we found that these receptors accounted for NE recruitment to the lungs of infected mice. In addition, Rip2-dependent responses were important for cytokine production and bacterial clearance. Collectively, these studies show that Nod1, Nod2, and Rip2 account for generation of innate immune responses in vivo, which are important for NE recruitment and bacterial clearance in a murine model of Legionnaires diseases. (C) 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Phosphoinositide-3 Kinase gamma Activity Contributes to Sepsis and Organ Damage by Altering Neutrophil Recruitment

Rationale Sepsis is a leading cause of death in the intensive care unit, characterized by a systemic inflammatory response (SIRS) and bacterial infection, which can often induce multiorgan damage and failure. Leukocyte recruitment, required to limit bacterial spread, depends on phosphoinositide-3 kinase gamma (PI3K gamma) signaling in vitro; however, the role of this enzyme in polymicrobial sepsis has remained unclear. Objectives: This study aimed to determine the specific role of the kinase activity of PI3K gamma in the pathogenesis of sepsis and multiorgan damage. Methods. PI3K gamma wild-type, knockout, and kinase-dead mice were exposed to cecal ligation and perforation induced sepsis and assessed for survival; pulmonary, hepatic, and cardiovascular damage; coagulation derangements; systemic inflammation; bacterial spread; and neutrophil recruitment. Additionally, wild-type mice were treated either before or after the onset of sepsis with a PI3K gamma inhibitor and assessed for survival, neutrophil recruitment, and bacterial spread. Measurements and Main Results: Both genetic and pharmaceutical PI3K gamma kinase inhibition significantly improved survival, reduced multiorgan damage, and limited bacterial decompartmentalization, while modestly affecting SIRS. Protection resulted from both neutrophil-independent mechanisms, involving improved cardiovascular function, and neutrophil-dependent mechanisms, through reduced susceptibility to neutrophil migration failure during severe sepsis by maintaining neutrophil surface expression of the chemokine receptor, CXCR2. Furthermore, PI3K gamma pharmacological inhibition significantly decreased mortality and improved neutrophil migration and bacterial control, even when administered during established septic shock. Conclusions: This study establishes PI3K gamma as a key molecule in the pathogenesis of septic infection and the transition from SIRS to organ damage and identifies it as a novel possible therapeutic target.

Bedside estimation of recruitable alveolar collapse and hyperdistension by electrical impedance tomography

To present a novel algorithm for estimating recruitable alveolar collapse and hyperdistension based on electrical impedance tomography (EIT) during a decremental positive end-expiratory pressure (PEEP) titration. Technical note with illustrative case reports. Respiratory intensive care unit. Patients with acute respiratory distress syndrome. Lung recruitment and PEEP titration maneuver. Simultaneous acquisition of EIT and X-ray computerized tomography (CT) data. We found good agreement (in terms of amount and spatial location) between the collapse estimated by EIT and CT for all levels of PEEP. The optimal PEEP values detected by EIT for patients 1 and 2 (keeping lung collapse < 10%) were 19 and 17 cmH(2)O, respectively. Although pointing to the same non-dependent lung regions, EIT estimates of hyperdistension represent the functional deterioration of lung units, instead of their anatomical changes, and could not be compared directly with static CT estimates for hyperinflation. We described an EIT-based method for estimating recruitable alveolar collapse at the bedside, pointing out its regional distribution. Additionally, we proposed a measure of lung hyperdistension based on regional lung mechanics.

Decreased AIRE Expression and Global Thymic Hypofunction in Down Syndrome

The Down syndrome (DS) immune phenotype is characterized by thymus hypotrophy, higher propensity to organ-specific autoimmune disorders, and higher susceptibility to infections, among other features. Considering that AIRE (autoimmune regulator) is located on 21q22.3, we analyzed protein and gene expression in surgically removed thymuses from 14 DS patients with congenital heart defects, who were compared with 42 age-matched controls with heart anomaly as an isolated malformation. Immunohistochemistry revealed 70.48 +/- 49.59 AIRE-positive cells/mm(2) in DS versus 154.70 +/- 61.16 AIRE-positive cells/mm(2) in controls (p < 0.0001), and quantitative PCR as well as DNA microarray data confirmed those results. The number of FOXP3-positive cells/mm(2) was equivalent in both groups. Thymus transcriptome analysis showed 407 genes significantly hypoexpressed in DS, most of which were related, according to network transcriptional analysis (FunNet), to cell division and to immunity. Immune response-related genes included those involved in 1) Ag processing and presentation (HLA-DQB1, HLA-DRB3, CD1A, CD1B, CD1C, ERAP) and 2) thymic T cell differentiation (IL2RG, RAG2, CD3D, CD3E, PRDX2, CDK6) and selection (SH2D1A, CD74). It is noteworthy that relevant AIRE-partner genes, such as TOP2A, LAMNB1, and NUP93, were found hypoexpressed in DNA microarrays and quantitative real-time PCR analyses. These findings on global thymic hypofunction in DS revealed molecular mechanisms underlying DS immune phenotype and strongly suggest that DS immune abnormalities are present since early development, rather than being a consequence of precocious aging, as widely hypothesized. Thus, DS should be considered as a non-monogenic primary immunodeficiency. The Journal of Immunology, 2011, 187: 3422-3430.

Crucial Role of TNF Receptors 1 and 2 in the Control of Polymicrobial Sepsis

Sepsis is still a major cause of mortality in the intensive critical care unit and results from an overwhelming immune response to the infection. TNF signaling pathway plays a central role in the activation of innate immunity in response to pathogens. Using a model of polymicrobial sepsis by i.p. injection of cecal microflora, we demonstrate a critical role of TNFR1 and R2 activation in the deregulated immune responses and death associated with sepsis. A large and persistent production of TNF was found in wild-type (B6) mice. TNFR1/R2-deficient mice, compared with B6 mice, survive lethal polymicrobial infection with enhanced neutrophil recruitment and bacterial clearance in the peritoneal cavity. Absence of TNFR signaling leads to a decreased local and systemic inflammatory response with diminished organ injury. Furthermore, using TNFR1/R2-deficient mice, TNF was found to be responsible for a decrease in CXCR2 expression, explaining reduced neutrophil extravasation and migration to the infectious site, and in neutrophil apoptosis. In line with the clinical experience, administration of Enbrel, a TNF-neutralizing protein, induced however only a partial protection in B6 mice, with no improvement of clinical settings, suggesting that future TNF immunomodulatory strategies should target TNFR1 and R2. In conclusion, the present data suggest that the endogenous TNFR1/R2 signaling pathway in polymicrobial sepsis reduces neutrophil recruitment contributing to mortality and as opposed to pan-TNF blockade is an important therapeutic target for the treatment of polymicrobial sepsis. The Journal of Immunology, 2009, 182: 7855-7864.

Targeting endothelin ET(A) and ET(B) receptors inhibits antigen-induced neutrophil migration and mechanical hypernociception in mice

Endothelin may contribute to the development of inflammatory events such as leukocyte recruitment and nociception. Herein, we investigated whether endothelin-mediated mechanical hypernociception (decreased nociceptive threshold, evaluated by electronic pressure-meter) and neutrophil migration (myeloperoxidase activity) are inter-dependent in antigen challenge-induced Th1-driven hind-paw inflammation. In antigen challenge-induced inflammation, endothelin (ET) ET(A) and ET(B) receptor antagonism inhibited both hypernociception and neutrophil migration. Interestingly, ET-1 peptide-induced hypernociception was not altered by inhibiting neutrophil migration or endothelin ET(B) receptor antagonism, but rather by endothelin ET(A) receptor antagonism. Furthermore, endothelin ET(A), but not ET(B), receptor antagonism inhibited antigen-induced PGE(2) production, whereas either selective or combined blockade of endothelin ET(A) and/or ET(B) receptors reduced hypernociception and neutrophil recruitment caused by antigen challenge. Concluding, this study advances knowledge into the role for endothelin in inflammatory mechanisms and further supports the potential of endothelin receptor antagonists in controlling inflammation.

Endothelins modulate inflammatory reaction in zymosan-induced arthritis: participation of LTB4, TNF-alpha, and CXCL-1

Endothelins (ETs) are involved in inflammatory events, including pain, fever, edema, and cell migration. ET-1 levels are increased in plasma and synovial membrane of rheumatoid arthritis (RA) patients, but the evidence that ETs participate in RA physiopathology is limited. The present study investigated the involvement of ETs in neutrophil accumulation and edema formation in the murine model of zymosan-induced arthritis. Intra-articular (i.a.) administration of selective ETA or ETB receptor antagonists (BQ-123 and BQ-788, respectively; 15 pmol/cavity) prior to i.a. zymosan injection (500 mu g/cavity) markedly reduced knee-joint edema formation and neutrophil influx to the synovial cavity 6 h and 24 h after stimulation. Histological analysis showed that ETA or ETB receptor blockade suppressed zymosan-induced neutrophil accumulation in articular tissue at 6 h. Likewise, dual blockade of ETA/ETB with bosentan (10 mg/kg, i.v.) also reduced edema formation and neutrophil counts 6 h after zymosan stimulation. Pretreatment with BQ-123 or BQ-788 (i.a.; 15 pmol/cavity) also decreased zymosan-induced TNF-alpha production within 6 h, keratinocyte-derived chemokine/CXCL1 production within 24 h, and leukotriene B-4 at both time-points. Consistent with the demonstration that ET receptor antagonists inhibit zymosan-induced inflammation, i.a. injection of ET-1 (1-30 pmol/cavity) or sarafotoxin S6c (0.1-30 pmol/cavity) also triggered edema formation and neutrophil accumulation within 6 h. Moreover, knee-joint synovial tissue expressed ETA and ETB receptors. These findings suggest that endogenous ETs contribute to knee-joint inflammation, acting through ETA and ETB receptors and modulating edema formation, neutrophil recruitment, and production of inflammatory mediators.

Anti-inflammatory effects of red pepper (Capsicum baccatum) on carrageenan- and antigen-induced inflammation

Inflammation is a pivotal component of a variety of diseases, such as atherosclerosis and tumour progression. Various naturally occurring phytochemicals exhibit anti-inflammatory activity and are considered to be potential drug candidates against inflammation-related pathological processes. Capsicum baccatum L. var. pendulum (Willd.) Eshbaugh (Solanaceae) is the most consumed species in Brazil, and its compounds, such as capsaicinoids, have been found to inhibit the inflammatory process. However, the anti-inflammatory effects of C. baccatum have not been characterized. Thus, this study was designed to evaluate the effects of C. baccatum juice in animal models of acute inflammation induced by carrageenan and immune inflammation induced by methylated bovine serum albumin. Pretreatment (30 min) of rats with pepper juice (0.25-2.0 g kg(-1)) significantly decreased leucocyte and neutrophil migration, exudate volume and protein and LDH concentration in pleural exudates of a pleurisy model. This juice also inhibited neutrophil migration and reduced the vascular permeability on carrageenan-induced peritonitis in mice. C. baccatum juice also reduced neutrophil recruitment and exudate levels of pro-inflammatory cytokines TNF-alpha, and IL-1 beta in mouse inflammatory immune peritonitis. Furthermore, we demonstrated that the main constituent of C. baccatum juice, as extracted with chloroform, is capsaicin. In agreement with this, capsaicin was able to inhibit the neutrophil migration towards the inflammatory focus. To our knowledge, this is the first demonstration of the anti-inflammatory effect of C. baccatum juice and our data suggest that this effect may be induced by capsaicin. Moreover, the anti-inflammatory effect induced by red pepper may be by inhibition of pro-inflammatory cytokine production at the inflammatory site.

A crucial role for TNF-alpha in mediating neutrophil influx induced by endogenously generated or exogenous chemokines, KC/CXCL1 and LIX/CXCL5

Background and purpose: Chemokines orchestrate neutrophil recruitment to inflammatory foci. In the present study, we evaluated the participation of three chemokines, KC/CXCL1, MIP-2/CXCL2 and LIX/CXCL5, which are ligands for chemokine receptor 2 (CXCR2), in mediating neutrophil recruitment in immune inflammation induced by antigen in immunized mice. Experimental approach: Neutrophil recruitment was assessed in immunized mice challenged with methylated bovine serum albumin, KC/CXCL1, LIX/CXCL5 or tumour necrosis factor (TNF)-alpha. Cytokine and chemokine levels were determined in peritoneal exudates and in supernatants of macrophages and mast cells by elisa. CXCR2 and intercellular adhesion molecule 1 (ICAM-1) expression was determined using immunohistochemistry and confocal microscopy. Key results: Antigen challenge induced dose- and time-dependent neutrophil recruitment and production of KC/CXCL1, LIX/CXCL5 and TNF-alpha, but not MIP-2/CXCL2, in peritoneal exudates. Neutrophil recruitment was inhibited by treatment with reparixin (CXCR1/2 antagonist), anti-KC/CXCL1, anti-LIX/CXCL5 or anti-TNF-alpha antibodies and in tumour necrosis factor receptor 1-deficient mice. Intraperitoneal injection of KC/CXCL1 and LIX/CXCL5 induced dose- and time-dependent neutrophil recruitment and TNF-alpha production, which were inhibited by reparixin or anti-TNF-alpha treatment. Macrophages and mast cells expressed CXCR2 receptors. Increased macrophage numbers enhanced, while cromolyn sodium (mast cell stabilizer) diminished, LIX/CXCL5-induced neutrophil recruitment. Macrophages and mast cells from immunized mice produced TNF-alpha upon LIX/CXCL5 stimulation. Methylated bovine serum albumin induced expression of ICAM-1 on mesenteric vascular endothelium, which was inhibited by anti-TNF-alpha or anti-LIX/CXCL5. Conclusion and implications: Following antigen challenge, CXCR2 ligands are produced and act on macrophages and mast cells triggering the production of TNF-alpha, which synergistically contribute to neutrophil recruitment through induction of the expression of ICAM-1.

Home range and habitat use by the roadside hawk, Rupornis magnirostris (Gmelin, 1788) (Aves: Falcaniformes) in southeastern Brazil

Although most raptor species are found mainly in the tropics, information on their home range and spatial requirements in the Neotropics is still scarce. In this study, we used radio telemetry to evaluate the home range and the habitat use and selection of five Roadside hawks, Rupornis magnirostris (Gmelin, 1788) in a heterogeneous landscape in southeastern Brazil. The average home range size calculated using the adaptive kernel method (95% isopleth) was 126.1ha (47.4-266.7ha), but using the minimum convex polygon method (95% isopleth) it was 143.54ha (32.6-382.3ha). The roadside hawk explored a wide variety of habitats, most of them opportunistically, as suggested in the literature. Despite this, habitat quality could influence home range size and promote habitat selection. The observation of habitat use as expected, as well as the relatively small home range size, could be related to the generalist/opportunistic behaviour of the roadside hawk.

SIZE AS A LINE OF LEAST RESISTANCE II: DIRECT SELECTION ON SIZE OR CORRELATED RESPONSE DUE TO CONSTRAINTS?

Evolutionary change in New World Monkey (NWM) skulls occurred primarily along the line of least resistance defined by size (including allometric) variation (g(max)). Although the direction of evolution was aligned with this axis, it was not clear whether this macroevolutionary pattern results from the conservation of within population genetic covariance patterns (long-term constraint) or long-term selection along a size dimension, or whether both, constraints and selection, were inextricably involved. Furthermore, G-matrix stability can also be a consequence of selection, which implies that both, constraints embodied in g(max) and evolutionary changes observed on the trait averages, would be influenced by selection Here, we describe a combination of approaches that allows one to test whether any particular instance of size evolution is a correlated by-product due to constraints (g(max)) or is due to direct selection on size and apply it to NWM lineages as a case study. The approach is based on comparing the direction and amount of evolutionary change produced by two different simulated sets of net-selection gradients (beta), a size (isometric and allometric size) and a nonsize set. Using this approach it is possible to distinguish between the two hypotheses (indirect size evolution due to constraints or direct selection on size), because although both may produce an evolutionary response aligned with g(max), the amount of change produced by random selection operating through the variance/covariance patterns (constraints hypothesis) will be much smaller than that produced by selection on size (selection hypothesis). Furthermore, the alignment of simulated evolutionary changes with g(max) when selection is not on size is not as tight as when selection is actually on size, allowing a statistical test of whether a particular observed case of evolution along the line of least resistance is the result of selection along it or not. Also, with matrix diagonalization (principal components [PC]) it is possible to calculate directly the net-selection gradient on size alone (first PC [PC1]) by dividing the amount of phenotypic difference between any two populations by the amount of variation in PC1, which allows one to benchmark whether selection was on size or not

Molecular characterization of human polyomavirus JC in Brazilian AIDS patients with and without progressive multifocal leukoencephalopathy

Background: JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), is classified in 8 different genotypes. Previous reports have suggested a positive association between specific genotypes and PML. Objective: To compare genotypes and adaptive mutations of JCV strains from Brazilian AIDS patients with and without PML. Study design: The VP1 region of JCV was amplified by polymerase chain reaction from cerebrospinal fluid samples from 51 patients with PML and from urine samples of 47 patients with AIDS without central nervous system disease. Genotyping was done by phylogenetic analysis. Amino acid replacement and selection pressures were also investigated. Results: JCV genotype frequency distributions showed that genotypes 2 (32.7%), 1 (26.5%) and 3 (23.5%) were the most prevalent. Genotype 1 had a positive association (p < 0.0001) and genotype 3 showed an inverse association (p < 0.001) with PML. A previously undescribed point mutation at residue 91 (L/I or L/V) and (L/P), non-genotype-associated, was found in 5/49 (10.2%) and 2/47 (4.3%) JCV sequences from PML and non-PML patients, respectively. This mutation was under positive selection only in PML patients. A previously described substitution of T-A in position 128 showed a significant difference between PML and non-PML cases (70% versus 16%, respectively, p < 0.0005). Conclusion: In Brazilian patients with AIDS, JCV genotype 1 showed a strong association with PML (p < 0.0001) and JCV genotype 3 showed an inverse association with PML. The possible association of aminoacids substitution in residues 91 and 128 with PML in patients with AIDS must be further investigated. (C) 2010 Elsevier B.V. All rights reserved.