Impact of phenylalanine on Hanseniaspora vineae aroma metabolism during wine fermentation.

Hanseniaspora vineae exhibits extraordinary positive oenological characteristics contributing to the aroma and texture of wines, especially by its ability to produce great concentrations of benzenoid and phenylpropanoid compounds compared with conventional Saccharomyces yeasts. Consequently, in practice, sequential inoculation of H. vineae and Saccharomyces cerevisiae allows to improve the aromatic quality of wines. In this work, we evaluated the impact on wine aroma produced by increasing the concentration of phenylalanine, the main amino acid precursor of phenylpropanoids and benzenoids. Fermentations were carried out using a Chardonnay grape juice containing 150 mg N/L yeast assimilable nitrogen. Fermentations were performed adding 60 mg/L of phenylalanine without any supplementary addition to the juice. Musts were inoculated sequentially using three different H. vineae strains isolated from Uruguayan vineyards and, after 96 h, S. cerevisiae was inoculated to complete the process. At the end of the fermentation, wine aromas were analysed by both gas chromatography-mass spectrometry and sensory evaluation through a panel of experts. Aromas derived from aromatic amino acids were differentially produced depending on the treatments. Sensory analysis revealed more floral character and greater aromatic complexity when compared with control fermentations without phenylalanine added. Moreover, fermentations performed in synthetic must with pure H. vineae revealed that even tyrosine can be used in absence of phenylalanine, and phenylalanine is not used by this yeast for the synthesis of tyrosine derivatives.

Exploring the effects of the fermentation method on the quality of Lycium barbarum and Polygonatum cyrtonema compound wine based on LC-MS metabolomics.

This study aimed to examine the effect of fermentation methods on the quality of Lycium barbarum and Polygonatum cyrtonema compound wine (LPW) by combining non-targeted metabolomic approaches with chemometrics and path profiling to determine the chemical and metabolic properties of LPW. The results demonstrated that SRA had higher leaching rates of total phenols and flavonoids, reaching 4.20 ± 0.10 v/v ethanol concentration. According to LC-MS non-targeting genomics, the metabolic profiles of LPW prepared by different mixtures of fermentation methods (Saccharomyces cerevisiae RW; Debaryomyces hansenii AS2.45) of yeast differed significantly. Amino acids, phenylpropanoids, flavonols, etc., were identified as the differential metabolites between different comparison groups. The pathways of tyrosine metabolism, biosynthesis of phenylpropanoids, and metabolism of 2-oxocarboxylic acids enriched 17 distinct metabolites. SRA stimulated the production of tyrosine and imparted a distinctive saucy aroma to the wine samples, providing a novel research concept for the microbial fermentation-based production of tyrosine.

M. leprae HSP18 suppresses copper (II) mediated ROS generation: Effect of redox stress on its structure and function.

Mycobacterium leprae, causative organism of leprosy, is known to counter redox stress generated by reactive oxygen species (ROS) during its survival inside host macrophages. But, the involvement of any antigenic protein(s) for countering such redox stress is still unknown. Interestingly, M. leprae HSP18, an important antigenic protein that helps in the growth and survival of M. leprae pathogen inside host macrophages, is induced under redox stress. Moreover, HSP18 also interacts with Cu2+. Copper (II) can induce redox stress via Fenton reaction. But, whether HSP18 suppresses Cu2+ mediated ROS generation, is still far from clear. Also, the effect of redox stress on its structure and function is not known. In this study, we show that HSP18 efficiently suppresses Cu2+ mediated generation of ROS and also prevents the redox mediated aggregation of a client protein (γD-crystallin). Upon exposure to substantial redox stress, irreversible perturbation in the secondary and tertiary structure of HSP18 and the tryptophan and tyrosine oxidation are evidenced. Interestingly, HSP18 retains a considerable amount of functionality even after being exposed to substantial redox stress. Perhaps, the redox scavenging ability as well as the chaperone function of HSP18 may possibly help M. leprae pathogen to counter redox stress inside host macrophages.

Putative 3-nitrotyrosine detoxifying genes identified in the yeast Debaryomyces hansenii: in silico search of regulatory sequences responsive to salt and nitrogen stress

Background: During salt stress, the yeast Debaryomyces hansenii synthesizes tyrosine as a strategy to avoid the oxidation of proteins. Tyrosine reacts with nitrogen radicals to form 3-nitrotyrosine. 3-nitrotyrosine prevents the effects of associated oxidative stress and thus contributes to the high halotolerace of the yeast. However, the mechanism of how D. hansenii counteracts the presence of this toxic compound is unclear. In this work, we evaluated D. hansenii's capacity to assimilate 3-nitrotyrosine as a unique nitrogen source and measured its denitrase activity under salt stress. To identify putative genes related to the assimilation of 3-nitrotyrosine, we performed an in silico search in the promoter regions of D. hansenii genome. Results: We identified 15 genes whose promoters had binding site sequences for transcriptional factors of sodium, nitrogen, and oxidative stress with oxidoreductase and monooxygenase GO annotations. Two of these genes, DEHA2E24178g and DEHA2C00286g, coding for putative denitrases and having GATA sequences, were evaluated by RT-PCR and showed high expression under salt and nitrogen stress. Conclusions: D. hansenii can grow in the presence of 3-nitrotyrosine as the only nitrogen source and has a high specific denitrase activity to degrade 3-nitrotyrosine in 1 and 2 M NaCl stress conditions. The results suggest that given the lack of information on transcriptional factors in D. hansenii, the genes identified in our in silico analysis may help explain 3-nitrotyrosine assimilation mechanisms.

Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction.

Controlled mechanical ventilation (CMV) is associated with the development of diaphragm atrophy and contractile dysfunction, and respiratory muscle weakness is thought to contribute significantly to delayed weaning of patients. Therefore, therapeutic strategies for preventing these processes may have clinical benefit. The aim of the current study was to investigate the role of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in CMV-mediated diaphragm wasting and weakness in rats. CMV-induced diaphragm atrophy and contractile dysfunction coincided with marked increases in STAT3 phosphorylation on both tyrosine 705 (Tyr705) and serine 727 (Ser727). STAT3 activation was accompanied by its translocation into mitochondria within diaphragm muscle and mitochondrial dysfunction. Inhibition of JAK signaling during CMV prevented phosphorylation of both target sites on STAT3, eliminated the accumulation of phosphorylated STAT3 within the mitochondria, and reversed the pathologic alterations in mitochondrial function, reduced oxidative stress in the diaphragm, and maintained normal diaphragm contractility. In addition, JAK inhibition during CMV blunted the activation of key proteolytic pathways in the diaphragm, as well as diaphragm atrophy. These findings implicate JAK/STAT3 signaling in the development of diaphragm muscle atrophy and dysfunction during CMV and suggest that the delayed extubation times associated with CMV can be prevented by inhibition of Janus kinase signaling.-Smith, I. J., Godinez, G. L., Singh, B. K., McCaughey, K. M., Alcantara, R. R., Gururaja, T., Ho, M. S., Nguyen, H. N., Friera, A. M., White, K. A., McLaughlin, J. R., Hansen, D., Romero, J. M., Baltgalvis, K. A., Claypool, M. D., Li, W., Lang, W., Yam, G. C., Gelman, M. S., Ding, R., Yung, S. L., Creger, D. P., Chen, Y., Singh, R., Smuder, A. J., Wiggs, M. P., Kwon, O.-S., Sollanek, K. J., Powers, S. K., Masuda, E. S., Taylor, V. C., Payan, D. G., Kinoshita, T., Kinsella, T. M. Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction.

DhARO4 induction and tyrosine nitration in response to reactive radicals generated by salt stress in Debaryomyces hansenii.

It has been previously reported that growth of Debaryomyces hansenii in 2 M NaCl induced the expression of ARO4. This gene codifies for DhAro4p, involved in the synthesis of the amino acid tyrosine. In this work we studied the activity of DhAro4p upon salt stress; a higher activity was observed in cells grown with 2 M NaCl, but tyrosine levels were not increased. On the other hand, the addition of tyrosine to the saline medium significantly enhanced the growth of D. hansenii. It was found that the oxidized form of tyrosine, 3-nitrotyrosine, increased in the presence of salt. Since NaCl protects against oxidative stress in D. hansenii (Navarrete et al., 2009), we propose that a protective pathway is the de novo synthesis of tyrosine and its immediate oxidation to 3-nitrotyrosine to counteract oxidative stress generated by salt stress, so we measured the production of reactive oxygen species (ROS) and nitric oxide (NO⁻) in D. hansenii after growing in 2 M NaCl. Results showed the presence of NO⁻ and the increased production of ROS; this is probably due to an increased respiratory activity in the cells grown in the presence of salt. Our results demonstrate that upon salt stress D hansenii responds to oxidative stress via the transcriptional activation of specific genes such as DhARO4.

Adeno-associated virus type 2-mediated gene transfer: role of cellular T-cell protein tyrosine phosphatase in transgene expression in established cell lines in vitro and transgenic mice in vivo.

The use of adeno-associated virus type 2 (AAV) vectors has gained attention as a potentially useful alternative to the more commonly used retrovirus and adenovirus vectors for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein (FKBP52), interacts with the single-stranded D sequence within the AAV inverted terminal repeats, inhibits viral second-strand DNA synthesis, and consequently limits high-efficiency transgene expression (K. Qing, J. Hansen, K. A. Weigel-Kelley, M. Tan, S. Zhou, and A. Srivastava, J. Virol., 75: 8968-8976, 2001). FKBP52 can be phosphorylated at both tyrosine and serine/threonine residues, but only the phosphorylated forms of FKBP52 interact with the D sequence. Furthermore, the tyrosine-phosphorylated FKBP52 inhibits AAV second-strand DNA synthesis by greater than 90%, and the serine/threonine-phosphorylated FKBP52 causes approximately 40% inhibition, whereas the dephosphorylated FKBP52 has no effect on AAV second-strand DNA synthesis. In the present study, we have identified that the tyrosine-phosphorylated form of FKBP52 is a substrate for the cellular T-cell protein tyrosine phosphatase (TC-PTP). Deliberate overexpression of the murine wild-type (wt) TC-PTP gene, but not that of a cysteine-to-serine (C-S) mutant, caused tyrosine dephosphorylation of FKBP52, leading to efficient viral second-strand DNA synthesis and resulting in a significant increase in AAV-mediated transduction efficiency in HeLa cells in vitro. Both wt and C-S mutant TC-PTP expression cassettes were also used to generate transgenic mice. Primitive hematopoietic stem/progenitor cells from wt TC-PTP-transgenic mice, but not from C-S mutant TC-PTP-transgenic mice, could be successfully transduced by recombinant AAV vectors. These studies corroborate the fact that tyrosine phosphorylation of the cellular FKBP52 protein strongly influences AAV transduction efficiency, which may have important implications in the optimal use of AAV vectors in human gene therapy.

Expression of inducible nitric oxide synthase and nitrotyrosine in borderline leprosy lesions.

BACKGROUND: In the response to T-helper cell (Th1)-type cytokines and interactions with pathogens, high levels of nitric oxide (NO) are produced by activated macrophages expressing the inducible NO synthase (iNOS). The role and importance of reactive nitrogen intermediates (RNIs) such as NO and peroxynitrite in the host response to diseases caused by intracellular pathogens such as Mycobacterium leprae and M. tuberculosis is unclear. OBJECTIVES: The aim of this study was to investigate the presence of local production of NO and peroxynitrite in borderline leprosy by using antibodies against iNOS and the product of peroxynitrite, nitrotyrosine (NT). METHODS: We detected the presence of iNOS and NT in skin biopsies from borderline leprosy patients, with and without reversal reaction (RR), by immunohistochemistry (n = 26). RESULTS: In general, the granulomas from borderline leprosy lesions with and without RR showed high and specific expression of iNOS and NT. Moreover, strong immunoreactivity to iNOS and NT was observed in granulomas surrounding and infiltrating dermal nerves. The expression of iNOS and NT was also strong in keratinocytes, fibroblasts and endothelial cells in close relation to the granulomatous reaction. In contrast, normal human skin showed no expression of iNOS and NT in these cells. CONCLUSIONS: We conclude that iNOS and NT are expressed in granulomas from borderline leprosy patients with and without RR and propose that RNIs might be involved in the nerve damage following RR in leprosy.

Intracellular signals triggered during association of Mycobacterium leprae and Mycobacterium bovis BCG with human monocytes.

To gain a better understanding of mycobacteria-host cell interaction, the present study compared the signal transduction events triggered during the interaction of Mycobacterium leprae (the causative agent of leprosy) and of Mycobacterium bovis BCG (an attenuated strain used as a vaccine against leprosy and tuberculosis) with human monocytes. The assays consisted of pre-treating or not THP-1 cells (a human monocytic cell line) with different kinase inhibitors, followed by incubation with fluorescein-labelled bacteria and analysis of bacterial association via fluorescence microscopy. The specific tyrosine kinase (TK) inhibitor tyrphostin AG126 provided the highest rates of association inhibition (>90% for BCG and >65% for M. leprae). The early activation of TKs during mycobacteria-host cell interaction was confirmed by immunoblot analysis, demonstrating that in several host cell proteins mycobacteria stimulated tyrosine phosphorylation. The use of the drugs wortmannin and bisindolylmaleimide I which, respectively, inhibit phosphatidylinositide 3-kinase (PI 3-kinase) and protein kinase C (PKC), produced lower but consistent results within a 35--60% association inhibition range for both bacteria. Dose response curves with these inhibitors were obtained. Similar results were obtained when primary human monocytes were used as host cells, strongly suggesting that TK, PKC and PI 3-kinase signals are activated during the interaction of human monocytes with both pathogenic and attenuated species of mycobacteria.

Pigment producing yeasts involved in the brown surface discoloration of ewes' cheese.

The appearance of a brown surface discoloration on Portuguese ewes' cheese has never previously been reported on. The regular occurrence of this defect over the past few years has caused serious financial losses to producers, which has led to growing interest in its study. This paper describes a preliminary approach to the problem, based on the hypothesis that pigment producing yeasts are involved. From a group of 51 yeast strains isolated from a number of brown cheese rinds, it was possible to distinguish four pigment producing groups: group I (12 strains), produced an extracellular brown pigment from tyrosine and alkalised the tested media; group II (21 strains), produced a diffusible, reddish-brown pigment from resorcinol and alkalised the tested media; group III (three strains), alkalised the tested media without producing any pigments; group IV (15 strains), neither produced pigments nor alkalised the media. Yarrowia lipolytica and Candida catenulata type strains were also tested and their behaviour was similar to the strains in groups I and IV, respectively. The Filobasidiella neoformans type strain was distinct from all the other groups. The identification methods used for some strains in groups I, II and III suggest that Yarrowia lipolytica species is common to all strains in group I, and that Debaryomyces hansenii is present in both groups II and III. A study of the effect of several metal ions on the production of the brown pigment from tyrosine indicated Mn2+ to be a strong activator. Evidence is provided suggesting that the browning process may be related to tyrosine Yarrowia lipolytica metabolism.

Unusual effects of reducing agents on 0-diphenoloxidase of Mycobacterium leprae.

Reducing agents had no effect on the oxidation of 3,4-dihydroxyphenylalanine (DOPA) to quinone by Mycobacterium leprae; no quinone formation by o-diphenoloxidase of mammalian or plant origin was detected under similar experimental conditions. Ascorbic acid and reduced glutathione prevented further oxidation and polymerization of the quinone to melanin by M. leprae; cysteine was less effective. In the presence of reducing agents, the quinone (indole-5,6-quinone) formed from DOPA by M. leprae was not reduced back to diphenol. On the other hand, the quinone (dopachrome) produced from DOPA by mammalian or plant phenolase was rapidly decolorized by reducing agents. Oxidized glutathione and cystine had little effect on o-diphenoloxidase from all of the three sources. Cyanide, which completely inhibited mammalian and plant phenolases, had only a partial effect on the enzyme in the bacilli. Various lines of evidence suggest that the properties of o-diphenoloxidase in M. leprae are different from those of similar enzymes obtained from other sources.