Ceramide is the key mediator of oxidative stress-induced apoptosis in retinal photoreceptor cells

Nitric oxide and reactive oxygen species play a critical role in photoreceptor apoptosis. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. Here, we demonstrate that the sphingolipid ceramide is the key mediator of oxidative stress-induced apoptosis in 661W retinal photoreceptor cells. Treatment of 661W cells with the nitric oxide donor, sodium nitroprusside, activates acid sphingomyelinase. As a result, sphingomyelin is hydrolysed, which leads to an increase in the concentration of ceramide. We also show that ceramide is responsible for the activation of the mitochondrial apoptotic pathway in 661W photoreceptor cells and subsequent activation of the caspase cascade. Furthermore, we show for the first time that ceramide is responsible for the increased Ca2+ levels in the mitochondria and cytosol that precedes activation of the calpain-mediated apoptotic pathway. Additionally, we provide evidence that ceramide also activates the endolysosomal protease cathepsin D pathway. In summary, our findings show that ceramide controls the cell death decisions in photoreceptor cells and highlight the relevance of acid sphingomyelinase as a potential therapeutic target for the treatment of retinal pathologies.     

NADPH Oxidase-generated Hydrogen Peroxide Induces DNA Damage in Mutant FLT3-expressing Leukemia Cells

Internal tandem duplication of the FMS-like tyrosine kinase (FLT3-ITD) receptor is present in 20% of acute myeloid leukemia (AML) patients and it has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (DSBs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Our group has previously reported that inhibition of FLT3-ITD signaling results in post-translational down-regulation of p22^phox, a small membrane-bound subunit of the NADPH oxidase (NOX) complex. Here we demonstrated that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD, have a higher protein level of p22^phox and p22^phox-interacting NOX isoforms than 32D cells transfected with the wild type FLT3 receptor (FLT3-WT). The inhibition of NOX proteins, p22^phox, and NOX protein knockdowns caused a reduction in ROS, as measured with a hydrogen peroxide (H₂O₂)-specific dye, peroxy orange 1 (PO1), and nuclear H₂O₂, as measured with nuclear peroxy emerald 1 (NucPE1). These reductions in the level of H₂O₂ following the NOX knockdowns were accompanied by a decrease in the number of DNA DSBs. We showed that 32D cells that express FLT3-ITD have a higher level of both oxidized DNA and DNA DSBs than their wild type counterparts. We also observed that NOX4 and p22^phox localize to the nuclear membrane in MV4–11 cells expressing FLT3-ITD. Taken together these data indicate that NOX and p22^phox mediate the ROS production from FLT3-ITD that signal to the nucleus causing genomic instability.     

An autonomous replicating element within the KSHV genome

Members of the herpesviridae family including Kaposi's sarcoma-associated herpesvirus (KSHV) persist latently in their hosts and harbor their genomes as closed circular episomes. Propagation of the KSHV genome into new daughter cells requires replication of the episome once every cell division and is considered critically dependent on expression of the virus encoded latency-associated nuclear antigen (LANA). This study demonstrates a LANA-independent mechanism of KSHV latent DNA replication. A cis-acting DNA element within a discreet KSHV genomic region termed the long unique region (LUR) can initiate and support replication of plasmids lacking LANA-binding sequences or a eukaryotic replication origin. The human cellular replication machinery proteins ORC2 and MCM3 associated with the LUR element and depletion of cellular ORC2 abolished replication of the plasmids indicating that recruitment of the host cellular replication machinery is important for LUR-dependent replication. Thus, KSHV can initiate replication of its genome independent of any trans-acting viral factors.     

Two‐dimensional reference map for the basic proteome of the human dorsolateral prefrontal cortex (dlPFC) of the prefrontal lobe region of the brain

We describe a 2‐DE proteomic reference map containing 227 basic proteins in the dorsolateral prefrontal cortex region of the human brain. Proteins were separated in the first dimension on pH 6–11 IPG strips using paper‐bridge loading and on 12% SDS‐PAGE in the second dimension. Proteins were subsequently identified by MS and spectra were analyzed using an in‐house proteomics data analysis platform, Proline. The 2‐DE reference map is available via the UCD 2‐DE Proteome Database ( http://proteomics‐portal.ucd.ie:8082 ) and can also be accessed via the WORLD‐2DPAGE Portal ( http://www.expasy.ch/world‐2dpage/ ). The associated protein identification data have been submitted to the PRIDE database (accession numbers 10018–10033). Separation of proteins in the basic region resolves more membrane associated proteins relevant to the synaptic pathology central to many neurological disorders. The 2‐DE reference map will aid with further characterisation of neurological disorders such as bipolar and schizophrenia.     

Morphological and molecular characterization of populations of plains bristlegrass (<i>Setaria macrostachya</i> Kunth) in Chihuahua,México

Plains bristlegrass (Setaria macrostachya Kunth) is a native grass with forage value. However, due to the lack of grazing management practices, populations and thus genetic diversity, have been reduced. Morphological and genetic variability were analyzed on 44 populations of plains bristlegrass in the State of Chihuahua. Plants were transplanted in a common area under natural conditions. Two years later, morphological characterization was evaluated measuring nine variables, and genetic variability using AFLP molecular markers. The principal components analysis (PC) showed that the three first principal components explained 73.74% of the variation. The variables with the greatest contribution to the variance in PC1 were plant height and inflorescence length; in CP2, tiller number and leaf width; and in PC3, tiller thickness. Application of four pairs of primers, presented 186 total bands, from which 87.10% showed polymorphism and 12.90% monomorphism. The combination of EcoRI-AGG MseI-CAG primers detected the highest percentage (93%) of polymorphism with 40 polymorphic bands. The cluster analysis and Dice coefficient indicated that populations clump into two groups. The wide genetic variability and morphological characteristics detected among populations represent the basis for the selection of populations that could be used with different purposes in the rehabilitation of ecosystems. In addition, this study will allow establishment of in situ conservation strategies.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

The ability to cleave 28S ribosomal RNA during apoptosis is a cell-type dependent trait unrelated to DNA fragmentation

Discrete cleavages within 28S rRNA divergent domains have previously been found to coincide with DNA fragmentation during apoptosis. Here we show that rRNA and DNA cleavages can occur independently in apoptotic cells, i.e. that the previously observed correlation is likely to be coincidental. In HL-60 cells, apoptosis with massive DNA fragmentation could be induced without any signs of rRNA cleavage. The opposite situation; rRNA cleavage without concomitant internucleosomal DNA fragmentation, was found in okadaic acid-treated Molt-4 cells. Other leukemia cell lines underwent apoptosis either without (K562 and Molt-3) or with (U937) both forms of polynucleotide cleavage. In K562 cells transfected with a temperature-sensitive p53 mutant, internucleosomal DNA fragmentation but not 28S rRNA cleavage was inducible by wild-type p53 expression. The absence of apoptotic rRNA cleavage in some cell types suggests that this phenomenon is tightly regulated and unrelated to DNA fragmentation or a presumed scheme for general macromolecular degradation in apoptotic cells.     

Carbonylation of glycolytic proteins is a key response to drug-induced oxidative stress and apoptosis

Recent work has highlighted the importance of protein post-translational modifications such as phosphorylation (enzymatic) and nitrosylation (nonenzymatic) in the early stages of apoptosis. In this study, we have investigated the levels of protein carbonylation, a nonenzymatic protein modification that occurs in conditions of cellular oxidative stress, during etopside-induced apoptosis of HL60 cells. Within 1 h of VP16 treatment, a number of proteins underwent carbonylation due to oxidative stress. This was inhibited by the antioxidant N-acetyl-L-cysteine. Among the proteins found to be carbonylated were glycolytic enzymes. Subsequently, we found that the rate of glycolysis was significantly reduced, probably due to a carbonylation mediated reduction in enzymatic activity of glycolytic enzymes. Our work demonstrates that protein carbonylation can be rapidly induced through cytotoxic drug treatment and may specifically inhibit the glycolytic pathway. Given the importance of glycolysis as a source of cellular ATP, this has severe implications for cell function.     

Bioengineered nisin derivatives with enhanced activity in complex matrices

Nisin A is the best known and most extensively characterized lantibiotic. As it is ribosomally synthesized, bioengineering‐based strategies can be used to generate variants. We have previously demonstrated that bioengineering of the hinge region of nisin A can result in the generation of variants with enhanced anti‐microbial activity against Gram‐positive pathogens. Here we created a larger bank of hinge variant producers and screened for producers that exhibit enhanced bioactivity as assessed by agar‐based assays against a selection of target strains. Further analysis of 12 ‘lead’ variants reveals that in many cases enhanced bioactivity is not attributable to enhanced specific activity but is instead as a consequence of an enhanced ability to diffuse through complex polymers. In the case of two variants, which contain the residues SVA and NAK, respectively, within the hinge region, we demonstrate that this enhanced trait enables the peptides to dramatically outperform nisin A with respect to controlling Listeria monocytogenes in commercially produced chocolate milk that contains carrageenan as a stabilizer.