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.

Hormonal modulation of photomorphogenesis-controlled anthocyanin accumulation in tomato (Solanum lycopersicum L. cv Micro-Tom) hypocotyls: Physiological and genetic studies

Hormones are likely to be important factors modulating the light-dependent anthocyanin accumulation. Here we analyzed anthocyanin contents in hypocotyls of near isogenic Micro-Tom (MT) tomato lines carrying hormone and phytochrome mutations, as single and double-mutant combinations. In order to recapitulate mutant phenotype, exogenous hormone applications were also performed Anthocyanin accumulation was promoted by exogenous abscisic acid (ABA) and inhibited by gibberellin (GA), in accordance to the reduced anthocyanin contents measured in ABA-deficient (notabills) and GA-constitutive response (procera) mutants. Exogenous cytokinin also enhanced anthocyanin levels in MT hypocotyls. Although auxin-insensitive chageotropica mutant exhibited higher anthocyanin contents, pharmacological approaches employing exogenous auxin and a transport inhibitor did not support a direct role of the hormone in anthocyanin accumulation Analysis of mutants exhibiting increased ethylene production (epwastic) or reduced sensitivity (Never ripe), together with pharmacological data obtained from plants treated with the hormone, indicated a limited role for ethylene in anthocyanin contents. Phytochrome-deficiency (aurea) and hormone double-mutant combinations exhibited phenotypes suggesting additive or synergistic interactions, but not fully espistatic ones, in the control of anthocyanin levels in tomato hypocotyls. Our results indicate that phytochrome-mediated anthocyanin accumulation in tomato hypocotyls is modulated by distinct hormone classes via both shared and independent pathways. (C) 2010 Elsevier Ireland Ltd. All rights reserved

Brassinosteroids interact negatively with jasmonates in the formation of anti-herbivory traits in tomato

Given the susceptibility of tomato plants to pests, the aim of the present study was to understand how hormones are involved in the formation of tomato natural defences against insect herbivory. Tomato hormone mutants, previously introgressed into the same genetic background of reference, were screened for alterations in trichome densities and allelochemical content. Ethylene, gibberellin, and auxin mutants indirectly showed alteration in trichome density, through effects on epidermal cell area. However, brassinosteroids (BRs) and jasmonates (JAs) directly affected trichome density and allelochemical content, and in an opposite fashion. The BR-deficient mutant dpy showed enhanced pubescence, zingiberene biosynthesis, and proteinase inhibitor expression; the opposite was observed for the JA-insensitive jai1-1 mutant. The dpyxjai1-1 double mutant showed that jai1-1 is epistatic to dpy, indicating that BR acts upstream of the JA signalling pathway. Herbivory tests with the poliphagous insect Spodoptera frugiperda and the tomato pest Tuta absoluta clearly confirmed the importance of the JA-BR interaction in defence against herbivory. The study underscores the importance of hormonal interactions on relevant agricultural traits and raises a novel biological mechanism in tomato that may differ from the BR and JA interaction already suggested for Arabidopsis.

Probing the hormonal activity of fractionated molecular humic components in tomato auxin mutants

We progressively reduced the complexity of humic matter by a mild sequential removal of unbound or free components, weakly, and strongly bound molecules. The auxin-like response of residues from each step was tested using tomato (cv. Micro-Tom) seedlings expressing DR5 auxin synthetic promoter fused to the beta-glucuronidase (GUS) reporter gene and the low auxin-sensitivity diageotropica (dgt) mutant. Both exogenous auxin and humic matter promoted lateral root emergence in the control, but failed to induce lateral roots in the dgt mutant. When strongly bound components were removed from humic matter by breaking the ester and ether bonds, the humic residues lost their ability to induce the DR5

Cloning, expression, molecular modelling and docking analysis of glutathione transferase from Saccharum officinarum

Sugarcane yield and quality are affected by a number of biotic and abiotic stresses. In response to such stresses, plants may increase the activities of some enzymes such as glutathione transferase (GST), which are involved in the detoxification of xenobiotics. Thus, a sugarcane GST was modelled and molecular docked using the program LIGIN to investigate the contributions of the active site residues towards the binding of reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). As a result, W13 and I119 were identified as key residues for the specificity of sugarcane GSTF1 (SoGSTF1) towards CDNB. To obtain a better understanding of the catalytic specificity of sugarcane GST (SoGSTF1), two mutants were designed, W13L and I119F. Tertiary structure models and the same docking procedure were performed to explain the interactions between sugarcane GSTs with GSH and CDNB. An electron-sharing network for GSH interaction was also proposed. The SoGSTF1 and the mutated gene constructions were cloned and expressed in Escherichia coli and the expressed protein purified. Kinetic analyses revealed different Km values not only for CDNB, but also for GSH. The Km values were 0.2, 1.3 and 0.3 mM for GSH, and 0.9, 1.2 and 0.5 mM for CDNB, for the wild type, W13L mutant and I119F mutant, respectively. The V(max) values were 297.6, 224.5 and 171.8 mu mol min(-1) mg(-1) protein for GSH, and 372.3, 170.6 and 160.4 mu mol min(-1) mg(-1) protein for CDNB.

Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic beta-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP`s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable beta-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.

TonB-dependent outer-membrane proteins and siderophore utilization in Pseudomonas fluorescens Pf-5

The soil bacterium Pseudomonas fluorescens Pf-5 produces two siderophores, a pyoverdine and enantio-pyochelin, and its proteome includes 45 TonB-dependent outer-membrane proteins, which commonly function in uptake of siderophores and other substrates from the environment. The 45 proteins share the conserved beta-barrel and plug domains of TonB-dependent proteins but only 18 of them have an N-terminal signaling domain characteristic of TonB-dependent transducers (TBDTs), which participate in cell-surface signaling systems. Phylogenetic analyses of the 18 TBDTs and 27 TonB-dependent receptors (TBDRs), which lack the N-terminal signaling domain, suggest a complex evolutionary history including horizontal transfer among different microbial lineages. Putative functions were assigned to certain TBDRs and TBDTs in clades including well-characterized orthologs from other Pseudomonas spp. A mutant of Pf-5 with deletions in pyoverdine and enantio-pyochelin biosynthesis genes was constructed and characterized for iron-limited growth and utilization of a spectrum of siderophores. The mutant could utilize as iron sources a large number of pyoverdines with diverse structures as well as ferric citrate, heme, and the siderophores ferrichrome, ferrioxamine B, enterobactin, and aerobactin. The diversity and complexity of the TBDTs and TBDRs with roles in iron uptake clearly indicate the importance of iron in the fitness and survival of Pf-5 in the environment.

Capacity of the extracellular matrix of the bone marrow to induce proliferation of myeloid cells in vitro in model of protein malnutrition in mice

The aim of this study was to verify the capacity of the extracellular matrix (ECM) obtained from bone marrow of malnourished mice to sustain survival and to induce the proliferation of myeloid cells. We also verified the capacity of the tests to interact with in vitro hematopoietic cytokines. Male ""Swiss"" mice were submitted to protein malnutrition with a diet contents of 4% casein until they lost 20% of the original weight, while the group-control was kept with a diet content of 14% of casein. The bone marrow was extracted with 1.0 mg of aprotinin/mL in PBS. The proliferation tests were carried out with myeloid cell line FDCP-1, by the colorimetric method of reduction of the MTT. The obtained ECM from nourished and undernourished mice induced cellular proliferation in vitro. Tests performed with Il-3 and GM-CSF cytokines in a concentration of 10 and 500 rho g/mL displayed synergic and regulatory effects respectively. The ECM obtained from the malnourished group submitted to the binding to GM-CSF demonstrated higher cellular proliferation than the ECM obtained from the control group (p<0.05). The results suggest that the alterations in the composition of ECM of bone marrow caused by malnutrition might lead to modification of the GM-CSF activity modulation.

Screening of Variables Influencing the Clavulanic Acid Production by Streptomyces DAUFPE 3060 Strain

Clavulanic acid (CA) is a beta-lactam antibiotic, which has a potent beta-lactamase inhibiting activity. The influence of five variables, namely pH (6.0, 6.4, and 6.8), temperature (28A degrees C, 30A degrees C, and 32A degrees C), agitation intensity (150, 200, and 250 rpm), glycerol concentration (5.0, 7.5, and 10 g/L) and soybean flour concentration (5.0, 12.5, and 20 g/L), on CA production by a new isolate of Streptomyces (DAUFPE 3060) was investigated in 250-mL Erlenmeyer flasks using a fractional factorial design. Temperature and soybean flour concentration were shown to be the two variables that exerted the most important effects on the production of CA at 95% confidence level. The highest CA concentration (494 mg/L) was obtained after 48 h at 150 rpm, 32A degrees C, pH 6.0, 5.0 g/L glycerol, and 20 g/L soybean flour concentrations. Under these conditions, the yields of biomass and product on consumed substrate were 0.26 g(X)/g(S) and 64.3 mg(P)/g(S), respectively. Fermentations performed in 3.0-L bench-scale fermenter allowed increasing the CA production by about 60%.

The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis

p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras(C118S)) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-inserisitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG. (C) 2008 Elsevier Inc. All rights reserved.

In vitro characterization of rosiglitazone metabolites and determination of the kinetic parameters employing rat liver microsomal fraction

Rosiglitazone (RSG), a thiazolidinedione antidiabetic drug, is metabolized by CYP450 enzymes into two main metabolites: N-desmethyl rosiglitazone (N-Dm-R) and rho-hydroxy rosiglitazone (rho-OH-R). In humans, CYP2C8 appears to have a major role in RSG metabolism. On the other hand, the in vitro metabolism of RSG in animals has not been described in literature yet. Based on these concerns, the kinetic metabolism study of RSG using rat liver microsomal fraction is described for the first time. Maximum velocity (V (max)) values of 87.29 and 51.09 nmol/min/mg protein were observed for N-Dm-R and rho-OH-R, respectively. Michaelis-Menten constant (K (m)) values were of 58.12 and 78.52 mu M for N-Dm-R and rho-OH-R, respectively. Therefore, these results demonstrated that this in vitro metabolism model presents the capacity of forming higher levels of N-Dm-R than of rho-OH-R, which also happens in humans. Three other metabolites were identified employing mass spectrometry detection under positive electrospray ionization: ortho-hydroxy-rosiglitazone (omicron-OH-R) and two isomers of N-desmethyl hydroxy-rosiglitazone. These metabolites have also been observed in humans. The results observed in this study indicate that rats could be a satisfactory model for RSG metabolism.

Molecular characterization of the Aspergillus fumigatus NCS-1 homologue, NcsA

Here, we characterize the Aspergillus homologue ncsA Neuronal Calcium Sensor. We that ncsA is not an essential gene and Delta ncsA growth decreased in the presence of EGTA and SDS. the Delta ncsA mutant is more resistant to calcium NcsA: mRFP localizes to the cytoplasm and its localization is not affected by the cellular response to calcium chloride or EGTA. The Delta ncsA mutant strain more sensitive to voriconazole, itraconazole, and Polar growth in the Delta ncsA mutant was also more aVected by lovastatin than in the wild type The Spitzenkorper can be visualized in both strains although the vacuolar system does not seem to be very different, there is an increase in the staining intensity on the germling surface of the Delta ncsA strain. NcsA promotes pmcA and pmcB expression and therefore there is a reduced expression of these ion pumps in the Delta ncsA mutant background, and also of other genes involved in the response to calcium in A. fumigatus. The ncsA inactivation mutation is not causing loss of virulence in a low dose murine infection when compared to the corresponding wild type strain.

Farnesol induces the transcriptional accumulation of the Aspergillus nidulans Apoptosis-Inducing Factor (AIF)-like mitochondrial oxidoreductase

Farnesol (FOH) is a non-sterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, a catabolite of the cholesterol biosynthetic pathway. These isoprenoids inhibit proliferation and induce apoptosis. It has been shown previously that FOH triggers morphological features characteristic of apoptosis in the filamentous fungus Aspergillus nidulans. Here, we investigate which pathways are influenced through FOH by examining the transcriptional profile of A. nidulans exposed to this isoprenoid. We observed decreased mRNA abundance of several genes involved in RNA processing and modification, transcription, translation, ribosomal structure and biogenesis, amino acid transport and metabolism, and ergosterol biosynthesis. We also observed increased mRNA expression of genes encoding a number of mitochondrial proteins and characterized in detail one of them, the aifA, encoding the Apoptosis-Inducing Factor (AIF)-like mitochondrial oxidoreductase. The Delta aifA mutant is more sensitive to FOH (about 8.0% and 0% survival when exposed to 10 and 100 mu M FOH respectively) than the wild type (about 97% and 3% survival when exposed to 10 and 100 mu M FOH respectively). These results suggest that AifA is possibly important for decreasing the effects of FOH and reactive oxygen species. Furthermore, we showed an involvement of autophagy and protein kinase C in A. nidulans FOH-induced apoptosis.

The effect of the dibenzylbutyrolactolic lignan (-)-cubebin on doxorubicin mutagenicity and recombinogenicity in wing somatic cells of Drosophila melanogaster

The dibenzylbutyrolactolic lignan (-)-cubebin was isolated from dry seeds of Piper cubeba L (Piperaceae). (-)-Cubebin possesses anti-inflammatory, analgesic and antimicrobial activities. Doxorubicin (DXR) is a topoisomerase-interactive agent that may induce single- and double-strand breaks, intercalate into the DNA and generate oxygen free radicals. Here, we examine the mutagenicity and recombinogenicity of different concentrations of (-)-cubebin alone or in combination with DXR using standard (ST) and high bioactivation (HB) crosses of the wing Somatic Mutation And Recombination Test in Drosophila melanogaster. The results from both crosses were rather similar. (-)-Cubebin alone did not induce mutation or recombination. At lower concentrations, (-)-cubebin statistically reduced the frequencies of DXR-induced mutant spots. At higher concentrations, however, (-)-cubebin was found to potentiate the effects of DXR, leading to either an increase in the production of mutant spots or a reduction, due to toxicity. These results suggest that depending on the concentration, (-)-cubebin may interact with the enzymatic system that catalyzes the metabolic detoxification of DXR, inhibiting the activity of mitochondria! complex 1 and thereby scavenging free radicals. Recombination was found to be the major effect of the treatments with DXR alone. The combined treatments reduced DXR mutagenicity but did not affect DXR recombinogenicity. (C) 2011 Elsevier Ltd. All rights reserved.

Functional characterization of the Aspergillus fumigatus PHO80 homologue

Phosphate is an ion that is essential for fungal growth. The systems for inorganic phosphate (Pi) acquisition in eukaryotic cells (PHO) have been characterized as a low-affinity (that assures a supply of Pi at normal or high external Pi concentrations) and a high-affinity (activated in response to Pi starvation). Here, as an initial step to understand the PHO pathway in Aspergillus fumigatus, we characterized the PH080 homologue, PhoB(PHO80). We show that the Delta phoB(PHO80) mutant has a polar growth defect (i.e., a delayed germ tube emergence) and, by phenotypic and phosphate uptake analyses, establish a link between PhoB(PHO80), calcineurin and calcium metabolism. Microarray hybridizations carried out with RNA obtained from wild-type and Delta phoB(PHO80) mutant cells identify Afu4g03610 (phoD(PHO84)), Afu7g06350 (phoE(PHO89)), Afu4g06020 (phoC(PHO81)), and Afu2g09040 (vacuolar transporter Vtc4) as more expressed both in the Delta phoB(PHO80) mutant background and under phosphate-limiting conditions of 0.1 mM P-i. Epifluorescence microscopy revealed accumulation of poly-phosphate in Delta phoB(PHO80) vacuoles, which was independent of extracellular phosphate concentration. Surprisingly, a phoD(PHO84) deletion mutant is indistinguishable phenotypically from the corresponding wild-type strain. mRNA analyses suggest that protein kinase A absence supports the expression of PHO genes in A. fumigatus. Furthermore, Delta phoB(PHO80) and Delta phoD(PHO84) mutant are fully virulent in a murine low dose model for invasive aspergillosis. (C) 2008 Elsevier Inc. All rights reserved.