• Isolation and characterisation of a bipotential haematopoietic cell line.

      Dexter, T Michael; Allen, Terence D; Scott, David; Teich, N; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1979-02-08)
    • Isolation and characterization of 5T4, a tumour-associated antigen

      Hole, Nicholas; Stern, Peter L; Department of Cancer Biology, Salk Institute, San Diego, CA, USA (1990)
    • Isolation and characterization of a human homologue of the latrophilin gene from a region of 1p31.1 implicated in breast cancer.

      White, Gavin R M; Varley, Jennifer; Heighway, Jim; CRC Section of Molecular Genetics, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1998-12-31)
      We have identified a region of chromosome 1p31.1 that shows high frequency loss of heterozygosity (LOH) in human breast cancer. This region forms part of a 7 Mb YAC/BAC contig. In order to identify candidate sequences, mutation of which might contribute to the development of disease, we have carried out mapping studies of ESTs localized to 1p31.1. This analysis, coupled with library screening and a modified 5' RACE-PCR strategy, resulted in the identification and characterization of a novel gene (LPHH1) which is located adjacent to the smallest region of overlapping loss (SRO) seen in tumours. The 4209 bp open reading frame of the 7 kb LPHH1 transcript encodes a peptide which shows approximately 65% identity to rat latrophilin, a G-coupled, seven span transmembrane protein, which binds alpha-latrotoxin. In the human sequence, whilst conservation of the transmembrane domain is high, the intra- and extracellular domains show two regions of variable structure, which are presumably generated by alternative splicing. Surprisingly, while expression of the rat gene is tightly restricted to neurological and perhaps some endocrine cells, the human sequence appears to be expressed very widely in all normal tissues tested. Northern and RT-PCR analysis of a panel of tumour cell lines showed that LPHH1 expression was variable, apparently elevated in some lines and absent or markedly reduced in others. Furthermore, characterization of the range of transcripts encoded in a breast tumour cell line, compared to normal breast, suggested that gene product variability was higher in the tumour.
    • Isolation and characterization of human mammary stem cells.

      Clarke, Robert B; Breast Biology Group, Division of Cancer Studies, University of Manchester, Christie Hospital, Wilmslow Road, Withington, Manchester, M20 4BX, UK. robert.clarke@manchester.ac.uk (2005-12)
      Since stem cells are present throughout the lifetime of an organism, it is thought that they may accumulate mutations, eventually leading to cancer. In the breast, tumours are predominantly oestrogen and progesterone receptor-positive (ERalpha/PR+). We therefore studied the biology of ERalpha/PR-positive cells and their relationship to stem cells in normal human mammary epithelium. We demonstrated that ERalpha/PR-positive cells co-express the putative stem cell markers p21(CIP1/WAF1), cytokeratin (CK) 19 and Musashi-1 when examined using dual label immunofluorescence on tissue sections. Next, we isolated a Hoechst dye-effluxing 'side population' (SP) from the epithelium using flow cytometry and demonstrated them to be undifferentiated cells by lack of expression of myoepithelial and luminal cell-specific antigens such as CALLA and MUC1. Epithelial SP cells were shown to be enriched for the putative stem cell markers p21(CIP1/WAF1), Musashi-1 and ERalpha/PR-positive cells. Lastly, SP cells, compared to non-SP, were highly enriched for the capacity to produce colonies containing multiple lineages in 3D basement membrane (Matrigel) culture. We conclude that breast stem cells include two populations: a primitive ERalpha/PR-negative stem cell necessary for development and a shorter term ERalpha/PR-positive stem cell necessary for adult tissue homeostasis during menstrual cycling. We speculate these two basic stem cell types may therefore be the cells of origin for ERalpha-positive and -negative breast tumours.
    • Isolation and characterization of progenitor-like cells from human renal proximal tubules.

      Lindgren, D; Boström, A; Nilsson, K; Hansson, J; Sjölund, J; Möller, C; Jirström, K; Nilsson, E; Landberg, Göran; Axelson, H; et al. (2011-02)
      The tubules of the kidney display a remarkable capacity for self-renewal on damage. Whether this regeneration is mediated by dedifferentiating surviving cells or, as recently suggested, by stem cells has not been unequivocally settled. Herein, we demonstrate that aldehyde dehydrogenase (ALDH) activity may be used for isolation of cells with progenitor characteristics from adult human renal cortical tissue. Gene expression profiling of the isolated ALDH(high) and ALDH(low) cell fractions followed by immunohistochemical interrogation of renal tissues enabled us to delineate a tentative progenitor cell population scattered through the proximal tubules (PTs). These cells expressed CD24 and CD133, previously described markers for renal progenitors of Bowman's capsule. Furthermore, we show that the PT cells, and the glomerular progenitors, are positive for KRT7, KRT19, BCL2, and vimentin. In addition, tubular epithelium regenerating on acute tubular necrosis displayed long stretches of CD133(+)/VIM(+) cells, further substantiating that these cells may represent a progenitor cell population. Furthermore, a potential association of these progenitor cells with papillary renal cell carcinoma was discovered. Taken together, our data demonstrate the presence of a previously unappreciated subset of the PT cells that may be endowed with a more robust phenotype, allowing increased resistance to acute renal injury, enabling rapid repopulation of the tubules.
    • Isolation and characterization of the integral glycosaminoglycan constituents of human amyloid A and monoclonal light-chain amyloid fibrils.

      Nelson, S R; Lyon, Malcolm; Gallagher, John T; Johnson, E A; Pepys, M B; Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, U.K. (1991-04-01)
      Amyloid fibrils were isolated by extraction in water from the livers and spleens of four patients who had died of monoclonal, light-chain (AL)-type, systemic amyloidosis and one with reactive systemic, amyloid A protein (AA)-type amyloidosis. Each fibril preparation contained 1-2% by weight of glycosaminoglycan (GAG) which was tightly associated with the fibrils and not just co-isolated from the tissues with them. After exhaustive digestion of the fibrils with papain and Pronase, the GAGs were specifically precipitated with cetylpyridinium chloride and were identified by cellulose acetate electrophoresis and selective susceptibility to specific glycosidases. All the preparations contained approximately equal amounts of heparan sulphate and dermatan sulphate. There was no evidence for the presence of chondroitin sulphate or other GAGs. Fine structural analysis by oligosaccharide mapping in gradient polyacrylamide gels, following partial digestion with specific glycosidases, showed very similar structures among the heparan sulphates and the dermatan sulphates, respectively. GAGs were also extracted by solubilizing amyloid fibrils in 4 M-guanidinium chloride followed by CsCl density-gradient ultracentrifugation. Although a minor proportion of the GAG material obtained in this way was apparently in the form of proteoglycan molecules, most of it was free GAG chains. The presence in amyloid fibrils of different types, in different organs and from different patients of particular GAG classes with similar structures supports the view that these molecules may be of pathogenic significance.
    • Isolation and partial characterisation of a Chinese hamster O6-alkylguanine-DNA alkyltransferase cDNA.

      Rafferty, Joseph A; Elder, Rhoderick H; Watson, Amanda J; Cawkwell, L; Potter, P M; Margison, Geoffrey P; CRC Department of Chemical Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (1992-04-25)
      The cDNA encoding Chinese hamster O6-alkylguanine-DNA-alkyltransferase (ATase) has been isolated from a library prepared from RNA isolated from V79 lung fibroblasts which had an upregulated level of this repair activity following stepwise selection with a chloroethylating agent (1, 2). Expression of the cDNA in E. coli produced functionally active ATase at levels of 2.5% of total cellular protein as determined by in vitro assay. The recombinant hamster protein has a molecular weight of 28 kDa as estimated by SDS-PAGE and fluorography and this was identical to that in the upregulated cells. The characteristic PCHRV pentapeptide of the alkyl acceptor site has been identified and there is a 68 amino acid residue region which is 90% conserved across all the mammalian proteins so far analysed: in contrast, the N- and C-terminal domains diverge by as much as 50% between species. Polyclonal antibodies to the human and rat ATases hybridised to the hamster protein on western analysis suggesting at least one common epitope shared across species. However, in antibody inhibition experiments neither of the antisera cross reacted with the hamster ATase in a way which interfered with functional activity whereas the anti-human antibodies inhibited the human ATase and the anti-rat antibodies inhibited the rat and mouse ATases. There may therefore be significant tertiary structural differences between the hamster protein and the other mammalian ATases.
    • Isolation and partial characterization of murine O6-alkylguanine-DNA-alkyltransferase: comparative sequence and structural properties.

      Santibanez-Koref, Mauro F; Elder, Rhoderick H; Fan, Chun-Yang; Cawkwell, Lynn; McKie, J H; Douglas, K T; Margison, Geoffrey P; Rafferty, Joseph A; Department of Cancer Genetics, University of Manchester, United Kingdom. (1992)
      A cDNA encoding murine O6-alkylguanine-DNA-alkyltransferase (ATase) has been sequenced after isolation from total liver RNA by the polymerase chain reaction using oligonucleotide primers derived from the rat ATase cDNA sequence. Functionally active murine ATase protein has been expressed in Escherichia coli at high levels (about 2% of total protein) and purified to apparent homogeneity (molecular mass 26 kDa). In liquid hybridization experiments, anti-human ATase polyclonal antibodies inhibited human but not rat or mouse ATase, whereas anti-rat polyclonal antibodies inhibited rat and mouse but not human ATase. Both antibodies detected all mammalian ATases tested by western analysis so far. These results indicate some common epitopes and at least one unique human epitope. We compared the amino-acid sequence of the murine ATase with those of other mammalian and bacterial ATases. The proteins of this family all have a large domain (approximately 70 amino acids) of highly conserved residues flanking the sequence PCHRV, which contains the alkyl-accepting cysteine residue of the active site. No evidence was found in the sequences for helix-turn-helix, leucine-zipper, or zinc-finger motifs for DNA recognition and binding. Nuclear localization signals (basic-residue-rich regions) could not be uniquely identified in the mammalian members of the family. Outside of the conserved PCHRV region, there were major differences between prokaryotic and eukaryotic proteins at the primary structure level: there was a series of proline-rich motifs, but these also varied between sequences.
    • Isolation of a cDNA encoding 5T4 oncofetal trophoblast glycoprotein. An antigen associated with metastasis contains leucine-rich repeats.

      Myers, Kevin A; Rahi-Saund, Veena; Davison, M D; Young, J A; Cheater, Amanada J; Stern, Peter L; Cancer Research Campaign Department of Immunology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, United Kingdom. (1994-03-25)
      The monoclonal antibody 5T4 defines a human oncotrophoblast antigen expressed by a variety of carcinomas but with a restricted pattern of expression in normal adult tissues. The 5T4 antigen has been isolated from term placenta as a 72-kDa glycoprotein consisting of a 42-kDa core protein with extensive N-linked glycosylation. A cDNA has been isolated from a human placental library using pools of oligonucleotides based on amino acid sequence obtained from purified 5T4 molecules. The predicted open reading frame encodes a protein of 420 amino acids with a molecular mass of 46 kDa and 8 potential N-glycosylation sites. There are N- and C-terminal hydrophobic segments corresponding to putative signal and membrane anchorage sequences, respectively. Northern analysis has demonstrated a major 2.5-kilobase mRNA present in cell lines serologically reactive with the monoclonal antibody 5T4. Comparison of the 5T4 protein sequence with current sequence data bases has identified the presence of leucine-rich repeats, which are found in a variety of proteins from yeast, insects, and mammals. The 5T4 antigen expression is strongly associated with metastasis in colorectal and gastric cancer, and, hence, the possible functions of the gene product and its relationship to tumor growth and progression are discussed.
    • Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

      Shaw, David M; Embleton, Jim; Westwater, C; Ryan, Matthew G; Myers, Kevin A; Kingsman, Susan M; Carroll, M W; Stern, Peter L; CRC Immunology Group, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (2000-12-15)
      The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.
    • Isolation of a human genomic fragment, co-amplified with c-Ki-ras, that affects plasmid supercoiling in E. coli.

      Heighway, Jim; Geurts van Kessel, A H (1987-04-24)
      Amplification of cellular proto-oncogenes has been implicated in the development of human malignancies. A library was constructed from genomic DNA extracted from a lung tumour, previously shown to carry an amplified c-Ki-ras 2 gene. Using a v-Ki-ras probe, a fragment with ras homology was isolated and shown to be amplified in the original tumour DNA to the same level as c-Ki-ras. Studies with human hamster hybrids demonstrated that it is normally located on human chromosome 12 (as is c-Ki-ras). The restriction map of the fragment is different from that of the known Ha, Ki or N-ras genes and its sequence shows evolutionary conservation, as demonstrated by hybridisation to the genomic DNA of several mammalian species. A pUC19 subclone (pK42), carrying a 1.3kb insert, shows supercoil heterogeneity in plasmid preparations, as does a second compatible plasmid introduced into the same bacterial host with pK42. It appears therefore that the subclone is encoding a product that affects DNA topoisomerase activity in E. coli.
    • Isolation of E-1-(4'-Hydroxyphenyl)-but-1-en-3-one from Scutellaria barbata.

      Ducki, Sylvie W; Hadfield, John A; Lawrence, N J; Liu, C Y; McGown, Alan T; Zhang, X; Department of Chemistry, UMIST, Manchester, U.K. (1996-04)
    • Isolation of paeonol from Arisaema erubescens.

      Ducki, Sylvie W; Hadfield, John A; Lawrence, Nicholas J; Zhang, X; McGown, Alan T; Cancer Research Campaign Department of Drug Development, Christie Hospital NHS Trust, Manchester, U.K. (1995-12)
    • Isolation of tumour antigen specific scFv's using a chimeric antigen receptor bicistronic retroviral vector in a mammalian screening protocol.

      Lipowska-Bhalla, Grazyna; Gilham, David E; Hawkins, Robert E; Rothwell, Dominic G; University of Manchester, CIMML, Manchester, United Kingdom (2013-09-12)
      The clinical potential of chimeric antigen receptors in adoptive cellular therapy is beginning to be realised with several recent clinical trials targeting CD19 showing promising results in advanced B cell malignancies. This increased efficacy corresponds with improved engineering of the chimeric receptors with the latest generation receptors eliciting greater signalling and proliferation potential. However, the antigen binding scFv domain of the receptors is critical in determining the activity of the chimeric receptor expressing T cells; as this determines specificity and affinity to the tumour antigen. In this study, we describe a mammalian T-cell line screening protocol employing a 2A-based bicistronic retroviral vector to isolate functional scFv's. This approach involves expression of the scFv library in a CAR, and is based on selection of clones capable of stimulating CD69 upregulation in a T cell line and has a number of advantages over previously described methods in that the use of a 2A-cassette ensures the exclusion of non-expressing scFv's and the screening using a chimeric receptor in a mammalian T-cell line ensures selection in the optimum context for therapeutic use. Proof of principle experiments show that the protocol was capable of a 105-fold enrichment of positive clones following three rounds of selection. Furthermore, an antigen specific clone was successfully isolated from a partially enriched scFv library confirming the strength of the protocol. This approach has the potential to identify novel scFv's of use in adoptive T cell therapy and, potentially, wider antibody-based applications.
    • The isolation, culture and therapeutic application of pluripotent stem cells derived from human embryos

      Ward, Christopher M; Immunology Group, Paterson Institute for Cancer Research, Wilmslow Road, Manchester, M20 4BX, UK (2002)
    • Issues on fit-for-purpose validation of a panel of ELISAs for application as biomarkers in clinical trials of anti-Angiogenic drugs.

      Brookes, K E; Cummings, Jeffrey; Backen, Alison C; Greystoke, Alastair; Ward, Timothy H; Jayson, Gordon C; Dive, Caroline; Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, UK. (2010-05-11)
      BACKGROUND: Successful introduction of new anticancer agents into the clinic is often hampered by a lack of qualified biomarkers. Studies have been conducted of 17 ELISAs representing a potential panel of pharmacodynamic/predictive biomarkers for drugs targeted to tumour vasculature. METHODS: The fit-for-purpose approach to method validation was used. Stability studies were performed using recombinant proteins in surrogate matrices, endogenous analytes in healthy volunteer and cancer patient plasma. The impact of platelet depletion was investigated. RESULTS: Method validation focused on measuring precision and showed that 15 of the 17 assays were within acceptable limits. Stability at -80 degrees C was shown for 3 months with all recombinant proteins in surrogate matrices, whereas under the same conditions instability was observed with KGF in platelet-rich and platelet-depleted plasma, and with PDGF-BB in platelet-depleted plasma from cancer patients. For measurement of extracellular circulating analytes, platelet depletion should be conducted before freezing of plasma to prevent release of PDGF-BB, FGFb and VEGF-A. A protocol was developed to remove >90% platelets from plasma requiring centrifugation at 2000 g for 25 min. CONCLUSIONS: These studies highlight the need for assay validation and crucial assessment of sample handling issues before commencement of biomarker analysis in clinical trials.
    • Ixekizumab for the treatment of psoriasis: up-to-date

      Craig, Sarah; Warren, RB; Skin cancer and Ageing group, Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK (2020)
      Introduction: Ixekizumab (an IL-17A antagonist) is a biologic therapeutic licensed for use in moderate-to-severe plaque psoriasis and psoriatic arthritis. IL-17 antagonists (also including Secukinumab and Brodalumab) represent a new generation of biologic therapy with rapid and high response rates, quickly becoming a crucial part of the psoriasis treatment armamentarium.Areas covered: In this review, we describe how IL-17A antagonists disrupt inflammatory cascades in psoriasis and summarize results from clinical trials examining the safety and efficacy of ixekizumab against placebo and comparators.Expert opinion: Ixekizumab induces a 75% reduction in psoriasis area severity index (PASI 75) in 89% of patients after 12 weeks and after 1 year, PASI 75 is maintained in 80% of patients. Ixekizumab is superior to both etanercept and ustekinumab, however, further comparator trials are needed to determine superiority between newer agents. Network meta-analysis suggests that ixekizumab is one of the most rapid and efficacious agents for treating psoriasis, but ideally more long-term real-world data are needed to determine the persistence of response. Candida may be commonly encountered during treatment and IL-17 agents should be avoided in patients with inflammatory bowel disease. Overall, ixekizumab represents an efficacious and well-studied therapeutic that can offer biologic-naïve and bio-failure patients durable disease control.
    • JNK signaling in cancer cell survival

      Wu, Q; Wu, W; Fu, B; Shi, Lei; Wang, X; Kuca, K; College of Life Science, Yangtze University, Jingzhou, China (2019)
      c-Jun N-terminal kinase (JNK) is involved in cancer cell apoptosis; however, emerging evidence indicates that this Janus signaling promotes cancer cell survival. JNK acts synergistically with NF-?B, JAK/STAT, and other signaling molecules to exert a survival function. JNK positively regulates autophagy to counteract apoptosis, and its effect on autophagy is related to the development of chemotherapeutic resistance. The prosurvival effect of JNK may involve an immune evasion mechanism mediated by transforming growth factor-?, toll-like receptors, interferon-?, and autophagy, as well as compensatory JNK-dependent cell proliferation. The present review focuses on recent advances in understanding the prosurvival function of JNK and its role in tumor development and chemoresistance, including a comprehensive analysis of the molecular mechanisms underlying JNK-mediated cancer cell survival. There is a focus on the specific "Yin and Yang" functions of JNK1 and JNK2 in the regulation of cancer cell survival. We highlight recent advances in our knowledge of the roles of JNK in cancer cell survival, which may provide insight into the distinct functions of JNK in cancer and its potential for cancer therapy.
    • JNK suppresses tumor formation via a gene-expression program mediated by ATF2.

      Gozdecka, Malgorzata; Lyons, Steve; Kondo, S; Taylor, J; Li, Yaoyong; Walczynski, J; Thiel, G; Breitwieser, Wolfgang; Jones, Nic; Department of Cell Regulation, CRUK Manchester Institute, Paterson Building, University of Manchester, Manchester (2014-11-20)
      JNK and p38 phosphorylate a diverse set of substrates and, consequently, can act in a context-dependent manner to either promote or inhibit tumor growth. Elucidating the functions of specific substrates of JNK and p38 is therefore critical for our understanding of these kinases in cancer. ATF2 is a phosphorylation-dependent transcription factor and substrate of both JNK and p38. Here, we show ATF2 suppresses tumor formation in an orthotopic model of liver cancer and cellular transformation in vitro. Furthermore, we find that suppression of tumorigenesis by JNK requires ATF2. We identify a transcriptional program activated by JNK via ATF2 and provide examples of JNK- and ATF2-dependent genes that block cellular transformation. Significantly, we also show that ATF2-dependent gene expression is frequently downregulated in human cancers, indicating that amelioration of JNK-ATF2-mediated suppression may be a common event during tumor development.
    • JNK-mediated activation of ATF2 contributes to dopaminergic neurodegeneration in the MPTP mouse model of Parkinson's disease.

      Huang, Q; Du, X; He, X; Yu, Q; Hu, K; Breitwieser, Wolfgang; Shen, Q; Ma, S; Li, M; Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2(nd) Road, Guangzhou 510080, China (2015-10-26)
      The c-Jun. N-terminal kinase (JNK)/c-Jun. pathway is a known critical regulator of dopaminergic neuronal death in Parkinson's disease (PD) and is considered a potential target for neuroprotective therapy. However, whether JNK is activated within dopaminergic neurons remains controversial, and whether JNK acts through downstream effectors other than c-Jun. to promote dopaminergic neuronal death remains unclear. In this study, we confirm that JNK but not p38 is activated in dopaminergic neurons after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxication. Furthermore, within the dopaminergic neurons of the substantia nigra in MPTP-treated mice, JNK2/3 phosphorylates threonine 69 (Thr69) of Activating transcription factor-2 (ATF2), a transcription factor of the ATF/CREB family, whereas the phosphorylation of Thr71 is constitutive and remains unchanged. The increased phosphorylation of ATF2 on Thr69 by JNK in the MPTP mouse model suggests a functional relationship between the transcriptional activation of ATF2 and dopaminergic neuron death. By using dopaminergic neuron-specific conditional ATF2 mutant mice, we found that either partial or complete deletion of the ATF2 DNA-binding domain in dopaminergic neurons markedly alleviates the MPTP-induced dopaminergic neurodegeneration, indicating that the activation of ATF2 plays a detrimental role in neuropathogenesis in PD. Taken together, our findings demonstrate that JNK-mediated ATF2 activation contributes to dopaminergic neuronal death in an MPTP model of PD.