Penicillium chrysogenum glucose oxidase -- a study on its antifungal effects

AIMS: Purification and characterization of the high molecular mass Candida albicans-killing protein secreted by Penicillium chrysogenum. METHODS AND RESULTS: The protein was purified by a combination of ultrafiltration, chromatofocusing and gel filtration. Enzymological characteristics [relative molecular mass (M(r)) = 155 000, subunit structure alpha(2) with M(r,alpha) = 76 000, isoelectric point (pI) = 5.4] were determined using SDS-PAGE and 2D-electrophoresis. N-terminal amino acid sequencing and homology search demonstrated that the antifungal protein was the glucose oxidase (GOX) of the fungus. The enzyme was cytotoxic for a series of bacteria, yeasts and filamentous fungi. Vitamin C (1.0 mg ml(-1)) prevented oxidative cell injuries triggered by 0.004 U GOX in Emericella nidulans cultures but bovine liver catalase was ineffective even at a GOX : catalase activity ratio of 0.004 : 200 U. A secondary inhibition of growth in E. nidulans cultures by the oxygen-depleting GOX-catalase system was likely to replace the primary inhibition exerted by H(2)O(2). CONCLUSIONS: Penicillium chrysogenum GOX possesses similar enzymological features to those described earlier for other Penicillium GOXs. Its cytotoxicity was dependent on the inherent antioxidant potential of the test micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Penicillium chrysogenum GOX may find future applications in glucose biosensor production, the disinfection of medical implants or in the food industry as an antimicrobial and/or preservative agent.     

Stimulation of the cyanide-resistant alternative respiratory pathway by oxygen in Acremonium chrysogenum correlates with the size of the intracellular peroxide pool

The relationship between oxygen input and activity of the cyanide-resistant alternative respiration of submerged cultures of Acremonium crysogenum was investigated. The volumetric oxygen transfer coefficient of the respective cultures correlated positively within almost two ranges of magnitude with the size of the intracellular peroxide pool, which in turn, correlated with the activity of the cyanide-resistant alternative respiratory pathway. Increased aeration also stimulated the glucose uptake rate but had no effect on the total respiration rate or the growth rate. Addition of the lipid peroxyl radical scavenger DL-alpha-tocopherol to A. chrysogenum cultures decreased the rate of intracellular peroxide production as well as glucose uptake. An increase in the cyanide-resistant fraction of total respiration was observed, while growth and the total respiratory activity remained unchanged. We conclude that intracellular peroxides may stimulate the alternative respiration in A. chrysogenum.     

A Penicillium expansum glucose oxidase-encoding gene, GOX2, is essential for gluconic acid production and acidification during colonization of deciduous fruit

Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest maceration of fruit through secretion of total, d-gluconic acid (GLA). Two P. expansum glucose oxidase (GOX)-encoding genes, GOX1 and GOX2, were analyzed. GOX activity and GLA accumulation were strongly related to GOX2 expression, which increased with pH to a maximum at pH 7.0, whereas GOX1 was expressed at pH 4.0, where no GOX activity or extracellular GLA were detected. This differential expression was also observed at the leading edge of the decaying tissue, where GOX2 expression was dominant. The roles of the GOX genes in pathogenicity were further studied through i) development of P. expansum goxRNAi mutants exhibiting differential downregulation of GOX2, ii) heterologous expression of the P. expansum GOX2 gene in the nondeciduous fruit-pathogen P. chrysogenum, and iii) modulation of GLA production by FeSO(4) chelation. Interestingly, in P. expansum, pH and GLA production elicited opposite effects on germination and biomass accumulation: 26% of spores germinated at pH 7.0 when GOX activity and GLA were highest whereas, in P. chrysogenum at the same pH, when GLA did not accumulate, 72% of spores germinated. Moreover, heterologous expression of P. expansum GOX2 in P. chrysogenum resulted in enhanced GLA production and reduced germination, suggesting negative regulation of spore germination and GLA production. These results demonstrate that pH modulation, mediated by GLA accumulation, is an important factor in generating the initial signal or signals for fungal development leading to host-tissue colonization by P. expansum.     

Two-stage batch process for the production of ajmalicine by Catharanthus roseus: The link between growth and production stage

The link between the growth stage and the production stage in a two-stage batch process was investigated using (filtered) inocula from different periods of the stationary phase of the growth cycle. In the production stage, ajmalicine production by Catharanthus roseus in a 3-L stirred tank reactor was induced with a high glucose concentration (80 g/L). Ajmalicine production in cultures started with cells from the late stationary phase was five times higher than in cultures started with cells from the early stationary phase. After transfer to the production stage, cells from the early stationary phase showed a transient increase in respiration and enzyme induction, followed by culture browning. In contrast, cells in the late stationary phase showed a typical induction pattern: constant respiration, and permanent enzyme induction. A striking similarity between the geraniol-10-hydroxylase (G10H) activity and the ajmalicine accumulation profile could be observed in all cultures, suggesting that G 10H regulated ajmalicine production in this investigation. The intracellular nitrate concentration was significantly higher in the inoculum showing a high ajmalicine production than in the inoculum with a low production. Consequently, nitrate may act as a marker for the start of the production stage: as soon as the nitrate is depleted in the growth medium secondary metabolism can be induced. (c) 1995 John Wiley & Sons, Inc.     

Identification of the Streptococcus mutans LytST two-component regulon reveals its contribution to oxidative stress tolerance

ABSTRACT: BACKGROUND: The S. mutans LrgA/B holin-like proteins have been shown to affect biofilm formation and oxidative stress tolerance, and are regulated by oxygenation, glucose levels, and by the LytST two-component system. In this study, we sought to determine if LytST was involved in regulating lrgAB expression in response to glucose and oxygenation in S. mutans. RESULTS: Real-time PCR revealed that growth phase-dependent regulation of lrgAB expression in response to glucose metabolism is mediated by LytST under low-oxygen conditions. However, the effect of LytST on lrgAB expression was less pronounced when cells were grown with aeration. RNA expression profiles in the wild-type and lytS mutant strains were compared using microarrays in early exponential and late exponential phase cells. The expression of 40 and 136 genes in early-exponential and late exponential phase, respectively, was altered in the lytS mutant. Although expression of comYB, encoding a DNA binding-uptake protein, was substantially increased in the lytS mutant, this did not translate to an effect on competence. However, a lrgA mutant displayed a substantial decrease in transformation efficiency, suggestive of a previously-unknown link between LrgA and S. mutans competence development. Finally, increased expression of genes encoding antioxidant and DNA recombination/repair enzymes was observed in the lytS mutant, suggesting that the mutant may be subjected to increased oxidative stress during normal growth. Although the intracellular levels of reaction oxygen species (ROS) appeared similar between wild-type and lytS mutant strains after overnight growth, challenge of these strains with hydrogen peroxide (H2O2) resulted in increased intracellular ROS in the lytS mutant. CONCLUSIONS: Overall, these results: (1) Reinforce the importance of LytST in governing lrgAB expression in response to glucose and oxygen, (2) Define a new role for LytST in global gene regulation and resistance to H2O2, and (3) Uncover a potential link between LrgAB and competence development in S. mutans.     

Growth inhibition by glucose oxidase system of enterotoxic Escherichia coli and Salmonella derby: in vitro studies

The antibacterial effect of different glucose oxidase (GOX)/glucose combinations was studied on two food-poisoning organisms, enterotoxic Escherichia coli PM 015 and Salmonella derby BP 177. Growth of E. coli in nutrient broth (NB) was clearly inhibited by 4.0 mg/ml glucose after 24 h when combined with 2.0 U/ml GOX and after 48 h when combined with 0.5 or 1.0 U/ml GOX. Salmonella derby was more resistant than E. coli, but showed clear growth inhibition only after 48 h when inoculated in tubes where 2 mg glucose/ml and 2 U GOX/ml (or 4 mg glucose/ml and 1 U GOX/ml) were combined. In order to understand if the enzyme effect on microbial growth can be attributed to hydrogen peroxide or to pH decrease as a result of the production of gluconic acid, catalase (CAT) was added to GOX/glucose system. Since CAT decomposes H₂O₂ to H₂O and O₂, the antibacterial effect was ascribed to a pH decrease as a result of gluconic acid in the system.     

A role for the myoglobin redox cycle in the induction of endothelial cell apoptosis

This study investigates the potential role of the ferric/ferryl redox cycle of myoglobin (Mb) in the development of endothelial cell injury. Bovine aortic endothelial cells were incubated with ferric Mb (0.5-100 micro M) in the presence or absence of low steady states of H(2)O(2) (3-4 micro M) generated by glucose oxidase (GOX). The reaction of ferric Mb with H(2)O(2) generated ferryl Mb as monitored spectrophotometrically. Ferryl Mb formation correlated with the induction of apoptosis as indicated by morphological criteria, caspase 3 activation, phosphatidylserine (PS) externalization, and nuclear condensation by Hoechst 33342 staining. The addition of ascorbate or catalase inhibited the formation of ferryl Mb and the onset of apoptosis, whereas apoptosis was enhanced in cells depleted of intracellular glutathione by pretreatment with buthionine sulfoximine. Mb and Mb/GOX suppressed cell cycle progression, but only Mb/GOX produced significant cell loss revealed by the accumulation of sub G1 events. These results suggest a role for the Mb redox cycle in the induction of endothelial cell apoptosis, which may be relevant in the pathophysiology of diseases characterized by the release of Mb from damaged muscle.     

A superoxide anion generator, pyrogallol induces apoptosis in As4.1 cells through the depletion of intracellular GSH content

We investigated the involvement of ROS such as H2O2 and O2*-, and GSH in As4.1 cell death induced by pyrogallol. The intracellular H2O2 levels were decreased or increased depending on the concentration and incubation time of pyrogallol. The levels of O2*- were significantly increased. Pyrogallol reduced the intracellular GSH content. And ROS scavengers, Tempol, Tiron, Trimetazidine and NAC could not significantly down-regulate the production of H2O2 and O2*-. However, these ROS scavengers slightly inhibited apoptosis. Interestingly, Tempol showing the recovery of GSH depletion induced by pyrogallol significantly decreased apoptosis without the significant reduction of intracellular O2*- levels. SOD and catalase did not change the level of H2O2 but decreased the level of O2*-. The inhibition of GSH depletion by these was accompanied with the decrease of apoptosis, as evidenced by sub-G1 DNA content, annexin V staining, mitochondria membrane potential (DeltaPsi(m)) and Western data. In addition, ROS scavengers and SOD did not alter a G2 phase accumulation of the cell cycle induced by pyrogallol. However, catalase changed the cell cycle distributions of pyrogallol-treated cells to those of pyrogallol-untreated cells. In summary, we have demonstrated that pyrogallol potently generates ROS, especially O2*-, in As4.1 JG cells, and Tempol, SOD and catalase could rescue to a lesser or greater extent cells from pyrogallol-induced apoptosis through the up-regulation of intracellular GSH content.     

Measurement of autolysis in submerged batch cultures of Penicillium chrysogenum

The process of cellular autolysis was studied in an industrial strain of Penicillium chrysogenum by a range of methods, including assessment of biomass decline, NH+4 release, changes in culture apparent viscosity, and by means of a quantitative assessment of changes in micromorphology using a computerized image analysis system. The pattern of total intracellular proteolytic and beta-1, 3-glucanolytic activity in the culture was also examined. The overall aim was to identify a suitable method, or methods, for examining the extent of autolysis in fungal cultures. Autolysis was studied in submerged batch processes, where DOT was maintained above 40% saturation (non-O2-limited), and, under O2-limited conditions. Both N and O2 limitation promoted extensive culture autolysis. Image analysis techniques were perhaps the most sensitive method of assessing the progress of autolysis in the culture. Autolytic regions within some hyphae were apparent even during growth phase, but became much more widespread as the process proceeded. The early stages of autolysis involved continued energy source consumption, increased carbon dioxide evolution rate, degradation of penicillin, and decreased broth filterability. Later stages involved widespread mycelial fragmentation, with some regrowth (cryptic growth) occurring in non-O2-limited cultures. Intracellular proteolytic activity showed two peaks, one during the growth phase, and the other during autolysis. Autolysis was also associated with a distinct peak in beta-1,3-glucanolytic activity, indicating that degradation of cell wall matrix polymers may be occurring during autolysis in this strain of P. chrysogenum.     

The use of culture redox potential and oxygen uptake rate for assessing glucose and glutamine depletion in hybridoma cultures

Culture redox potential (CRP) and oxygen uptake rate (OUR) were monitored on-line during glucose- and glutamine-limited batch cultures of a murine hybridoma cell line that secretes a neutralizing monoclonal antibody specific to toxin 2 of the scorpion Centruroides noxius Hoffmann. It was found that OUR and CRP can be used for assessing the viable cell concentration and growth phases of the culture. Before nutrient depletion, OUR increased exponentially with viable cell concentration, whereas CRP decreased monotonically until cell viability started to decrease. During the death phase, CRP gradually increased. A sudden decrease in OUR occurred upon glucose or glutamine depletion. CRP traced the dissolved oxygen profile during a control action or an operational eventuality, however, during nutrient depletion it did not follow the expected behavior of a system composed mainly by the O(2)/H(2)O redox couple. Such a behavior was not due to the accumulated lactate or ammonia, nor to possible intracellular redox potential changes caused by nutrient depletion, as inferred from respiration inhibition by rotenone or uncoupled respiration by 2,4-dinitrophenol. As shown in this study, operational eventualities can be erroneously interpreted as changes in OUR when using algorithms based solely on oxygen balances. However, simultaneous measurements of CRP and OUR may be used to discriminate real metabolic events from operational failures. The results presented here can be used in advanced real-time algorithms for controling glucose and glutamine at low concentrations, avoiding under- or over-feeding them in hybridoma cultures, and consequently reducing the accumulation of metabolic wastes and improving monoclonal antibody production. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 555-563, 1997.     

Stem cell factor and H2O2 induce GLUT1 translocation in M07e cells

This work aims to elucidate the mechanisms involved in the early activation of glucose transport in hematopoietic M07e cells by stem cell factor (SCF) and a reactive oxygen species (ROS) as H2O2. SCF and H2O2 increase Vmax for glucose transport; this enhancement is due to a higher content in GLUT1 in plasma membranes, possibly through a translocation from intracellular stores. Inhibitors of tyrosine kinases or phospholipase C (PLC) remove glucose transport enhancement and prevent translocation. The inhibitory effect of STI-571 suggests a role for c-kit tyrosine kinase on glucose transport activation not only by SCF, but also by H2O2. On the other hand, neither protein kinase C nor phosphoinositide-3-kinase appear to be involved in the acute activation of glucose transport. Our data suggest that i) in M07e cells, SCF and exogenous H2O2 elicit a short-term activation of glucose transport through a translocation of GLUT1 from intracellular stores to plasma membranes; ii) both stimuli could share at least some signaling pathways leading to glucose uptake activation, involving protein tyrosine kinases and PLC iii) H2O2 could act increasing the level of tyrosine phosphorylation through the inhibition of tyrosine phosphatases and mimicking the regulation role of endogenous ROS.     

ROS production and Glut1 activity in two human megakaryocytic cell lines

Reactive oxygen species (ROS) has been increasingly recognised as intracellular messengers in signal transduction following receptor activation by a variety of bioactive peptides including growth factors, cytokines and hormones. In this study ROS production and glucose transport activity were evaluated in the growth factor dependent M07e cells and in B1647 cells, not requiring additional hematopoietic cytokines for growth: the aim was to investigate whether ROS could be involved in the regulation of Glut1-mediated glucose uptake in both cell lines. The effect of the synthetic superoxide and hydrogen peroxide scavenger EUK-134 on DOG uptake activity and intracellular ROS formation supports the concept of reactive oxygen species as signalling molecules. In order to investigate ROS generation sources, diphenyleneiodonium, an inhibitor of flavoprotein centres and apocynin, an inhibitor of NAD(P)H oxidase, were used: they inhibit both ROS production and glucose uptake activation. All these data support the hypothesis that ROS can contribute to the regulation of glucose transport, not only in M07e cells but also in B1647 cells; we could speculate that one possible source of ROS, linked somehow with Glut1 activity, can be a NAD(P)H oxidase similar to that one present in phagocytic cells.     

Involvement of Na+/K+-ATPase in hydrogen peroxide-induced hypertrophy in cardiac myocytes

We have shown that increased production of reactive oxygen species (ROS) was required for ouabain-induced hypertrophy in cultured cardiac myocytes. In the present study we assessed whether long-term exposure of myocytes to nontoxic ROS stress alone is sufficient to induce hypertrophy. A moderate amount of H2O2 was continuously generated in culture media by glucose oxidase. This resulted in a steady increase in intracellular ROS in cultured cardiac myocytes for at least 12 h. Such sustained, but not transient, increase in intracellular ROS at a level comparable to that induced by ouabain was sufficient to stimulate protein synthesis, increase cell size, and change the expression of several hypertrophic marker genes. Like ouabain, glucose oxidase increased intracellular Ca2+ and activated extracellular signal-regulated kinases 1 and 2 (ERK1/2). These effects of glucose oxidase were additive to ouabain-induced cellular changes. Furthermore, glucose oxidase stimulated endocytosis of the plasma membrane Na+/K+-ATPase, resulting in significant inhibition of sodium pump activity. While inhibition of ERK1/2 abolished glucose oxidase-induced increases in protein synthesis, chelating intracellular Ca2+ by BAPTA-AM showed no effect. These results, taken together with our prior observations, suggest that ROS may cross talk with Na+/K+-ATPase, leading to the activation of hypertrophic pathways in cardiac myocytes.     

Yeast-like cell formation and glutathione metabolism in autolysing cultures of Penicillium chrysogenum

The bulk formation of yeast-like (arthrospore-like) cells were typical in carbon-depleted submerged cultures of the high beta-lactam producer Penicillium chrysogenum NCAIM 00237 strain independently of the nitrogen-content of the culture medium. This morphogenetic switch was still quite common in carbon-starving cultures of the low-penicillin-producer strain P. chrysogenum ATCC 28089 (Wis 54-1255) when the nitrogen-content of the medium was low but was a very rare event in wild-type P. chrysogenum cultures. The mycelium-->yeast-like cell transition correlated well with a relatively high glutathione concentration and a reductive glutathione/glutathione disulfite (GSH/GSSG) redox balance in autolysing cultures, which was a consequence of industrial strain development. Paradoxically, the development of high beta-lactam productivity resulted in a high intracellular GSH level and, concomitantly, in an increased y-glutamyltranspeptidase (i.e. GSH-decomposing) activity in the autolytic phase of growth of P. chrysogenum NCAIM 00237. The hypothesized causal connection between GSH metabolism and cell morphology, if verified, may help us in future metabolic engineering of industrially important filamentous fungi.     

Reactive oxygen species stimulated human hepatoma cell proliferation via cross-talk between PI3-K/PKB and JNK signaling pathways

Reactive oxygen species (ROS) are important for intracellular signaling mechanisms regulating many cellular processes. Manganese superoxide dismutase (MnSOD) may regulate cell growth by changing the level of intracellular ROS. In our study, we investigated the effect of ROS on 7721 human hepatoma cell proliferation. Treatment with H2O2 (1-10 microM) or transfection with antisense MnSOD cDNA constructs significantly increased the cell proliferation. Recently, the mitogen-activated protein kinases (MAPK) and the protein kinase B (PKB) were proposed to be involved in cell growth. Accordingly, we assessed the ability of ROS to activate MAPK and PKB. PKB and extracellular signal-regulated kinase (ERK) were both rapidly and transiently activated by 10 microM H2O2, but the activities of p38 MAPK and JNK were not changed. ROS-induced PKB activation was abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002, suggesting that PI3-K is an upstream mediator of PKB activation in 7721 cells. Transfection with sense PKB cDNA promoted c-fos and c-jun expression in 7721 cells, suggesting that ROS may regulate c-fos and c-jun expression via the PKB pathway. Furthermore we found that exogenous H2O2 could stimulate the proliferation of PKB-AS7721 cells transfected with antisense PKB cDNA, which was partly dependent on JNK activation, suggesting that H2O2 stimulated hepatoma cell proliferation via cross-talk between the PI3-K/PKB and the JNK signaling pathways. However, insulin could stimulate 7721 cell proliferation, which is independent of cross-talk between PI3-K/PKB and JNK pathways. In addition, H2O2 did not induce the cross-talk between the PI3-K/PKB and the JNK pathways in normal liver cells. Taken together, we found that ROS regulate hepatoma cell growth via specific signaling pathways (cross-talk between PI3-K/PKB and JNK pathway) which may provide a novel clue to elucidate the mechanism of hepatoma carcinogenesis.     

The requirement of both intracellular reactive oxygen species and intracellular calcium elevation for the induction of heparin-binding EGF-like growth factor in vascular endothelial cells and smooth muscle cells.

Heparin-binding EGF-like growth factor (HB-EGF), which is a potent mitogen for vascular smooth muscle cells (SMC) and fibroblasts, has been reported to be strongly implicated in atherosclerosis and wound healing. HB-EGF mRNA is known to be induced by thrombin, angiotensin-II, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and HB-EGF itself in SMC. In vascular endothelial cells (EC), its mRNA is induced by tumor necrosis factor-alpha and interleukin-1beta. Only phorbol 12-myristate 13-acetate is a common inducer for HB-EGF mRNA. The present study shows that calcium ionophore A23187 also induced HB-EGF mRNA in both SMC and in EC and that both intracellular reactive oxygen species (ROS) and an increase in calcium levels were essential for the induction of this growth factor mRNA. While HB-EGF caused an increase in both intracellular ROS and calcium in SMC, it increased only calcium, but not the intracellular ROS in EC. When the intracellular ROS was elevated by treatment with hydrogen peroxide (H2O2) or by depletion of glutathione by buthionine sulfoxamine, both HB-EGF and thrombin were observed to upregulate HB-EGF mRNA in EC. These data suggest that H2O2, produced by activated leukocytes in inflammatory lesions, upregulates HB-EGF mRNA by cooperating with thrombin, angiotensin-II, and the above growth factors. Since activated macrophages under the EC are thought to elevate the ROS in neighboring EC, this mechanism might play a major role in the progression of atherosclerosis and for wound healing.     

Melanin content and hydroperoxide metabolism in human melanoma cells

Human melanoma cells were grown to exponential and stationary phases showing melanin contents of 4.2 +/- 0.3 and 11.3 +/- 0.6 micrograms/10(6) cells, respectively. The cells were separated in four subpopulations by a Percoll gradient; the subpopulation of density 1.07 (g/ml) was the most enriched in pigmented cells and produced 28 and 58% of the cells in exponential and stationary phases, respectively. Melanoma cells had similar superoxide dismutase and glutathione peroxidase activities in exponential and stationary phases. Moreover melanoma cells exhibited a higher catalase activity in the stationary phase: whole homogenate and cytosol activities were 7.0 +/- 0.3 and 10.8 +/- 0.6 U/mg protein, whereas in exponential phase the activities were 4.9 +/- 0.1 and 7.6 +/- 0.3 U/mg protein for whole homogenate and cytosol, respectively. The intracellular H2O2 steady-state concentration was 3.3 +/- 0.2 and 2.1 +/- 0.2 microM H2O2 for exponential and stationary phases, respectively. The spontaneous chemiluminescence of the two culture phases was 169 +/- 27 cps/10(6) cells (exponential) and 78 +/- 24 cps/10(6) cells (stationary). The cytotoxicity of H2O2 generated extracellularly by glucose oxidase was determined after 60 min of exposure. IC50 values for exponential and stationary cell cultures were 0.9 and 2.4 mU/ml of glucose oxidase, respectively. The increased catalase activities in the stationary phase as compared with the exponential phase are consistent with the decreased intracellular H2O2, with the decreased spontaneous chemiluminescence, and with the increased resistance to exogenous H2O2.     

Ras-dependent and -independent regulation of reactive oxygen species by mitogenic growth factors and TGF-beta1.

Mitogenic growth factors and transforming growth factor beta1 (TGF-beta1) induce the generation of reactive oxygen species (ROS) in nonphagocytic cells, but their enzymatic source(s) and regulatory mechanisms are largely unknown. We previously reported on the ability of TGF-beta1 to activate a cell surface-associated NADH:flavin:O(2) oxidoreductase (NADH oxidase) that generates extracellular H(2)O(2). In this study, we compared the ROS-generating enzymatic systems activated by mitogenic growth factors and TGF-beta1 with respect to the primary reactive species produced (O(2)(.-) vs. H(2)O(2)), the site of generation (intracellular vs. extracellular) and regulation by Ras. We find that the mitogenic growth factors PDGF-BB, FGF-2, and TGF-alpha (an EGF receptor ligand) are able to rapidly (within 5 min) induce the generation of intracellular O(2)(.-) without detectable NADH oxidase activity or extracellular H(2)O(2) release. In contrast, TGF-beta1 does not stimulate intracellular O(2)(.-) production and the delayed induction of extracellular H(2)O(2) release is not associated with O(2)(.-) production. Expression of dominant-negative Ras (N17Ras) protein by herpes simplex virus-mediated gene transfer blocks mitogen-stimulated intracellular O(2)(.-) generation but has no effect on TGF-beta1-induced NADH oxidase activation/H(2)O(2) production. These results demonstrate that there are at least two distinctly different ROS-generating enzymatic systems in lung fibroblasts regulated by mitogenic growth factors and TGF-beta1 via Ras-dependent and -independent mechanisms, respectively. In addition, these findings suggest that endogenous production of ROS by growth factors/cytokines may have different biological effects depending on the primary reactive species generated and site of production.     

Crosstalk between ROS homeostasis and secondary metabolism in S. natalensis ATCC 27448: modulation of pimaricin production by intracellular ROS

Streptomyces secondary metabolism is strongly affected by oxygen availability. The increased culture aeration enhances pimaricin production in S. natalensis, however the excess of O(2) consumption can lead to an intracellular ROS imbalance that is harmful to the cell. The adaptive physiological response of S. natalensis upon the addition of exogenous H(2)O(2) suggested that the modulation of the intracellular ROS levels, through the activation of the H(2)O(2) inducible catalase during the late exponential growth phase, can alter the production of pimaricin. With the construction of defective mutants on the H(2)O(2) related enzymes SodF, AhpCD and KatA1, an effective and enduring modulation of intracellular ROS was achieved. Characterization of the knock-out strains revealed different behaviours regarding pimaricin production: whilst the superoxide dismutase defective mutant presented low levels of pimaricin production compared to the wild-type, the mutants defective on the H(2)O(2)-detoxifying enzymes displayed a pimaricin overproducer phenotype. Using physiological and molecular approaches we report a crosstalk between oxidative stress and secondary metabolism regulatory networks. Our results reveal that the redox-based regulation network triggered by an imbalance of the intracellular ROS homeostasis is also able to modulate the biosynthesis of pimaricin in S. natalensis.