Choline metabolism underpins macrophage IL-4 polarization and RELMα up-regulation in helminth infection.

Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated up-regulation of choline transport compared to naïve macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELMα (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naïve or mice infected with the intestinal helminth Heligmosomoides polygyrus were treated with the choline kinase α inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELMα expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus, with increased egg burden. Together, these data demonstrate that choline metabolism is required for macrophage RELMα induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity.

Metabolomics and computational analysis of the role of monoamine oxidase activity in delirium and SARS-COV-2 infection.

Delirium is an acute change in attention and cognition occurring in ~ 65% of severe SARS-CoV-2 cases. It is also common following surgery and an indicator of brain vulnerability and risk for the development of dementia. In this work we analyzed the underlying role of metabolism in delirium-susceptibility in the postoperative setting using metabolomic profiling of cerebrospinal fluid and blood taken from the same patients prior to planned orthopaedic surgery. Distance correlation analysis and Random Forest (RF) feature selection were used to determine changes in metabolic networks. We found significant concentration differences in several amino acids, acylcarnitines and polyamines linking delirium-prone patients to known factors in Alzheimer's disease such as monoamine oxidase B (MAOB) protein. Subsequent computational structural comparison between MAOB and angiotensin converting enzyme 2 as well as protein-protein docking analysis showed that there potentially is strong binding of SARS-CoV-2 spike protein to MAOB. The possibility that SARS-CoV-2 influences MAOB activity leading to the observed neurological and platelet-based complications of SARS-CoV-2 infection requires further investigation.

Identification of pannexin 1-regulated genes, interactome, and pathways in rhabdomyosarcoma and its tumor inhibitory interaction with AHNAK.

Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, is an aggressive cancer with a poor prognosis. Despite current management, the 5-year survival rate for patients with metastatic RMS is ∼30%; underscoring the need to develop better treatment strategies. We have recently reported that pannexin 1 (PANX1) levels are downregulated in RMS and that restoring its expression inhibits RMS progression. Here, we have surveyed and characterized the molecular changes induced by PANX1 re-expression in RMS. We cataloged transcriptomic changes in this context by RNA sequencing. At the protein level, we unveiled PANX1 interactors using BioID, complemented by co-immunoprecipitation coupled to high-performance liquid chromatography/electrospray ionization tandem mass spectrometry performed in PANX1-enriched fractions. Using these data, we generated searchable public databases for the PANX1 interactome and changes to the RMS transcriptome occurring when PANX1 expression is restored. STRING network analyses revealed a PANX1 interactome involving plasma membrane and cytoskeleton-associated proteins including the previously undescribed interactor AHNAK. Indeed, AHNAK knockdown abrogated the PANX1-mediated reduction in RMS cell viability and migration. Using these unbiased approaches, we bring insight to the mechanisms by which PANX1 inhibits RMS progression, identifying the cell migration protein AHNAK as a key modifier of PANX1-mediated changes in RMS malignant properties.

Growth environment and organ specific variation in in-vitro cytoprotective activities of Picea mariana in PC12 cells exposed to glucose toxicity: a plant used for treatment of diabetes symptoms by the Cree of Eeyou Istchee (Quebec, Canada).

BACKGROUND: The Cree of Eeyou Istchee (James Bay area of northern Quebec) suffer from a high rate of diabetes and its complications partly due to the introduction of the western lifestyle within their culture. As part of a search for alternative medicine based on traditional practice, this project evaluates the biological activity of Picea mariana (Mill.) Britton, Sterns & Poggenb. needle, bark, and cone, in preventing glucose toxicity to PC12-AC cells in vitro (a diabetic neurophathy model) and whether habitat and growth environment influence this activity. METHODS: Three different organs (needle, bark, and cone) of P. mariana were collected at different geographical locations and ecological conditions and their 80% ethanolic extracts were prepared. Extracts were then tested for their ability to protect PC12-AC cells from hyperglycaemic challenge at physiologically relevant concentrations of 0.25, 0.5, 1.0 and 2.0 µg/mL. Folin-Ciocalteu method was used to determine the total phenolic content of P. mariana extracts. RESULTS: All extracts were well-tolerated in vitro exhibiting LD50 of 25 µg/mL or higher. Extracts from all tested organs showed a cytoprotective concentration-dependent response. Furthermore, the cytoprotective activity was habitat- and growth environment-dependent with plants grown in bog or forest habitats in coastal or inland environments exhibiting different cytoprotective efficacies. These differences in activity correlated with total phenolic content but not with antioxidant activity. In addition, this paper provides the first complete Ultra-Performance Liquid Chromatography-quadrupole time-of-flight (UPLC-QTOF) mass spectrometry analysis of Picea mariana's bark, needles and cones. CONCLUSIONS: Together, these results provide further understanding of the cytoprotective activity of Canadian boreal forest plants identified by the Cree healers of Eeyou Istchee in a cell model of diabetic neuropathy. Their activity is relevant to diabetic peripheral neuropathic complications and shows that their properties can be optimized by harvesting in optimal growth environments.

Choline transport links macrophage phospholipid metabolism and inflammation.

Choline is an essential nutrient that is required for synthesis of the main eukaryote phospholipid, phosphatidylcholine. Macrophages are innate immune cells that survey and respond to danger and damage signals. Although it is well-known that energy metabolism can dictate macrophage function, little is known as to the importance of choline homeostasis in macrophage biology. We hypothesized that the uptake and metabolism of choline are important for macrophage inflammation. Polarization of primary bone marrow macrophages with lipopolysaccharide (LPS) resulted in an increased rate of choline uptake and higher levels of PC synthesis. This was attributed to a substantial increase in the transcript and protein expression of the choline transporter-like protein-1 (CTL1) in polarized cells. We next sought to determine the importance of choline uptake and CTL1 for macrophage immune responsiveness. Chronic pharmacological or CTL1 antibody-mediated inhibition of choline uptake resulted in altered cytokine secretion in response to LPS, which was associated with increased levels of diacylglycerol and activation of protein kinase C. These experiments establish a previously unappreciated link between choline phospholipid metabolism and macrophage immune responsiveness, highlighting a critical and regulatory role for macrophage choline uptake via the CTL1 transporter.

A Regulatory Network Involving ß-Catenin, e-Cadherin, PI3k/Akt, and Slug Balances Self-Renewal and Differentiation of Human Pluripotent Stem Cells In Response to Wnt Signaling.

The mechanisms underlying disparate roles of the canonical Wnt signaling pathway in maintaining self-renewal or inducing differentiation and lineage specification in embryonic stem cells (ESCs) are not clear. In this study, we provide the first demonstration that self-renewal versus differentiation of human ESCs (hESCs) in response to Wnt signaling is predominantly determined by a two-layer regulatory circuit involving ß-catenin, E-cadherin, PI3K/Akt, and Slug in a time-dependent manner. Short-term upregulation of ß-catenin does not lead to the activation of T-cell factor (TCF)-eGFP Wnt reporter in hESCs. Instead, it enhances E-cadherin expression on the cell membrane, thereby enhancing hESC self-renewal through E-cadherin-associated PI3K/Akt signaling. Conversely, long-term Wnt activation or loss of E-cadherin intracellular ß-catenin binding domain induces TCF-eGFP activity and promotes hESC differentiation through ß-catenin-induced upregulation of Slug. Enhanced expression of Slug leads to a further reduction of E-cadherin that serves as a ß-catenin "sink" sequestering free cytoplasmic ß-catenin. The formation of such a framework reinforces hESCs to switch from a state of temporal self-renewal associated with short-term Wnt/ß-catenin activation to definitive differentiation. Stem Cells 2015;33:1419-1433.

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) protects neurons from oxidative death via an Nrf2 astrocyte-specific mechanism independent of PPARγ.

Astrocytes are critical for the antioxidant support of neurons. Recently, we demonstrated that low level hydrogen peroxide (H(2) O(2) ) facilitates astrocyte-dependent neuroprotection independent of the antioxidant transcription factor Nrf2, leaving the identity of the endogenous astrocytic Nrf2 activator to question. In this study, we show that an endogenous electrophile, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), non-cell autonomously protects neurons from death induced by depletion of the major antioxidant glutathione. Nrf2 knockdown in astrocytes abrogated 15d-PGJ2's neuroprotective effect as well as 15d-PGJ2 facilitated Nrf2-target gene induction. In contrast, knockdown of the transcription factor peroxisome proliferator activated-receptor gamma (PPARγ), a well-characterized 15d-PGJ2 target, did not alter 15d-PGJ2 non-cell autonomous neuroprotection. In addition, several PPARγ agonists of the thiazolidinedione (TZD) family failed to induce neuroprotection. Unexpectedly, however, the TZD troglitazone (which contains a chromanol moiety found on vitamin E) induced astrocyte-mediated neuroprotection, an effect which was mimicked by the vitamin E analogs alpha-tocopherol or alpha-tocotrienol. Our findings lead to two important conclusions: (i) 15d-PGJ2 induces astrocyte-mediated neuroprotection via an Nrf2 but not PPARγ mediated pathway, suggesting that 15d-PGJ2 is a candidate endogenous modulator of Nrf2 protective pathways in astrocytes; (ii) selective astrocyte treatment with analogs or compounds containing the chromanol moiety of vitamin E facilitates non-cell autonomous neuroprotection.

Characterizing the cytoprotective activity of Sarracenia purpurea L., a medicinal plant that inhibits glucotoxicity in PC12 cells.

BACKGROUND: The purple pitcher plant, Sarracenia purpurea L., is a widely distributed species in North America with a history of use as both a marketed pain therapy and a traditional medicine in many aboriginal communities. Among the Cree of Eeyou Istchee in northern Québec, the plant is employed to treat symptoms of diabetes and the leaf extract demonstrates multiple anti-diabetic activities including cytoprotection in an in vitro model of diabetic neuropathy. The current study aimed to further investigate this activity by identifying the plant parts and secondary metabolites that contribute to these cytoprotective effects. METHODS: Ethanolic extracts of S. purpurea leaves and roots were separately administered to PC12 cells exposed to glucose toxicity with subsequent assessment by two cell viability assays. Assay-guided fractionation of the active extract and fractions was then conducted to identify active principles. Using high pressure liquid chromatography together with mass spectrometry, the presence of identified actives in both leaf and root extracts were determined. RESULTS: The leaf extract, but not that of the root, prevented glucose-mediated cell loss in a concentration-dependent manner. Several fractions elicited protective effects, indicative of multiple active metabolites, and, following subfractionation of the polar fraction, hyperoside (quercetin-3-O-galactoside) and morroniside were isolated as active constituents. Phytochemical analysis confirmed the presence of hyperoside in the leaf but not root extract and, although morroniside was detected in both organs, its concentration was seven times higher in the leaf. CONCLUSION: Our results not only support further study into the therapeutic potential and safety of S. purpurea as an alternative and complementary treatment for diabetic complications associated with glucose toxicity but also identify active principles that can be used for purposes of standardization and quality control.

Expression and detrimental role of hematopoietic prostaglandin D synthase in spinal cord contusion injury.

Prostaglandin D(2) (PGD(2) ) is a potent inflammatory mediator, which is implicated in both the initiation and resolution of inflammation in peripheral non-neural tissues. Its role in the central nervous system has not been fully elucidated. Spinal cord injury (SCI) is associated with an acute inflammatory response, which contributes to secondary tissue damage that worsens functional loss. We show here, with the use of hematopoietic prostaglandin D synthase (HPGDS) deficient mice and a HPGDS selective inhibitor (HQL-79), that PGD(2) plays a detrimental role after SCI. We also show that HPGDS is expressed in macrophages in the injured mouse spinal cord and contributes to the increase in PGD(2) in the contused spinal cord. HPGDS(-/-) mice also show reduced secondary tissue damage and reduced expression of the proinflammatory chemokine CXCL10 as well as an increase in IL-6 and TGFß-1 expression in the injured spinal cord. This was accompanied by a reduction in the expression of the microglia/macrophage activation marker Mac-2 and an increase in the antioxidant metallothionein III. Importantly, HPGDS deficient mice exhibit significantly better locomotor recovery after spinal cord contusion injury than wild-type (Wt) mice. In addition, systemically administered HPGDS inhibitor (HQL-79) also enhanced locomotor recovery after SCI in Wt mice. These data suggest that PGD(2) generated via HPGDS has detrimental effects after SCI and that blocking the activity of this enzyme can be beneficial.

Lyso-form fragment ions facilitate the determination of stereospecificity of diacyl glycerophospholipids.

In this work we report the development of a novel methodology for the determination of stereospecificity of diacyl glycerophospholipids, including glycerophosphatidic acids (PA), glycerophosphoserines (PS), glycerophosphoglycerols (PG), glycerophosphoinositols (PI), and glycerophosphoethanolamines (PE), which can be conventionally ionized in negative ion mode. This methodology uses MS(2) recorded on a hybrid quadrupole time-of-flight mass spectrometer to determine the stereospecificity of diacyl glycerophospholipids based on the lyso-form fragment ions, attributed to the neutral loss of fatty acyl moieties. The fragmentation patterns of a variety of diacyl glycerophospholipid standards were first fully examined over a wide range of collision energy. We observed that lyso-form fragment ions corresponding to the neutral loss of fatty acyl moieties attached to the sn2 position as free fatty acids ([M-Sn2](-) ) and as ketenes ([M-(Sn2-H(2) O)](-) ) exhibited consistently higher intensity than their counterpart ions due to the neutral loss of fatty acyl moieties attached to the sn1 position ([M-Sn1](-) and [M-(Sn1-H(2) O)](-) ). Therefore, we concluded that an empirical fragmentation rule can be used to precisely determine the stereospecificity of diacyl glycerophospholipids, primarily on the basis of relative abundance of the lyso-form fragment ions. We then examined the product ion spectra of diacyl glycerophospholipids recorded from lipid extracts of rat hepatoma cells, where the stereospecific information of these lipids was conclusively determined. Combining the novel methodology reported in this work with the currently widely practiced mass spectrometric techniques such as multiple precursor ion scans (MPIS), fatty acyl scans (FAS), and multidimensional mass spectrometry based shotgun lipidomics (MDMS-SL), should enable a reliable and convenient platform for comprehensive glycerophospholipid profiling.

Inhibition of advanced glycation end product formation by medicinal plant extracts correlates with phenolic metabolites and antioxidant activity.

Nonenzymatic formation of advanced glycation end products (AGEs) is accelerated under hyperglycemic conditions characteristic of type 2 diabetes mellitus and contributes to the development of vascular complications. As such, inhibition of AGE formation represents a potential therapeutic target for the prevention and treatment of diabetic complications. In the present study, ethanolic extracts of 17 medicinal plants were assessed for inhibitory effects on in vitro AGE formation through fluorometric and immunochemical detection of fluorescent AGEs and N(ε)-(carboxymethyl)lysine adducts of albumin (CML-BSA), respectively. Most extracts inhibited fluorescent AGE formation with IC (50) values ranging from 0.4 to 38.6 µg/mL and all extracts reduced CML-BSA formation but to differing degrees. Results obtained through both methods were highly correlated. Antiglycation activities were positively correlated with total phenolic content, free radical scavenging activity and reduction in malonyldiadehyde levels following oxidation of low-density lipoprotein, but negatively correlated with lag time to formation of conjugated dienes. Together, these results provide evidence that antioxidant phenolic metabolites mediate the antiglycation activity of our medicinal plant collection, a relationship that likely extends to other medicinal and food plants.

Evaluation of the antidiabetic potential of selected medicinal plant extracts from the Canadian boreal forest used to treat symptoms of diabetes: part II.

Among the Cree of northern Quebec, the disproportionately high rate of diabetic complications is largely due to the cultural inadequacy of modern therapies for type 2 diabetes. To establish culturally adapted antidiabetic treatments, our team identified several candidate plant species used by the Cree to treat symptoms of diabetes. An initial study focused on 8 species and revealed that most possess significant in vitro antidiabetic activity. The purpose of the present study was to assess a further 9 species identified through the ethnobotanical survey. Crude plant extracts were screened for (i) potentiation of basal and insulin-stimulated glucose uptake by skeletal muscle cells (C2C12) and adipocytes (3T3-L1); (ii) potentiation of glucose-stimulated insulin secretion by pancreatic beta cells (betaTC); (iii) potentiation of adipogenesis in 3T3-L1 cells; (iv) protection against glucose toxicity and glucose deprivation in PC12-AC neuronal precursor cells; and (v) diphenylpicrylhydrazyl (DPPH) oxygen free radical scavenging. Four species potentiated basal glucose uptake in muscle cells or adipocytes, one species being as potent as metformin. Adipogenesis was accelerated by 4 species with a potency roughly half that of rosiglitazone. Five species protected PC12-AC cells against glucose toxicity and 4 protected against glucose deprivation. Five species exhibited antioxidant activity comparable to ascorbic acid. However, no species increased insulin secretion. The present study revealed that Gaultheria hispidula, Rhododendron tomentosum, and Vaccinium vitis-idaea exhibit a promising profile of antidiabetic potential and are good candidates for more in-depth evaluation.

Identification of lysophosphatidylcholine (LPC) and platelet activating factor (PAF) from PC12 cells and mouse cortex using liquid chromatography/multi-stage mass spectrometry (LC/MS3).

Lipids play essential roles in cellular structural support, energy storage and signal transduction. Recently, mass spectrometry (MS) has been used to produce three-dimensional maps that elucidate the lipid composition of complex cellular lysates. The identification of individual lipids within these maps is slow and requires the synthesis and spiking of each candidate lipid. We present a novel MS-based technique that rapidly elucidates the atomic connectivity of the fatty acid/alcohol substituent on the sn-1 position of several different families of glycerophosphocholine-containing lipids within the confines of a chromatographic separation. Sodiated lipid species were fragmented to produce radical cations which lost successive methylene groups upon further collisional activation to reveal the identity of the parent molecule. This approach was demonstrated to be effective on isobaric members of the lysophosphatidylcholine (LPC) and platelet activating factor (PAF) families of glycerophospholipids. We demonstrate the application of this technique to unambiguously identify these species within complex cellular lysates and tissue extracts.

Differential regulation of wild-type and mutant alpha-synuclein binding to synaptic membranes by cytosolic factors.

BACKGROUND: Alpha-Synuclein (alpha-syn), a 140 amino acid protein associated with presynaptic membranes in brain, is a major constituent of Lewy bodies in Parkinson's disease (PD). Three missense mutations (A30P, A53T and E46K) in the alpha-syn gene are associated with rare autosomal dominant forms of familial PD. However, the regulation of alpha-syn's cellular localization in neurons and the effects of the PD-linked mutations are poorly understood. RESULTS: In the present study, we analysed the ability of cytosolic factors to regulate alpha-syn binding to synaptic membranes. We show that co-incubation with brain cytosol significantly increases the membrane binding of normal and PD-linked mutant alpha-syn. To characterize cytosolic factor(s) that modulate alpha-syn binding properties, we investigated the ability of proteins, lipids, ATP and calcium to modulate alpha-syn membrane interactions. We report that lipids and ATP are two of the principal cytosolic components that modulate Wt and A53T alpha-syn binding to the synaptic membrane. We further show that 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) is one of the principal lipids found in complex with cytosolic proteins and is required to enhance alpha-syn interaction with synaptic membrane. In addition, the impaired membrane binding observed for A30P alpha-syn was significantly mitigated by the presence of protease-sensitive factors in brain cytosol. CONCLUSION: These findings suggest that endogenous brain cytosolic factors regulate Wt and mutant alpha-syn membrane binding, and could represent potential targets to influence alpha-syn solubility in brain.

Plant phenolics regulate neoplastic cell growth and survival: a quantitative structure-activity and biochemical analysis.

The anti-tumour activities of many plant phenolics at high concentrations (>100 micromol/L) suggest their potential use as dietary supplements in cancer chemoprevention and cancer chemotherapy. However, it is not clear what impact phenolic compounds have at the physiological concentrations obtained through consumption of high phenolic diets on neoplastic cells. In the present study, 54 naturally occurring phenolics were evaluated at physiologically relevant concentrations for their capacity to alter PC12 cell viability in response to serum deprivation, the chemotherepeutic agent etoposide, and the apoptogen C2-ceramide. Surprisingly, novel mitogenic, cytoprotective, and antiapoptotic activities were detected. Quantitative structure-activity relationship modelling indicated that many of these activities could be predicted by compound lipophilicity, steric bulk, and (or) antioxidant capacity, with the exception of inhibition of ceramide-induced apoptosis. Where quantitative structure-activity relationship analysis was insufficient, biochemical assessment demonstrated that the benzoate orsellinic acid blocked downstream caspase-12 activation following ceramide challenge. These findings demonstrate substantive mitogenic, cytoprotective, and antiapoptotic biological activities of plant phenolics on neoplastic cells at physiologically relevant dietary concentrations that should be considered in chemopreventive and chemotherapeutic strategies.

Identification and quantitation of changes in the platelet activating factor family of glycerophospholipids over the course of neuronal differentiation by high-performance liquid chromatography electrospray ionization tandem mass spectrometry.

Glycerophospholipids are important structural lipids in membranes with changes associated with progressive neurodegenerative disorders such as Alzheimer disease. Synthesis of the platelet activating factor (PAF) glycerophospholipid subclass is implicated in the control of neuronal differentiation and death. In this article, we combine nanoflow HPLC and mass spectrometry to screen, identify, and quantitate changes in glycerophospholipid subspecies, specifically PAF family members, over the course of neuronal differentiation. Furthermore, precursor ion scans for fragments characteristic of PAF phosphocholine family members and the standard additions of PAF subspecies were combined to perform absolute quantitation of PAF lipids in undifferentiated and differentiated PC12 cells. Surprisingly, a marked asymmetry was detected in the two predominant PAF species (C16:0, C18:0) over the course of differentiation. These results describe a new technique for the sensitive analysis of lipids combining nanoflow HPLC, ESI-MS, and precursor ion scan. Limits of detection of as little as 2 pg of PAF and LPC were obtained, and analysis of the lipidome of as little as 70,000 cells was performed on this system. Furthermore, application to the PC12 model identified a quantifiable difference between PAF molecular species produced over the course of neuronal differentiation.

Platelet activating factor-induced neuronal apoptosis is initiated independently of its G-protein coupled PAF receptor and is inhibited by the benzoate orsellinic acid.

The bioactive lipid mediator platelet activating factor (PAF) is recognized as a key effecter of neuronal apoptosis, yet it is not clear whether its G-protein coupled receptor (PAFR) initiates or prevents PAF neurotoxicity. Using PAFR-/- and congenic wild-type mice, we show that PAF triggers caspase-3/7 activity and neuronal death in PAFR-/- but not PAFR+/+ cerebellar granule neurons. Restoring receptor expression by recombinant adenoviral infection protected cells from PAF challenge. Neuronal death was not mediated by nitric oxide or N-methyl-d-aspartate receptor signaling given that N-nitro-l-arginine methyl ester and MK-801 did not inhibit PAF-induced neuronal loss in PAFR-/- neurons. To intervene in PAFR-independent neurotoxicity, the anti-apoptotic actions of three structurally distinct PAF antagonists were compared to a panel of plant and fungal benzoic acid derivatives. We found that the PAF antagonist BN 52021 but not FR 49175 or CV 3988 inhibited PAFR-independent neurotoxicity. Orsellinic acid, a fungal-derived benzoic acid, blocked PAF-mediated neuronal apoptosis without affecting PAFR-mediated neuroprotection. These findings demonstrate that PAF can transduce apoptotic death in primary neurons independently of its G-protein coupled receptor, that PAFR activation is neuroprotective, and that orsellinic acid effectively attenuates PAFR-independent neuronal apoptosis.

HIV protease inhibitors modulate apoptosis signaling in vitro and in vivo.

HIV protease inhibitors are an integral part of effective anti-HIV therapy. The drugs block HIV protease, prevent proper packaging of HIV virions, and decrease the HIV viral burden in the peripheral blood of infected individuals. In addition to direct anti-viral effects, the HIV protease inhibitors also modulate apoptosis. A growing body of work demonstrates the anti-apoptotic effects of HIV protease inhibitors on CD4+ and CD8+ T cells during HIV infection. The mechanism of this apoptosis inhibition is supported by several proposed hypotheses for how they alter the fate of the cell, including preventing adenine nucleotide translocator pore function, which consequently prevents loss of mitochondrial transmembrane potential. More recently, the anti-apoptotic effects of the HIV protease inhibitors have been tested in non-HIV, non-immune cell, whereby protease inhibitors prevent apoptosis, and disease in animal models of sepsis, hepatitis, pancreatitis and stroke. Interestingly, when HIV protease inhibitors are used at supra-therapeutic concentrations, they exert pro-apoptotic effects. This has been demonstrated in a number of tumor models. Although it is unclear how HIV protease inhibitors can induce apoptosis at increased concentrations, future research will define the targets of the immunomodulation and reveal the full clinical potential of this intriguing class of drugs.

Selected plant species from the Cree pharmacopoeia of northern Quebec possess anti-diabetic potential.

Type II diabetes is a major health problem worldwide. Some populations, such as aboriginal peoples, are particularly at risk for this disease. In the Cree Nation of Quebec, Canada, prevalence in adults is approaching 20%, and the consequences are compounded by low compliance with modern medicine. In 2003, we conducted an ethnobotanical study of Cree medicinal plants used for the treatment of symptoms of diabetes. This served as the basis for a project designed to identify efficacious complementary treatment options more readily accepted by this population. The present study assesses the in vitro anti-diabetic potential of extracts from the 8 most promising plants to emerge from the ethnobotanical study. Cell-based bioassays were employed to screen for (i) potentiation of glucose uptake by skeletal muscle cells (C2C12) and adipocytes (3T3-L1); (ii) potentiation of glucose-stimulated insulin secretion (GSIS) and insulin production by pancreatic beta cells (INS 832/13); (iii) potentiation of triglyceride accumulation in differentiating 3T3-L1 cells; (iv) protection against glucose toxicity and glucose deprivation in pre-sympathetic neurons (PC12-AC). Additionally, anti-oxidant activity was measured biochemically by the diphenylpicrylhydrazyl (DPPH) reduction assay. All plant extracts potentiated basal or insulin-stimulated glucose uptake to some degree in muscle cells or adipocytes. Adipocyte differentiation was accelerated by 4 extracts. Five extracts conferred protection in PC12 cells. Three extracts displayed free radical scavenging activity similar to known anti-oxidants. None of the plant extracts enhanced GSIS or insulin content in INS 832/13 beta cells. It is concluded that the Cree pharmacopoeia contains several plants with significant anti-diabetic potential.

Apoptosis-inducing factor is a key factor in neuronal cell death propagated by BAX-dependent and BAX-independent mechanisms.

Mitochondria release proteins that propagate both caspase-dependent and caspase-independent cell death pathways. AIF (apoptosis-inducing factor) is an important caspase-independent death regulator in multiple neuronal injury pathways. Presently, there is considerable controversy as to whether AIF is neuroprotective or proapoptotic in neuronal injury, such as oxidative stress or excitotoxicity. To evaluate the role of AIF in BAX-dependent (DNA damage induced) and BAX-independent (excitotoxic) neuronal death, we used Harlequin (Hq) mice, which are hypomorphic for AIF. Neurons carrying double mutations for Hq/Apaf1-/- (apoptosis proteases-activating factor) are impaired in both caspase-dependent and AIF-mediated mitochondrial cell death pathways. These mutant cells exhibit extended neuroprotection against DNA damage, as well as glutamate-induced excitotoxicity. Specifically, AIF is involved in NMDA- and kainic acid- but not AMPA-induced excitotoxicity. In vivo excitotoxic studies using kainic acid-induced seizure showed that Hq mice had significantly less hippocampal damage than wild-type littermates. Our results demonstrate an important role for AIF in both BAX-dependent and BAX-independent mechanisms of neuronal injury.