A frameshift mutation and alternate splicing in human brain generate a functional form of the pseudogene cytochrome P4502D7 that bemethylates codeine to morphine

A frameshift mutation 138delT generates an open reading frame in the pseudogene, cytochrome P4502D7 (CYP2D7), and an alternate spliced functional transcript of CYP2D7 containing partial inclusion of intron 6 was identified in human brain but not in liver or kidney from the same individual. mRNA and protein of the brain variant CYP2D7 were detected in 6 of 12 human autopsy brains. Genotyping revealed the presence of the frameshift mutation 138delT only in those human subjects who expressed the brain variant CYP2D7. Genomic DNA analysis in normal volunteers revealed the presence of functional CYP2D7 in 4 of 8 individuals. In liver, the major organ involved in drug metabolism, a minor metabolic pathway mediated by CYP2D6 metabolizes codeine (pro-drug) to morphine (active drug), whereas norcodeine is the major metabolite. In contrast, when expressed in Neuro2a cells, brain variant CYP2D7 metabolized codeine to morphine with greater efficiency compared with the corresponding activity in cells expressing CYP2D6. Morphine binds to μ-opioid receptors in certain regions of the central nervous system, such as periaqueductal gray, and produces pain relief. The brain variant CYP2D7 and μ-opioid receptor colocalize in neurons of the periaqueductal gray area in human brain, indicating that metabolism of codeine to morphine could occur at the site of opioid action. Histio-specific isoforms of P450 generated by alternate splicing, which mediate selective metabolism of pro-drugs within tissues, particularly the brain, to generate active drugs may play an important role in drug action and provide newer insights into the genetics of metabolism

Drug Metabolism in Human Brain: High Levels of Cytochrome P4503A43 in Brain and Metabolism of Anti-Anxiety Drug Alprazolam to Its Active Metabolite

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more α-hydroxy alprazolam (α-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both α-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of α-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of α-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action

Bioactivity, biocompatibility and antimicrobial properties of a chitosan-mineral composite for periodontal tissue regeneration

A composite membrane of the polymer, chitosan, and the silver-exchanged mineral phase, tobermorite, was prepared by solvent casting and characterised by scanning electron microscopy and Fourier transform infrared spectroscopy. The in vitro bioactivity, cytocompatibility and antimicrobial activity of the composite were evaluated with respect to its potential application as a guided tissue regeneration (GTR) membrane. The in vitro bioactivity was verified by the formation of hydroxyapatite on the surface of the membrane in simulated body fluid and its cytocompatibility was established using MG63 human osteosarcoma cells. The presence of silver ions conferred significant antimicrobial activity against S. aureus, P. aeruginosa and E. coli. The findings of this investigation have indicated that the chitosansilver-tobermorite composite is a prospective candidate for GTR applications

Resveratrol-induced cytotoxicity in human Burkitt's lymphoma cells is coupled to the unfolded protein response

<p>Abstract</p> <p>Background</p> <p>Resveratrol (RES), a natural phytoalexin found at high levels in grapes and red wine, has been shown to induce anti-proliferation and apoptosis of human cancer cell lines. However, the underlying molecular mechanisms are at present only partially understood.</p> <p>Method</p> <p>The effects of RES on activation of unfolded protein responses (UPR) were evaluated using Western blotting, semi-quantitative and real-time RT-PCR. Cell death was evaluated using Annexin V/PI staining and subsequent FACS.</p> <p>Results</p> <p>Similar as tunicamycin, treatment with RES lead to the activation of all 3 branches of the UPR, with early splicing of XBP-1 indicative of IRE1 activation, phosphorylation of eIF2α consistent with ER resident kinase (PERK) activation, activating transcription factor 6 (ATF6) splicing, and increase in expression levels of the downstream molecules GRP78/BiP, GRP94 and CHOP/GADD153 in human Burkitt's lymphoma Raji and Daudi cell lines. RES was shown to induce cell death, which could be attenuated by thwarting upregulation of CHOP.</p> <p>Conclusions</p> <p>Our data suggest that activation of the apoptotic arm of the UPR and its downstream effector CHOP/GADD153 is involved, at least in part, in RES-induced apoptosis in Burkitt's lymphoma cells.</p

PINK1 Is Necessary for Long Term Survival and Mitochondrial Function in Human Dopaminergic Neurons

Parkinson's disease (PD) is a common age-related neurodegenerative disease and it is critical to develop models which recapitulate the pathogenic process including the effect of the ageing process. Although the pathogenesis of sporadic PD is unknown, the identification of the mendelian genetic factor PINK1 has provided new mechanistic insights. In order to investigate the role of PINK1 in Parkinson's disease, we studied PINK1 loss of function in human and primary mouse neurons. Using RNAi, we created stable PINK1 knockdown in human dopaminergic neurons differentiated from foetal ventral mesencephalon stem cells, as well as in an immortalised human neuroblastoma cell line. We sought to validate our findings in primary neurons derived from a transgenic PINK1 knockout mouse. For the first time we demonstrate an age dependent neurodegenerative phenotype in human and mouse neurons. PINK1 deficiency leads to reduced long-term viability in human neurons, which die via the mitochondrial apoptosis pathway. Human neurons lacking PINK1 demonstrate features of marked oxidative stress with widespread mitochondrial dysfunction and abnormal mitochondrial morphology. We report that PINK1 plays a neuroprotective role in the mitochondria of mammalian neurons, especially against stress such as staurosporine. In addition we provide evidence that cellular compensatory mechanisms such as mitochondrial biogenesis and upregulation of lysosomal degradation pathways occur in PINK1 deficiency. The phenotypic effects of PINK1 loss-of-function described here in mammalian neurons provides mechanistic insight into the age-related degeneration of nigral dopaminergic neurons seen in PD

NRF2 Activation Restores Disease Related Metabolic Deficiencies in Olfactory Neurosphere-Derived Cells from Patients with Sporadic Parkinson's Disease

Extent: 14p.Background: Without appropriate cellular models the etiology of idiopathic Parkinson’s disease remains unknown. We recently reported a novel patient-derived cellular model generated from biopsies of the olfactory mucosa (termed olfactory neurosphere-derived (hONS) cells) which express functional and genetic differences in a disease-specific manner. Transcriptomic analysis of Patient and Control hONS cells identified the NRF2 transcription factor signalling pathway as the most differentially expressed in Parkinson’s disease. Results: We tested the robustness of our initial findings by including additional cell lines and confirmed that hONS cells from Patients had 20% reductions in reduced glutathione levels and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium, inner salt] metabolism compared to cultures from healthy Control donors. We also confirmed that Patient hONS cells are in a state of oxidative stress due to higher production of H2O2 than Control cultures. siRNA-mediated ablation of NRF2 in Control donor cells decreased both total glutathione content and MTS metabolism to levels detected in cells from Parkinson’s Disease patients. Conversely, and more importantly, we showed that activation of the NRF2 pathway in Parkinson’s disease hONS cultures restored glutathione levels and MTS metabolism to Control levels. Paradoxically, transcriptomic analysis after NRF2 pathway activation revealed an increased number of differentially expressed mRNAs within the NRF2 pathway in L-SUL treated Patient-derived hONS cells compared to L-SUL treated Controls, even though their metabolism was restored to normal. We also identified differential expression of the PI3K/AKT signalling pathway, but only post-treatment. Conclusions: Our results confirmed NRF2 as a potential therapeutic target for Parkinson’s disease and provided the first demonstration that NRF2 function was inducible in Patient-derived cells from donors with uniquely varied genetic backgrounds. However, our results also demonstrated that the response of PD patient-derived cells was not co-ordinated in the same way as in Control cells. This may be an important factor when developing new therapeutics.Anthony L. Cook, Alejandra M. Vitale, Sugandha Ravishankar, Nicholas Matigian, Greg T. Sutherland, Jiangou Shan, Ratneswary Sutharsan, Chris Perry, Peter A. Silburn, George D. Mellick, Murray L. Whitelaw, Christine A. Wells, Alan Mackay-Sim and Stephen A. Woo

The ongoing pursuit of neuroprotective therapies in Parkinson disease

Many agents developed for neuroprotective treatment of Parkinson disease (PD) have shown great promise in the laboratory, but none have translated to positive results in patients with PD. Potential neuroprotective drugs, such as ubiquinone, creatine and PYM50028, have failed to show any clinical benefits in recent high-profile clinical trials. This 'failure to translate' is likely to be related primarily to our incomplete understanding of the pathogenic mechanisms underlying PD, and excessive reliance on data from toxin-based animal models to judge which agents should be selected for clinical trials. Restricted resources inevitably mean that difficult compromises must be made in terms of trial design, and reliable estimation of efficacy is further hampered by the absence of validated biomarkers of disease progression. Drug development in PD dementia has been mostly unsuccessful; however, emerging biochemical, genetic and pathological evidence suggests a link between tau and amyloid-β deposition and cognitive decline in PD, potentially opening up new possibilities for therapeutic intervention. This Review discusses the most important 'druggable' disease mechanisms in PD, as well as the most-promising drugs that are being evaluated for their potential efficiency in treatment of motor and cognitive impairments in PD

Chapitre 14: Phytopathogènes et stratégies de contrôle en aquaponie

peer reviewedAmong the diversity of plant diseases occurring in aquaponics, soil-borne pathogens, such as Fusarium spp., Phytophthora spp. and Pythium spp., are the most problematic due to their preference for humid/aquatic environment conditions. Phytophthora spp. and Pythium spp. which belong to the Oomycetes pseudo-fungi require special attention because of their mobile form of dispersion, the so-called zoospores that can move freely and actively in liquid water. In coupled aquaponics, curative methods are still limited because of the possible toxicity of pesticides and chemical agents for fish and beneficial bacteria (e.g. nitrifying bacteria of the biofilter). Furthermore, the development of biocontrol agents for aquaponic use is still at its beginning. Consequently, ways to control the initial infection and the progression of a disease are mainly based on preventive actions and water physical treatments. However, suppressive action (suppression) could happen in aquaponic environment considering recent papers and the suppressive activity already highlighted in hydroponics. In addition, aquaponic water contains organic matter that could promote establishment and growth of heterotrophic bacteria in the system or even improve plant growth and viability directly. With regards to organic hydroponics (i.e. use of organic fertilisation and organic plant media), these bacteria could act as antagonist agents or as plant defence elicitors to protect plants from diseases. In the future, research on the disease suppressive ability of the aquaponic biotope must be increased, as well as isolation, characterisation and formulation of microbial plant pathogen antagonists. Finally, a good knowledge in the rapid identification of pathogens, combined with control methods and diseases monitoring, as recommended in integrated plant pest management, is the key to an efficient control of plant diseases in aquaponics.Cos

Tissue-enhanced plasma proteomic analysis for disease stratification in amyotrophic lateral sclerosis

Motor Neurone Disease Association (Malaspina/Apr13/817–791). Wellcome Trust support to a parallel study (Pathfinder Award, grant number 103208)