Inhibition of interferon-gamma-induced nitric oxide production in endotoxin-activated macrophages by cytolethal distending toxin

Introduction: Cytolethal distending toxin (CDT) is a DNA-targeting agent produced by certain pathogenic gram-negative bacteria such as the periodontopathogenic organism Aggregatibacter actinomycetemcomitans. CDT targets lymphocytes and other cells causing cell cycle arrest and apoptosis, impairing the host immune response and contributing to the persistence of infections caused by this microorganism. In this study we explored the effects of CDT on the innate immune response, by investigating how it affects production of nitric oxide (NO) by macrophages. Methods: Murine peritoneal macrophages were stimulated with Escherichia coli sonicates and NO production was measured in the presence or not of active CDT. Results: We observed that CDT promptly and significantly inhibited NO production by inducible nitric oxide synthase (iNOS) in a dose-dependent manner. This inhibition is directed towards interferon-gamma-dependent pathways and is not mediated by either interleukin-4 or interleukin-10. Conclusion: This mechanism may constitute an important aspect of the immunosuppression mediated by CDT and may have potential clinical implications in A. actinomycetemcomitans infections.

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) with a broad range of 3HV content at high yields by Burkholderia sacchari IPT 189

The biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from sucrose and propionic acid by Burkholderia sacchari IPT 189 was studied using a two-stage bioreactor process. In the first stage, this bacterium was cultivated in a balanced culture medium until sucrose exhaustion. In the second stage, a solution containing sucrose and propionic acid as carbon source was fed to the bioreactor at various sucrose/propionic acid (s/p) ratios at a constant specific flow rate. Copolymers with 3HV content ranging from 40 down to 6.5 (mol%) were obtained with 3HV yield from propionic acid (Y-3HV/prop) increasing from 1.10 to 1.34 g g(-1). Copolymer productivity of 1 g l(-1) h(-1) was obtained with polymer biomass content rising up to 60% by increasing a specific flow rate at a constant s/p ratio. Increasing values of 3HV content were obtained by varying the s/p ratios. A simulation of production costs considering Y-3HV/prop obtained in the present work indicated that a reduction of up to 73% can be reached, approximating US$ 1.00 per kg which is closer to the value to produce P3HB from sucrose (US$ 0.75 per kg).

Relative populations and toxin production by Aspergillus flavus and Fusarium verticillioides in artificially inoculated corn at various stages of development under field conditions

The presence, development and production of mycotoxins by Aspergillus flavus and Fusarium verticillioides were studied in corn ears under field conditions after artificial contamination of corn silks. The planted area was divided into five treatments: T1, inoculated with A.flavus solution containing 1 x 10(8) spores, ears covered; T2, inoculated with F. verticillioides solution containing 1 X 10(8) spores, ears covered; T3, inoculated with E verticillioides plus A. flavus solution containing 1 x 10(8) spores of each, ears covered; T4, sprayed with sterile phosphate-buffered saline, ears covered; TS, non-sprayed silks, uncovered ears. Soil and air samples were also collected and analysed for the occurrence of fungi. Water activity, relative air humidity, rainfall and temperature were determined to assess the correlation between abiotic factors and the presence of fungi in the samples. Contamination with the inoculated fungus predominated in T1 and T2. In the other treatments, F. verticillioides was the most frequently isolated contaminant irrespective of treatment. Considering the production of mycotoxins, a positive relation between the production of fumonisins B-1 and B-2 and the frequency of F. verticillioides was statistically verified in all treatments. (C) 2007 Society of Chemical Industry.

Influence of macro- and micronutrient fertilization on fungal contamination and fumonisin production in corn grains

The aim of this study was to investigate the effects of nutrients (nitrogen, zinc and boron) on fungal growth and fumonisins production in corn samples obtained at the beginning of grain formation and at harvest. Three nitrogen doses were applied to the corn plants through soil in combination with three zinc doses and two boron doses during sowing. Mycological analysis of grains, using Dichloran Rose-Bengal Chloramphenicol Agar, collected at the beginning of formation demonstrated a fungal population predominantly of yeasts. Analysis of freshly harvested corn revealed a higher frequency of Penicillium spp. (72%) and F verticillioides (27%). High Performance Liquid Chromatography analysis revealed that 100% of grains were contaminated with fumonisins B, at levels ranging from 0.3 to 24.3 mg/kg and 93% contaminated with fumonisin B(2) at levels ranging from 0.05 to 5.42 mg/kg. Nitrogen (50 kg/ha) in combination with boron (0.5 kg/ha) resulted in an increased fumonisin B2 production. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular characterization and fumonisin production by Fusarium verticillioides isolated from corn grains of different geographic origins in Brazil

Gibberella moniliformis is most commonly associated with maize worldwide and produces high levels of fumonisins, some of the most agriculturally important mycotoxins. Studies demonstrate that molecular methods can be helpful for a rapid identification of Fusarium species and their levels of toxin production. The purpose of this research was to apply molecular methods (AFLP, TEF-1 alpha partial gene sequencing and PCR based on MAT alleles) for the identification of Fusarium species isolated from Brazilian corn and to verify if real time RT-PCR technique based on FUM1 and FUM19 genes is appropriated to estimate fumonisins B(1) and B(2) production levels. Among the isolated strains, 96 were identified as Fusarium verricillioides, and four as other Fusarium species. Concordant phylogenies were obtained by AFLP and TEF-1 alpha sequencing, permitting the classification of the different species into distinct clades. Concerning MAT alleles, 70% of the F. verricillioides isolates carried the MAT-1 and 30% MAT-2. A significant correlation was observed between the expression of the genes and toxin production r=0.95 and r=0.79 (correlation of FUM1 with FB(1) and FB(2), respectively, P < 0.0001): r=0.93 and r =0.78 (correlation of FUM19 with FB(1) and FB(2). respectively, P < 0.0001). Molecular methods used in this study were found to be useful for the rapid identification of Fusarium species. The high and significant correlation between FUM1 and FUM19 expression and fumonisins production suggests that real time RT-PCR is suitable for studies considering the influence of abiotic and biotic factors on expression of these genes. This is the first report concerning the expression of fumonisin biosynthetic genes in Fusarium strains isolated from Brazilian agricultural commodity. (c) 2010 Elsevier B.V. All rights reserved.

Collagenase production and hemolytic activity related to 16S rRNA variability among Parvimonas micra oral isolates

Parvimonas micra are gram positive anaerobic cocci isolated from the oral cavity and frequently related to polymicrobial infections in humans. Despite reports about phenotypic differences, the genotypic variation of P. micra and its role in virulence are still not elucidated. The aim of this study was to determine the genotypic diversity of P. micra isolates obtained from the subgingival biofilm of subjects with different periodontal conditions and to correlate these findings with phenotypic traits. Three reference strains and 35 isolates of P. micro were genotyped by 16S rRNA PCR-RFLP and phenotypic traits such as collagenase production, elastolytic and hemolytic activities were evaluated. 16S rRNA PCR-RFLP showed that P. micra could be grouped into two main clusters: C1 and C2; cluster C1 harbored three genotypes (HG1259-like, HG1467-like and ICBM0583-like) while cluster C2 harbored two genotypes (ATC03270-like and ICBM036). A wide variability in collagenolytic activity intensities was observed among all isolates, while elastolytic activity was detected in only two isolates. There was an association between hemolytic activity in rabbit erythrocytes and cluster C2. There was an association between hemolytic activity in rabbit erythrocytes and cluster C1. Although these data suggest a possible association between P. micra genetic diversity and their pathogenic potential, further investigations are needed to confirm this hypothesis. (C) 2009 Elsevier Ltd. All rights reserved.

Effects of gamma radiation on the growth of Alternaria alternata and on the production of alternariol and alternariol monomethyl ether in sunflower seeds

The objective of the present study was to evaluate the effects of different gamma radiation doses on the growth of Alternaria alternata and on the production of toxins alternariol (AOH), and alternariol monomethyl ether (AME) in sunflower seed samples. After irradiation with 2, 5 and 7 kGy, the spore mass was resuspended in sterile distilled water and the suspension was inoculated into sunflower seeds. The number of colony-forming units per gram (CFU/g) was determined after culture on Dichloran Rose Bengal Chloramphenicol and Dichloran Chloramphenicol Malt Extract Agar. The presence of AOH and AME was investigated by liquid chromatography coupled to mass spectrometry. The radiation doses used resulted in a reduction of the number of A. alternata CFU/g and of AOH and AME levels when compared to the nonirradiated control group. Maximum reduction of the fungus (98.5%) and toxins (99.9%) was observed at a dose of 7 and 5 kGy, respectively. Under the present conditions, gamma radiation was found to be an alternative for the control of A. alternata and, consequently, of AOH and AME production in sunflower seeds. (C) 2009 Elsevier Ltd. All rights reserved.

Cloning and overexpression of the xylose isomerase gene from Burkholderia sacchari and production of polyhydroxybutyrate from xylose

A different organization for the xyl operon was found in different genomes of Burkholderia and Pseudomomas species. Degenerated primers were designed based on Burkholderia genomes and used to amplify the xylose isomerase gene (xylA) from Burkholderia sacchari IPT101 The gene encoded a protein of 329 amino acids, which showed the highest similarity (90%) to the homologous gene of Burkholderia dolosa. It was cloned in the broad host range plasmid pBBR1MCS-2, which partially restored growth and polyhydroxybutyrate production capability in xylose to a B. sacchari xyl(-) mutant. When xylA was overexpressed in the wild-type strain, it was not able to increase growth and polyhydroxybutyrate production, suggesting that XylA activity is not limiting for xylose utilization in B. sacchari.

Polyhydroxyalkanoates production by actinobacteria isolated from soil

Polyhydroxyalkanoates (PHAs) are biodegradable and renewable polymers produced by a wide range of bacterial groups. New microbial bioprospection approaches have become an important way to find new PHA producers and new synthesized polymers. Over the past years, bacteria belonging to actinomycetes group have become known as PHA producers, such as Nocardia and Rhodococcus species, Kineosphaera limosa Liu et a]. 2002, and, more recently, Streptomyces species. In this paper, we disclose that there are more actinobacteria PHA producers in addition to the genera cited. Some unusual genera, such as Streptoalloteichus, and some genera frequently present in soil, such as Streptacidiphilus, have been found. Thirty-four isolates were able to accumulate poly(3-hydroxybutyrate) and a number of these have traces of poly(3-hydroxyvalerate) when cultivated on glucose or glucose and casein as carbon source. Furthermore, some strains showed traces of medium chain length PHA. Transmission electron microscopy demonstrated that the PHA accumulation occurs in hyphae and spores.

Heme Impairs Prostaglandin E(2) and TGF-beta Production by Human Mononuclear Cells via Cu/Zn Superoxide Dismutase: Insight into the Pathogenesis of Severe Malaria

In many hemolytic disorders, such as malaria, the release of free heme has been involved in the triggering of oxidative stress and tissue damage. Patients presenting with severe forms of malaria commonly have impaired regulatory responses. Although intriguing, there is scarce data about the involvement of heme on the regulation of immune responses. In this study, we investigated the relation of free heme and the suppression of anti-inflammatory mediators such as PGE(2) and TGF-beta in human vivax malaria. Patients with severe disease presented higher hemolysis and higher plasma concentrations of Cu/Zn superoxide dismutase (SOD-1) and lower concentrations of PGE(2) and TGF-beta than those with mild disease. In addition, there was a positive correlation between SOD-1 concentrations and plasma levels of TNF-alpha. During antimalaria treatment, the concentrations of plasma SOD-1 reduced whereas PGE(2) and TGF-beta increased in the individuals severely ill. Using an in vitro model with human mononuclear cells, we demonstrated that the heme effect on the impairment of the production of PGE(2) and TGF-beta partially involves heme binding to CD14 and depends on the production of SOD-1. Aside from furthering the current knowledge about the pathogenesis of vivax malaria, the present results may represent a general mechanism for hemolytic diseases and could be useful for future studies of therapeutic approaches. The Journal of Immunology, 2010, 185: 1196-1204.

Use of L-Proline and ATP Production by Trypanosoma cruzi Metacyclic Forms as Requirements for Host Cell Invasion

The process of host cell invasion by Trypanosoma cruzi depends on parasite energy. What source of energy is used for that event is not known. To address this and other questions related to T. cruzi energy requirements and cell invasion, we analyzed metacyclic trypomastigote forms of the phylogenetically distant CL and G strains. For both strains, the nutritional stress experienced by cells starved for 24, 36, or 48 h in phosphate-buffered saline reduced the ATP content and the ability of the parasite to invade HeLa cells proportionally to the starvation time. Inhibition of ATP production by treating parasites with rotenone plus antimycin A also diminished the infectivity. Nutrient depletion did not alter the expression of gp82, the surface molecule that mediates CL strain internalization, but increased the expression of gp90, the negative regulator of cell invasion, in the G strain. When L-proline was given to metacyclic forms starved for 36 h, the ATP levels were restored to those of nonstarved controls for both strains. Glucose had no such effect, although this carbohydrate and L-proline were transported in similar fashions. Recovery of infectivity promoted by L-proline treatment of starved parasites was restricted to the CL strain. The profile of restoration of ATP content and gp82-mediated invasion capacity by L-proline treatment of starved Y-strain parasites was similar to that of the CL strain, whereas the Dm28 and Dm30 strains, whose infectivity is downregulated by gp90, behaved like the G strain. L-Proline was also found to increase the ability of the CL strain to traverse a gastric mucin layer, a property important for the establishment of T. cruzi infection by the oral route. Efficient translocation of parasites through gastric mucin toward the target epithelial cells in the stomach mucosa is an essential requirement for subsequent cell invasion. By relying on these closely associated ATP-driven processes, the metacyclic trypomastigotes effectively accomplish their internalization.

High frequency of immature dendritic cells and altered in situ production of interleukin-4 and tumor necrosis factor-alpha in lung cancer

Introduction Antigen-presenting cells, like dendritic cells (DCs) and macrophages, play a significant role in the induction of an immune response and an imbalance in the proportion of macrophages, immature and mature DCs within the tumor could affect significantly the immune response to cancer. DCs and macrophages can differentiate from monocytes, depending on the milieu, where cytokines, like interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce DC differentiation and tumor necrosis factor (TNF)-alpha induce DC maturation. Thus, the aim of this work was to analyze by immunohistochemistry the presence of DCs (S100+ or CD1a+), macrophages (CD68+), IL-4 and TNF-alpha within the microenvironment of primary lung carcinomas. Results Higher frequencies of both immature DCs and macrophages were detected in the tumor-affected lung, when compared to the non-affected lung. Also, TNF-alpha-positive cells were more frequent, while IL-4-positive cells were less frequent in neoplastic tissues. This decreased frequency of mature DCs within the tumor was further confirmed by the lower frequency of CD14-CD80+ cells in cell suspensions obtained from the same lung tissues analyzed by flow cytometry. Conclusion These data are discussed and interpreted as the result of an environment that does not oppose monocyte differentiation into DCs, but that could impair DC maturation, thus affecting the induction of effective immune responses against the tumor.

Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos

Myostatin is described as a negative regulator of the skeletal muscle growth. Genetic engineering, in order to produce animals with double the muscle mass and that can transmit the characteristic to future progeny, may be useful. In this context, the present study aimed to analyse the feasibility of lentiviral-mediated delivery of short hairpin RNA (shRNA) targeting of myostatin into in vitro produced transgenic bovine embryos. Lentiviral vectors were used to deliver a transgene that expressed green fluorescent protein (GFP) and an shRNA that targeted myostatin. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR. The lentiviral vector was microinjected into the perivitellinic space of in vitro matured oocytes. Non-microinjected oocytes were used as the control. After injection, oocytes were fertilized and cultured in vitro. Blastocysts were evaluated by epifluorescence microscopy. Results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells, as the transducted group had a less amount of myostatin mRNA after 72 h of differentiation (p < 0.05) and had less myotube formation than the non-transduced group (p < 0.05). There was no difference in cleavage and blastocyst rates between the microinjected and control groups. After hatching, 3.07% of the embryos exhibited GFP expression, indicating that they expressed shRNA targeting myostatin. In conclusion, we demonstrate that a lentiviral vector effectively performed shRNA myostatin gene knockdown and gene delivery into in vitro produced bovine embryos. Thus, this technique can be considered a novel option for the production of transgenic embryos and double muscle mass animals.

Chronic exercise decreases cytokine production in healthy rat skeletal muscle

Skeletal muscle is the source of pro- and anti-inflammatory cytokines, and recently, it has been recognized as an important source of interleukin-6 (IL-6). Acute physical exercise is known to induce a pro-inflammatory cytokine profile in the plasma. However, the effect of chronic physical exercise in the production of pro- and anti-inflammatory cytokines by the skeletal muscle has never been examined. We assessed IL-6, TNF-alpha, IL-1 beta and IL-10 levels in the skeletal muscle of rats submitted to endurance training. Animals were randomly assigned to either a Sedentary group (S, n = 7) or an endurance exercise trained group (T, n = 8). Trained rats ran on a treadmill for 5 days week(-1) for 8 weeks (60% VO(2max)). Detection of IL-6, TNF-alpha, IL-1 beta and IL-10 protein expression was carried out by ELISA. We found decreased expression of IL-1 beta, IL-6, TNF-alpha and IL-10 (28%, 27%. 32% and 37%, respectively, p < 0.05) in the extensor digital longus (EDL) from T, when compared with S. In the soleus, IL-1 beta, TNF-alpha and IL-10 protein levels were similarly decreased (34%, 42% and 50%, respectively, p < 0.05) in T in relation to S, while IL-6 expression was not affected by the training protocol. In conclusion, exercise training induced decreased cytokine protein expression in the skeletal muscle. These data show that in healthy rats, 8-week moderate-intensity aerobic training down regulates skeletal muscle production of cytokines involved in the onset, maintenance and regulation of inflammation, and that the response is heterogeneous according to fibre composition. Copyright (C) 2009 John Wiley & Sons, Ltd.

Infeasibility handling in genetic algorithm using nested domains for production planning

In this paper we present a genetic algorithm with new components to tackle capacitated lot sizing and scheduling problems with sequence dependent setups that appear in a wide range of industries, from soft drink bottling to food manufacturing. Finding a feasible solution to highly constrained problems is often a very difficult task. Various strategies have been applied to deal with infeasible solutions throughout the search. We propose a new scheme of classifying individuals based on nested domains to determine the solutions according to the level of infeasibility, which in our case represents bands of additional production hours (overtime). Within each band, individuals are just differentiated by their fitness function. As iterations are conducted, the widths of the bands are dynamically adjusted to improve the convergence of the individuals into the feasible domain. The numerical experiments on highly capacitated instances show the effectiveness of this computational tractable approach to guide the search toward the feasible domain. Our approach outperforms other state-of-the-art approaches and commercial solvers. (C) 2009 Elsevier Ltd. All rights reserved.