The loss of vital cells within healthy tissues contributes to the development, progression and treatment outcomes of many human disorders, including neurological and infectious diseases as well as environmental and medical toxicities. Conversely, the abnormal survival and accumulation of damaged or superfluous cells drives human pathologies, including cancers and autoimmune diseases. Apoptosis is an evolutionarily conserved cell death pathway that is responsible for the proper culling of cells during normal eukaryotic development and the maintenance of organismal homeostasis. This pathway is controlled by the BCL-2 (B Cell Lymphoma 2) family of proteins, which contains both pro-apoptotic and pro-survival members that balance the decision between cellular life and death. Recent insights into the dynamic interactions between the BCL-2 proteins and how they control apoptotic cell death in healthy and diseased cells have uncovered novel opportunities for therapeutic intervention. Importantly, the development of both positive and negative small molecule modulators of apoptosis are now enabling researchers to translate the discoveries that have been made in the laboratory into clinical practice to positively impact human health.

Since its initial identification as one of the genes most highly upregulated upon T-cell activation, osteopontin (or Eta-1, as it was designated then) has been demonstrated to have many roles in the regulation of the immune response on multiple levels. It contributes to the development of immune-mediated and inflammatory diseases, and it regulates the host response to infection. In some cases, the mechanisms of these effects have been elucidated, while other mechanistic functions of the protein remain obscure. The protein itself makes these analyses complex, since it binds to a series of different integrins, and in addition to its classically secreted form, an intracellular form of osteopontin has been identified, which participates in several aspects of immune regulation. In this review, we focus on the role of osteopontin in a series of immune-related diseases, particularly those where significant advances have been made in recent years: multiple sclerosis, rheumatoid arthritis, lupus and related diseases, Sjögren's disease, colitis, and 1 area of inflammatory pathology, alcoholic and nonalcoholic liver diseases. A recurring theme in these diseases is a link between osteopontin and pathogenic T cells, particularly T helper 17 cells, where osteopontin produced by dendritic cells supports IL-17 expression, contributing to pathology. In addition, a role for osteopontin in B-cell differentiation is becoming clear. In general, osteopontin contributes to pathology in these diseases, but there are examples where it has a protective role; deciphering the mechanisms underlying these differences and the specific receptors for osteopontin will be a research challenge for the future. Aside from its newly discovered role in the development of Sjögren's disease, the role of osteopontin in inflammatory conditions in the oral cavity is still poorly understood. Elucidation of this role will be of interest.

BackgroundScopolamine is a well-known cholinergic antagonist that causes amnesia in human and animal models. Scopolamine-induced amnesia in rodent models has been widely used to understand the molecular, biochemical, behavioral changes, and to delineate therapeutic targets of memory impairment. Although this has been linked to the decrease in central cholinergic neuronal activity following the blockade of muscarinic receptors, the underlying molecular and cellular mechanism(s) particularly the effect on neuroplasticity remains elusive. In the present study, we have investigated (i) the effects of scopolamine on the molecules involved in neuronal and glial plasticity both in vivo and in vitro and (ii) their recovery by alcoholic extract of Ashwagandha leaves (i-Extract).Methodology/Principal FindingsAs a drug model, scopolamine hydrobromide was administered intraperitoneally to mice and its effect on the brain function was determined by molecular analyses. The results showed that the scopolamine caused downregulation of the expression of BDNF and GFAP in dose and time dependent manner, and these effects were markedly attenuated in response to i-Extract treatment. Similar to our observations in animal model system, we found that the scopolamine induced cytotoxicity in IMR32 neuronal and C6 glioma cells. It was associated with downregulation of neuronal cell markers NF-H, MAP2, PSD-95, GAP-43 and glial cell marker GFAP and with upregulation of DNA damage- γH2AX and oxidative stress- ROS markers. Furthermore, these molecules showed recovery when cells were treated with i-Extract or its purified component, withanone.ConclusionOur study suggested that besides cholinergic blockade, scopolamine-induced memory loss may be associated with oxidative stress and Ashwagandha i-Extract, and withanone may serve as potential preventive and therapeutic agents for neurodegenerative disorders and hence warrant further molecular analyses.

Lifelong dietary restriction (DR) is known to have many potential beneficial effects on brain function as well as delaying the onset of neurological diseases. In the present investigation, the effect of late-onset short-term intermittent fasting dietary restriction (IF-DR) regimen was studied on motor coordination and cognitive ability of ageing male rats. These animals were further used to estimate protein carbonyl content and mitochondrial complex I-IV activity in different regions of brain and peripheral organs, and the degree of age-related impairment and reversion by late-onset short-term IF-DR was compared with their levels in 3-month-old young rats. The results of improvement in motor coordination by rotarod test and cognitive skills by Morris water maze in IF-DR rats were found to be positively correlated with the decline in the oxidative molecular damage to proteins and enhanced mitochondrial complex IV activity in different regions of ageing brain as well as peripheral organs. The work was further extended to study the expression of synaptic plasticity-related proteins, such as synaptophysin, calcineurin and CaM kinase II to explore the molecular basis of IF-DR regimen to improve cognitive function. These results suggest that even late-onset short-term IF-DR regimen have the potential to retard age-associated detrimental effects, such as cognitive and motor performance as well as oxidative molecular damage to proteins.

Background and PurposeWithanolides are naturally occurring chemical compounds. They are secondary metabolites produced via oxidation of steroids and structurally consist of a steroid-backbone bound to a lactone or its derivatives. They are known to protect plants against herbivores and have medicinal value including anti-inflammation, anti-cancer, adaptogenic and anti-oxidant effects. Withaferin A (Wi-A) and Withanone (Wi-N) are two structurally similar withanolides isolated from Withania somnifera, also known as Ashwagandha in Indian Ayurvedic medicine. Ashwagandha alcoholic leaf extract (i-Extract), rich in Wi-N, was shown to kill cancer cells selectively. Furthermore, the two closely related purified phytochemicals, Wi-A and Wi-N, showed differential activity in normal and cancer human cells in vitro and in vivo. We had earlier identified several genes involved in cytotoxicity of i-Extract in human cancer cells by loss-of-function assays using either siRNA or randomized ribozyme library.Methodology/Principal FindingsIn the present study, we have employed bioinformatics tools on four genes, i.e., mortalin, p53, p21 and Nrf2, identified by loss-of-function screenings. We examined the docking efficacy of Wi-N and Wi-A to each of the four targets and found that the two closely related phytochemicals have differential binding properties to the selected cellular targets that can potentially instigate differential molecular effects. We validated these findings by undertaking parallel experiments on specific gene responses to either Wi-N or Wi-A in human normal and cancer cells. We demonstrate that Wi-A that binds strongly to the selected targets acts as a strong cytotoxic agent both for normal and cancer cells. Wi-N, on the other hand, has a weak binding to the targets; it showed milder cytotoxicity towards cancer cells and was safe for normal cells. The present molecular docking analyses and experimental evidence revealed important insights to the use of Wi-A and Wi-N for cancer treatment and development of new anti-cancer phytochemical cocktails.

PTH regulates osteoblastic function by activating PTH/PTHrP receptors (PTH1Rs), which trigger several signaling pathways in parallel, including cAMP/protein kinase A (PKA) and, via both phospholipase-C (PLC)-dependent and PLC-independent mechanisms, protein kinase C (PKC). These signaling functions have been mapped to distinct domains within PTH(1-34), but their roles in mediating the anabolic effect of intermittent PTH in vivo are unclear. We compared the anabolic effects in mice of hPTH(1-34) with those of two analogs having restricted patterns of PTH1R signaling. [G 1 ,R 19 ]hPTH(1-28) lacks the 29-34 domain of hPTH(1-34) needed for PLCindependent PKC activation, incorporates a Gly 1 mutation that prevents PLC activation, and stimulates only cAMP/PKA signaling. [G 1 ,R 19 ]hPTH(1-34) retains the 29-34 domain and activates both cAMP/PKA and PLC-independent PKC.Human PTH(1-34) (40 μg/kg), [G 1 ,R 19 ]hPTH(1-34) (120 μg/kg), and [G 1 ,R 19 ]hPTH(1-28) (800 μg/kg), at doses equipotent in elevating blood cAMP at 10 min and cAMP-dependent gene expression in bone at 6 h after s.c. injection, were administered to 10 week old female C57BL/6J mice 5 days/ week for 4 weeks. Acute blood cAMP responses, retested after 4 weeks, were not reduced by the preceding PTH treatment. The three PTH peptides induced equivalent increases in distal femoral bone mineral density (BMD), and, by microCT analysis, distal femoral and vertebral bone volume and trabecular thickness and mid-femoral cortical endosteal apposition. [G 1 ,R 19 ]hPTH(1-34) and hPTH(1-34) increased distal femoral BMD more rapidly and augmented total-body BMD and bone volume of proximal tibial trabeculi to a greater extent than did [G 1 ,R 19 ]hPTH(1-28),.We conclude that cAMP/PKA signaling is the dominant mechanism for the anabolic actions of PTH in trabecular bone and PLC-independent PKC signaling, attributable to the PTH(29-34) sequence, appears to accelerate the trabecular response and augment BMD at some skeletal sites. PTH1R PLC signaling pathway is not required for an anabolic effect of intermittent PTH(1-34) on bone.

Background Trypanosoma congolense are extracellular and intravascular blood parasites that cause debilitating acute or chronic disease in cattle and other domestic animals. Diminazene aceturate (Berenil) has been widely used as a chemotherapeutic agent for trypanosomiasis in livestock since 1955. As in livestock, treatment of infected highly susceptible BALB/c mice with Berenil leads to rapid control of parasitemia and survival from an otherwise lethal infection. The molecular and biochemical mechanisms of action of Berenil are still not very well defined and its effect on the host immune system has remained relatively unstudied. Here, we investigated whether Berenil has, in addition to its trypanolytic effect, a modulatory effect on the host immune response to Trypanosoma congolense.Methodology/Principal FindingsBALB/c and C57BL/6 mice were infected intraperitoneally with T. congolense, treated with Berenil and the expression of CD25 and FoxP3 on splenic cells was assessed directly ex vivo. In addition, serum levels and spontaneous and LPS-induced production of pro-inflammatory cytokines by splenic and hepatic CD11b+ cells were determined by ELISA. Berenil treatment significantly reduced the percentages of CD25+ cells, a concomitant reduction in the percentage of regulatory (CD4+Foxp3+) T cells and a striking reduction in serum levels of disease exacerbating pro-inflammatory cytokines including IL-6, IL-12, TNF and IFN-γ. Furthermore, Berenil treatment significantly suppressed spontaneous and LPS-induced production of inflammatory cytokines by splenic and liver macrophages and significantly ameliorated LPS-induced septic shock and the associated cytokine storm.Conclusions/SignificanceCollectively, these results provide evidence that in addition to its direct trypanolytic effect, Berenil also modulates the host immune response to the parasite in a manner that dampen excessive immune activation and production of pathology-promoting pro-inflammatory cytokines, suggesting that this drug may also be beneficial for treatment of disease conditions caused by excessive production of inflammatory cytokines.

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