AQUEOUS BARK EXTRACT OF TERMINALIA ARJUNA PROTECTS AGAINST PHENYLHYDRAZINE INDUCED OXIDATIVE DAMAGE IN GOAT RED BLOOD CELL MEMBRANE BOUND AND METABOLIC ENZYMES

Objective: The objective of the present study is to determine the phenylhydrazine (PHZ) induced oxidative stress mediated alteration in the metabolic status and morphology of the red blood cells (RBC) and amelioration of the same by aqueous bark extract of Terminalia arjuna (TA).Methods: Fresh goat blood collected from local Kolkata Corporation approved slaughter house, was used for the present study. Packed cells were prepared from the freshly collected goat blood and were divided into four groups as follows for further studies i. e Group I: Control (CON), Group II: TA bark extract treated, named T5C (5 mg/ml, incubation mixture; positive control), Group III: PHZ treated (1 mM), Group IV: PHZ treated+TA bark extract at a dose of 5 mg/ml, named as P+T5. ROS, superoxide anion radical, and hydroxyl radical scavenging activity were determined. Intracellular iron and intracellular nitrate concentration were estimated. Activities of various membrane-bound enzymes like Na+/K+-ATPase, Mg2+-ATPase and Ca2+-ATPase and Ach-E were determined. Moreover, the activities of some metabolic enzymes like glucose 6-phosphate dehydrogenase (G6PDH), hexokinase, aldolase, lactase dehydrogenase were also studied. In addition, the morphological structure of RBCs was also determined.Results: PHZ treatment caused significant alterations in RBC morphology as well as altered the activities of membrane-bound as well as metabolic enzymes. All these changes following oxidative stress were found to be ameliorated when the RBCs were co-treated with PHZ and aqueous bark extract of TA. However, aqueous bark extract of TA alone did not exhibit any such changes in RBC.Conclusion: The aqueous bark extract of TA ameliorates PHZ-induced oxidative damages in goat RBC possibly by an antioxidant mechanism(s). The aqueous bark extract of TA may have future therapeutic relevance in oxidative stress-induced damages in RBCs.Keywords: Antioxidant enzymes, Aqueous bark extract, Oxidative stress, Phenylhydrazine, Red blood cells, Terminalia arjun

SILYMARIN PROTECTS AGAINST COPPER-ASCORBATE INDUCED INJURY TO GOAT CARDIAC MITOCHONDRIA IN VITRO: INVOLVEMENT OF ANTIOXIDANT MECHANISM(S)

Silymarin, 'one of the component of the Milk thistle seeds Silybum marianum (L.) is used in traditional food and medicine in India. In the present study, we investigated the antioxidant activities of Silymarin against copper-ascorbate induced toxic injury to mitochondria obtained from goat heart, in vitro. Incubation of isolated cardiac mitochondria with copper-ascorbate resulted in elevated levels of lipid peroxidation and protein carbonylation of the mitochondrial membrane, a reduced level of mitochondrial GSH and altered status of antioxidant enzymes as well as decreased activities of pyruvate dehydrogenase and the Kreb's cycle enzymes, altered mitochondrial morphology, mitochondrial swelling and di-tyrosine level. All these changes were found to be ameliorated when the cardiac mitochondria were co-incubated with copper-ascorbate and Silymarin, in vitro. Silymarin, in our in vitro experiments, was found to scavenge hydrogen peroxide, superoxide anion free radicals, hydroxyl radicals and DPPH radical, in a chemically defined system, indicating that this compound may provide protection to cardiac mitochondria against copper-ascorbate induced toxic injury through its antioxidant activities. The results of this study suggest that Silymarin may be considered as a future therapeutic antioxidant and may be used singly or as a co-therapeutic in the treatment of diseases associated with mitochondrial oxidative stress

Plasticity of Lgr5-Negative Cancer Cells Drives Metastasis in Colorectal Cancer

Colorectal cancer stem cells (CSCs) express Lgr5 and display extensive stem cell-like multipotency and self-renewal and are thought to seed metastatic disease. Here, we used a mouse model of colorectal cancer (CRC) and human tumor xenografts to investigate the cell of origin of metastases. We found that most disseminated CRC cells in circulation were Lgr5- and formed distant metastases in which Lgr5+ CSCs appeared. This p

Simultaneous Inhibition of Ceramide Hydrolysis and Glycosylation Synergizes to Corrupt Mitochondrial Respiration and Signal Caspase Driven Cell Death in Drug-Resistant Acute Myeloid Leukemia

Acute myelogenous leukemia (AML), the most prevalent acute and aggressive leukemia diagnosed in adults, often recurs as a difficult-to-treat, chemotherapy-resistant disease. Because chemotherapy resistance is a major obstacle to successful treatment, novel therapeutic intervention is needed. Upregulated ceramide clearance via accelerated hydrolysis and glycosylation has been shown to be an element in chemotherapy-resistant AML, a problem considering the crucial role ceramide plays in eliciting apoptosis. Herein we employed agents that block ceramide clearance to determine if such a "reset" would be of therapeutic benefit. SACLAC was utilized to limit ceramide hydrolysis, and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) was used to block the glycosylation route. The SACLAC D-threo-PDMP inhibitor combination was synergistically cytotoxic in drug-resistant, P-glycoprotein-expressing (P-gp) AML but not in wt, P-gp-poor cells. Interestingly, P-gp antagonists that can limit ceramide glycosylation via depression of glucosylceramide transit also synergized with SACLAC, suggesting a paradoxical role for P-gp in the implementation of cell death. Mechanistically, cell death was accompanied by a complete drop in ceramide glycosylation, concomitant, striking increases in all molecular species of ceramide, diminished sphingosine 1-phosphate levels, resounding declines in mitochondrial respiratory kinetics, altered Akt, pGSK-3β, and Mcl-1 expression, and caspase activation. Although ceramide was generated in wt cells upon inhibitor exposure, mitochondrial respiration was not corrupted, suggestive of mitochondrial vulnerability in the drug-resistant phenotype, a potential therapeutic avenue. The inhibitor regimen showed efficacy in an in vivo model and in primary AML cells from patients. These results support the implementation of SL enzyme targeting to limit ceramide clearance as a therapeutic strategy in chemotherapy-resistant AML, inclusive of a novel indication for the use of P-gp antagonists.This work was supported by grants from the National Institutes of Health (National Cancer Institute NIH P01 CA171983 (T.P.L., M.K., M.C.C.), DOD-W81XWH-19-1-0213 (K.H.F.-W.), Spanish Ministry of Science and Innovation PID2020-113813RB-100 (G.F.), and the Brody Brothers Foundation (K.H.F.-W.), Kinston, NC.Peer reviewe

Simultaneous Inhibition of Ceramide Hydrolysis and Glycosylation Synergizes to Corrupt Mitochondrial Respiration and Signal Caspase Driven Cell Death in Drug-Resistant Acute Myeloid Leukemia

Acute myelogenous leukemia (AML), the most prevalent acute and aggressive leukemia diagnosed in adults, often recurs as a difficult-to-treat, chemotherapy-resistant disease. Because chemotherapy resistance is a major obstacle to successful treatment, novel therapeutic intervention is needed. Upregulated ceramide clearance via accelerated hydrolysis and glycosylation has been shown to be an element in chemotherapy-resistant AML, a problem considering the crucial role ceramide plays in eliciting apoptosis. Herein we employed agents that block ceramide clearance to determine if such a “reset” would be of therapeutic benefit. SACLAC was utilized to limit ceramide hydrolysis, and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) was used to block the glycosylation route. The SACLAC D-threo-PDMP inhibitor combination was synergistically cytotoxic in drug-resistant, P-glycoprotein-expressing (P-gp) AML but not in wt, P-gp-poor cells. Interestingly, P-gp antagonists that can limit ceramide glycosylation via depression of glucosylceramide transit also synergized with SACLAC, suggesting a paradoxical role for P-gp in the implementation of cell death. Mechanistically, cell death was accompanied by a complete drop in ceramide glycosylation, concomitant, striking increases in all molecular species of ceramide, diminished sphingosine 1-phosphate levels, resounding declines in mitochondrial respiratory kinetics, altered Akt, pGSK-3β, and Mcl-1 expression, and caspase activation. Although ceramide was generated in wt cells upon inhibitor exposure, mitochondrial respiration was not corrupted, suggestive of mitochondrial vulnerability in the drug-resistant phenotype, a potential therapeutic avenue. The inhibitor regimen showed efficacy in an in vivo model and in primary AML cells from patients. These results support the implementation of SL enzyme targeting to limit ceramide clearance as a therapeutic strategy in chemotherapy-resistant AML, inclusive of a novel indication for the use of P-gp antagonists

mRNA and Protein levels of rat pancreas specific protein disulphide isomerase are downregulated during Hyperglycemia

100-107<span style="mso-fareast-font-family: Calibri;mso-ansi-language:EN-IN;mso-fareast-language:EN-IN">Diabetes (Type I and Type II) which affects nearly every organ in the body is a multi-factorial non-communicable disorder. Hyperglycemia is the most characteristic feature of this disease. Loss of beta cells is common in both types of diabetes whose detailed cellular and molecular mechanisms are yet to be elucidated. As this disease is complex, identification of specific biomarkers for its early detection, management and devising new therapies is challenging. Based on the fact that functionally defective proteins provide the biochemical basis for many diseases, in this study, we tried to identify differentially expressed proteins during hyperglycemia.<span style="mso-fareast-font-family:Calibri; mso-ansi-language:EN-IN;mso-fareast-language:EN-IN"> For that, hyperglycemia was induced in overnight fasted rats by intra-peritoneal injection of streptozotocin (STZ). The pancreas was isolated from control and treated rats for subsequent analyses. The 2D-gel electrophoresis followed by MALDI-TOF-MS-MS analyses revealed several up- and down- regulated proteins in hyperglycemic rat pancreas including the downregulation of a pancreas specific isoform of protein disulphide isomerase a2 (Pdia2).This observation was validated by western blot. Quantitative PCR experiments showed that the level of Pdia2 mRNA is also proportionally reduced in hyperglycemic pancreas. </span

Para-Phenylenediamine Induces Apoptotic Death of Melanoma Cells and Reduces Melanoma Tumour Growth in Mice

Melanoma is one of the most aggressive forms of cancer, usually resistant to standard chemotherapeutics. Despite a huge number of clinical trials, any success to find a chemotherapeutic agent that can effectively destroy melanoma is yet to be achieved. Para-phenylenediamine (p-PD) in the hair dyes is reported to purely serve as an external dyeing agent. Very little is known about whether p-PD has any effect on the melanin producing cells. We have demonstrated p-PD mediated apoptotic death of both human and mouse melanoma cells in vitro. Mouse melanoma tumour growth was also arrested by the apoptotic activity of intraperitoneal administration of p-PD with almost no side effects. This apoptosis is shown to occur primarily via loss of mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS), and caspase 8 activation. p-PD mediated apoptosis was also confirmed by the increase in sub-G0/G1 cell number. Thus, our experimental observation suggests that p-PD can be a potential less expensive candidate to be developed as a chemotherapeutic agent for melanoma

Flow Cytometry of Mouse and Human Adipocytes for the Analysis of Browning and Cellular Heterogeneity

Summary: Adipocytes, once considered simple lipid-storing cells, are rapidly emerging as complex cells with many biologically diverse functions. A powerful high-throughput method for analyzing single cells is flow cytometry. Several groups have attempted to analyze and sort freshly isolated adipocytes; however, using an adipocyte-specific reporter mouse, we demonstrate that these studies fail to detect the majority of white adipocytes. We define critical settings required for adipocyte flow cytometry and provide a rigid strategy for analyzing and sorting white and brown adipocyte populations. The applicability of our protocol is shown by sorting mouse adipocytes based on size or UCP1 expression and demonstrating that a subset of human adipocytes lacks the β2-adrenergic receptor, particularly in the insulin-resistant state. In conclusion, the present study confers key technological insights for analyzing and sorting mature adipocytes, opening up numerous downstream research applications. : Freshly isolated adipocytes are a notoriously difficult cell type to study. Hagberg et al. provide a detailed flow cytometry protocol for the analysis and sorting of mouse and human adipocytes by defining the critical factors and conditions required for studying this specialized cell type and pinpointing common pitfalls. Keywords: adipocyte, adipose tissue, flow cytometry, FACS, mouse, human, uncoupled protein 1, beta 2 adrenergic recepto