An increased risk of HIV-1 associated dementia (HAD) has been observed in patients abusing methamphetamine (METH). Since both HIV viral proteins (gp120, Tat) and METH induce oxidative stress, drug abusing patients are at a greater risk of oxidative stress-induced damage. The objective of this study was to determine if N-acetylcysteine amide (NACA) protects the blood brain barrier (BBB) from oxidative stress-induced damage in animals exposed to gp120, Tat and METH. To study this, CD-1 mice pre-treated with NACA/saline, received injections of gp120, Tat, gp120 + Tat or saline for 5 days, followed by three injections of METH/saline on the fifth day, and sacrificed 24 h after the final injection. Various oxidative stress parameters were measured, and animals treated with gp120+Tat+Meth were found to be the most challenged group, as indicated by their GSH and MDA levels. Treatment with NACA significantly rescued the animals from oxidative stress. Further, NACA-treated animals had significantly higher expression of TJ proteins and BBB permeability as compared to the group treated with gp120+Tat+METH alone, indicating that NACA can protect the BBB from oxidative stress-induced damage in gp120, Tat and METH exposed animals, and thus could be a viable therapeutic option for patients with HAD.

The era of highly active antiretroviral therapy (HAART) has controlled AIDS and its related disorders considerably; however, prevalence of HIV-1-associated neurocognitive disorders (HAND) has been on the rise in the post-HAART era. In view of these developments, we investigated whether a HAART drug combination of 3’-Azido-2’, 3’-deoxythymidine (AZT) and Indinavir (IDV) can alter the functionality of the blood-brain barrier (BBB) endothelial cells, thereby exacerbating this condition. Viability of hCMEC/D3 cells (in vitro model of BBB) that were exposed to these drugs was significantly reduced after 72 hr treatment, in a dose-dependent manner. Reactive oxygen species (ROS) were highly elevated after the exposure, indicating that mechanisms that induce oxidative stress were involved. Measures of oxidative stress parameters, such as glutathione (GSH) and malondialdehyde (MDA), were found to be altered in the treated groups. Loss of mitochondrial membrane potential (Ψm), as assessed with fluorescent microscopy and decreased levels of ATP, indicated that cytoxicity was mediated through mitochondrial dysfunction. Furthermore, AZT + IDV treatment caused apoptosis in endothelial cells, as assessed by the expression of cytochrome c and procaspase-3 proteins. Pretreatment with thiol antioxidant N-acetylcysteine amide (NACA) reversed some of the pro-oxidant effects of AZT+IDV. Results from our in vitro studies indicate that the AZT + IDV combination may affect the BBB in HIV infected individuals treated with HAART drugs.

A disintegrin and metalloproteinase domain 10 (Adam10), a member of the ADAM family of cell membrane-anchored proteins, has been linked to the regulation of the Notch, EGF, E-cadherin, and other signaling pathways. However, it is unclear what role Adam10 has in the kidney in vivo. In this study, we showed that Adam10 deficiency in ureteric bud (UB) derivatives leads to a decrease in urinary concentrating ability, polyuria, and hydronephrosis in mice. Furthermore, Adam10 deficiency led to a reduction in the percentage of aquaporin 2 (Aqp2) + principal cells (PCs) in the collecting ducts that was accompanied by a proportional increase in the percentage of intercalated cells (ICs). This increase was more prominent in type A ICs than in type B ICs. Foxi1, a transcription factor important for the differentiation of ICs, was upregulated in the Adam10 mutants. The observed reduction of Notch activity in Adam10 mutant collecting duct epithelium and the similar reduction of PC/IC ratios in the collecting ducts in mice deficient for mindbomb E3 ubiquitin protein ligase 1, a key regulator of the Notch and Wnt/receptor-like tyrosine kinase signaling pathways, suggest that Adam10 regulates cell fate determination through the activation of Notch signaling, probably through the regulation of Foxi1 expression. However, phenotypic differences between the Adam10 mutants, the Mib1 mutants, and the Foxi1 mutants suggest that the functions of Adam10 in determining the fate of collecting duct cells are more complex than those of a simple upstream factor in a linear pathway involving Notch and Foxi1.

NFAT upregulation has been linked to cellular transformation intrinsically, but it is unclear whether and how tissue cells with NFAT activation change the local environment for tumor initiation and progression. Direct evidence showing NFAT activation initiates primary tumor formation in vivo is also lacking. Using inducible transgenic mouse systems, we show that tumors form in a subset of, but not all, tissues with NFATc1 activation, indicating that NFAT oncogenic effects depend on cell types and tissue contexts. In NFATc1-induced skin and ovarian tumors, both cells with NFATc1 activation and neighboring cells without NFATc1 activation have significant upregulation of c-Myc and activation of Stat3. Besides known and suspected NFATc1 targets, such as Spp1 and Osm, we have revealed the early upregulation of a number of cytokines and cytokine receptors, as key molecular components of an inflammatory microenvironment that promotes both NFATc1+ and NFATc1− cells to participate in tumor formation. Cultured cells derived from NFATc1-induced tumors were able to establish a tumorigenic microenvironment, similar to that of the primary tumors, in an NFATc1-dependent manner in nude mice with T cell deficiency, revealing an addiction of these tumors to NFATc1 activation and downplaying a role for T cells in the NFATc1-induced tumorigenic microenvironment. These findings collectively suggest that beyond the cell autonomous effects on the upregulation of oncogenic proteins, NFATc1 activation has non-cell autonomous effects through the establishment of a promitogenic microenvironment for tumor growth. This study provides direct evidence for the ability of NFATc1 in inducing primary tumor formation in vivo and supports targeting NFAT signaling in anti-tumor therapy.

A reverse-phase HPLC method incorporating dithiothreitol (DTT) reduction for quantitative determination of oxidized glutathione (GSSG) in biological samples is described here. This method is based on our previous enzymatic reduction technique that uses N-1-(pyrenyl) maleimide (NPM) as a derivatizing agent. In our earlier method, glutathione disulfide (GSSG) was measured by first reducing it to GSH with glutathione reductase (GR) in the presence of NADPH. However, this is a very costly and time-consuming technique. The method described here employs a common and inexpensive thiol-disulfide exchanging agent, DTT, for reduction of GSSG to GSH, followed by derivatization with NPM. The calibration curves are linear over a concentration range of 25-1250 nm (r(2) > 0.995). The coefficients of variations for intra-run precision and inter-run precision range from 0.49 to 5.10% with an accuracy range of 1.78-6.15%. The percentage of relative recovery ranges from 97.3 to 103.2%. This new method provides a simple, efficient, and cost-effective way of determining glutathione disulfide levels with a 2.5 nm limit of detection per 5 microL injection volume.