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BACKGROUND: Free radical-induced oxidative stress is the root cause for many human diseases. Naturally occurring antioxidant supplements from plants are vital to counter the oxidative damage in cells. The main objective of the present study was to characterize the antioxidant and antiproliferative potential of rice bran extracted from an important Indian rice variety, Njavara and to compare the same with two commercially available basmati rice varieties: Vasumathi, Yamini and a non medicinal variety, Jyothi. METHODS: Methanolic extracts of rice bran from four varieties; Vasumathi, Yamini, Jyothi and Njavara were used to study their total phenolic and flavonoid contents, in vitro antioxidant activities including total antioxidant activity, scavenging of nitric oxide and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical, reducing power and cytotoxic activity in C6 glioma cells. Correlation coefficient and regression analysis were done by using Sigmastat version 3.1 and Stata statistical package respectively. RESULTS: Rice bran methanolic extract from Njavara showed the highest antioxidant and cell cytotoxic properties compared to the other three rice varieties. IC50 values for scavenging DPPH and nitric oxide were in the range of 30.85-87.72 microg/ml and 52.25-107.18 microg/ml respectively. Total antioxidant activity and reducing power were increased with increasing amounts of the extract. Total phenolic and flavonoid contents were in the range of 3.2-12.4 mg gallic acid-equivalent (GAE)/g bran and 1.68-8.5 mg quercetin-equivalent (QEE)/g bran respectively. IC50 values of cytotoxic assay (MTT assay) were 17.53-57.78 microg/ml. Correlation coefficient and regression analysis of phenolic content with DPPH and NO scavenging, MTT (-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, total antioxidant assay and reducing power showed a highly significant correlation coefficient values (96-99%) and regression values (91-98%). CONCLUSION: The results of the present study show that the crude methanolic extract from Njavara rice bran contains significantly high polyphenolic compounds with superior antioxidant activity as evidenced by scavenging of free radicals including DPPH and NO. Njavara extracts also showed highest reducing power activity, anti-proliferative property in C6 glioma cells. In conclusion, it is conceivable that the Njavara rice variety could be exploited as one of the potential sources for plant--based pharmaceutical products. [ABSTRACT FROM AUTHOR]

Demyelination is a common sign of peripheral nerve injuries (PNIs) caused by damage to the myelin sheath surrounding axons in the sciatic nerve. There are not many methods to induce demyelination in the peripheral nervous system (PNS) using animal models. This study describes a surgical approach using a single partial sciatic nerve suture to induce demyelination in young male Sprague Dawley (SD) rats. After the post-sciatic nerve injury (p-SNI) to the sciatic nerve, histology and immunostaining show demyelination or myelin loss in early to severe phases with no self-recovery. The rotarod test confirms the loss of motor function in nerve-damaged rats. Transmission electron microscopic (TEM) imaging of nerve-damaged rats reveals axonal atrophy and inter-axonal gaps. Further, administration of Teriflunomide (TF) to p-SNI rats resulted in the restoration of motor function, repair of axonal atrophies with inter-axonal spaces, and myelin secretion or remyelination. Taken together, our findings demonstrate a surgical procedure that can induce demyelination in the rat sciatic nerve, which is then remyelinated after TF treatment.

The physiological and metabolic functions of PIMT/TGS1, a third-generation transcriptional apparatus protein, in glucose homeostasis sustenance are unclear. Here, we observed that the expression of PIMT was upregulated in the livers of short-term fasted and obese mice. Lentiviruses expressing Tgs1-specific shRNA or cDNA were injected into wild-type mice. Gene expression, hepatic glucose output, glucose tolerance, and insulin sensitivity were evaluated in mice and primary hepatocytes. Genetic modulation of PIMT exerted a direct positive impact on the gluconeogenic gene expression program and hepatic glucose output. Molecular studies utilizing cultured cells, in vivo models, genetic manipulation, and PKA pharmacological inhibition establish that PKA regulates PIMT at post-transcriptional/translational and post-translational levels. PKA enhanced 3'UTR-mediated translation of TGS1 mRNA and phosphorylated PIMT at Ser656, increasing Ep300-mediated gluconeogenic transcriptional activity. The PKA-PIMT-Ep300 signaling module and associated PIMT regulation may serve as a key driver of gluconeogenesis, positioning PIMT as a critical hepatic glucose sensor., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Glioblastoma (GBM) is a highly aggressive brain tumor. Resistance mechanisms in GBM present an array of challenges to understand its biology and to develop novel therapeutic strategies. We investigated the role of a TSG, MTUS1 /ATIP1 in glioma. Glioma specimen, cells and low passage GBM sphere cultures (GSC) were analyzed for MTUS1 /ATIP1 expression at the RNA and protein level. Methylation analyses were done by bisulfite sequencing (BSS). The consequence of chemotherapy and irradiation on ATIP1 expression and the influence of different cellular ATIP1 levels on survival was examined in vitro and in vivo. MTUS1 /ATIP1 was downregulated in high-grade glioma (HGG), GSC and GBM cells and hypermethylation at the ATIP1 promoter region seems to be at least partially responsible for this downregulation. ATIP1 overexpression significantly reduced glioma progression by mitigating cell motility, proliferation and facilitate cell death. In glioma-bearing mice, elevated MTUS1 /ATIP1 expression prolonged their survival. Chemotherapy, as well as irradiation, recovered ATIP1 expression both in vitro and in vivo. Surprisingly, ATIP1 overexpression increased irradiation-induced DNA-damage repair, resulting in radio-resistance. Our findings indicate that MTUS1 /ATIP1 serves as TSG-regulating gliomagenesis, progression and therapy resistance. In HGG, higher MTUS1 /ATIP1 expression might interfere with tumor irradiation therapy.

Background: Low grade gliomas (LGG) are most often noted with the unpredictable overall survival and progression to higher grades. Objective: In the present study, we analyze the clinicopathological features influencing the prognostic outcomes and compared the features with criteria developed by EORTC., Materials and Methods: We observed the 130 LGG clinical cases in single institute and maintained the follow-up for more than 5 years. In addition, the molecular details were confirmed with markers as IDH, 1p/19q codeletion, p53 and ATRX mutations., Results: The mean age of patients as 37.67 years and male population contributing to 70%. We observed biased incidence among the male population with dominating occurrence at frontal and parietal lobes in the brain. 40.8% patients had oligodendroglioma, 33.8% astrocytoma, 19.2% oligoastrocytoma and 2.3% gemistocytic astrocytoma pathology. Patients who were subjected to chemotherapy and radiotherapy were noted with average survival of 29 months. Oligodendroglial tumors were found with progression free survival (PFS) of 25 months, oligoastrocytoma cases with 32 months, diffuse astrocytoma cases with 23 months while the gemistocytic astrocytoma cases had 22 months. The PFS for LGG cases was 4.7 years while the overall survival was 4.9 years. Mean survival of patients with KPS score <70 and >70 was 1.5 & 4.9 years respectively. 64 patients were observed with the tumor size >5 cm. In total, 72.3% of the patients were underwent GTR, 23.3% STR and 3.8% underwent biopsy., Conclusion: Taken together, the clinical symptoms, expression of molecular markers and the prognosis details provided by our results can help for better management of LGG cases. We further propose to use following five factors to accurately describe the prognosis and tumor recurrence: 1) Age >50 years, 2) tumor size >5 cm, 3) MIB index >5%, 4) KPS score < 70 and 5) gemistocytic pathology., Competing Interests: None

Statins are first-line therapy drugs for cholesterol lowering. While they are highly effective at lowering cholesterol, they have propensity to induce hyperglycemia in patients. Only limited studies have been reported which studied the impact of statins on (a) whether they can worsen glucose tolerance in a high sucrose fed animal model and (b) if so, what could be the molecular mechanism. We designed studies using high sucrose fed animals to explore the above questions. The high sucrose fed animals were treated with atorvastatin and simvastatin, the two most prescribed statins. We examined the effects of statins on hyperglycemia, glucose tolerance, fatty acid accumulation and insulin signaling. We found that chronic treatment with atorvastatin made the animals hyperglycemic and glucose intolerant in comparison with diet alone. Treatment with both statins lead to fatty acid accumulation and inhibition of insulin signaling in the muscle tissue at multiple points in the pathway.

Gliomas are incurable solid tumors with extremely high relapse rate and definite mortality. As gliomas readily acquire resistance to only approved drug, temozolomide (TMZ), there is increasing need to overcome drug resistance by novel therapeutics or by repurposing the existing therapy. In the current study, we investigated antitumor efficacy of roscovitine, a Cdk inhibitor, in combination with TMZ in vitro (U87, U373, LN 18 and C6 cell lines) and in vivo (orthotopic glioma model in Wistar rats) glioma models. We observed that TMZ treatment following a pre-treatment with RSV significantly enhanced chemo-sensitivity and suppressed the growth of glioma cells by reducing Cdk-5 activity and simultaneous induction of autophagy and Caspase-3 mediated apoptosis. Additionally, reduced expression of Ki67, GFAP and markers of angiogenesis (CD31, VEGF) was observed in case of TMZ + RSV treatments. Also, presence of reactive astrocytes in peri-tumoral areas and in areas around blood vessels was completely diminished in TMZ + RSV treated brain sections. Taken together, results in the current study provide evidence that RSV in conjunction with TMZ restricts glioma growth, reduces angiogenesis and also eliminates reactive astrocytes thereby preventing the spread of glioma to adjacent healthy brain tissues and thus might be more potent therapeutic option for glioma.

Astrocytoma constitutes the most noted malignancies of the central nervous system with worse clinical outcomes in grade IV astrocytoma or glioblastoma multiforme. Owing to poor clinical outcomes with existing therapeutic regime, there is a need to revisit the initial course of treatment. Statistical information of clinicopathological parameters could be used to understand the spread of disease and, in turn, to formulate updated treatment management. In the present study, we have seen anatomic distribution of astrocytoma subtypes in a group of 479 patients and correlated it with survival outcomes. Anatomic location was confirmed by MRI (magnetic resonance imaging) images. A registry of patients was maintained with clinicopathological details as tumor type, location, age/sex, and survival after surgery. We have observed overall survival particulars in patients diagnosed with astrocytoma. Our findings highlight that in total cases, tumor location was anatomically dominated by frontal and temporal lobes. Survival analysis in high-grade (grade III, p  = 0.03; grade IV, p  = 0.01) astrocytic tumors confirms poor outcomes with temporal, parietal, and occipital location as compared to frontal lobe. Overall survival study demonstrates glioblastoma multiforme (GBM) was associated with worse prognosis as compared to astrocytoma subtypes ( p  < 0.0001). In high-grade astrocytomas, anaplastic astrocytoma was found with 34 months of median survival age while 14 months in the case of patients with glioblastoma multiforme. In conclusion, we report dismal prognosis in parietal, temporal, and occipital lobes in grade II, grade III, and grade IV astrocytoma patients. Among astrocytoma subtypes, patients with glioblastoma multiforme were associated with worse survival outcomes. We uniquely feature the survival of astrocytoma patients for the first time and observe GBM patients have slightly longer survival., Competing Interests: The authors declare that they have no conflict of interest.The present studies involving human participants were approved by the Institutional Ethics Committee (ICE), University of Hyderabad and KIMS Foundation Research Centre (KFRC), KIMS, Secunderabad, India. All subjects participating were completely anonymized.Informed consent was obtained from all individual participants included in the study.

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.

Nutritional abundance associated with chronic inflammation and dyslipidemia impairs the functioning of endoplasmic reticulum (ER) thereby hampering cellular responses to insulin. PHLPP1 was identified as a phosphatase which inactivates Akt, the master regulator of insulin mediated glucose homeostasis. Given the suggestive role of PHLPP1 phosphatase in terminating insulin signalling pathways, deeper insights into its functional role in inducing insulin resistance are warranted. Here, we show that PHLPP1 expression is enhanced in skeletal muscle of insulin resistant rodents which also displayed ER stress, an important mediator of insulin resistance. Using cultured cells and PHLPP1 knockdown mice, we demonstrate that PHLPP1 facilitates the development of ER stress. Importantly, shRNA mediated ablation of PHLPP1 significantly improved glucose clearance from systemic circulation with enhanced expression of glucose transporter 4 (GLUT-4) in skeletal muscle. Mechanistically, we show that endogenous PHLPP1 but not PP2Cα interacts with and directly dephosphorylates AMPK Thr 172 in myoblasts without influencing its upstream kinase, LKB1. While the association between endogenous PHLPP1 and AMPK was enhanced in ER stressed cultured cells and soleus muscle of high fat diet fed mice, the basal interaction between PP2Ac and AMPK was minimally altered. Further, we show that PHLPP1α is phosphorylated by ERK1/2 at Ser 932 under ER stress which is required for its ability to interact with and dephosphorylate AMPK and thereby induce ER stress. Taken together, our data position PHLPP1 as a key regulator of ER stress., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Interleukin-12 (IL-12) is formed by the interaction of IL-12p35 and IL-12p40 expressed independently from IL-12A and IL-12B genes. This interleukin plays prominent role in the T-helper type-1 (Th1) response against intracellular pathogens. Variations in IL-12B gene causes disruption of various activities one of them is suppression of Th1 response and is one of the characteristic features observed in patients with active tuberculosis. Hence, in the present study IL-12B gene status was evaluated in 50 new sputum smear-positive pulmonary tuberculosis patients (NSP-PTB) as identified by Ziehl-Nielsen (ZN) staining and 50 apparently healthy control subjects (HCS) who were sputum smear-negative. The sequence analysis showed novel missense mutations p.Ser205Ile, p.Leu206Glu, p.Pro207Ser, p.Glu209Lys, p.Val210Ser, p.(Ser205&#95;Cys327delinsIleGlu) and p.(Lys217&#95;Leu218delinsIle) were found in exon 5 of the IL-12B gene in nine patients resulting formation of inactive IL-12 and three patients showed novel frame shift mutations p.(Asn222Leufs∗23) in exon 5 of causing the formation of truncated protein. Several mutations were noted in intron 2 of the IL-12B gene in 5 patients and in 13 patients mutations were observed in 3' UTR region. All together 30/50 patients (60%) showed mutations in IL-12B gene. Decreased levels of interferon-gamma (IFN-γ) and IL-12 as determined by ELISA and flow cytometry were observed in the peripheral blood mononuclear cell culture supernatants in TB patients having mutations compared with control subjects. Further, in silico analysis revealed due to frame shift mutations in exon 5 at Asn222 resulted in deletion of functional fibronectin type-III (FN3) domain which leads to formation of inactive IL-12 in these patients., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cerebral malaria (CM) is a neurological complication arising due to Plasmodium falciparum or Plasmodium vivax infection. Minocycline, a semi-synthetic tetracycline, has been earlier reported to have a neuroprotective role in several neurodegenerative diseases. In this study, we investigated the effect of minocycline treatment on the survivability of mice during experimental cerebral malaria (ECM). The currently accepted mouse model, C57BL/6 mice infected with Plasmodium berghei ANKA, was used for the study. Infected mice were treated with an intra-peritoneal dose of minocycline hydrochloride, 45mg/kg daily for ten days that led to parasite clearance in blood, brain, liver and spleen on 7th day post-infection; and the mice survived until experiment ended (90days) without parasite recrudescence. Evans blue extravasation assay showed that blood-brain barrier integrity was maintained by minocycline. The tumor necrosis factor-alpha protein level and caspase activity, which is related to CM pathogenesis, was significantly reduced in the minocycline-treated group. Fluoro-Jade® C and hematoxylin-eosin staining of the brains of minocycline group revealed a decrease in degenerating neurons and absence of hemorrhages respectively. Minocycline treatment led to decrease in gene expressions of inflammatory mediators like interferon-gamma, CXCL10, CCL5, CCL2; receptors CXCR3 and CCR2; and hence decrease in T-cell-mediated cerebral inflammation. We also proved that this reduction in gene expressions is irrespective of the anti-parasitic property of minocycline. The distinct ability of minocycline to modulate gene expressions of CXCL10 and CXCR3 makes it effective than doxycycline, a tetracycline used as chemoprophylaxis. Our study shows that minocycline is highly effective in conferring neuroprotection during ECM., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Phagocytosis of malaria pigment (hemozoin) induces increased activity of matrix metalloproteinase (MMP)-9, an endopeptidase involved in cytokine regulation. In this study, we examined whether a common functional MMP-9 promoter polymorphism (rs3918242) affects Plasmodium falciparum infection in pregnancy. Eighteen percent of Ghanaian primiparae carried the minor T allele. It was associated with reduced odds of placental hemozoin and of placental as well as peripheral blood parasitemia. The results indicate that a common MMP-9 polymorphism protects against placental malaria indicating that this endopeptidase is involved in susceptibility to P. falciparum., (© The American Society of Tropical Medicine and Hygiene.)

Frozen shrimp continued to be the single largest item of export from India in terms of value accounting for about 44% of the total marine export earnings. Headless, peeled frozen shrimp is a common and dominant item in the market and there is need for differentiating peeled Penaeus sp from Metapenaeus, Parapenopsis and Macrobrachium sp as consumer preference and price vary. Furthermore, there is need to find out original species used in value addition of shrimp products. Hence, it is essential for development of simple and consumer friendly technique for the identification of shrimp and their products in the market. Two monoclonal antibodies (MAbs) C-15 (IgG3) and C-52 (IgG2a) reacting with 65 and 47 kD proteins of Penaeus monodon respectively in the Western blot were selected. In epitope analysis by immunodot, the two MAbs reacted and recognized specific proteins of P. monodon, Fenneropenaeus indicus and Littopenaeus vannamei and not that of Metapenaeus, Parapenopsis, Macrobrachium rosenbergii, crabs and fishes. The immunodot required 120 min for completion. The sensitivity of the immunodot to detect proteins of P. monodon was 0.225 mg with MAb C-15 and 0.028 mg with MAb C-52. The MAb based immunodot developed, could be used for identifying and differentiating meat of P. monodon, F. indicus, and L. vannamei from that of Metapenaeus, Parapenopsis, M. rosenbergii, crabs and fishes.

PHLPP1 is a novel tumor suppressor, but its role in the regulation of innate immune responses, which are frequently dysregulated in cancer, is unexplored. Here, we report that LPS attenuated PHLPP1 expression at mRNA and protein levels in immune cells, suggesting its involvement in immune responses. To test this, we overexpressed PHLPP1 in RAW 264.7 macrophages and observed a dramatic reduction in LPS/IFN-γ-induced iNOS expression. Conversely, silencing of PHLPP1 by siRNA or by shRNA robustly augmented LPS/IFN-γ-induced iNOS expression. qPCR and iNOS promoter reporter experiments showed that PHLPP1 inhibited iNOS transcription. Mechanistic analysis revealed that PHLPP1 suppressed LPS/IFN-γ-induced phosphorylation of ser 727 STAT1; however, the underlying mechanisms differed. PHLPP1 reduced IFN-γ-stimulated but not LPS-induced ERK1/2 phosphorylation, and inhibition of ERK1/2 abolished IFN-γ-induced ser 727 STAT1 phosphorylation and iNOS expression. In contrast, PHLPP1 knockdown augmented LPS-induced but not IFN-γ-elicited p38 phosphorylation. Blockade of p38 abolished LPS-stimulated phosphorylation of ser 727 STAT1 and iNOS expression. Furthermore, PHLPP1 suppressed LPS-induced phosphorylation of tyr 701 STAT1 by dampening p38-dependent IFN-β feedback. Collectively, our data demonstrate for the first time that PHLPP1 plays a vital role in restricting innate immune responses of macrophages, and further studies may show it to be a potential therapeutic target within the context of dysregulated macrophage activity., (© 2014 Society for Leukocyte Biology.)

PRIP-Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB-binding protein (CBP)/ E1A binding protein p300 (Ep300) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERK2 phosphorylates PIMT at Ser(298) and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMT(S298D)) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMT(S298D) but not PIMT(S298A) augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U0126, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T4 hormone to rats activated ERK1/2 resulting in enhanced PIMT ser(298) phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERK2-mediated phosphorylation of PIMT at Ser(298) is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia.

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., (Copyright © 2012 S. Karger AG, Basel.)

Infiltration of leukocytes into post-ischemic cerebrum is a well-described phenomenon in stroke injury. Because CD-8(+) T-lymphocytes secrete cytotoxic proteases, including granzyme-b (Gra-b) that exacerbates post-ischemic brain damage, we investigated roles of Gra-b in human stroke. To study the role of Gra-b in stroke, ischemic and non-ischemic tissues (from post-mortem stroke patients) were analyzed using immunoblotting, co-immunoprecipitation, terminal deoxy uridine nick end labeling (TUNEL) and Annexin-V immunostaining, and in vitro neuron survival assays. Activated CG-SH cells and supernatants were used to model leukocyte-dependent injury. Non-ischemic brain tissues were used as non-pathological controls. Non-activated CG-SH cells and supernatants were used as controls for in vitro experiments. Human stroke (ischemic) samples contained significantly higher levels of Gra-b and interferon-gamma inducible protein-10 (IP-10/CXCL10) than non-ischemic controls. In stroke, poly (ADP-ribose) polymerase-1 and heat shock protein-70 were cleaved to canonical proteolytic "signature" fragments by Gra-b. Gra-b was also found to bind to Bid and caspase-3. Gra-b also co-localized with Annexin-V(+) /TUNEL(+) in degenerating neurons. Importantly, Gra-b inhibition protected both normal and ischemia-reperfused neurons against in vitro neurotoxicity mediated by activated CG-SH cells and supernatants. These results suggest that increased leukocyte infiltration and elevated Gra-b levels in the post-stroke brain can induce contact-dependent and independent post-ischemic neuronal death to aggravate stroke injury., (© 2010 The Authors; Brain Pathology © 2010 International Society of Neuropathology.)

The normal function of poly (ADP-ribose) polymerase-1 (PARP-1) is the routine repair of DNA damage by adding poly (ADP ribose) polymers in response to a variety of cellular stresses. Recently, it has become widely appreciated that PARP-1 also participates in diverse physiological and pathological functions from cell survival to several forms of cell death and has been implicated in gene transcription, immune responses, inflammation, learning, memory, synaptic functions, angiogenesis and aging. In the CNS, PARP inhibition attenuates injury in pathologies like cerebral ischemia, trauma and excitotoxicity demonstrating a central role of PARP-1 in these pathologies. PARP-1 is also a preferred substrate for several 'suicidal' proteases and the proteolytic action of suicidal proteases (caspases, calpains, cathepsins, granzymes and matrix metalloproteinases (MMPs)) on PARP-1 produces several specific proteolytic cleavage fragments with different molecular weights. These PARP-1 signature fragments are recognized biomarkers for specific patterns of protease activity in unique cell death programs. This review focuses on specific suicidal proteases active towards PARP-1 to generate signature PARP-1 fragments that can identify key proteases and particular forms of cell death involved in pathophysiology. The roles played by some of the PARP-1 fragments and their associated binding partners in the control of different forms of cell death are also discussed.

Background: Carbon tetra chloride (CCl4), an industrial solvent, is a hepatotoxic agent and it is the well established animal model for free radical-induced liver injury. The present investigation was carried out to establish the protective effect of natansnin, a novel dibenzoyl glycoside from Salvinia natans against CCl4 induced oxidative stress and cellular degeneration in rat liver., Results: CCl4 significantly increased the levels of lipid peroxides, oxidized glutathione and decreased the levels of reduced glutathione, SOD and CAT. CCl4 induce marked histopathological changes and increase in the levels of apoptotic proteins. CCl4 treatment significantly increased the levels of apoptotic proteins such as caspases-3, PARP, Bax, Bid and cytochrome C and also increased the levels of inflammatory mediators iNos and Cox-2. Natansnin treatment significantly decreased the levels of CCl4 induced apoptotic proteins and inflammatory mediators. Further natansinin treatment significantly inhibited the CCl4 induced apoptosis which was evident form the reduced TUNEL positive cells., Conclusions: In conclusion, our study demonstrated the protective effect of natansnin against CCl4 induced oxidative stress and cellular degeneration in rat liver tissue. This protective effect of natansnin can be correlated to its direct antioxidant effect.

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, beta-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.

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 (micro) 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 approximately 89 kDa and approximately 24 kDa fragments, into other fragments of different molecular weights. Caspase mediated cell death may lead to appearance of larger approximately 89 kDa fragment and smaller approximately 24 kDa fragment indicating existence of apoptosis in the tumors. However, other fragments corresponding to approximately 64 kDa, approximately 54 kDa, and approximately 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.

The iron-induced model of post-traumatic chronic focal epilepsy in rats was studied by depth-electrode mapping to investigate the spread of epileptiform activity into subcortical brain structures after its onset in the cortical epileptic focus. Electrical seizure activity was recorded in the hippocampal CA1 and CA3 areas, amygdala and caudate-putamen, in rats with iron-induced chronic cortical focal epilepsy. These experiments showed that the epileptiform activity with its onset in the cortical focus synchronously propagated into the studied subcortical brain areas. Seizure behaviours seemed to increase in correspondence with the spread of the epileptic electrographic activity in subcortical areas. Comparison of the cortical focus electroencephalographic and associated multiple-unit action potential recordings with those from the subcortical structures showed that the occurrence and evolution of the epileptiform activity in the subcortical structures were in parallel with that in the cortical focus. The intracerebral anatomic progression and delineation of seizure spread (mapped by field potential (EEG) and multiple-unit action potentials (MUA) recordings) indicated participation of these regions in the generalization of seizure activity in this model of epilepsy. The seizure-induced activation of the hippocampus appeared to evolve into an epileptic focus independent of the cortical focus. The present study demonstrates the propagation of epileptic activity from the cortical focus into the limbic and basal ganglia regions. Treatment of iron-induced epileptic rats with ethosuximide, an anti-absence drug, resulted in suppression of the epileptiform activity in the cortical focus as well as in the subcortical brain areas.