Decreased histone deacetylase 2 impairs Nrf2 activation by oxidative stress

Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in cellular defence against oxidative stress by inducing the expression of multiple anti-oxidant genes. However, where high levels of oxidative stress are observed, such as chronic obstructive pulmonary disease (COPD), Nrf2 activity is reduced, although the molecular mechanism for this defect is uncertain. Here, we show that down-regulation of histone deacetylase (HDAC) 2 causes Nrf2 instability, resulting in reduced anti-oxidant gene expression and increase sensitivity to oxidative stress. Although Nrf2 protein was clearly stabilized after hydrogen peroxide (H₂O₂) stimulation in a bronchial epithelial cell line (BEAS2B), Nrf2 stability was decreased and Nrf2 acetylation increased in the presence of an HDAC inhibitor, trichostatin A (TSA). TSA also reduced Nrf2-regulated heme-oxygenase-1 (HO-1) expression in these cells, and this was confirmed in acute cigarette-smoke exposed mice in vivo. HDAC2 knock-down by RNA interference resulted in reduced H₂O₂-induced Nrf2 protein stability and activity in BEAS2B cells, whereas HDAC1 knockdown had no effect. Furthermore, monocyte-derived macrophages obtained from healthy volunteers (non-smokers and smokers) and COPD patients showed a significant correlation between HDAC2 expression and Nrf2 expression (r = 0.92, p < 0.0001). Thus, reduced HDAC2 activity in COPD may account for increased Nrf2 acetylation, reduced Nrf2 stability and impaired anti oxidant defences.     

Binding to syntenin-1 protein defines a new mode of ubiquitin-based interactions regulated by phosphorylation

Syntenin-1 is a PDZ domain-containing adaptor that controls trafficking of transmembrane proteins including those associated with tetraspanin-enriched microdomains. We describe the interaction of syntenin-1 with ubiquitin through a novel binding site spanning the C terminus of ubiquitin, centered on Arg(72), Leu(73), and Arg(74). A conserved LYPSL sequence in the N terminus, as well as the C-terminal region of syntenin-1, are essential for binding to ubiquitin. We present evidence for the regulation of this interaction through syntenin-1 dimerization. We have also established that syntenin-1 is phosphorylated downstream of Ulk1, a serine/threonine kinase that plays a critical role in autophagy and regulates endocytic trafficking. Importantly, Ulk1-dependent phosphorylation of Ser(6) in the LYPSL prevents the interaction of syntenin-1 with ubiquitin. These results define an unprecedented ubiquitin-dependent pathway involving syntenin-1 that is regulated by Ulk1.     

Endoplasmic reticulum stress-induced apoptosis in Leishmania through Ca2+-dependent and caspase-independent mechanism

Numerous reports have shown that mitochondrial dysfunctions play a major role in apoptosis of Leishmania parasites, but the endoplasmic reticulum (ER) stress-induced apoptosis in Leishmania remains largely unknown. In this study, we investigate ER stress-induced apoptotic pathways in Leishmania major using tunicamycin as an ER stress inducer. ER stress activates the expression of ER-localized chaperone protein BIP/GRP78 (binding protein/identical to the 78-kDa glucose-regulated protein) with concomitant generation of intracellular reactive oxygen species. Upon exposure to ER stress, the elevation of cytosolic Ca(2+) level is observed due to release of Ca(2+) from internal stores. Increase in cytosolic Ca(2+) causes mitochondrial membrane potential depolarization and ATP loss as ablation of Ca(2+) by blocking voltage-gated cation channels with verapamil preserves mitochondrial membrane potential and cellular ATP content. Furthermore, ER stress-induced reactive oxygen species (ROS)-dependent release of cytochrome c and endonuclease G from mitochondria to cytosol and subsequent translocation of endonuclease G to nucleus are observed. Inhibition of caspase-like proteases with the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone or metacaspase inhibitor antipain does not prevent nuclear DNA fragmentation and phosphatidylserine exposure. Conversely, significant protection in tunicamycin-induced DNA degradation and phosphatidylserine exposure was achieved by either pretreatment of antioxidants (N-acetyl-L-cysteine, GSH, and L-cysteine), chemical chaperone (4-phenylbutyric acid), or addition of Ca(2+) chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethyl ester). Taken together, these data strongly demonstrate that ER stress-induced apoptosis in L. major is dependent on ROS and Ca(2+)-induced mitochondrial toxicity but independent of caspase-like proteases.     

Virulence Factors and Anti Fungal Sensitivity Pattern of<Emphasis Type="Italic"> Candida</Emphasis> Sp. Isolated from HIV and TB Patients

The study comprised of 60 Candida spp., 50 isolates from HIV and TB positive individuals (immunocompromised) and 10 isolates from non-HIV and -TB patients (immunocompetent). Among the 60 Candidal isolates, 83.3% were identified as C. albicans, 11.6% as C. glabrata and rest 5% as C. krusei. There is no study in production pattern of extracellular enzymes of Candida spp. isolated from HIV and TB patients in comparison with non-HIV and -TB patients in India. The comparison of phospholipase activities showed that there was a significant difference between the groups at (P = 0.001). The non-HIV and -TB groups of C. glabrata and C. krusei did not show detectable phospholipase activity when compared to the HIV and TB groups. The mean difference in the phospholipase activities of these two groups was significant (P = <0.001). Candida spp. of both the groups do not possess the ability to hydrolyze gelatin. All the strains possessed the ability to show alpha haemolysis. Even though it had shown alpha haemolysis, the significant difference in haemolytic activity was observed only in C. albicans (P = <0.001). None of the isolates from the two groups possessed the ability to hydrolyze gelatin. In the resistance profile of Candida spp., C. albicans of HIV and TB groups had shown resistance to fluconazole, Itraconazole, ketaconazole, nystatin but showed 100% sensitivity towards amphotericin-B. The isolates of C. krusei and C. glabrata showed no resistance to any of the drugs tested. In the case of, non-HIV and -TB patients the resistance pattern was low.     

CNS Targets Support and Sustain Differentiation of Cultured Neuronal and Retinal Progenitor Cells

Superior colliculus (SC) is the target of retinal neurons, allowing them to form connections. Cultured stem cells/progenitors can potentially be used as donor tissue to reconstruct degenerated retina including perhaps replacing lost ganglion cells in glaucoma. In which case, it will be essential for these cells to integrate with the central nervous system targets. Here, we have investigated if the mid-brain region containing superior colliculus (SC) provides a permissive environment for the survival and differentiation of neural progenitors, including retinal progenitor cells propagated in cultures. Neural (NPCs) and retinal progenitor cells (RPCs) from green fluorescent protein (GFP) transgenic mice were cultured. Passage two through four neural and retinal progenitor cells were subsequently cocultured with the SC organotypic slices and maintained in culture for 17 and eight days respectively. Differentiation of the neurons was studied by immunocytochemistry for retinotypic neuronal markers. Retinal progenitor cells cocultured with SC slices were able to differentiate into various neuronal morphologies. Some cocultured progenitor cells differentiated into neurons as suggested by class III β tubulin immunoreactivity. In addition, specific retinotypic neuronal differentiation of RPC was detected by immunoreactivity for calbindin and PKC. SC provides a permissive environment that supports survival and differentiation of the progenitor cells.     

Mechanistic control of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) splice isoforms by the heterogeneous nuclear ribonuclear proteins hnRNP L, hnRNP A1, and hnRNP M

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.     

Pollination and Pollen-pistil Interaction in Oil Palm, Elaeis guineensis

Structural and cytochemical aspects of the pistil and detailsof pollination and pollen-pistil interaction were investigatedin the African oil palm (Elaeis guineensis Jacq.), an importantperennial oil crop. The stigma is trilobed, wet and papillate.The branched papillae are confined to a narrow linear zone oneach stigmatic lobe. Each stigmatic lobe harbours a deep stigmaticgroove, which runs adaxially along the surface. The stigmaticgroove is bordered by a well-defined layer of glandular cells,each of which has a pectinaceous cap on the inner tangentialwall. The style is hollow. The canal cells show thickeningson the inner tangential wall. The stigmatic groove and stylarcanal contain an extracellular matrix secreted by the canalcells which is rich in proteins, acidic polysaccharides andpectins. The canal cells at the base of the style are papillateand loosely fill the stylar canal. The stigma becomes receptivewhen the stigmatic lobes separate, and remains so for 24 h.Pollination is mediated by weevils as well as by the wind. Undernatural conditions the pollination efficiency was 100%. Pollinationinduces additional secretion in the stigmatic groove and stylarcanal. During post-pollination secretion, the pectinaceous capsof the cells lining the stigmatic groove are degraded. Pollengrains germinate on the stigmatic papillae and tubes grow onthe surface of the papillae, entering the stigmatic groove andadvancing along it into the stylar canal to eventually gainaccess to the locules. Pollen tubes are seen in the ovules 18–20h after pollination. Copyright 2001 Annals of Botany Company Arecaceae, Elaeis guineensis, African oil palm, pollination, stigmatic grove, stylar canal, Tenera hybrid, weevil