Impact of the lectin chaperone calnexin on the stress response, virulence and proteolytic secretome of the fungal pathogen Aspergillus fumigatus

Calnexin is a membrane-bound lectin chaperone in the endoplasmic reticulum (ER) that is part of a quality control system that promotes the accurate folding of glycoproteins entering the secretory pathway. We have previously shown that ER homeostasis is important for virulence of the human fungal pathogen Aspergillus fumigatus, but the contribution of calnexin has not been explored. Here, we determined the extent to which A. fumigatus relies on calnexin for growth under conditions of environmental stress and for virulence. The calnexin gene, clxA, was deleted from A. fumigatus and complemented by reconstitution with the wild type gene. Loss of clxA altered the proteolytic secretome of the fungus, but had no impact on growth rates in either minimal or complex media at 37°C. However, the ΔclxA mutant was growth impaired at temperatures above 42°C and was hypersensitive to acute ER stress caused by the reducing agent dithiothreitol. In contrast to wild type A. fumigatus, ΔclxA hyphae were unable to grow when transferred to starvation medium. In addition, depleting the medium of cations by chelation prevented ΔclxA from sustaining polarized hyphal growth, resulting in blunted hyphae with irregular morphology. Despite these abnormal stress responses, the ΔclxA mutant remained virulent in two immunologically distinct models of invasive aspergillosis. These findings demonstrate that calnexin functions are needed for growth under conditions of thermal, ER and nutrient stress, but are dispensable for surviving the stresses encountered in the host environment.     

Abscisic acid – an intraleaf water-stress signal

Physiological aspects of abscisic acid (ABA) as a drought signal directed specifically at guard cells are topical research foci. Most investigations concentrate on the importance of remote sources of ABA against the background knowledge that leaves are also a source of ABA. Foliar compartmentation of ABA and water-stress-induced release of cellular ABA into the apoplast imply additional levels of complexity. In the present study, we established that the pre-existing foliar ABA pool can be moved to guard cells. We detached leaflets of Vicia faba L. to eliminate an external source of ABA. The leaflets were then pretreated with cycloheximide (CHI), an inhibitor of protein biosynthesis that abolished ABA accumulation in the leaflets. Next, the leaflets were water stressed. After incubation, leaflets were snap-frozen and freeze-dried. Guard cells were individually dissected from the leaflets; such guard-cell samples contain both apoplastic and symplastic ABA. The ABA content of guard cells of CHI-treated, water-stressed leaflets was less than that of control water-stressed leaflets, but higher than that of control water-sufficient leaflets, indicating that guard cells are targets of intraleaf ABA redistribution under stress.     

Sensitization of taxol-induced apoptosis by curcumin involves down-regulation of nuclear factor-kappaB and the serine/threonine kinase Akt and is independent of tubulin polymerization

Taxol is the best anticancer agent that has ever been isolated from plants, but its major disadvantage is its dose-limiting toxicity. In this study, we report with mechanism-based evidence that curcumin, a nontoxic food additive commonly used by the Indian population, sensitizes tumor cells more efficiently to the therapeutic effect of Taxol. A combination of 5 nm Taxol with 5 microm curcumin augments anticancer effects more efficiently than Taxol alone as evidenced by increased cytotoxicity and reduced DNA synthesis in HeLa cells. Furthermore, our results reveal that this combination at the cellular level augments activation of caspases and cytochrome c release. This synergistic effect was not observed in normal cervical cells, 293 cells (in which Taxol down-regulates nuclear factor-kappaB (NF-kappaB)), or HeLa cells transfected with inhibitor kappaBalpha double mutant (IkappaBalpha DM), although the transfection itself sensitized the cells to Taxol-induced cytotoxicity. Evaluation of signaling pathways common to Taxol and curcumin reveals that this synergism was in part related to down-regulation of NF-kappaB and serine/threonine kinase Akt pathways by curcumin. An electrophoretic mobility shift assay revealed that activation of NF-kappaB induced by Taxol is down-regulated by curcumin. We also noted that curcumin-down-regulated Taxol induced phosphorylation of the serine/threonine kinase Akt, a survival signal which in many instances is regulated by NF-kappaB. Interestingly, tubulin polymerization and cyclin-dependent kinase Cdc2 activation induced by Taxol was not affected by curcumin. Altogether, our observations indicate that Taxol in combination with curcumin may provide a superior therapeutic index and advantage in the clinic for the treatment of refractory tumors.     

Differential activation of Smads in HeLa and SiHa cells that differ in their response to transforming growth factor-beta

We assessed the responsiveness of six human cervical cancer cell lines to transforming growth factor (TGF)-beta with p3TP-lux reporter assay and found that HeLa and SiHa cells were highly responsive to TGF-beta. However, when pSBE4-BV/Luc reporter with four Smad binding elements was used, only the SiHa, not the HeLa, cells showed Smad activation. Smad DNA binding activity was relatively more in SiHa than in HeLa cells upon TGF-beta treatment, and the active complex contained Smad 2 and Smad 4. In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, HeLa cells treated with 5 ng/ml of TGF-beta for 24 h showed proliferation, whereas SiHa cells showed growth inhibition under the same conditions. TGF-beta treatment resulted in G(0)/G(1) arrest with a reduction in S-phase only in SiHa cells. A chemical inhibitor of Smad activation (SB203580) blocked the growth inhibitory effect of TGF-beta in SiHa, whereas the proliferative response in HeLa was unaffected. TGF-beta-induced translocation of phospho-Smad 2 was relatively less in HeLa than in SiHa cells. MAPK activation occurred within 5 min and persisted up to 15 min upon TGF-beta treatment in HeLa but was negligible in SiHa cells. TGF-beta activated JNK in HeLa, but SiHa cells showed a down-regulation of its activity. When an inhibitor of MAPK (U0126) was used, the TGF-beta-mediated proliferative response in HeLa cells was completely abolished. SB203580 did not affect MAPK activation induced by TGF-beta in HeLa cells. We report for the first time an activation, presumably independent of Smad activation, of TGF-beta-dependent MAPK within 5 min of treatment that resulted in cell cycle progression in a cervical adenocarcinoma cell line, HeLa.     

FAIR: A server for internal sequence repeats

An Internet computing server has been developed to identify all the occurrences of the internal sequence repeats in a protein and DNA sequences. Further, an option is provided for the users to check the occurrence(s) of the resultant sequence repeats in the other sequence and structure (Protein Data Bank) databases. The databases deployed in the proposed computing engine are up-to-date and thus the users will get the latest information available in the respective databases. The server is freely accessible over the World Wide Web (WWW). AVAILABILITY: http://bioserver1.physics.iisc.ernet.in/fair/     

Breaking the HxNy outbreak

The latest emergence of influenza A (H1N1) virus outbreak demonstrated how swiftly a new strain of flu can evolve and spread around the globe. The A/H1N1 flu has been spreading at unprecedented speed, and further spread within the countries being affected and to other adjacent or far way countries is considered inevitable due to the rapid emigration of infected individuals across the world. In this bioinformation, we discuss the mechanism of evolution of a new HxNy strain and the essential criteria for potentially breaking the outbreak of these extremely harmful and rapidly evolving viral strains in the near future by taking the recent H1N1 pandemic as a classical paradigm.     

S-glutathionylation activates STIM1 and alters mitochondrial homeostasis

Oxidant stress influences many cellular processes, including cell growth, differentiation, and cell death. A well-recognized link between these processes and oxidant stress is via alterations in Ca²⁺ signaling. However, precisely how oxidants influence Ca²⁺ signaling remains unclear. Oxidant stress led to a phenotypic shift in Ca²⁺ mobilization from an oscillatory to a sustained elevated pattern via calcium release–activated calcium (CRAC)–mediated capacitive Ca²⁺ entry, and stromal interaction molecule 1 (STIM1)– and Orai1-deficient cells are resistant to oxidant stress. Functionally, oxidant-induced Ca²⁺ entry alters mitochondrial Ca²⁺ handling and bioenergetics and triggers cell death. STIM1 is S-glutathionylated at cysteine 56 in response to oxidant stress and evokes constitutive Ca²⁺ entry independent of intracellular Ca²⁺ stores. These experiments reveal that cysteine 56 is a sensor for oxidant-dependent activation of STIM1 and demonstrate a molecular link between oxidant stress and Ca²⁺ signaling via the CRAC channel.     

Expedient synthesis of coumarin-coupled triazoles via ‘click chemistry’ leading to the formation of coumarin-triazole-sugar hybrids

Coumarin-based triazoles were synthesized from 3-azidomethylcoumarin and a terminal acetylenic compound. Uncatalysed thermal conditions result in a mixture of both 1,4- and 1,5-regioisomers or the thermodynamically more stable 1,4-regioisomer, whereas the Cu(I)-catalysed reaction affords only the favourable 1,4-regioisomer. B3LYP/6-31G(d) level of theory has been used to calculate geometry and frequency features of the reactants, transition states (TSs) and products. Computational studies further reveal that 1,4-regioisomeric products are more favourable and also thermodynamically more stable compared to the 1,5-regioisomers.