NADPH oxidase mediates the oxygen-glucose deprivation/reperfusion-induced increase in the tyrosine phosphorylation of the N-methyl-D-aspartate receptor NR2A subunit in retinoic acid differentiated SH-SY5Y Cells

ABSTRACT: BACKGROUND: Evidence exists that oxidative stress promotes the tyrosine phosphorylation of N-methyl-D-aspartate receptor (NMDAR) subunits during post-ischemic reperfusion of brain tissue. Increased tyrosine phosphorylation of NMDAR NR2A subunits has been reported to potentiate receptor function and exacerbate NMDAR-induced excitotoxicity. Though the effect of ischemia on tyrosine phosphorylation of NMDAR subunits has been well documented, the oxidative stress signaling cascades mediating the enhanced tyrosine phosphorylation of NR2A subunits remain unclear. RESULTS: We report that the reactive oxygen species (ROS) generator NADPH oxidase mediates an oxidative stress-signaling cascade involved in the increased tyrosine phosphorylation of the NR2A subunit in post-ischemic differentiated SH-SY5Y neuroblastoma cells. Inhibition of NADPH oxidase attenuated the increased tyrosine phosphorylation of the NMDAR NR2A subunit, while inhibition of ROS production from mitochondrial or xanthine oxidase sources failed to dampen the post-ischemic increase in tyrosine phosphorylation of the NR2A subunit. Additionally, inhibition of NADPH oxidase blunted the interaction of activated Src Family Kinases (SFKs) with PSD-95 induced by ischemia/reperfusion. Lastly, inhibition of NADPH oxidase also markedly reduced cell death in post-ischemic SH-SY5Y cells stimulated by NMDA. CONCLUSIONS: These data indicate that NADPH oxidase has a key role in facilitating NMDAR NR2A tyrosine phosphorylation via SFK activation during post-ischemic reperfusion.     

Preeclampsia: multiple approaches for a multifactorial disease

Preeclampsia is a pregnancy-specific disorder characterized by hypertension and excess protein excretion in the urine. It is an important cause of maternal and fetal morbidity and mortality worldwide. The disease is almost exclusive to humans and delivery of the pregnancy continues to be the only effective treatment. The disorder is probably multifactorial, although most cases of preeclampsia are characterized by abnormal maternal uterine vascular remodeling by fetally derived placental trophoblast cells. Numerous in vitro and animal models have been used to study aspects of preeclampsia, the most common being models of placental oxygen dysregulation, abnormal trophoblast invasion, inappropriate maternal vascular damage and anomalous maternal-fetal immune interactions. Investigations into the pathophysiology and treatment of preeclampsia continue to move the field forward, albeit at a frustratingly slow pace. There remains a pressing need for novel approaches, new disease models and innovative investigators to effectively tackle this complex and devastating disorder.     

Global and Local Concerns: What Attitudes and Beliefs Motivate Farmers to Mitigate and Adapt to Climate Change?

In response to agriculture''s vulnerability and contribution to climate change, many governments are developing initiatives that promote the adoption of mitigation and adaptation practices among farmers. Since most climate policies affecting agriculture rely on voluntary efforts by individual farmers, success requires a sound understanding of the factors that motivate farmers to change practices. Recent evidence suggests that past experience with the effects of climate change and the psychological distance associated with people''s concern for global and local impacts can influence environmental behavior. Here we surveyed farmers in a representative rural county in California''s Central Valley to examine how their intention to adopt mitigation and adaptation practices is influenced by previous climate experiences and their global and local concerns about climate change. Perceived changes in water availability had significant effects on farmers'' intention to adopt mitigation and adaptation strategies, which were mediated through global and local concerns respectively. This suggests that mitigation is largely motivated by psychologically distant concerns and beliefs about climate change, while adaptation is driven by psychologically proximate concerns for local impacts. This match between attitudes and behaviors according to the psychological distance at which they are cognitively construed indicates that policy and outreach initiatives may benefit by framing climate impacts and behavioral goals concordantly; either in a global context for mitigation or a local context for adaptation.     

Yield response of dual-toxin Bt cotton to Helicoverpa zea infestations

Field cage experiments were conducted to determine the impact of bollworms, Helicoverpa zea (Boddie), on yields of Bollgard II and Widestrike cotton, Gossypium hirsutum L. One-day-old bollworm larvae were infested in white flowers of Bollgard II and in white flowers and terminals of Widestrike cotton. The infestation levels included 0, 50, and 100% of white flowers for each type of cotton. Terminal infestations included one or two larvae per terminal on Widestrike cotton. Larvae were placed in flowers of Bollgard II cotton each day for 1 to 4 wk during the first 4 wk of flowering during 2003, 2004, and 2005 seasons and in the flowers or terminals of Widestrike cotton each day for 1 to 3 wk. Averaged across years and durations of infestation, yields of Bollgard II cotton were significantly reduced compared with noninfested Bollgard II cotton when 100% of white flowers were infested. For Widestrike cotton, there was a reduction in yield when 100% of white flowers were infested in 2005, but not in 2006. There was a significant relationship for cumulative numbers of white flowers infested on seedcotton yield of Bollgard II during one of the 3 yr of the experiment. The regression equation during that year had a slope of -0.77. No significant relationships were observed for cumulative numbers of white flowers infested on yields of Widestrike cotton. Results of the current experiment suggest bollworms will rarely cause yield losses of Bollgard II and Widestrike cotton. Future research will need to focus on developing specific thresholds for bollworms on Bollgard II and Widestrike cotton.     

c-Rel is essential for the development of innate and T cell-induced colitis

Inflammatory bowel disease is a chronic inflammatory response of the gastrointestinal tract mediated in part by an aberrant response to intestinal microflora. Expression of IL-23 subunits p40 and p19 within cells of the innate immune system plays a central role in the development of lower bowel inflammation in response inflammatory challenge. The NF-kappaB subunit c-Rel can regulate expression of IL-12/23 subunits suggesting that it could have a critical role in mediating the development of chronic inflammation within the lower bowel. In this study, we have analyzed the role of c-Rel within the innate immune system in the development of lower bowel inflammation, in two well-studied models of murine colitis. We have found that the absence of c-Rel significantly impaired the ability of Helicobacter hepaticus to induce colitis upon infection of RAG-2-deficient mice, and ameliorated the ability of CD4(+)CD45RB(high) T cells to induce disease upon adoptive transfer into RAG-deficient mice. The absence of c-Rel interfered with the expression of IL-12/23 subunits both in cultured primary macrophages and within the colon. Thus, c-Rel plays a critical role in regulating the innate inflammatory response to microflora within the lower bowel, likely through its ability to modulate expression of IL-12/23 family members.     

A novel proteolytic cascade generates an extracellular matrix-derived chemoattractant in chronic neutrophilic inflammation

Chronic neutrophilic inflammation is a manifestation of a variety of lung diseases including cystic fibrosis (CF). There is increasing evidence that fragments of extracellular matrix proteins, such as collagen and elastin, play an important role in inflammatory cell recruitment to the lung in animal models of airway inflammation. Unfortunately, the association of these peptides with human disease and the identification of therapeutic targets directed toward these inflammatory pathways have remained elusive. In this study, we demonstrate that a novel extracellular matrix-derived neutrophil chemoattractant, proline-glycine-proline (PGP), acts through CXC receptors 1 and 2 on neutrophils, similar to N-acetylated proline-glycine-proline (N-alpha-PGP). We describe the specific multistep proteolytic pathway involved in PGP generation from collagen, involving matrix metalloproteases 8 and 9 and prolyl endopeptidase, a serine protease for which we identify a novel role in inflammation. PGP generation correlates closely with airway neutrophil counts after administration of proteases in vivo. Using CF as a model, we show that CF sputum has elevated levels of PGP peptides and that PGP levels decline during the course of CF inpatient therapy for acute pulmonary exacerbation, pointing to its role as a novel biomarker for this disease. Finally, we demonstrate that CF secretions are capable of generating PGP from collagen ex vivo and that this generation is significantly attenuated by the use of inhibitors directed toward matrix metalloprotease 8, matrix metalloprotease 9, or prolyl endopeptidase. These experiments highlight unique protease interactions with structural proteins regulating innate immunity and support a role for these peptides as novel biomarkers and therapeutic targets for chronic, neutrophilic lung diseases.     

Structural abnormalities in neurons are sufficient to explain the clinical disease and fatal outcome of experimental rabies in yellow fluorescent protein-expressing transgenic mice

Under natural conditions and in some experimental models, rabies virus infection of the central nervous system causes relatively mild histopathological changes, without prominent evidence of neuronal death despite its lethality. In this study, the effects of rabies virus infection on the structure of neurons were investigated with experimentally infected transgenic mice expressing yellow fluorescent protein (YFP) in neuronal subpopulations. Six-week-old mice were inoculated in the hind-limb footpad with the CVS strain of fixed virus or were mock infected with vehicle (phosphate-buffered saline). Brain regions were subsequently examined by light, epifluorescent, and electron microscopy. In moribund CVS-infected mice, histopathological changes were minimal in paraffin-embedded tissue sections, although mild inflammatory changes were present. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and caspase-3 immunostaining showed only a few apoptotic cells in the cerebral cortex and hippocampus. Silver staining demonstrated the preservation of cytoskeletal integrity in the cerebral cortex. However, fluorescence microscopy revealed marked beading and fragmentation of the dendrites and axons of layer V pyramidal neurons in the cerebral cortex, cerebellar mossy fibers, and axons in brainstem tracts. At an earlier time point, when mice displayed hind-limb paralysis, beading was observed in a few axons in the cerebellar commissure. Toluidine blue-stained resin-embedded sections from moribund YFP-expressing animals revealed vacuoles within the perikarya and proximal dendrites of pyramidal neurons in the cerebral cortex and hippocampus. These vacuoles corresponded with swollen mitochondria under electron microscopy. Vacuolation was also observed ultrastructurally in axons and in presynaptic nerve endings. We conclude that the observed structural changes are sufficient to explain the severe clinical disease with a fatal outcome in this experimental model of rabies.     

MicroRNAs in the miR-106b family regulate p21/CDKN1A and promote cell cycle progression

microRNAs in the miR-106b family are overexpressed in multiple tumor types and are correlated with the expression of genes that regulate the cell cycle. Consistent with these observations, miR-106b family gain of function promotes cell cycle progression, whereas loss of function reverses this phenotype. Microarray profiling uncovers multiple targets of the family, including the cyclin-dependent kinase inhibitor p21/CDKN1A. We show that p21 is a direct target of miR-106b and that its silencing plays a key role in miR-106b-induced cell cycle phenotypes. We also show that miR-106b overrides a doxorubicin-induced DNA damage checkpoint. Thus, miR-106b family members contribute to tumor cell proliferation in part by regulating cell cycle progression and by modulating checkpoint functions.     

The enzymatic basis for pesticide bioremediation

Enzymes are central to the biology of many pesticides, influencing their modes of action, environmental fates and mechanisms of target species resistance. Since the introduction of synthetic xenobiotic pesticides, enzymes responsible for pesticide turnover have evolved rapidly, in both the target organisms and incidentally exposed biota. Such enzymes are a source of significant biotechnological potential and form the basis of several bioremediation strategies intended to reduce the environmental impacts of pesticide residues. This review describes examples of enzymes possessing the major activities employed in the bioremediation of pesticide residues, and some of the strategies by which they are employed. In addition, several examples of specific achievements in enzyme engineering are considered, highlighting the growing trend in tailoring enzymatic activity to a specific biotechnologically relevant function.