ADHII in Aspergillus nidulans is induced by carbon starvation stress

In Aspergillus nidulans there are three NAD(+)-dependent alcohol dehydrogenases (ADHs) that are capable of utilizing ethanol as a substrate. ADHI is the physiological enzyme of ethanol catabolism and ADHIII is induced under conditions of anaerobiosis. The physiological role of ADHII (structural gene alcB) is unknown. We have measured beta-galactosidase in a transformant with an alcB::lacZ fusion and have shown that alcB is maximally expressed under conditions of carbon starvation. The behavior of the alcB::lacZ transformant suggests a hierarchy of repressing carbon sources characteristic of repression by the general carbon catabolite repressor protein, CreA, but in a creA(d)30 background the transformant shows only partial derepression of beta-galactosidase on 1% glucose compared to the creA+ strain. Our results suggest that, in addition to carbon catabolite repression acting via CreA, a CreA-independent mechanism is involved in induction of alcB on carbon starvation.     

Novel reporter gene expression systems for monitoring activation of the Aspergillus nidulans HOG pathway

The Aspergillus nidulans high-osmolarity glycerol response (AnHOG) pathway is involved in osmoadaptation. We found that fludioxonil, a fungicide, causes improper activation of HogA mitogen-activated protein kinase (MAPK) in A. nidulans. Here we present novel reporter systems for monitoring activation of the AnHOG pathway. The promoter region of gfdB (glycerol-3-phosphate dehydrogenase), whose expression depends on the presence of HogA, was fused to a beta-glucuronidase uidA gene (GUS) to construct the reporter, which was introduced into A. nidulans wild type and hogADelta. Increased GUS activity was detected in the wild type only when it was treated with high osmolarity or fludioxonil, while reporter activity was scarcely stimulated in the hogADelta mutant. These results indicate that the reporter activity is controlled via HogA activation. Furthermore, we present possible applications of the reporter systems in screening new antifungal compounds.     

Oxidative stress-induced calcium signalling in Aspergillus nidulans

The effects of oxidative stress on levels of calcium ion (Ca(2+)) in Aspergillus nidulans were measured using strains expressing aequorin in the cytoplasm (Aeq(cyt)) and mitochondria (Aeq(mt)). When oxidative stress was induced by exposure to 10-mM H(2)O(2), the mitochondrial calcium response (Ca(mt)(2+)) was greater than the change in cytoplasmic calcium (Ca(c)(2+)). The Ca(mt)(2+) response to H(2)O(2) was dose dependent, while the increase in [Ca(c)(2+)] did not change with increasing H(2)O(2). The increase in both [Ca(c)(2+)] and [Ca(mt)(2+)] in response to oxidative stress was enhanced by exposure of cells to Ca(2+). The presence of chelator in the external medium only partially inhibited the Ca(mt)(2+) and Ca(c)(2+) responses to oxidative stress. Reagents that alter calcium fluxes had varied effects on the Ca(mt)(2+) response to peroxide. Ruthenium red blocked the increase in [Ca(mt)(2+)], while neomycin caused an even greater increase in [Ca(mt)(2+)]. Treatment with ruthenium red and neomycin had no effect on the Ca(c)(2+) response. Bafilomycin A and oligomycin had no effect on either the mitochondrial or cytoplasmic response. Inhibitors of both voltage-regulated calcium channels and intracellular calcium release channels inhibited the Ca(2+)-dependent component of the Ca(mt)(2+) response to oxidative stress. We conclude that the more significant Ca(2+) response to oxidative stress occurs in the mitochondria and that both intracellular and extracellular calcium pools can contribute to the increases in [Ca(c)(2+)] and [Ca(mt)(2+)] induced by oxidative stress.     

A two-component histidine kinase of the rice blast fungus is involved in osmotic stress response and fungicide action

We isolated and characterized a histidine kinase gene (HIK1) from the rice blast fungus, Pyricularia oryzae (Magnaporthe grisea). The deduced amino acid sequence of HIK1 showed highest similarity (85.7%) to a hybrid-type histidine kinase, Os-1/Nik-1 of Neurospora crassa. Disruption of HIK1 caused no defect in cell growth on normal media and in pathogenicity to rice plants. The Deltahik1 strain acquired resistance to three groups of fungicides (phenylpyrroles, dicarboximides, and aromatic hydrocarbons) similar to os-1 mutants of N. crassa. The Deltahik1 strain showed increased sensitivity to high concentrations of sugars although its salt sensitivity was not elevated, suggesting that the rice blast fungus can distinguish osmostresses caused by high sugar concentrations and high salt concentrations. In contrast, os-1 mutants of N. crassa are sensitive to high concentrations of both salts and sugars. These findings suggest that P. oryzae and N. crassa partially differ in their os (osmosensitive) signal transduction pathway.     

The yeast two-component SLN1 branch of the HOG pathway and the scaffolding activity of Pbs2 modulate the response to endoplasmic reticulum stress induced by tunicamycin

International Microbiology - In addition to the UPR pathway, yeast cells require components of the HOG pathway to respond to ER stress. In this work, we found that unphosphorylated Sln1 and Ssk1...     

Spermatozoal response to osmotic stress

The process of sperm cryopreservation imparts on sperm cells the stress of low-temperature and drastic osmotic change. Damage to the cell plasma membrane results in cell injury in a number of cellular structures and associated functions. Studies in the author's laboratory have focused upon the various mechanisms of osmotic and thermal injury including plasma membrane lipid structure, mitochondrial membrane potential, and intracellular signaling. We have determined that cryoinjury to sperm, as for somatic cells, is a multi-factorial event and some of these events are reversible while some are not.     

Dissection of the HOG pathway activated by hydrogen peroxide in Saccharomyces cerevisiae

Cells usually cope with oxidative stress by activating signal transduction pathways. In the budding yeast Sacchromyces cerevisiae, the high osmolarity glycerol (HOG) pathway has long been implicated in transducing the oxidative stress‐induced signal, but the underlying mechanisms are not well defined. Based on phosphorylation of the mitogen‐activated protein kinase (MAPK) Hog1, we reveal that the signal from hydrogen peroxide (H₂O₂) flows through Ssk1, the response regulator of the two‐component system of the HOG pathway. Downstream signal transduction into the HOG MAPK cascade requires the MAP kinase kinase kinase (MAP3K) Ssk2 but not its paralog Ssk22 or another MAP3K Ste11 of the pathway, culminating in Hog1 phosphorylation via the MAP2K Pbs2. When overexpressed, Ssk2 is also activated in an Ssk1‐independent manner. Unlike in mammals, H₂O₂ does not cause endoplasmic reticulum stress, which can activate Hog1 through the conventional unfolded protein response. Hog1 activated by H₂O₂ is retained in the cytoplasm, but is still able to activate the cAMP‐ or stress‐responsive elements by unknown mechanisms.     

Chloride channels activated by osmotic stress in T lymphocytes

We have used whole-cell and perforated-patch recording techniques to characterize volume-sensitive Cl- channels in T and B lymphocytes. Positive transmembrane osmotic pressure (intracellular osmolality > extracellular osmolality) triggers the slow induction of a Cl- conductance. Membrane stretch caused by cellular swelling may underlie the activation mechanism, as moderate suction applied to the pipette interior can reversibly oppose the induction of Cl- current by an osmotic stimulus. Intracellular ATP is required for sustaining the Cl- current. With ATP-free internal solutions, the inducibility of Cl- current declines within minutes of whole-cell recording, while in whole- cell recordings with ATP or in perforated-patch experiments, the current can be activated for at least 30 min. The channels are anion selective with a permeability sequence of I- > SCN- > NO3-, Br- > Cl- > MeSO3- > acetate, propionate > ascorbate > aspartate and gluconate. GCl does not show voltage- and time-dependent gating behavior at potentials between -100 and +100 mV, but exhibits moderate outward rectification in symmetrical Cl- solutions. Fluctuation analysis indicates a unitary chord conductance of approximately 2 pS at -80 mV in the presence of symmetrical 160 mM Cl-. The relationship of mean current to current variance during the osmotic activation of Cl- current implies that each cell contains on the order of 10(4) activatable Cl- channels, making it the most abundant ion channel in lymphocytes yet described. The current is blocked in a voltage-dependent manner by DIDS and SITS (Ki = 17 and 89 microM, respectively, at +40 mV), the degree of blockade increasing with membrane depolarization. The biophysical and pharmacological properties of this Cl- channel are consistent with a role in triggering volume regulation in lymphocytes exposed to hyposmotic conditions.     

Two new genes involved in signalling ambient pH in Aspergillus nidulans

Two new genes, palH and palI, where mutations mimic the effects of acidic growth pH have been identified in Aspergillus nidulans. A palH mutation is phenotypically indistinguishable from mutations in the palA, palB, palC, and palF genes, whereas palI mutations differ only in that they allow some growth at pH 8. Mutations in palA, B, C, F, and H are epistatic to a palI mutation and the significance of this epistasis is discussed. Additionally, palE and palB mutations have been shown to be allelic. Thus, a total of six genes where mutations mimic acidic growth conditions has been identified.     

The pH-induced glycosylation of secreted phosphatases is mediated in Aspergillus nidulans by the regulatory gene pacC-dependent pathway

In this communication, we show that the pacC(c)14 mutation drastically reduced the mannose and N-acetylglycosamine content of the pacA-encoded acid phosphatase secreted by the fungus Aspergillus nidulans when grown at 22 degrees C, pH 5.0, compared to a control strain. The staining after PAGE was not observed for the pacA-encoded acid phosphatase, while the palD-encoded Pi-repressible alkaline phosphatase had an altered electrophoretic mobility. In addition, the secreted acid phosphatase also had a reduced number of isoforms visualized by staining after IEF and glycosylation had a protective effect against its heat inactivation. We also show that a full-length version of gene pacC-1 cloned from Neurospora crassa complemented the pacC(c)14 mutation of A. nidulans, including the remediation of both the acid and alkaline Pi-repressible phosphatases secreted at pH 5.0, which indicates that glycosylation of secreted phosphatases is mediated in A. nidulans by the conserved PacC pathway that governs pH-responsive gene expression.     

Genetic analysis of the TOR pathway in Aspergillus nidulans

We identified five genes encoding components of the TOR signaling pathway within Aspergillus nidulans. Unlike the situation in Saccharomyces cerevisiae, there is only a single Tor kinase, as in plant and animal systems, and mutant phenotypes suggest that the TOR pathway plays only a minor role in regulating nitrogen metabolism.     

Mutants of the pentose phosphate pathway in Aspergillus nidulans

Mutants of the pentose phosphate pathway have been isolated in Aspergillus nidulans. These fail to grow on a variety of carbohydrates that are catabolized through the pentose phosphate pathway. They also grow poorly on nitrate and nitrite as sole nitrogen sources. The pentose phosphate pathway mutations have been assigned to two unlinked genes. Mutants with lesions in the pppB locus have reduced activities of four enzymes of the pentose phosphate pathway, of glucose-phosphate isomerase, and of mannitol-1-phosphate dehydrogenase. pppA(-) mutants have elevated activities of these same enzymes except for transaldolase, for which they have much reduced activity. Both classes of mutants accumulate sedoheptulose-7-phosphate to an extent that is increased considerably when nitrate is present in the medium. Nitrate does not cause an increase in accumulation of sedoheptulose-7-phosphate in double mutants which, in addition to the pppA1 mutation, carry a mutation that leads to the lack of nitrate reductase activity. These last results suggest that nitrate stimulates the flux through the oxidative pentose phosphate pathway, but that this stimulation depends upon the metabolism of nitrate.