Enriched Environment Prevents Hypobaric Hypoxia Induced Neurodegeneration and is Independent of Antioxidant Signaling

Hypobaric hypoxia (HH) induced neurodegeneration has been attributed to several factors including increased oxidative stress, glutamate excitotoxicity, decreased growth factors, apoptosis, etc. Though enriched environment (EE) has been known to have beneficial effects in various neurological disorders, its effect on HH mediated neurodegeneration remains to be studied. Therefore, the present study was conducted to explore the effect of EE on HH induced neurodegeneration. Male Sprague-Dawley rats were placed in enriched and standard conditions during exposure to HH (7 days) equivalent to an altitude of 25,000 ft. The effect of EE on oxidative stress markers, apoptosis, and corticosterone level in hippocampus was investigated. EE during exposure to HH was found to decrease neurodegeneration as evident from decreased caspase 3 expression and LDH leakage. However, no significant changes were observed in ROS, MDA, and antioxidant status of hippocampus. HH elevates corticosterone level and affected the diurnal corticoid rhythm which may contribute to neurodegeneration, whereas EE ameliorate this effect. Because of the association of neurotrophins and stress and/or corticosterone the BDNF and NGF levels were also examined and it was found that HH decreases their level but concurrent exposure to EE maintains their level. Moreover, inhibition of Tyrosine kinase receptor (Trk) with K252a nullifies the protective effect of EE, whereas Trk activation with agonist, amitriptyline showed protective effect similar to EE. Taken together, we conclude that EE has a potential to ameliorate HH mediated neuronal degeneration which may act through antioxidant independent pathway by modulation of neurotrophins.     

Antitumor potential of Castanopsis indica (Roxb. ex Lindl.) A. DC. leaf extract against Ehrlich's ascites carcinoma cell

Methanol extract of C. indica (MECI) leaves showed direct cytotoxicity on Ehrlich ascites carcinoma (EAC) cell in a dose dependant manner and there was significant decrease in the tumor volume, viable cell count, tumor weight and elevated the life span of EAC tumor bearing mice. Hematological profile and biochemical estimations were significantly restored to normal levels in MECI treated as compared to EAC control mice. MECI treatment significantly modulated the tissue antioxidant assay parameters as compared to the EAC control mice. The results revealed that MECI possesses significant dose dependent antitumor potential which may be due to its cytotoxicity and antioxidant properties.     

EGFR and c-Met Cross Talk in Glioblastoma and Its Regulation by Human Cord Blood Stem Cells

Receptor tyrosine kinases (RTK) and their ligands control critical biologic processes, such as cell proliferation, migration, and differentiation. Aberrant expression of these receptor kinases in tumor cells alters multiple downstream signaling cascades that ultimately drive the malignant phenotype by enhancing tumor cell proliferation, invasion, metastasis, and angiogenesis. As observed in human glioblastoma (hGBM) and other cancers, this dysregulation of RTK networks correlates with poor patient survival. Epidermal growth factor receptor (EGFR) and c-Met, two well-known receptor kinases, are coexpressed in multiple cancers including hGBM, corroborating that their downstream signaling pathways enhance a malignant phenotype. The integration of c-Met and EGFR signaling in cancer cells indicates that treatment regimens designed to target both receptor pathways simultaneously could prove effective, though resistance to tyrosine kinase inhibitors continues to be a substantial obstacle. In the present study, we analyzed the antitumor efficacy of EGFR inhibitors erlotinib and gefitinib and c-Met inhibitor PHA-665752, along with their respective small hairpin RNAs (shRNAs) alone or in combination with human umbilical cord blood stem cells (hUCBSCs), in glioma cell lines and in animal xenograft models. We also measured the effect of dual inhibition of EGFR/c-Met pathways on invasion and wound healing. Combination treatments of hUCBSC with tyrosine kinase inhibitors significantly inhibited invasion and wound healing in U251 and 5310 cell lines, thereby indicating the role of hUCBSC in inhibition of RTK-driven cell behavior. Further, the EGFR and c-Met localization in glioma cells and hGBM clinical specimens indicated that a possible cross talk exists between EGFR and c-Met signaling pathway.     

Appraisal of a Leishmania major Strain Stably Expressing mCherry Fluorescent Protein for Both In Vitro and In Vivo Studies of Potential Drugs and Vaccine against Cutaneous Leishmaniasis

Background

Leishmania major cutaneous leishmaniasis is an infectious zoonotic disease. It is produced by a digenetic parasite, which resides in the phagolysosomal compartment of different mammalian macrophage populations. There is an urgent need to develop new therapies (drugs) against this neglected disease that hits developing countries. The main goal of this work is to establish an easier and cheaper tool of choice for real-time monitoring of the establishment and progression of this pathology either in BALB/c mice or in vitro assays. To validate this new technique we vaccinated mice with an attenuated Δhsp70-II strain of Leishmania to assess protection against this disease.

Methodology

We engineered a transgenic L. major strain expressing the mCherry red-fluorescent protein for real-time monitoring of the parasitic load. This is achieved via measurement of fluorescence emission, allowing a weekly record of the footpads over eight weeks after the inoculation of BALB/c mice.

Results

In vitro results show a linear correlation between the number of parasites and fluorescence emission over a range of four logs. The minimum number of parasites (amastigote isolated from lesion) detected by their fluorescent phenotype was 10,000. The effect of antileishmanial drugs against mCherry+L. major infecting peritoneal macrophages were evaluated by direct assay of fluorescence emission, with IC₅₀ values of 0.12, 0.56 and 9.20 µM for amphotericin B, miltefosine and paromomycin, respectively. An experimental vaccination trial based on the protection conferred by an attenuated Δhsp70-II mutant of Leishmania was used to validate the suitability of this technique in vivo.

Conclusions

A Leishmania major strain expressing mCherry red-fluorescent protein enables the monitoring of parasitic load via measurement of fluorescence emission. This approach allows a simpler, faster, non-invasive and cost-effective technique to assess the clinical progression of the infection after drug or vaccine therapy.     

SPARC overexpression inhibits cell proliferation in neuroblastoma and is partly mediated by tumor suppressor protein PTEN and AKT

Secreted protein acidic and rich in cysteine (SPARC) is also known as BM-40 or Osteonectin, a multi-functional protein modulating cell-cell and cell-matrix interactions. In cancer, SPARC is not only linked with a highly aggressive phenotype, but it also acts as a tumor suppressor. In the present study, we sought to characterize the function of SPARC and its role in sensitizing neuroblastoma cells to radio-therapy. SPARC overexpression in neuroblastoma cells inhibited cell proliferation in vitro. Additionally, SPARC overexpression significantly suppressed the activity of AKT and this suppression was accompanied by an increase in the tumor suppressor protein PTEN both in vitro and in vivo. Restoration of neuroblastoma cell radio-sensitivity was achieved by overexpression of SPARC in neuroblastoma cells in vitro and in vivo. To confirm the role of the AKT in proliferation inhibited by SPARC overexpression, we transfected neuroblastoma cells with a plasmid vector carrying myr-AKT. Myr-AKT overexpression reversed SPARC-mediated PTEN and increased proliferation of neuroblastoma cells in vitro. PTEN overexpression in parallel with SPARC siRNA resulted in decreased AKT phosphorylation and proliferation in vitro. Taken together, these results establish SPARC as an effector of AKT-PTEN-mediated inhibition of proliferation in neuroblastoma in vitro and in vivo.     

Kaposi's sarcoma-associated herpesvirus forms a multimolecular complex of integrins (alphaVbeta5, alphaVbeta3, and alpha3beta1) and CD98-xCT during infection of human dermal microvascular endothelial cells, and CD98-xCT is essential for the postentry stage of infection

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface heparan sulfate (HS) and α3β1 integrin during the early stages of infection of human dermal microvascular endothelial cells (HMVEC-d) and human foreskin fibroblasts (HFF), and these interactions are followed by virus entry overlapping with the induction of preexisting host cell signal pathways. KSHV also utilizes the amino acid transporter protein xCT for infection of adherent cells, and the xCT molecule is part of the cell surface heterodimeric membrane glycoprotein CD98 (4F2 antigen) complex known to interact with α3β1 and αVβ3 integrins. KSHV gB mediates adhesion of HMVEC-d, CV-1, and HT-1080 cells and HFF via its RGD sequence. Anti-αV and -β1 integrin antibodies inhibited the cell adhesion mediated by KSHV-gB. Variable levels of neutralization of HMVEC-d and HFF infection were observed with antibodies against αVβ3 and αVβ5 integrins. Similarly, variable levels of inhibition of virus entry into adherent HMVEC-d, 293 and Vero cells, and HFF was observed by preincubating virus with soluble α3β1, αVβ3, and αVβ5 integrins, and cumulative inhibition was observed with a combination of integrins. We were unable to infect HT1080 cells. Virus binding and DNA internalization studies suggest that αVβ3 and αVβ5 integrins also play roles in KSHV entry. We observed time-dependent temporal KSHV interactions with HMVEC-d integrins and CD98/xCT with three different patterns of association and dissociation. Integrin αVβ5 interaction with CD98/xCT predominantly occurred by 1 min postinfection (p.i.) and dissociated at 10 min p.i., whereas α3β1-CD98/xCT interaction was maximal at 10 min p.i. and dissociated at 30 min p.i., and αVβ3-CD98/xCT interaction was maximal at 10 min p.i. and remained at the observed 30 min p.i. Fluorescence microscopy also showed a similar time-dependent interaction of αVβ5-CD98. Confocal-microscopy studies confirmed the association of CD98/xCT with α3β1 and KSHV. Preincubation of KSHV with soluble heparin and α3β1 significantly inhibited this association, suggesting that the first contact with HS and integrin is an essential element in subsequent CD98-xCT interactions. Anti-CD98 and xCT antibodies did not block virus binding and entry and nuclear delivery of viral DNA; however, viral-gene expression was significantly inhibited, suggesting that CD98-xCT play roles in the post-entry stage of infection, possibly in mediating signal cascades essential for viral-gene expression. Together, these studies suggest that KSHV interacts with functionally related integrins (αVβ3, α3β1, and αVβ5) and CD98/xCT molecules in a temporal fashion to form a multimolecular complex during the early stages of endothelial cell infection, probably mediating multiple roles in entry, signal transduction, and viral-gene expression.     

Membrane degradation, accumulation of Phosphatidic acid, stimulation of catalase activity and nuclear DNA fragmentation during 2,4-d-induced leaf senescence in mustard

We investigated 2,4-D-induced leaf senescence in young mustard seedlings. A set of morphometric, biochemical and molecular parameters were analyzed to characterize senescence markers. In accordance with earlier reports, chloroplast-membrane degradation marked the early phase of leaf senescence based on the analysis of the galactolipid fraction. Degradation of grana occurred earlier to that of the envelope, as revealed by the relative level of their specific galactolipids, namely, monogalactosyl diglyceride and digalactosyl diglyceride. Phospholipids showed extensive degradation resulting in the accumulation of lyso-derivatives of major phospholipids and phosphatidic acid (PA) in senescing leaves. Catalase activity was stimulated by 2,4-D and reflected scavenging of reactive oxygen species. Nuclear DNA degradation, a previously known death signal that represented a point of no return from progression of senescence, occurred late on the 4th day subsequent to 2,4-D supplementation. AgNO₃, an inhibitor of ethylene biosynthesis, inhibited leaf senescence by ca. 54% based on PA content Involvement of 2,4-D, ethylene and abscisic acid in leaf senescence is discussed in relation to hormonal interplay.