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We examined the potential expression and function of α7 nicotinic acetylcholine receptors (nAChRs) in leukocytes. RT-PCR with α7 specific primers revealed the presence of the receptor mRNA in leukocytes. Immunoblotting and immunofluorescence experiments demonstrated the expression of a protein that is recognized by α7 specific antibodies. However, nicotine and acetylcholine (ACh) failed to elicit current in leukocytes. Binding experiments with α-bungarotoxin rhodamine conjugated were negative, illustrating the absence of a high-affinity binding site. RT-PCR analysis revealed the selective expression of the dupα7 mRNA. These data indicate that leukocytes express in their membrane the dupα7 protein but its physiological role remains to be identified. [Copyright &y& Elsevier]

Reactive oxygen species (ROS) are key modulators of apoptosis and carcinogenesis. One of the important sources of ROS is NADPH oxidases (NOXs). The isoform NOX5 is highly expressed in lymphoid tissues, but it has not been detected in any common Hodgkin or non-Hodgkin lymphoma cell lines. In diverse, nonlymphoid malignant cells NOX5 exerts an antiapoptotic effect. Apoptosis suppression is the hallmark feature of a rare type of lymphoma, termed anaplastic lymphoma kinase-positive (ALK + ) anaplastic large-cell lymphoma (ALCL), and a major factor in the therapy resistance and relapse of ALK + ALCL tumors. We applied RT-PCR and Western blot analysis to detect NOX5 expression in three ALK + ALCL cell lines (Karpas-299, SR-786, SUP-M2). We investigated the role of NOX5 in apoptosis by small-interfering RNA (siRNA)-mediated gene silencing and chemical inhibition of NOX5 using FACS analysis and examining caspase 3 cleavage in Karpas-299 cells. We used immunohistochemistry to detect NOX5 in ALK + ALCL pediatric tumors. NOX5 mRNA was uniquely detected in ALK + ALCL cells, whereas cell lines of other lymphoma classes were devoid of NOX5. Transfection of NOX5-specific siRNA and chemical inhibition of NOX5 abrogated calcium-induced superoxide production and increased caspase 3-mediated apoptosis in Karpas-299 cells. Immunohistochemistry revealed focal NOX5 reactivity in pediatric ALK + ALCL tumor cells. These results indicate that NOX5-derived ROS contribute to apoptosis blockage in ALK + ALCL cell lines and suggest NOX5 as a potential pharmaceutical target to enhance apoptosis and thus to suppress tumor progression and prevent relapse in pediatric ALK + ALCL patients that resist classical therapeutic approaches. [ABSTRACT FROM AUTHOR]

Abstract: Ischemia-induced neuronal damage has been linked to elevated production of reactive oxygen species (ROS) both in animal models and in humans. NADPH oxidase enzymes (NOX-es) are a major enzymatic source of ROS, but their role in brain ischemia has not yet been investigated. The present study was carried out to examine the expression of NOX4, one of the new NADPH oxidase isoforms in a mouse model of focal permanent brain ischemia. We demonstrate that NOX4 is expressed in neurons using in situ hybridization and immunohistochemistry. Ischemia, induced by middle cerebral artery occlusion resulted in a dramatic increase in cortical NOX4 expression. Elevated NOX4 mRNA levels were detectable as early as 24 h after the onset of ischemia and persisted throughout the 30 days of follow-up period, reaching a maximum between days 7 and 15. The early onset suggests neuronal reaction, while the peak period corresponds to the time of neoangiogenesis occurring mainly in the peri-infarct region. The occurrence of NOX4 in the new capillaries was confirmed by immunohistochemical staining. In summary, our paper reports the presence of the ROS producing NADPH oxidase NOX4 in neurons and demonstrates an upregulation of its expression under ischemic conditions. Moreover, a role for NOX4 in ischemia/hypoxia-induced angiogenesis is suggested by its prominent expression in newly formed capillaries. [Copyright &y& Elsevier]