2-[2-(4-(trifluoromethyl)phenylamino)thiazol-4-yl]acetic acid (Activator-3) is a potent activator of AMPK

AMPK is considered as a potential high value target for metabolic disorders. Here, we present the molecular modeling, in vitro and in vivo characterization of Activator-3, 2-[2-(4-(trifluoromethyl)phenylamino)thiazol-4-yl]acetic acid, an AMP mimetic and a potent pan-AMPK activator. Activator-3 and AMP likely share common activation mode for AMPK activation. Activator-3 enhanced AMPK phosphorylation by upstream kinase LKB1 and protected AMPK complex against dephosphorylation by PP2C. Molecular modeling analyses followed by in vitro mutant AMPK enzyme assays demonstrate that Activator-3 interacts with R70 and R152 of the CBS1 domain on AMPK γ subunit near AMP binding site. Activator-3 and C2, a recently described AMPK mimetic, bind differently in the γ subunit of AMPK. Activator-3 unlike C2 does not show cooperativity of AMPK activity in the presence of physiological concentration of ATP (2 mM). Activator-3 displays good pharmacokinetic profile in rat blood plasma with minimal brain penetration property. Oral treatment of High Sucrose Diet (HSD) fed diabetic rats with 10 mg/kg dose of Activator-3 once in a day for 30 days significantly enhanced glucose utilization, improved lipid profiles and reduced body weight, demonstrating that Activator-3 is a potent AMPK activator that can alleviate the negative metabolic impact of high sucrose diet in rat model

Endoplasmic Reticulum Stress and Neurodegeneration in Experimental Cerebral Malaria

Experimental cerebral malaria (ECM) resulting from Plasmodium berghei ANKA (PbA) infection in mice results in neuronal cell death. However, the precise mechanisms leading to neuronal cell death in ECM have not been fully elucidated. In the present study, we report the presence of endoplasmic reticulum (ER) stress markers and activation of the unfolded protein response (UPR) in the brain during the pathogenesis of ECM. Specific findings included activation of PKR-like ERkinase, inositol-requiring enzyme 1 and cleavage of activating transcription factor (ATF) 6 indicating the activation of all three major arms of the UPR. Further, we found changes in the protein levels of phosphorylated eukaryotic initiation factor α (p-eIF2α), ATF4, growth arrest and DNA damage-inducible protein 34, B cell lymphoma protein 2 (BCL-2), BCL-2-associated X protein, caspase-7, cleavage of caspase-3, and caspase-12. Our results demonstrate that ER stress-induced neuronal cell death in PbA-infected mice is associated with the expression of the pro-apoptotic molecule CHOP and downregulation of anti-apoptotic ER quality control molecules binding immunoglobulin protein, calreticulin and calnexin. Further CHOP was found to be localized in neurons and plays an essential role in neuronal cell death as revealed by our Fluoro-Jade B double staining. These results implicate an imbalance between ER stress-mediated pro-apoptotic and anti-apoptotic/survival signalling as a critical determinant of neuronal cell death in ECM

Differential PARP cleavage: An indication for existence of multiple forms of cell death in human gliomas

Background: Gliomas represent a diverse range of clinical presentation, histological differentiation, and response to therapy. Altered cell proliferation and cell death signals in gliomas are of great interest to elucidate the key molecules involved and to find effective treatment modalities. By considering the role of different proteases in correlation with differential poly (ADP-ribose) polymerase (PARP) fragmentation we have studied the pattern of cell death in human glioma tissues. Materials and Methods: In our study, five different human glioma biopsies were collected and analyzed for the PARP cleavage pattern by using western immunoblotting. Samples were also analyzed for pro-caspase 3, calpain I (µ) and II (m), granzyme-B and apoptosis-inducing factor (AIF). Parallel sections of histologically confirmed astrocytoma and glioblastoma multiforme (GBM) were used for immunohistochemical analysis of cleaved caspase-3, granzyme B, AIF and cyclo-oxygenase -2 (cox-2). Results: We found PARP fragmentation, along with usual ~ 89 kDa and ~ 24 kDa fragments, into other fragments of different molecular weights. Caspase mediated cell death may lead to appearance of larger ~ 89 kDa fragment and smaller ~ 24 kDa fragment indicating existence of apoptosis in the tumors. However, other fragments corresponding to ~ 64 kDa, ~ 54 kDa, and ~ 40 kDa were observed concomitantly in all glial tumor tissues. Conclusions: These results may indicate, not only apoptosis and necrosis, but there occurs the co-existence of intermediate cell death pathways in human glial tumors

Isolation, characterization and differentiation potential of rat bone marrow stromal cells

Background: Bone marrow mesenchymal cells have been identified as a source of pluripotent stem cells with varying degrees of plasticity in humans. However, there are a few reports on rat-derived cells, which could be good models for the research purpose. We describe here a simple method of establishing the rat bone marrow stromal cells by the principle of adhesion and document their phenotype along with their differentiation potential to other lineages. Materials and Methods: Rat bone marrow stromal cells were isolated by three methods: direct plastic adherence, ficoll hypaque separation and a combination of both. The stromal cells obtained by these methods were characterized by fluorescent activating cell sorting (FACS) for established hematopoietic and non-hematopoietic markers. The cells obtained by combination method (combination of ficoll density gradient centrifugation and plastic adherence) were cultured and serially passaged. Transcriptional confirmation was done by reverse transcription polymerase chain reaction (RT-PCR) for vimentin and collagen type 1 alpha 1. Attempts were made to differentiate the marrow stromal cells into adipocytes, osteocytes and neuronal like cells. Results: Bone marrow samples from 10 rats yielded 4-5 million bone marrow mononuclear cells /ml per femur. Of the three methods tested, a combination method yielded good growth of spindle cells. The cells obtained by combined method showed high percentage of positivity for vimentin, fibronectin and CD90 and negative for hematopoietic markers. Further, RT-PCR confirmed vimentin and collagen type - 1 alpha 1 expression. Oil red O staining and Alizarin red staining confirmed adipocytic and osteogenic differentiation. On immunocytochemical analysis, the cells expressed nestin, β-tubulin III, neurofilament and synaptophysin. Conclusion: Adequate quantities of rat marrow stromal cell cultures can be established by a simple method based on adhesion properties. Their phenotypic characteristics and plasticity support the evidence that they are mesenchymal stem cells with a distinct tendency for neural lineage

Brainstem glioma: Clinical significance and prognostic evaluation

In the present study, we have determined the prognostic value and observed the clinicopathological features of brainstem glioma. Overall, we found more number of male patients with brainstem glioma (male:female ratio: 1:6). All patients were subjected to surgery followed by chemotherapy. The tumor was mainly located at brainstem and thalamus. We have recorded the symptoms associated with onset of lesions and predominated by headache, vomiting and limb weakness. We observed the overall survival pattern in brain stem glioma cases and further among low and high grade subgroups. Patients with high grade brainstem glioma were recorded with the median survival of 15 months. There was a statistically significant difference in the survival pattern of low grade vs high grade group (p = 0.015, hazard ratio: 0.29, 95% CI of ratio: 0.058 to 0.71) Taken together, the clinical presentation provides valuable information to enhance the therapeutic measures for the brainstem glioma cases. Keywords: Brainstem, Glioma, Surger