Normal Aerobic Conditions Research Articles

Relationship between seed germination physiological characteristics and germination percentages of direct-seeded hybrid Indica rice under low-temperature and anaerobic interaction

Low-temperature and anaerobic conditions are two major limiting factors in the germination of direct-seeded rice. Sixteen hybrid Indica rice varieties were screened and subjected to two different temperatures and oxygen levels. The results revealed that relative to anaerobic conditons, low-temperature is the key limiting factor for seed germination. For all varieties, relative to normal temperature (30°C) and aerobic conditions (89.0% germination), the germination percentages when seeds were germinated under anaerobic (normal temperature), low-temperature (15°C) or combined anaerobic and low-temperature conditions were 64.0, 46.0 and 23.2%, respectively. Under the three latter treatments, the soluble sugar (SS) and gibberellin (GA) was significantly decreased, the abscisic acid (ABA) had significantly increased by 14.6–251.8%, and the superoxide dismutase (SOD) initially increased by 30.2–49.7% and decreased thereafter by 34.7%. Seeds of 'Shen 9 you 28' and 'Jingliangyou 534' performed better than seeds of the other varieties, with high germination percentages, SS, SOD, GA and low ABA. Correlation analysis revealed that the germination percentages were significantly correlated with the SS, SOD, GA, ABA and GA/ABA. Elevated SS, GA and SOD, as well as reduced ABA content were thought to account for the high germination of rice varieties resistant to combined low-temperature and anaerobic stress.

Preparation and characterization of metal-substituted carotenoid cleavage oxygenases

Carotenoid cleavage oxygenases (CCO) are non-heme iron enzymes that catalyze oxidative cleavage of alkene bonds in carotenoid and stilbenoid substrates. Previously, we showed that the iron cofactor of CAO1, a resveratrol-cleaving member of this family, can be substituted with cobalt to yield a catalytically inert enzyme useful for trapping active site-bound stilbenoid substrates for structural characterization. Metal substitution may provide a general method for identifying the natural substrates for CCOs in addition to facilitating structural and biophysical characterization of CCO-carotenoid complexes under normal aerobic conditions. Here, we demonstrate the general applicability of cobalt substitution in a prototypical carotenoid cleaving CCO, apocarotenoid oxygenase (ACO) from Synechocystis. Among the non-native divalent metals investigated, cobalt was uniquely able to stably occupy the ACO metal binding site and inhibit catalysis. Analysis by X-ray crystallography and X-ray absorption spectroscopy demonstrate that the Co(II) forms of both ACO and CAO1 exhibit a close structural correspondence to the native Fe(II) enzyme forms. Hence, cobalt substitution is an effective strategy for generating catalytically inert but structurally intact forms of CCOs.

Improvement in the Photobiological Hydrogen Production of Aggregated Chlorella by Dimethyl Sulfoxide.

Photobiological hydrogen production plays a vital role in generating clean renewable energy owing to its low energy consumption and environmental friendliness. Although materials-induced Chlorella aggregates have been developed to achieve sustained photobiological hydrogen production under normal aerobic conditions, the yield is relatively low and equals only 0.42 % of the light-to-H2 energy-conversion efficiency. Herein, we report that only 0.5 vol % dimethyl sulfoxide in an aqueous environment significantly enhances the H2 yield produced by aggregated Chlorella, reaching 0.69 % of the light-to-H2 energy-conversion efficiency. This improvement can be attributed to an increase in the cellular respiration rate by dimethyl sulfoxide, which results in a decrease in the oxygen content inside the aggregates and, ultimately, to the activation of more hydrogenases. More generally, this strategy consists of a functional enhancement in organism-material hybrids by using small molecules.

Microwave assisted Suzuki-Miyaura and Mizoroki-Heck cross-couplings catalyzed by non-symmetric Pd(II) CNS pincers supported by iminophosphorane ligands

Two new PdII non-symmetrical CNS pincer type complexes [PdCl{C6H4(Ph2P=NC6H4SR-κ3-C,N,S)}] [R=Bn (3) and 4-C6H5NO2 (4)], supported by iminophosphorane ligands functionalized with a thioether moiety, were prepared and fully characterized. These compounds and the already known complexes 1 (R=Me) and 2 (R=Ph) were evaluated as catalysts in microwave assisted Suzuki-Miyaura and Mizoroki-Heck coupling reactions. Whereas compound 1 instantaneously decomposed under MW irradiation, as visible black Pd0 precipitated in both processes, compounds 2–4 showed good conversions without apparent decomposition. The coupling reactions were dramatically accelerated under MW radiation when compared to conventional heating, with 3 (R=4-C6H4NO2) showing the highest catalytic efficiency of all in both processes. The catalytic activity of complex 2 was further examined in the two cross-couplings with a series of para-substituted bromo-benzenes. Both catalyses proceeded under normal aerobic conditions and were not affected by an excess of elemental Hg. The Mizoroki-Heck reaction operated with high regioselectivity, yielding only the linear 1,2-products.

Abstract P5-05-05: Inhibition of enhanced glucose uptake and glycolysis by KU-55933 as a novel strategy against aggressive breast cancer

Abstract The ability of cancer cells to produce large amounts of lactate through aerobic glycosis (Warburg effect) is coupled to high rates of glucose uptake. Enhanced glucose uptake and glycolysis are closely correlated to increased breast tumor aggressiveness and poor prognosis. However, despite the importance of glucose uptake in supplying energy and preventing apoptosis of cancer cells, the majority of current efforts in searching for therapeutic agents targeting glucose metabolism have been aimed at modulating activities of different metabolic enzymes that are involved in glycolysis. Very limited studies have been done in developing novel therapeutic agents against glucose uptake in breast cancer cells. Ataxia-telangiectasia (A-T) is a monogenic, autosomal recessive disorder characterized by cerebellar ataxia and oculocutaneous telangiectasias. The gene mutated in this disease, ATM (A-T, mutated), encodes a 370-kDa protein kinase. Although ATM is traditionally considered to be a nuclear protein that functions as a signal transducer in the cellular response to DNA damage, it is now known that ATM is also present in the cytoplasm and has important cytoplasmic functions. We previously discovered that ATM activates Akt, a main regulator of glucose uptake, by stimulating its phosphorylation at Ser473 following insulin treatment. We also found that ATM participates in insulin-mediated glucose uptake in muscle cells, and KU-55933, a specific inhibitor of ATM, strongly inhibits this process. Recently, we found that KU-55933 inhibits cell proliferation by inducing apoptosis in MDA-MB-231, a triple-negative breast cancer cell line. We have also found that KU-55933 inhibits migration of MDA-MB-231 by a cell invasion assay. Furthermore, we found that these cancer cells exhibit enhanced glucose uptake in response to insulin and the addition of KU-55933 leads to a dramatic reduction of insulin-mediated glucose uptake in these cells. To further test whether KU-55933's ability to induce apoptosis is linked to its inhibition of glucose uptake, we performed a cell death ELISA assay in MDA-MB-231 cells treated with KU-55933 and different concentrations of glucose. Our results show that KU-55933 induces apoptosis of MDA-MB-231 cells, resulting in a similar degree of cell death as glucose starvation, while cells treated with glucose in conjunction with KU-55933 have decreased apoptosis. Moreover, we performed a cell migration assay and found that KU-55933 strongly inhibits the migration of MDA-MB-231 cells (similar to that caused by glucose starvation), which is almost fully rescued by the extra glucose supplemented in the cell culture medium. We have also established a positional isotope labeling-based targeted metabolomics method that can directly measure the conversion from glucose to lactate through glycolysis in cancer cells. Our results show strong production of lactate from glucose in MDA-MB-231 cells even under normal aerobic growth conditions, and KU-55933 strongly inhibits this process. Our findings may lead to the development of KU-55933 and its analogs as a new generation of therapeutic agents against aggressive breast cancer. Citation Format: Yang D-Q, Harris B, Jiang S, Li Y, Freund D, Hegeman A, Cleary M. Inhibition of enhanced glucose uptake and glycolysis by KU-55933 as a novel strategy against aggressive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-05-05.

The Warburg Effect as a VDAC‐Hexokinase‐Mediated Electrical Suppression of Mitochondrial Energy Metabolism

It is known that nearly half of tumor energy metabolism is provided by glycolysis under normal aerobic conditions (the Warburg Effect) that has been attributed to a respiratory impairment in cancer cells. In the last years, the mitochondrial impairment in cancer cells has been explained by the closure of mitochondrial outer membrane VDACs (porins) by free tubulin (as molecular corks) leading to a suppression of mitochondrial metabolism. Here, we further developed a concept that mitochondria in cancer cells are suppressed by the outer membrane potential (OMP) generated by the metabolically‐dependent mechanism involving the functioning of the VDAC‐hexokinase and/or the ANT(adenine nucleotide translocator)‐VDAC‐hexokinase complexes.These complexes and generated OMP allow initiation of glycolysis preferentially by ATP generated in mitochondria. High selectivity of channeling of mitochondrial energy into the cytosol of cancer cells, to only initiate glycolysis, is achieved due to the electrical closure of free VDACs (non‐bound to hexokinase) by the metabolically‐dependent OMP, by a mode preventing turbo acceleration of glycolysis by cytosolic ATP. Even at low values of OMP, not high enough to completely close VDACs, the ATP flux from the mitochondrial intermembrane space should be almost completely suppressed according to Goldman equation. The value of OMP directly generatedby the VDAC‐hexokinase complex, for example, depends on the Gibbs free energy of the hexokinase reaction. It means that VDAC might function as a physiologically important electrical switcher of tumor energy metabolism, allowing a clear explanation of the Warburg Effect mechanism. (Research grants #111852128625 and #520154531565, Colciencias).

Adaptive response of Amphibacillus xylanus to normal aerobic and forced oxidative stress conditions

Amphibacillus xylanus grows at the same rate and with the same cell yield under aerobic and anaerobic conditions. Under aerobic conditions, it exhibits vigorous oxygen consumption in spite of lacking a respiratory system and haem catalase. To understand the adaptive response of A. xylanus to oxidative stresses, a genomic analysis of A. xylanus was conducted. The analysis showed that A. xylanus has the genes of four metabolic systems: two pyruvate metabolic pathways, a glycolytic metabolic pathway and an NADH oxidase (Nox)-AhpC (Prx) system. A transcriptional study confirmed that A. xylanus has these metabolic systems. Moreover, genomic analysis revealed the presence of two genes for NADH oxidase (nox1 and nox2), both of which were identified in the transcriptional analysis. The nox1 gene in A. xylanus was highly expressed under normal aerobic conditions but that of nox2 was not. A purification study of NADH oxidases indicated that the gene product of nox1 is a primary metabolic enzyme responsible for metabolism of both oxygen and reactive oxygen species. A. xylanus was successfully grown under forced oxidative stress conditions such as 0.1 mM H2O2, 0.3 mM paraquat and 80 % oxygen. Proteomic analysis revealed that manganese SOD, Prx, pyruvate dehydrogenase complex E1 and E3 components, and riboflavin synthase β-chain are induced under normal aerobic conditions, and the other proteins except the five aerobically induced proteins were not induced under forced oxidative stress conditions. Taken together, the present findings indicate that A. xylanus has a unique defence system against forced oxidative stress.

Perturbation of Cytochrome c Maturation Reveals Adaptability of the Respiratory Chain in Mycobacterium tuberculosis

ABSTRACTMycobacterium tuberculosis depends on aerobic respiration for growth and utilizes an aa3-type cytochrome c oxidase for terminal electron transfer. Cytochrome c maturation in bacteria requires covalent attachment of heme to apocytochrome c, which occurs outside the cytoplasmic membrane. We demonstrate that in M. tuberculosis the thioredoxin-like protein Rv3673c, which we named CcsX, is required for heme insertion in cytochrome c. Inactivation of CcsX resulted in loss of c-type heme absorbance, impaired growth and virulence of M. tuberculosis, and induced cytochrome bd oxidase. This suggests that the bioenergetically less efficient bd oxidase can compensate for deficient cytochrome c oxidase activity, highlighting the flexibility of the M. tuberculosis respiratory chain. A spontaneous mutation in the active site of vitamin K epoxide reductase (VKOR) suppressed phenotypes of the CcsX mutant and abrogated the activity of the disulfide bond-dependent alkaline phosphatase, which shows that VKOR is the major disulfide bond catalyzing protein in the periplasm of M. tuberculosis.

Production of biohydrogen by heterologous expression of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase in Escherichia coli

Oxygen sensitivity of hydrogenase is a critical issue in efficient biological hydrogen production. In the present study, oxygen-tolerant [NiFe]-hydrogenase from the marine bacterium, Hydrogenovibrio marinus, was heterologously expressed in Escherichia coli, for the first time. Recombinant E. coli BL21 expressing H. marinus [NiFe]-hydrogenase actively produced hydrogen, but the parent strain did not. Recombinant H. marinus hydrogenase required both nickel and iron for biological activity. Compared to the recombinant E. coli [NiFe]-hydrogenase 1 described in our previous report, recombinant H. marinus [NiFe]-hydrogenase displayed 1.6- to 1.7-fold higher hydrogen production activity in vitro. Importantly, H. marinus [NiFe]-hydrogenase exhibited relatively good oxygen tolerance in analyses involving changes of surface aeration and oxygen proportion within a gas mixture. Specifically, recombinant H. marinus [NiFe]-hydrogenase produced ∼7- to 9-fold more hydrogen than did E. coli [NiFe]-hydrogenase 1 in a gaseous environment containing 5-10% (v/v) oxygen. In addition, purified H. marinus [NiFe]-hydrogenase displayed a hydrogen evolution activity of ∼28.8 nmol H₂/(minmg protein) under normal aerobic purification conditions. Based on these results, we suggest that oxygen-tolerant H. marinus [NiFe]-hydrogenase can be employed for in vivo and in vitro biohydrogen production without requirement for strictly anaerobic facilities.

A Murine Model to Study the Antibacterial Effect of Copper on Infectivity of Salmonella Enterica Serovar Typhimurium

This study investigated the effect of copper as an antibacterial agent on the infectivity of Salmonella enterica serovar Typhimurium. Mice were infected orally with a standardized dose of unstressed Salmonella Typhimurium and copper-stressed cells of Salmonella Typhimurium. Bacterial counts in ileum, blood, liver and spleen were observed up to 168 h under normal aerobic conditions. Serum sensitivity, phagocytosis, malondialdehyde levels and histopathology were studied for both set of animals. A decreased bacterial count in the organs with mild symptoms of infection and a complete recovery by 48 h was observed in mice infected with copper-stressed bacteria. Histopathological examination of ileum tissue demonstrated regeneration of damaged tissue post-infection with copper-stressed bacteria and no malondialdehyde levels were detected after 24 h in ileum, spleen and liver. Exposure to copper sensitized Salmonella Typhimurium to the lytic action of serum and intracellular killing by peritoneal macrophages. It can be concluded that copper stress confers a decrease in the infectivity of healthy Salmonella Typhimurium in normal mice. This study highlights the significance of use of copper as an antibacterial agent against Salmonella Typhimurium in reducing the risk of incidence of Salmonella infections from contaminated water.

Differential requirement of two homologous proteins encoded by sll1214 and sll1874 for the reaction of Mg protoporphyrin monomethylester oxidative cyclase under aerobic and micro-oxic growth conditions

The two open reading frames in the Synechocystis sp. PCC 6803 genome, sll1214 and sll1874, here designated cycI and cycII, respectively, encode similar proteins, which are involved in the Mg protoporphyrin monomethylester (MgProtoME) cyclase reaction. The impairment of tetrapyrrole biosynthesis was examined by separate inactivation of both cyclase encoding genes followed by analysis of chlorophyll contents, MgProtoME levels and several enzyme activities of tetrapyrrole biosynthesis. We additionally addressed the question, whether the two isoforms can complement cyclase deficiency under normal aerobic and micro-oxic growth conditions in light. A cycII knock-out mutant grew without any adverse symptoms at normal air conditions, but showed MgProtoME accumulation at growth under low oxygen conditions. A complete deletion of cycI failed in spite of mixotrophic growth and low light at both ambient and low oxygen, but resulted in accumulation of 150 and 28 times more MgProtoME, respectively, and circa 60% of the wild-type chlorophyll content. The CycI deficiency induced a feedback-controlled limitation of the metabolic flow in the tetrapyrrole biosynthetic pathway by reduced ALA synthesis and Fe chelatase activity. Ectopic expression of the CycI protein restored the wild-type phenotype in cycI− mutant cells under ambient air as well as micro-oxic growth conditions. Overexpressed CycII protein could not compensate for cycI− mutation under micro-oxic and aerobic growth conditions, but complemented the cycII knock-out mutant as indicated by wild-type MgProtoME and chlorophyll levels. Our findings indicate the essential contribution of CycI to the cyclase reaction at ambient and low oxygen conditions, while low oxygen conditions additionally require CycII for the cyclase activity.

NrdI, a flavodoxin involved in maintenance of the diferric-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is essential in all organisms. Class I RNRs consist of two homodimeric subunits: alpha2 and beta2. The alpha subunit contains the site of nucleotide reduction, and the beta subunit contains the essential diferric-tyrosyl radical (Y*) cofactor. Escherichia coli contains genes encoding two class I RNRs (Ia and Ib) and a class III RNR, which is active only under anaerobic conditions. Its class Ia RNR, composed of NrdA (alpha) and NrdB (beta), is expressed under normal aerobic growth conditions. The class Ib RNR, composed of NrdE (alpha) and NrdF (beta), is expressed under oxidative stress and iron-limited growth conditions. Our laboratory is interested in pathways of cofactor biosynthesis and maintenance in class I RNRs and modulation of Y* levels as a means of regulating RNR activity. Our recent studies have implicated a [2Fe2S]-ferredoxin, YfaE, in the NrdB diferric-Y* maintenance pathway and possibly in the biosynthetic and regulatory pathways. Here, we report that NrdI is a flavodoxin counterpart to YfaE for the class Ib RNR. It possesses redox properties unprecedented for a flavodoxin (E(ox/sq) = -264 +/- 17 mV and E(sq/hq) = -255 +/- 17 mV) that allow it to mediate a two-electron reduction of the diferric cluster of NrdF via two successive one-electron transfers. Data presented support the presence of a distinct maintenance pathway for NrdEF, orthogonal to that for NrdAB involving YfaE.

Involvement of oxidative stress response genes in redox homeostasis, the level of reactive oxygen species, and ageing in

Saccharomyces cerevisiae mutants lacking oxidative stress response genes were used to investigate which genes are required under normal aerobic conditions to maintain cellular redox homeostasis, using intracellular glutathione redox potential (glutathione E(h)) to indicate the redox environment of the cells. Levels of reactive oxygen species (ROS) and mitochondrial membrane potentials (MMP) were also assessed by FACS using dihydroethidium and rhodamine 123 as fluorescent probes. Cells became more oxidised as strains shifted from exponential growth to stationary phase. During both phases the presence of reduced thioredoxin and the activity of glutathione reductase were important for redox homeostasis. Thioredoxin reductase contributed less during exponential phase when there was a strong requirement for active Yap1p transcription factor, but was critical during stationary phase. The absence of ROS detoxification systems, such as catalases or superoxide dismutases, had a lesser effect on glutathione E(h), but a more pronounced effect on ROS levels and MMP. These results reflect the major shift in ROS generation as cells switch from fermentative to respiratory metabolism and also showed that there was not a strong correlation between ROS production, MMP and cellular redox environment. Heterogeneity was detected in populations of strains with compromised anti-oxidant defences, and as cells aged they shifted from one cell type with low ROS content to another with much higher intracellular ROS.

Epo protects SOD2‐deficient mouse astrocytes from damage by oxidative stress

Erythropoietin (Epo) expression, which regulates erythropoiesis, has been shown in rat and mouse brain after hypoxia. A previous study from our laboratory showed that astrocytes from manganese-superoxide dismutase (SOD2) homozygous knockout (SOD2(-/-)) mice can survive under 5% O(2), but not under normal aerobic conditions. However, the mechanism involved is not clear. Our preliminary study using reverse transcriptase-polymerase chain reaction showed increased Epo mRNA expression in astrocytes cultured with 5% hypoxia compared with astrocytes under normal conditions. After administration of anti-sense Epo, protection decreased with time. Dose-dependent administration of Epo to SOD2(-/-) mouse astrocytes improved their survivability under normal conditions. Survivability of heterozygous SOD2(-/+) mutant and wild-type mouse astrocyte cultures was the same under normal conditions but, after administration of 2 mM of paraquat, a reactive oxygen species generator, survivability of the SOD2(-/+) astrocytes decreased remarkably compared with the wild-type cells. Epo administration 24 h before exposure to paraquat significantly improved the survivability of the SOD2(-/+) astrocytes. Western blot studies suggest that Jak-Stat signal transduction pathways are involved in this process. Our study demonstrates an important role for Epo in the protection of astrocytes from reactive oxygen species. We suggest that Epo can compensate in part for the antioxidant properties of mitochondrial SOD2 deficiency.

Peroxiredoxin-null Yeast Cells Are Hypersensitive to Oxidative Stress and Are Genomically Unstable

Peroxiredoxins are a family of abundant peroxidases found in all organisms. Although these antioxidant enzymes are thought to be critically involved in cellular defense and redox signaling, their exact physiological roles are largely unknown. In this study, we took a genetic approach to address the functions of peroxiredoxins in budding yeast. We generated and characterized a yeast mutant lacking all five peroxiredoxins. The quintuple peroxiredoxin-null mutant was still viable, though the growth rate was lower under normal aerobic conditions. Although peroxiredoxins are not essential for cell viability, peroxiredoxin-null yeast cells were more susceptible to oxidative and nitrosative stress. In the complete absence of peroxiredoxins, the expression of other antioxidant proteins including glutathione peroxidase and glutathione reductase was induced. In addition, the quintuple mutant was hypersensitive to glutathione depletion. Thus, the glutathione system might cooperate with other antioxidant enzymes to compensate for peroxiredoxin deficiency. Interestingly, the peroxiredoxinnull yeast cells displayed an increased rate of spontaneous mutations that conferred resistance to canavanine. This mutator phenotype was rescued by yeast peroxiredoxin Tsa1p, but not by its active-site mutant defective for peroxidase activity. Our findings suggest that the antioxidant function of peroxiredoxins is important for maintaining genome stability in eukaryotic cells.

Oxidative damage to proteins in yeast cells exposed to adaptive levels of H2O2

When yeast cells are exposed to sublethal concentrations of oxidants, they adapt to tolerate subsequent lethal treatments. Here, we show that this adaptation involves tolerance of oxidative damage, rather than protection of cellular constituents. o- and m-tyrosine levels are used as a sensitive measure of protein oxidative damage and we show that such damage accumulates in yeast cells exposed to H2O2 at low adaptive levels. Glutathione represents one of the main cellular protections against free radical attack and has a role in adaptation to oxidative stress. Yeast mutants defective in glutathione metabolism are shown to accumulate significant levels of o- and m-tyrosine during normal aerobic growth conditions.

Abnormal growth of polyamine-deficient Escherichia coli mutant is partially caused by oxidative stress-induced damage

Polyamines participate in numerous cellular processes and are required for normal cell growth in Escherichia coli. In this study, we constructed a new polyamine-deficient E. coli mutant and investigated the physiological function of polyamines during normal aerobic growth conditions. We showed that the requirement for sulfur-containing, branched chain, and aromatic amino acids, which was exhibited in the sodA sodB double mutant faced with severe oxidative stress, was also true of the polyamine-deficient mutant during normal aerobic cell growth. Sorbitol, sucrose, mannose, 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron), an antioxidant that functions as an oxygen radical scavenger including O 2 −, and thiamine partially relieved the cell growth defect caused by polyamine depletion in a dose-dependent manner. As was the case for the cells treated with paraquat, the mutant had an elongated shape compared with the polyamine-proficient wild type. Decreased aeration also relieved the cell growth defect of the polyamine-deficient mutant. Finally, we confirmed that chloromethyl-2 ′,7 ′-dichlorofluorescin diacetate (DCFH-DA), which is oxidized in a fluorescent product in the presence of various oxidants, also fluoresce in the polyamine-deficient cells. These results showed that abnormal growth of the polyamine-deficient E. coli mutant results partially from oxidative stress-induced damage and the mutant thus exhibits the requirement for antioxidant or specific nutritional amino acid during normal aerobic growth.

A large-scale study of Yap1p-dependent genes in normal aerobic and H2O2-stress conditions: the role of Yap1p in cell proliferation control in yeast.

Yeast genes regulated by the transcriptional activator Yap1p were screened by two independent methods: (i) use of a LacZ-fused gene library and (ii) high-density membrane hybridization. Changes in transcriptome profile were examined in the presence and in the absence of Yap1p, as well as under normal and H2O2-mediated stress conditions. Both approaches gave coherent results, leading to the identification of many genes that appear to be directly or indirectly regulated by Yap1p. Promoter sequence analysis of target genes revealed that this regulatory effect is not always dependent upon the presence of a Yap1p binding site. The results show that the regulatory role of Yap1p is not restricted to the activation of stress response but that this factor can act as a positive or a negative regulator, both under normal and oxidative stress conditions. Among the targets, a few genes participating in growth control cascades were detected. In particular, the RPI1 gene, a repressor of the ras-cAMP pathway, was found to be downregulated by Yap1p during the early phase of growth, but upregulated in the stationary phase or after oxidative stress.

Increased susceptibility of Eucalyptus marginata to stem infection by Phytophthora cinnamomi resulting from root hypoxia

Eucalyptus marginata growing on rehabilitated bauxite mines may be exposed to waterlogging (hypoxia) at the roots, as well as ponding around the stems at the soil surface. This paper examines whether these conditions may predispose stems of E. marginata to infection by Phytophthora cinnamomi. Plants of E. marginata clones resistant and susceptible to P. cinnamomi were grown in an aeroponics system that could be sealed to allow the manipulation of oxygen levels in the root zone to simulate waterlogging. Plants grown under normal oxygen conditions were compared with those whose root zone was exposed to hypoxia (2 mg O2 L−1) before, during or after the stems were inoculated with zoospores of P. cinnamomi. Inoculation was achieved by constructing receptacles around the stems that could hold water and zoospores.Stomatal conductance increased in plants whose roots had been exposed to hypoxia. This effect lasted for at least 2 weeks after the resumption of normal oxygen conditions. P. cinnamomi entered and colonized ponded stems; however, there were no visible lesions on stems 14 days after inoculation. For any given clone of E. marginata, the extent of colonization was significantly greater in stems whose root zone had been exposed to hypoxia than in control stems. The activity of the plant defence‐associated enzymes PAL, 4‐CL and CAD and the concentrations of soluble phenolics were higher in the stems of plants whose roots were exposed to hypoxia, but the increase in activity in response to colonization was much greater for plants whose roots were under normal aerobic conditions. The greatest difference between colonized and noninoculated plants was observed at the colonization front. Peroxidase activity increased after tissues were colonized, rather than preceding the colonization as seen with the other enzymes.The stress induced by root hypoxia remained after roots were returned to normal oxygen conditions. Plants with root hypoxia showed greater stem colonization by P. cinnamomi and seemed less able to recognize the pathogen and switch on rapid defence responses.

Isolation and expression of the gene encoding mitochondrial ADP/ATP carrier (AAC) from the pathogenic yeast Candida parapsilosis.

A gene homologous to Saccharomyces cerevisiae AAC genes coding for mitochondrial ADP/ATP carriers has been cloned from the pathogenic yeast Candida parapsilosis. A probe obtained by PCR amplification from C. parapsilosis DNA, using primers derived from the conserved transmembrane region of yeast ADP/ATP carriers, was used for screening of the C. parapsilosis genomic library. The cloned gene was sequenced and found to encode a polypeptide of 303 amino acids that shows homology with other yeast and fungal mitochondrial ADP/ATP carriers. The gene was designated CpAAC1 and was able to complement the growth phenotypes of S. cerevisiae double deletion mutant (Deltaaac2; Deltaaac3). The expression of the CpAAC1 gene was reduced under semi-anaerobic conditions and it was affected at normal aerobic conditions by the nature of carbon sources used for growth. Hybridization experiments indicate that C. parapsilosis possesses a single gene encoding a mitochondrial ADP/ATP carrier.