Efficient Bioconversion of High Concentration Phytosterol Microdispersion to 4-Androstene-3,17-Dione (AD) by Mycobacterium sp. B3805.

Low solubility of sterols in aqueous media limits efficient steroid production mediated by biocatalytic microorganisms such as Mycobacterium. Sterol emulsion technologies have been developed with low success rates, largely due to the complexity of generating stable and bioavailable particles. In this study, several aqueous dispersions of sterols in-water of different particle sizes were bioconverted to 4-androstene-3,17-dione (AD) in a solvent-free environment, using a classic microorganism Mycobacterium sp. B3805 as a model system. According to our results, the high concentration (20 g/L) phytosterol dispersions with the smallest particle size tested (370 nm) achieved up to 54% (7.4 g/L) AD production yield in 11 days. Moreover, the use of 0.1 biomass/sterols ratio in a complex bioconversion media containing yeast extract, and a 1:1 glucose/microdispersion ratio in the presence of the surfactant DK-Ester P-160 (HLB16), allowed homogenization and increased microdispersion stability, thus achieving the best results using emulsion technologies to date.

Production and Biotransformation of Phytosterol Microdispersions to Produce 4-Androstene-3,17-Dione.

The current state of knowledge regarding phytosterols biotransformation to produce the steroid intermediate 4-androstene-3,17-dione (AD) shows different technologies. However, the initial concentration of phytosterols in batch cultures is limited due to its low solubility in aqueous media, causing serious difficulties for scaling up because of the low mass transfer. In this chapter, we describe a fermentation method of a phytosterol microdispersion with Mycobacterium sp. B3805 in the context of an integral technology for AD production. The microdispersion generation is based on a patent application that claims the production of an aqueous phytosterol microdispersion with an average size particle of 370 nm, and high stability and solubility in water at high phytosterols concentrations (Harting et al., 2012/US0046254). Our results indicate that up to 20 g/L phytosterols can be biotransformed with this technology allowing a good dispersion and stability of reactants in the fermentation broth.

Polarity protein Par3 controls B-cell receptor dynamics and antigen extraction at the immune synapse.

B-cell receptor (BCR) engagement with surface-tethered antigens leads to the formation of an immune synapse, which facilitates antigen uptake for presentation to T-lymphocytes. Antigen internalization and processing rely on the early dynein-dependent transport of BCR-antigen microclusters to the synapse center, as well as on the later polarization of the microtubule-organizing center (MTOC). MTOC repositioning allows the release of proteases and the delivery of MHC class II molecules at the synapse. Whether and how these events are coordinated have not been addressed. Here we show that the ancestral polarity protein Par3 promotes BCR-antigen microcluster gathering, as well as MTOC polarization and lysosome exocytosis, at the synapse by facilitating local dynein recruitment. Par3 is also required for antigen presentation to T-lymphocytes. Par3 therefore emerges as a key molecule in the coupling of the early and late events needed for efficient extraction and processing of immobilized antigen by B-cells.

Cardiac-oxidized antigens are targets of immune recognition by antibodies and potential molecular determinants in chagas disease pathogenesis.

Trypanosoma cruzi elicits reactive oxygen species (ROS) of inflammatory and mitochondrial origin in infected hosts. In this study, we examined ROS-induced oxidative modifications in the heart and determined whether the resultant oxidized cardiac proteins are targets of immune response and of pathological significance in Chagas disease. Heart biopsies from chagasic mice, rats and human patients exhibited, when compared to those from normal controls, a substantial increase in protein 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), carbonyl, and 3-nitrotyrosine (3-NT) adducts. To evaluate whether oxidized proteins gain antigenic properties, heart homogenates or isolated cardiomyocytes were oxidized in vitro and one- or two-dimensional gel electrophoresis (2D-GE)/Western blotting (WB) was performed to investigate the proteomic oxidative changes and recognition of oxidized proteins by sera antibodies in chagasic rodents (mice, rats) and human patients. Human cardiomyocytes exhibited LD(50) sensitivity to 30 µM 4-HNE and 100 µM H(2)O(2) at 6 h and 12 h, respectively. In vitro oxidation with 4-HNE or H(2)O(2) resulted in a substantial increase in 4-HNE- and carbonyl-modified proteins that correlated with increased recognition of cardiac (cardiomyocytes) proteins by sera antibodies of chagasic rodents and human patients. 2D-GE/Western blotting followed by MALDI-TOF-MS/MS analysis to identify cardiac proteins that were oxidized and recognized by human chagasic sera yielded 82 unique proteins. We validated the 2D-GE results by enzyme-linked immunosorbent assay (ELISA) and WB and demonstrated that oxidation of recombinant titin enhanced its immunogenicity and recognition by sera antibodies from chagasic hosts (rats and humans). Treatment of infected rats with phenyl-α-tert-butyl nitrone (PBN, antioxidant) resulted in normalized immune detection of cardiac proteins associated with control of cardiac pathology and preservation of heart contractile function in chagasic rats. We conclude that ROS-induced, cardiac-oxidized antigens are targets of immune recognition by antibodies and molecular determinants for pathogenesis during Chagas disease.

Defects of mtDNA replication impaired mitochondrial biogenesis during Trypanosoma cruzi infection in human cardiomyocytes and chagasic patients: the role of Nrf1/2 and antioxidant response.

BACKGROUND: Mitochondrial dysfunction is a key determinant in chagasic cardiomyopathy development in mice; however, its relevance in human Chagas disease is not known. We determined if defects in mitochondrial biogenesis and dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1)-regulated transcriptional pathways constitute a mechanism or mechanisms underlying mitochondrial oxidative-phosphorylation (OXPHOS) deficiency in human Chagas disease. METHODS AND RESULTS: We utilized human cardiomyocytes and left-ventricular tissue from chagasic and other cardiomyopathy patients and healthy donors (n>6/group). We noted no change in citrate synthase activity, yet mRNA and/or protein levels of subunits of the respiratory complexes were significantly decreased in Trypanosoma cruzi-infected cardiomyocytes (0 to 24 hours) and chagasic hearts. We observed increased mRNA and decreased nuclear localization of PGC-1-coactivated transcription factors, yet the expression of genes for PPARγ-regulated fatty acid oxidation and nuclear respiratory factor (NRF1/2)-regulated mtDNA replication and transcription machinery was enhanced in infected cardiomyocytes and chagasic hearts. The D-loop formation was normal or higher, but mtDNA replication and mtDNA content were decreased by 83% and 40% to 65%, respectively. Subsequently, we noted that reactive oxygen species (ROS), oxidative stress, and mtDNA oxidation were significantly increased, yet NRF1/2-regulated antioxidant gene expression remained compromised in infected cardiomyocytes and chagasic hearts. CONCLUSIONS: The replication of mtDNA was severely compromised, resulting in a significant loss of mtDNA and expression of OXPHOS genes in T cruzi-infected cardiomyocytes and chagasic hearts. Our data suggest increased ROS generation and selective functional incapacity of NRF2-mediated antioxidant gene expression played a role in the defects in mtDNA replication and unfitness of mtDNA for replication and gene expression in Chagas disease.

Hydroquinone and HO differentially affect the ultrastructure and expression of ligninolytic genes in the basidiomycete Ceriporiopsis subvermispora.

The biodegradation of lignin is a highly oxidative process in which various oxidases and peroxidases play a major role. During lignin decay, the generation of aromatic compounds and reactive oxygen species leads to oxidative stress. In this work, the effect of the oxidative compounds H(2)O(2) and hydroquinone in the ligninolytic fungus Ceriporiopsis subvermispora was studied, both at the ultrastructural and at the transcriptional level. Transmission electron microscopy revealed the presence of microvesicles and extensive cytoplasm degeneration after incubation with hydroquinone, but not with H(2)O(2). Studies of the intracellular redox state of the fungus showed that hydroquinone causes a transient decrease in the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and an increase in the glutathione-S-transferase mRNA levels. These results suggest that hydroquinone produces oxidative stress in this microorganism. On the other hand, it was observed that hydroquinone, but not H(2)O(2), affects Mn-dependent peroxide and laccase transcripts levels. We propose that the mechanism by which the fungus reacts against oxidative stress contributes to its selectivity toward lignin during wood decay.