136 results on '"MacManus JP"'
Search Results
2. Inhibition by thyrocalcitonin (calcitonin) of the cyclic AMP- mediated stimulation of thymocyte proliferation by epinephrine
- Author
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Whitfield Jf, MacManus Jp, and D. J. Gillan
- Subjects
Calcitonin ,Male ,Vasopressin ,medicine.medical_specialty ,Epinephrine ,Swine ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Parathyroid hormone ,Stimulation ,Thymus Gland ,Biology ,Biochemistry ,Endocrinology ,In vivo ,Internal medicine ,medicine ,Cyclic AMP ,Animals ,Lymphocytes ,Cells, Cultured ,Cell growth ,Biochemistry (medical) ,General Medicine ,Rats ,Kinetics ,Cardiovascular agent ,Drug Antagonism ,Cell Division ,medicine.drug - Abstract
Epinephrine stimulates the proliferation of rat thymic lymphocytes maintained in vitro by a cyclic AMP-mediated process. The mitogenic action of epinephrine is completely inhibited by low concentrations (6.0 to 8.0 mU per ml) of a highly purified sample of porcine thyrocalcitonin (calcitonin). In view of the fact that the same low levels of thyrocalcitonin are known to inhibit the cyclic AMP-mediated mitogenic action of vasopressin and the similar physiologically important mitogenic action of parathyroid hormone, it is proposed that thyrocalcitonin (calcitonin) may be general regulator of cyclic AMP-controlled cell proliferation in vivo. (Author)
- Published
- 1971
3. Parathyroid glands and mitotic stimulation in rat bone marrow after hemorrhage
- Author
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Perris, AD, primary, MacManus, JP, additional, Whitfield, JF, additional, and Weiss, LA, additional
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- 1971
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4. Cerebral ischemia causes dysregulation of synaptic adhesion in mouse synaptosomes.
- Author
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Costain WJ, Rasquinha I, Sandhu JK, Rippstein P, Zurakowski B, Slinn J, MacManus JP, and Stanimirovic DB
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- Agrin metabolism, Animals, Cell Death, Hippocampus metabolism, Hippocampus ultrastructure, Infarction, Middle Cerebral Artery pathology, Mice, Neuronal Plasticity, Neurons metabolism, Neurons ultrastructure, Prosencephalon metabolism, Prosencephalon ultrastructure, RNA, Messenger metabolism, Secretory Vesicles ultrastructure, Synapses ultrastructure, Synaptosomes ultrastructure, Time Factors, beta Catenin metabolism, Cell Adhesion Molecules metabolism, Infarction, Middle Cerebral Artery metabolism, Secretory Vesicles metabolism, Synapses metabolism, Synaptosomes metabolism
- Abstract
Synaptic pathology is observed during hypoxic events in the central nervous system in the form of altered dendrite structure and conductance changes. These alterations are rapidly reversible, on the return of normoxia, but are thought to initiate subsequent neuronal cell death. To characterize the effects of hypoxia on regulators of synaptic stability, we examined the temporal expression of cell adhesion molecules (CAMs) in synaptosomes after transient middle cerebral artery occlusion (MCAO) in mice. We focused on events preceding the onset of ischemic neuronal cell death (<48 h). Synaptosome preparations were enriched in synaptically localized proteins and were free of endoplasmic reticulum and nuclear contamination. Electron microscopy showed that the synaptosome preparation was enriched in spheres (approximately 650 nm in diameter) containing secretory vesicles and postsynaptic densities. Forebrain mRNA levels of synaptically located CAMs was unaffected at 3 h after MCAO. This is contrasted by the observation of consistent downregulation of synaptic CAMs at 20 h after MCAO. Examination of synaptosomal CAM protein content indicated that certain adhesion molecules were decreased as early as 3 h after MCAO. For comparison, synaptosomal Agrn protein levels were unaffected by cerebral ischemia. Furthermore, a marked increase in the levels of p-Ctnnb1 in ischemic synaptosomes was observed. p-Ctnnb1 was detected in hippocampal fiber tracts and in cornu ammonis 1 neuronal nuclei. These results indicate that ischemia induces a dysregulation of a subset of synaptic proteins that are important regulators of synaptic plasticity before the onset of ischemic neuronal cell death.
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- 2008
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5. Cerebral ischemia induces neuronal expression of novel VL30 mouse retrotransposons bound to polyribosomes.
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Costain WJ, Rasquinha I, Graber T, Luebbert C, Preston E, Slinn J, Xie X, and MacManus JP
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- Animals, Base Sequence genetics, Brain blood supply, Brain metabolism, Brain physiopathology, Brain Ischemia metabolism, Brain Ischemia physiopathology, Cerebral Infarction metabolism, Cerebral Infarction physiopathology, Disease Models, Animal, Infarction, Middle Cerebral Artery genetics, Infarction, Middle Cerebral Artery metabolism, Infarction, Middle Cerebral Artery physiopathology, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Polyribosomes metabolism, Promoter Regions, Genetic genetics, RNA, Messenger genetics, RNA, Messenger metabolism, Retroviridae genetics, Sequence Homology, Nucleic Acid, Brain Ischemia genetics, Cerebral Infarction genetics, Gene Expression Regulation genetics, Neurons metabolism, Polyribosomes genetics, Retroelements genetics
- Abstract
Mammalian genomes are burdened with a large heterogeneous group of endogenous replication defective retroviruses (retrotransposons). Previously, we identified a transcript resembling a virus-like 30S (VL30) retrotransposon increasing in mouse brain following transient cerebral ischemia. Paradoxically, this non-coding RNA was found bound to polyribosomes. Further analysis revealed that multiple retrotransposon species (BVL-1-like and mVL30-1-like) were bound to polyribosomes and induced by ischemia. These VL30 transcripts remained associated with polyribosomes in the presence of 0.5 M KCl, indicating that VL30 mRNA was tightly associated with ribosomal subunits. Furthermore, the profile of BVL-1 distribution on polyribosomal profiles was distinct from those of translated and translationally repressed mRNA. Consistent with expectations, 5.0 kb VL30 transcripts were detected in ischemic brain with a temporal pattern of expression that was distinct from c-fos. Expression of VL30 was localized in neurons using a combination of in situ hybridization and immunocytochemistry. 3'-RACE-PCR experiments yielded two unique sequences (VL30x-1 and VL30x-2) that were homologous to known VL30 genes. Phylogenetic analysis of VL30 promoter sequence (U3 region) resulted in the identification of two large VL30 subgroups. VL30x-1 and VL30x-2 were closely related and classified in a group that was distinct from the well-characterized VL30 genes BVL-1 and mVL30-1. The promoter regions of VL30x-1 and VL30x-2 did not possess the consensus sequences for either hypoxia or anoxia response elements, suggesting an alternative mechanism for induction. This is the first report that demonstrates ischemia-induced, neuronal expression of unique VL30 retrotransposons in mouse brain.
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- 2006
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6. Translation-state analysis of gene expression in mouse brain after focal ischemia.
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MacManus JP, Graber T, Luebbert C, Preston E, Rasquinha I, Smith B, and Webster J
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- 5' Untranslated Regions, Animals, Mice, Mice, Inbred C57BL, Oligonucleotide Array Sequence Analysis, Polyribosomes metabolism, RNA, Messenger metabolism, Reproducibility of Results, Brain Ischemia metabolism, Gene Expression Profiling, Gene Expression Regulation, Protein Biosynthesis
- Abstract
Confounding any genome-scale analysis of gene expression after cerebral ischemia is massive suppression of protein synthesis. This inefficient translation questions the utility of examining profiles of total transcripts. Our approach to such postischemic gene profiling in the mouse by microarray analysis was to concentrate on those mRNAs bound to polyribosomes. In our proof-of-principle study, polysomally bound and unbound mRNAs were subjected to microarray analysis: of the 1,161 transcripts that we found to increase after ischemia, only 36% were bound to polyribosomes. In addition to the expected increases in heat-shock proteins and metallothioneins, increases in several other bound transcripts involved in the promotion of cell survival or antiinflammatory behavior were noted, such as CD63 (Lamp3), Lcn2 (lipocalin-2), Msn (moesin), and UCP2 (uncoupling protein 2), all of which showed increases in cognate protein by Western blotting. The list of heretofore nonfunctionally annotated transcripts (RIKEN clones/ESTs) that increased appeared to be novel. How some transcripts are selected in ischemic brain for translation into protein, while others are rejected, is not clear. The length of the 5'-UTR in the ischemically induced transcripts that occur in the NCBI RefSeq database did not indicate any general tendency to be more than 200 nt, nor to be longer than the 5'-UTRs of the unbound transcripts. Thus, the presence of a complex 5'-UTR region with internal ribosome entry sites (IRES) or polypyrimidine tracts (TOP) does not appear to be the basis of selection for translation in ischemic brain.
- Published
- 2004
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7. Absence of the transcription factor E2F1 attenuates brain injury and improves behavior after focal ischemia in mice.
- Author
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MacManus JP, Jian M, Preston E, Rasquinha I, Webster J, and Zurakowski B
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- Animals, Brain pathology, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, E2F Transcription Factors, E2F1 Transcription Factor, In Situ Nick-End Labeling, Infarction, Middle Cerebral Artery, Mice, Mice, Inbred C57BL, Mice, Knockout, Neurons cytology, Neurons metabolism, Regional Blood Flow, Transcription Factors genetics, Behavior physiology, Brain metabolism, Brain Ischemia metabolism, Brain Ischemia pathology, Cell Cycle Proteins, Transcription Factors metabolism
- Abstract
Because of observations that cultured neurons from mice deficient in the transcription factor E2F1 exhibit resistance after treatment with a wide variety of cell-death inducers, the authors investigated whether resistance extended to a cerebral ischemic insult. No differences in cerebral blood flow or physiologic parameters were observed in the mutant E2F1 littermates after the focal ligation. After 2 hours of left middle cerebral artery occlusion and 1 day of reperfusion, a 33% smaller infarct (P < 0.05) was observed by 2,3,5-triphenyltetrazolium staining in the brains of E2F1-null mice compared with their E2F1+/+ and +/- littermates. A milder ischemic insult produced by 20 minutes of middle cerebral artery occlusion and 7 days of reperfusion produced a greater difference in the E2F1-null animals with a 71% smaller infarct (P < 0.001) compared to littermate controls. A decrease in neuronal damage after mild ischemia in E2F1-null mice was observed by immunohistochemical monitoring of the loss in neuronal-specific microtubule-associated protein 2 cytoskeletal protein and the appearance of nuclear DNA fragmentation by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling. This decreased brain damage was evidenced by improved behavior in motor function of E2F1 -/- mice compared with their E2F1 +/+ littermates by 7 days of reperfusion. In an effort to address the underlying molecular mechanism of the resistance of E2F1-null mice, the expression of several downstream proapoptotic target genes (p73, Apaf1, Arf) of the E2F1 transcription factor was measured by quantitative polymerase chain reaction. Although an attenuated increase in Hsp68 mRNA was found in E2F1 -/- mice, no changes in the proapoptotic transcripts were found after ischemia, and a mechanistic inference was not possible. The authors conclude that the transcription factor E2F1 does modulate neuronal viability in brain after cerebral ischemia and corroborates the findings with cultured neurons.
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- 2003
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8. Molecular mechanisms of cerebral ischemia-induced neuronal death.
- Author
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Hou ST and MacManus JP
- Subjects
- Animals, Brain Ischemia genetics, Brain Ischemia physiopathology, DNA Damage genetics, Gene Expression Regulation genetics, Humans, Mitochondria genetics, Mitochondria metabolism, Nerve Degeneration genetics, Nerve Degeneration physiopathology, Neurotoxins metabolism, Oxidative Stress genetics, Reperfusion Injury genetics, Reperfusion Injury physiopathology, Brain Ischemia metabolism, Cell Death genetics, Nerve Degeneration metabolism, Reperfusion Injury metabolism
- Abstract
The mode of neuronal death caused by cerebral ischemia and reperfusion appears on the continuum between the poles of catastrophic necrosis and apoptosis: ischemic neurons exhibit many biochemical hallmarks of apoptosis but remain cytologically necrotic. The position on this continuum may be modulated by the severity of the ischemic insult. The ischemia-induced neuronal death is an active process (energy dependent) and is the result of activation of cascades of detrimental biochemical events that include perturbion of calcium homeostasis leading to increased excitotoxicity, malfunction of endoplasmic reticulum and mitochondria, elevation of oxidative stress causing DNA damage, alteration in proapoptotic gene expression, and activation of the effector cysteine proteases (caspases) and endonucleases leading to the final degradation of the genome. In spite of strong evidence showing that brain infarction can be reduced by inhibiting any one of the above biochemical events, such as targeting excitotoxicity, up-regulation of an antiapoptotic gene, or inhibition of a down-stream effector caspase, it is becoming clear that targeting a single gene or factor is not sufficient for stroke therapeutics. An effective neuroprotective therapy is likely to be a cocktail aimed at all of the above detrimental events evoked by cerebral ischemia and the success of such therapeutic intervention relies upon the complete elucidation of pathways and mechanisms of the cerebral ischemia-induced active neuronal death.
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- 2002
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9. Attenuation of neurotoxicity in cortical cultures and hippocampal slices from E2F1 knockout mice.
- Author
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Gendron TF, Mealing GA, Paris J, Lou A, Edwards A, Hou ST, MacManus JP, Hakim AM, and Morley P
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- Animals, Calcium metabolism, Cell Hypoxia physiology, Cells, Cultured, E2F Transcription Factors, E2F1 Transcription Factor, Electric Stimulation, Fetus, Gene Expression Regulation, Glucose pharmacology, In Vitro Techniques, Kinetics, Male, Mice, Mice, Knockout, RNA, Messenger genetics, Receptors, N-Methyl-D-Aspartate genetics, Reverse Transcriptase Polymerase Chain Reaction, Synaptic Transmission physiology, Transcription Factors deficiency, Transcription, Genetic, Cell Cycle Proteins, Cerebral Cortex physiology, DNA-Binding Proteins, Evoked Potentials physiology, Hippocampus physiology, Neurons physiology, Transcription Factors genetics, Transcription Factors metabolism
- Abstract
The E2F1 transcription factor modulates neuronal apoptosis induced by staurosporine, DNA damage and beta-amyloid. We demonstrate E2F1 involvement in neuronal death induced by the more physiological oxygen-glucose deprivation (OGD) in mouse cortical cultures and by anoxia in mouse hippocampal slices. E2F1(+/+) and (-/-) cultures were comparable, in that they contained similar neuronal densities, responded with similar increases in intracellular calcium concentration ([Ca(2+)]i) to glutamate receptor agonists, and showed similar NMDA receptor subunit mRNA expression levels for NR1, NR2A and NR2B. Despite these similarities, E2F1(-/-) cultures were significantly less susceptible to neuronal death than E2F1(+/+) cultures 24 and 48 h following 120-180 min of OGD. Furthermore, the absence of E2F1 significantly improved the ability of CA1 neurons in hippocampal slices to recover synaptic transmission following a transient anoxic insult in vitro. These results, along with our finding that E2F1 mRNA levels are significantly increased following OGD, support a role for E2F1 in the modulation of OGD- and anoxia-induced neuronal death. These findings are consistent with studies showing that overexpression of E2F1 in postmitotic neurons causes neuronal degeneration and the absence of E2F1 decreases infarct volume following cerebral ischemia.
- Published
- 2001
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10. The transcription factor E2F1 promotes dopamine-evoked neuronal apoptosis by a mechanism independent of transcriptional activation.
- Author
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Hou ST, Cowan E, Walker T, Ohan N, Dove M, Rasqinha I, and MacManus JP
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- Animals, Apoptosis drug effects, Caspase 3, Caspases metabolism, Cell Line, Cell Survival drug effects, Cells, Cultured, Cerebellum cytology, DNA, Antisense pharmacology, DNA-Binding Proteins metabolism, E2F Transcription Factors, E2F1 Transcription Factor, Genes, Reporter, Green Fluorescent Proteins, Humans, Luciferases genetics, Luminescent Proteins genetics, Mice, Mice, Knockout, Neurons drug effects, Oxidopamine toxicity, Recombinant Fusion Proteins metabolism, Reverse Transcriptase Polymerase Chain Reaction, Transcription Factors deficiency, Transcriptional Activation, Transfection, Apoptosis physiology, Cell Cycle Proteins, Cerebellum physiology, Dopamine toxicity, Neurons cytology, Neurons physiology, Transcription Factors genetics, Transcription Factors metabolism
- Abstract
The E2F1 transcription factor plays an important role in promoting neuronal apoptosis; however, it is not clear how E2F1 does this. Here we show that E2F1 is involved in dopamine (DA)-evoked apoptosis in cerebellar granule neurons (CGNs). E2F1 -/- CGNs and CGNs expressing an antisense E2F1 cDNA were significantly protected from DA-toxicity relative to controls. The neuronal protection was accompanied by significantly reduced caspase 3 activity. E2F1-mediated neuronal apoptosis did not require activation of gene transcription because: (1) ectopic expression of E2F1 or its mutants lacking the transactivation domain induced neuronal apoptosis, whereas an E2F1 mutant lacking the DNA-binding domain did not; (2) under all of these conditions, known E2F1 target genes including cyclin A, cdc2 and p19(ARF) were not induced; and (3) DA-evoked neuronal apoptosis was associated with up-regulated E2F1, but not transcription of its target genes. Finally, E2F1-mediated neuronal apoptosis was associated with reduced nuclear factor (NF)-kappaB DNA-binding activity. Taken together, these data suggest that E2F1 promotes DA-evoked caspase 3-dependent neuronal apoptosis by a mechanism independent of gene transactivation, and this may possibly occur through inhibition of anti-apoptotic genes including NF-kappaB.
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- 2001
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11. Increased expression of the transcription factor E2F1 during dopamine-evoked, caspase-3-mediated apoptosis in rat cortical neurons.
- Author
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Hou ST, Cowan E, Dostanic S, Rasquinha I, Comas T, Morley P, and MacManus JP
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- Animals, Antioxidants pharmacology, Apoptosis drug effects, Caspase 3, Caspase Inhibitors, Cerebral Cortex cytology, Cysteine Proteinase Inhibitors pharmacology, E2F Transcription Factors, E2F1 Transcription Factor, Gene Expression physiology, Neurons enzymology, Oligopeptides pharmacology, RNA, Messenger analysis, Rats, Rats, Sprague-Dawley, Retinoblastoma-Binding Protein 1, Apoptosis physiology, Carrier Proteins, Caspases metabolism, Cell Cycle Proteins, DNA-Binding Proteins, Dopamine pharmacology, Neurons cytology, Transcription Factors genetics
- Abstract
The transcription factor E2F1 mRNA and protein levels increased in rat cortical neurons in response to dopamine (DA)- or 6-hydroxydopamine (OHDA)-evoked apoptosis. Increased E2F1 protein was detected in the nucleus of neurons by double fluorescent immunocytochemistry using antibodies to E2F1 and NeuN. DA and 6-OHDA induced caspase-3-mediated apoptosis of cortical neurons which was attenuated by the addition of antioxidants or caspase-3 inhibitors to the cultures. Antioxidants prevented DA-evoked neuronal apoptosis, and also attenuated the increase in E2F1 expression. These findings suggest that increased expression of the transcription factor E2F1 may serve as a death signal during DA-evoked neuronal apoptosis.
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- 2001
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12. Hyperglycemia enhances DNA fragmentation after transient cerebral ischemia.
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Li PA, Rasquinha I, He QP, Siesjö BK, Csiszár K, Boyd CD, and MacManus JP
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- Animals, Apoptosis, Caspase 3, Caspases metabolism, Corpus Striatum pathology, Cytochrome c Group metabolism, Dentate Gyrus pathology, Enzyme Activation, Hippocampus pathology, Hyperglycemia physiopathology, In Situ Nick-End Labeling, Ischemic Attack, Transient physiopathology, Male, Microscopy, Electron, Neocortex pathology, Neurons pathology, Rats, Rats, Wistar, Thalamus pathology, DNA Fragmentation, Hyperglycemia pathology, Ischemic Attack, Transient pathology
- Abstract
Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia (NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular weight DNA fragments were detected by PFGE, whereas low molecular weight DNA fragments were detected using LM-PCR techniques. The LM-PCR procedure was performed on DNA from test samples with blunt (without Klenow polymerase) and 3'-recessed ends (with Klenow polymerase). In addition, cytochrome c release and caspase-3 activation were studied by immunocytochemistry. Results show that HG causes cytochrome c release, activates caspase-3, and exacerbates DNA fragments induced by ischemia. Thus, in HG rats, but not in control or NGs, TUNEL-stained cells were found in the cingulate cortex, neocortex, thalamus, and dorsolateral crest of the striatum, where neuronal death was observed by conventional histopathology, and where both cytosolic cytochrome c and active caspase-3 were detected by confocal microscopy. In the neocortex, both blunt-ended and stagger-ended fragments were detected in HG, but not in NG rats. Electron microscopy (EM) analysis was performed in the cingulate cortex, where numerous TUNEL-positive neurons were observed. Although DNA fragmentation was detected by TUNEL staining and electrophoresis techniques, EM analysis failed to indicate apoptotic cell death. It is concluded that HG triggers a cell death pathway and exacerbates DNA fragmentation induced by ischemia.
- Published
- 2001
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13. Apoptosis after experimental stroke: fact or fashion?
- Author
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MacManus JP and Buchan AM
- Subjects
- Animals, Brain Ischemia metabolism, Brain Ischemia physiopathology, Caspases metabolism, Cell Nucleus metabolism, Cell Nucleus pathology, DNA Fragmentation physiology, Disease Models, Animal, Gene Expression Regulation physiology, Humans, Mitochondria metabolism, Necrosis, Stroke metabolism, Stroke physiopathology, Apoptosis genetics, Brain Ischemia genetics, Stroke genetics
- Abstract
This review examines the appearance of hallmarks of apoptosis following experimental stroke. The reviewed literature leaves no doubt that ischemic cell death in the brain is active, that is, requires energy; is gene directed, that is, requires new gene expression; and is capase-mediated, that is, uses apoptotic proteolytic machinery. However, sufficient differences to both classical necrosis and apoptosis exist which prevent easy mechanistic classification. It is concluded that ischemic cell death in the brain is neither necrosis nor apoptosis but is a chimera which appears on a continuum that has apoptosis and necrosis at the poles. The position on this continuum could be modulated by the intensity of the ischemic injury, the consequent availability of ATP and new protein synthesis, and both the age and context of the neuron in question. Thus the ischemic neuron may look necrotic but have actively died in an energy dependent manner with new gene expression and destruction via the apoptotic proteolytic machinery.
- Published
- 2000
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14. The transcription factor E2F1 modulates apoptosis of neurons.
- Author
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Hou ST, Callaghan D, Fournier MC, Hill I, Kang L, Massie B, Morley P, Murray C, Rasquinha I, Slack R, and MacManus JP
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- Adenoviridae genetics, Animals, Apoptosis drug effects, Caspase 3, Caspases metabolism, Cells, Cultured, Cerebral Cortex cytology, Cerebral Cortex embryology, DNA Fragmentation, E2F Transcription Factors, E2F1 Transcription Factor, Gene Expression, In Situ Nick-End Labeling, Neurons drug effects, Rats, Rats, Sprague-Dawley, Retinoblastoma-Binding Protein 1, Reverse Transcriptase Polymerase Chain Reaction, Staurosporine pharmacology, Transcription Factor DP1, Transcription Factors genetics, Transfection, Apoptosis physiology, Carrier Proteins, Cell Cycle Proteins, DNA-Binding Proteins, Neurons cytology, Transcription Factors physiology
- Abstract
The transcription factor E2F1 is known to mediate apoptosis in isolated quiescent and postmitotic cardiac myocytes, and its absence decreases the size of brain infarction following cerebral ischemia. To demonstrate directly that E2F1 modulates neuronal apoptosis, we used cultured cortical neurons to show a temporal association of the transcription and expression of E2F1 in neurons with increased neuronal apoptosis. Cortical neurons lacking E2F1 expression (derived from E2F1 -/- mice) were resistant to staurosporine-induced apoptosis as evidenced by the significantly lower caspase 3-like activity and a lesser number of cells with apoptotic morphology in comparison with cortical cultures derived from wild-type mice. Furthermore, overexpressing E2F1 alone using replication-deficient recombinant adenovirus was sufficient to cause neuronal cell death by apoptosis, as evidenced by the appearance of hallmarks of apoptosis, such as the threefold increase in caspase 3-like activity and increased laddered DNA fragmentation, in situ endlabeled DNA fragmentation, and numbers of neuronal cells with punctate nuclei. Taken together, we conclude that E2F1 plays a key role in modulating neuronal apoptosis.
- Published
- 2000
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15. Despite the internucleosomal cleavage of DNA, reactive oxygen species do not produce other markers of apoptosis in cultured neurons.
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Hill IE, Murray C, Richard J, Rasquinha I, and MacManus JP
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- Animals, Antioxidants pharmacology, Apoptosis drug effects, Biomarkers, Caspases metabolism, Cell Nucleus pathology, Cholinesterase Inhibitors pharmacology, Comet Assay, Cycloheximide pharmacology, Enzyme Inhibitors pharmacology, Female, Free Radical Scavengers pharmacology, Glutamic Acid pharmacology, Metalloporphyrins pharmacology, Molsidomine analogs & derivatives, Molsidomine pharmacology, Nerve Degeneration chemically induced, Nerve Degeneration metabolism, Nerve Tissue Proteins metabolism, Neurons enzymology, Oxidation-Reduction, Phenanthrolines pharmacology, Pregnancy, Protein Synthesis Inhibitors pharmacology, Rats, Rats, Sprague-Dawley, Staurosporine pharmacology, Uric Acid pharmacology, Apoptosis physiology, DNA Fragmentation drug effects, Neurons cytology, Nucleosomes metabolism, Reactive Oxygen Species metabolism
- Abstract
The cell death induced by hydroxyl radicals generated by Cu-phenanthroline and peroxynitrite generated by 3-morpholinosydnonimine hydrochloride (SIN-1) in rat primary cortical neuronal cultures was compared with the apoptotic death induced by staurosporine and the necrotic death induced by glutamate. Both SIN-1 and Cu-phenanthroline were capable of generating internucleosomal cleavage of DNA-a hallmark of apoptosis. Other characteristics of this cell death, such as nuclear morphology by light microscopy; DNA breaks by single-cell gel electrophoresis; the effects of the apoptotic inhibitors cycloheximide, aurintricarboxylic acid, and tosyl-l-lysine chloromethyl ketone; the measurement of caspase activity; and the effects of antioxidants, were then analyzed. The conclusion from these hallmarks of apoptosis is that the cell death induced by these reactive oxygen species is not apoptosis., (Copyright 2000 Academic Press.)
- Published
- 2000
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16. Decreased brain infarct following focal ischemia in mice lacking the transcription factor E2F1.
- Author
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MacManus JP, Koch CJ, Jian M, Walker T, and Zurakowski B
- Subjects
- Animals, Brain Ischemia physiopathology, Cerebrovascular Circulation, E2F Transcription Factors, E2F1 Transcription Factor, Etanidazole analogs & derivatives, Etanidazole metabolism, Hydrocarbons, Fluorinated metabolism, Hypoxia metabolism, Hypoxia pathology, Immunohistochemistry, Mice, Mice, Inbred C57BL, Mice, Inbred Strains, Retinoblastoma-Binding Protein 1, Transcription Factor DP1, Transcription Factors metabolism, Brain Ischemia complications, Brain Ischemia metabolism, Carrier Proteins, Cell Cycle Proteins, Cerebral Infarction etiology, Cerebral Infarction pathology, DNA-Binding Proteins, Transcription Factors deficiency
- Abstract
E2F1+/- mice subjected to 2 h middle cerebral artery occlusion developed an infarct of 77.0 +/- 3.2 mm3 (mean +/- s.e.m., n = 15) in the ischemic hemisphere after 24 h reperfusion. A significantly smaller infarct of 58.8 +/- 4.8 mm3 (n = 15; p < 0.01) was found in E2F1-/- animals. Both deficient and normal mice had similar cerebral angioarchitecture and intra-ischemic decreases in regional blood flow. Similar areas of hypoxia in both groups of ischemic animals were demonstrated directly by immunohistochemical detection of nitroimidazole adducts. It was concluded that all animals received the same ischemic insult, yet the subsequent damage was different in the mutant mice. This is the first indication that the E2F1 gene plays a role in ischemic death of post-mitotic neurons.
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- 1999
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17. Cerebral ischemia produces laddered DNA fragments distinct from cardiac ischemia and archetypal apoptosis.
- Author
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MacManus JP, Fliss H, Preston E, Rasquinha I, and Tuor U
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- Animals, Apoptosis physiology, Cells, Cultured, Hypertension pathology, Male, Myocardial Ischemia pathology, Neurons pathology, Polymerase Chain Reaction, Rats, Rats, Inbred SHR, Rats, Sprague-Dawley, DNA Fragmentation, Hypertension genetics, Myocardial Ischemia genetics
- Abstract
The electrophoretic pattern of laddered DNA fragments which has been observed after cerebral ischemia is considered to indicate that neurons are dying by apoptosis. Herein the authors directly demonstrate using ligation-mediated polymerase chain reaction methods that 99% of the DNA fragments produced after either global or focal ischemia in adult rats, or produced after hypoxia-ischemia in neonatal rats, have staggered ends with a 3' recess of approximately 8 to 10 nucleotides. This is in contrast to archetypal apoptosis in which the DNA fragments are blunt ended as seen during developmental programmed cell death in dying cortical neurons, neuroblastoma, or thymic lymphocytes. It is not simply ischemia that results in staggered ends in DNA fragments because ischemic myocardium is similar to archetypal apoptosis with a vast majority of blunt-ended fragments. It is concluded that the endonucleases that produce this staggered fragmentation of the DNA backbone in ischemic brain must be different than those of classic or type I apoptosis.
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- 1999
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18. Activation of DNA-dependent protein kinase may play a role in apoptosis of human neuroblastoma cells.
- Author
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Chakravarthy BR, Walker T, Rasquinha I, Hill IE, and MacManus JP
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- Brain Neoplasms pathology, Caspases metabolism, DNA Fragmentation, DNA-Activated Protein Kinase, Enzyme Activation, Enzyme Inhibitors pharmacology, Humans, Hydrazones pharmacology, Immunoblotting, Neuroblastoma pathology, Nuclear Proteins, Polymerase Chain Reaction, Protein Serine-Threonine Kinases antagonists & inhibitors, Pyridones pharmacology, Staurosporine antagonists & inhibitors, Staurosporine toxicity, Tumor Cells, Cultured, Ultraviolet Rays, Apoptosis physiology, Brain Neoplasms enzymology, DNA-Binding Proteins, Neuroblastoma enzymology, Protein Serine-Threonine Kinases metabolism
- Abstract
Treating SH-SY5Y human neuroblastoma cells with 1 microM staurosporine resulted in a three- to fourfold higher DNA-dependent protein kinase (DNA-PK) activity compared with untreated cells. Time course studies revealed a biphasic effect of staurosporine on DNA-PK activity: an initial increase that peaked by 4 h and a rapid decline that reached approximately 5-10% that of untreated cells by 24 h of treatment. Staurosporine induced apoptosis in these cells as determined by the appearance of internucleosomal DNA fragmentation and punctate nuclear morphology. The maximal stimulation of DNA-PK activity preceded significant morphological changes that occurred between 4 and 8 h (40% of total number of cells) and increased with time, reaching 70% by 48 h. Staurosporine had no effect on caspase-1 activity but stimulated caspase-3 activity by 10-15-fold in a time-dependent manner, similar to morphological changes. Similar time-dependent changes in DNA-PK activity, morphology, and DNA fragmentation occurred when the cells were exposed to either 100 microM ceramide or UV radiation. In all these cases the increase in DNA-PK activity preceded the appearance of apoptotic markers, whereas the loss in activity was coincident with cell death. A cell-permeable inhibitor of DNA-PK, OK-1035, significantly reduced staurosporine-induced punctate nuclear morphology and DNA fragmentation. Collectively, these results suggest an intriguing possibility that activation of DNA-PK may be involved with the induction of apoptotic cell death.
- Published
- 1999
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19. Increased Mdm2 expression in rat brain after transient middle cerebral artery occlusion.
- Author
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Tu Y, Hou ST, Huang Z, Robertson GS, and MacManus JP
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- Animals, Blotting, Western, Cerebral Arteries, Cerebral Cortex metabolism, Constriction, Immunosorbent Techniques, In Situ Hybridization, Kinetics, Male, Neurons metabolism, Polymerase Chain Reaction, Proto-Oncogene Proteins c-mdm2, RNA, Messenger, Rats, Rats, Inbred SHR, Rats, Sprague-Dawley, Reperfusion, Brain metabolism, Gene Expression, Ischemic Attack, Transient metabolism, Nuclear Proteins, Proto-Oncogene Proteins genetics
- Abstract
The negative regulator of p53 transactivation, Mdm2, increased in the ischemic territory after 90 minutes of transient middle cerebral artery occlusion in spontaneously hypertensive rats compared to sham controls. Increased mdm2 mRNA was detected by semiquantitative reverse transcriptase polymerase chain reaction by 6 hours of reperfusion in the ipsilateral hemisphere. In situ hybridization histochemistry was used to localize increases in mdm2 mRNA which occurred in neurons of ischemic cortex and dorsolateral striatum. The number of labeled neurons increased by approximately 20-fold and the cells displayed five-fold increases of mdm2 mRNA in the cortex. Immunohistochemical staining for Mdm2 revealed that its mRNA was efficiently translated in the ischemic cortex, but not striatum, by 8 to 24 hours of reperfusion. Western blotting confirmed 30- to 40-fold increases in the full-length protein of 90 kd at these time points without evidence of alternative splicing. Because Mdm2 is a negative regulator of the apoptosis promoting activity of p53, increased expression of Mdm2 may be a component of a repair response in injured neurons, and supports Mdm2 being an indicator of DNA damage in the brain early after an ischemic insult in a similar way to Gadd45.
- Published
- 1998
- Full Text
- View/download PDF
20. Gene expression induced by cerebral ischemia: an apoptotic perspective.
- Author
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MacManus JP and Linnik MD
- Subjects
- Animals, Humans, Apoptosis genetics, Brain Ischemia genetics, Brain Ischemia pathology, Gene Expression Regulation
- Published
- 1997
- Full Text
- View/download PDF
21. Glutamate-treated rat cortical neuronal cultures die in a way different from the classical apoptosis induced by staurosporine.
- Author
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MacManus JP, Rasquinha I, Black MA, Laferriere NB, Monette R, Walker T, and Morley P
- Subjects
- Animals, Cells, Cultured, DNA Fragmentation drug effects, Dose-Response Relationship, Drug, Protein Kinase C antagonists & inhibitors, Rats, Time Factors, Apoptosis drug effects, Cell Death drug effects, Cerebral Cortex cytology, Enzyme Inhibitors pharmacology, Glutamates pharmacology, Neurons cytology, Staurosporine pharmacology
- Abstract
The alkaloid protein kinase inhibitor staurosporine induced neuronal cell death with both the morphological and the biochemical characteristics of apoptosis. The punctate chromatin associated with apoptosis with retention of plasma membrane integrity was observed in neurons identified by colocalization of NeuN staining. Such cells had DNA fragmentation visualized by in situ end-labeling which was seen as a laddered pattern upon gel electrophoresis. In contrast cells treated with glutamate did not exhibit either of these morphological or biochemical hallmarks of apoptosis. Instead a much smaller and more compact pyknotic structure was observed associated with smeared DNA fragmentation patterns. A confocal time-lapse study of the appearance of the morphological changes in individual nuclei after staurosporine treatment showed collapse into punctate chromatin over a period of 10 min. In contrast, the collapse into small pyknotic nuclei after glutamate treatment was at least 10 times slower. It is concluded that excitotoxicity produced by glutamate did not induce cell death by an apoptotic mechanism in cultured cortical neurons.
- Published
- 1997
- Full Text
- View/download PDF
22. Detection of higher-order 50- and 10-kbp DNA fragments before apoptotic internucleosomal cleavage after transient cerebral ischemia.
- Author
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MacManus JP, Rasquinha I, Tuor U, and Preston E
- Subjects
- Animals, Animals, Newborn, Brain pathology, Decerebrate State, Electrophoresis, Agar Gel, Electrophoresis, Gel, Pulsed-Field, Hypoxia complications, Ischemic Attack, Transient complications, Male, Necrosis, Rats, Rats, Sprague-Dawley, Rats, Wistar, Apoptosis physiology, DNA Fragmentation, Ischemic Attack, Transient genetics, Ischemic Attack, Transient pathology, Nucleosomes physiology
- Abstract
DNA fragments of 50 and 10 kbp were found in ischemic brain in adult rats following two-vessel occlusion or in neonates following hypoxia-ischemia. These higher-order fragments were detected before any laddered oligonucleosomal DNA fragmentation characteristic of apoptosis. Both the 50- and 10-kbp fragments were also detected during necrosis produced by decapitation, but these led to smeared smaller fragments, not laddered patterns. End-group analysis showed the presence of both 3'-OH and 5'-OH ends in both the 50- and 10-kbp fragments but the predominance of 3'-OH ends in the laddered fragments. A higher proportion of 5'-OH to 3'-OH ends was found in the 10-kbp fragment compared to the larger 50-kbp fragment, suggesting a selective degradation of the 50-kbp DNA fragment to the laddered oligonucleosomal patterns. Overall, the mode of DNA fragmentation appeared different from that described in classic apoptosis of thymocytes.
- Published
- 1997
- Full Text
- View/download PDF
23. A comparison of cathepsin B processing and distribution during neuronal death in rats following global ischemia or decapitation necrosis.
- Author
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Hill IE, Preston E, Monette R, and MacManus JP
- Subjects
- Animals, Blotting, Western, Brain Ischemia pathology, Cell Death, Decerebrate State pathology, Immunohistochemistry, Male, Necrosis, Rats, Rats, Sprague-Dawley, Time Factors, Tissue Distribution, Brain Ischemia metabolism, Brain Ischemia physiopathology, Cathepsin B metabolism, Decerebrate State metabolism, Decerebrate State physiopathology, Neurons physiology, Protein Processing, Post-Translational
- Abstract
The objective of this study was to examine the possible role of the cysteine protease cathepsin B (E.C. 3.4.22.1) in the delayed neuronal death in rats subjected to the two-vessel occlusion model of global ischemia. Immunohistochemistry of the hippocampus showed an alteration in the distribution of cathepsin B in CA1 neurons from a lysosomal pattern to a more intense label redistributed into the cytoplasm. This change was not detected until the neurons had become morphologically altered with obvious shrinkage of the cytoplasmic region. Western blotting and enzyme activity measurements of subcellular fractions, including lysosomes and a cell soluble fraction, demonstrated that there was an overall decrease in cathepsin B activity at this time but an increase in the proenzyme form, particularly in the soluble fraction. This was found to be completely different from the marked loss of all forms of cathepsin B in necrotic neurons following decapitation.
- Published
- 1997
- Full Text
- View/download PDF
24. Apoptosis is restricted to the thalamus in thiamine-deficient rats.
- Author
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Matsushima K, MacManus JP, and Hakim AM
- Subjects
- Animals, Ataxia etiology, Ataxia pathology, Ataxia physiopathology, DNA Fragmentation, Male, Rats, Rats, Sprague-Dawley, Reference Values, Thalamus cytology, Thalamus physiopathology, Thiamine Deficiency physiopathology, Time Factors, Apoptosis, Thalamus pathology, Thiamine Deficiency pathology
- Abstract
Thiamine deficiency (TD) produces lesions in the thalamus, mamillary and medial geniculate nuclei, and inferior colliculus. To clarify the pathogenesis of these lesions, we examined the occurrence of hallmarks of apoptosis following TD in rat brain. Histological assessment showed apoptotic cells in the thalamus and medial geniculate nucleus but not in the inferior colliculus. We used terminal deoxynucleotidyl transferase-mediated deoxyuridine (dUPT)-biotin nick-end labelling (TUNEL) and gel electrophoresis to demonstrate that TD is associated with apoptotic cell death. In the thalamus, DNA fragmentation appeared from day 14 of deficiency and preceded the appearance of ataxia. The inferior colliculus and mamillary nucleus were without electrophoretic DNA fragments, and only rare TUNEL-positive labelling was observed. This model shows a rare combination of both apoptosis and necrosis in the same lesioned brain.
- Published
- 1997
25. Increases in DNA lesions and the DNA damage indicator Gadd45 following transient cerebral ischemia.
- Author
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Hou ST, Tu Y, Buchan AM, Huang Z, Preston E, Rasquinha I, Robertson GS, and MacManus JP
- Subjects
- Animals, Animals, Newborn, DNA Fragmentation, Genetic Markers, Intracellular Signaling Peptides and Proteins, Ischemic Attack, Transient metabolism, Male, Protein Biosynthesis, Rats, Rats, Inbred SHR, Rats, Sprague-Dawley, Rats, Wistar, GADD45 Proteins, DNA Damage, Ischemic Attack, Transient genetics, Proteins genetics, Proteins metabolism
- Abstract
Transient global or focal ischemia leads to the production of several types of lesions in the DNA backbone including alkali-labile sites, and both single-stranded (ss) and double-stranded (ds) breaks. The ds breaks result in high molecular weight fragments of 10-50 kbp that contain both 3'- and 5'-OH end-groups, suggesting that more than one endonuclease is involved. This lesioning of DNA is followed by the appearance of the damage-response indicator Gadd45 in the ischemic hemisphere following middle cerebral artery occlusion. By 6 h, gadd45 mRNA was shown to increase by semi-quantitative reverse transcriptase - polymerase chain reaction. In situ hybridization histochemistry indicated that these increases in gadd45 mRNA occurred in pyramidal neurons located on the edge of the infarcted cortex. Gadd45 immunostaining yielded similar findings with maximal protein staining detected at 18 h after occlusion. In neurons, in the infarct core with frank DNA fragmentation shown by in situ TdT-mediated dUTP-biotin nick end labeling (TUNEL) at 24 h, the Gadd45 immunostaining was not visible. Taken together, these findings suggest that Gadd45 responds to DNA damage following ischemia as part of a repair response mounted by brain cells attempting to survive the insult.
- Published
- 1997
26. Apoptotic human SH-SY5Y neuroblastoma cells have regularly spaced single strand DNA breaks and increased DNA-dependent protein kinase activity.
- Author
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MacManus JP, Rasquinha I, Walker T, and Chakravarthy B
- Subjects
- DNA analysis, DNA-Activated Protein Kinase, Electrophoresis, Agar Gel, Electrophoresis, Gel, Pulsed-Field, Humans, Nuclear Proteins, Tumor Cells, Cultured, Apoptosis physiology, DNA Damage physiology, DNA-Binding Proteins, Neuroblastoma pathology, Protein Serine-Threonine Kinases metabolism
- Abstract
SH-SY5Y human neuroblastoma cells died by apoptosis when treated with staurosporine or ceramide. The treated cells had both the nuclear morphology and patterns of DNA fragmentation which are characteristic of apoptosis. Higher order DNA fragments separable by pulse field gel electrophoresis were shown to contain regularly spaced single-strand nicks by producing a laddered pattern upon alkali treatment. Further evidence of DNA damage in treated cells was shown by increased activity of DNA-dependent protein kinase. This human cell model may prove useful in delineating the role of a cellular repair response to DNA damage prior to the irreversible steps of the cell death program.
- Published
- 1996
27. Differences in DNA fragmentation following transient cerebral or decapitation ischemia in rats.
- Author
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MacManus JP, Hill IE, Preston E, Rasquinha I, Walker T, and Buchan AM
- Subjects
- Animals, Brain metabolism, Brain pathology, Decerebrate State, Glial Fibrillary Acidic Protein metabolism, Ischemic Attack, Transient pathology, Male, Necrosis, Prosencephalon blood supply, Rats, Time Factors, DNA Damage, Ischemic Attack, Transient genetics
- Abstract
The time course of appearance of cells with DNA damage was studied in rats following transient severe forebrain ischemia. This DNA damage could be detected by in situ end-labeling on brain sections. The breaks in DNA appeared selectively by day 1 in the striatum and later in the CA1 region of the hippocampus. It was possible by double labeling to show that there was no DNA damage in astrocytes. The DNA breaks consisted of laddered DNA fragments indicative of an ordered apoptotic type of internucleosomal cleavage, which persisted without smearing for up to 7 days of reperfusion. In contrast, the DNA breaks following ischemia induced by decapitation were random and, after gel electrophoresis, consisted of smeared fragments of multiple sizes. There was some early regional cellular death, restricted to the dentate of the hippocampus, prior to the pannecrotic degeneration. It is concluded that transient forebrain ischemia leads to a type of neuronal destruction that is not random necrosis but that shares some component of the apoptotic cell death pathway.
- Published
- 1995
- Full Text
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28. Bifunctional fusion proteins consisting of a single-chain antibody and an engineered lanthanide-binding protein.
- Author
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MacKenzie CR, Clark ID, Evans SV, Hill IE, MacManus JP, Dubuc G, Bundle DR, Narang SA, Young NM, and Szabo AG
- Subjects
- Amino Acid Sequence, Base Sequence, Binding, Competitive, Calcium-Binding Proteins chemistry, Calcium-Binding Proteins immunology, Carrier Proteins metabolism, Cross-Linking Reagents chemistry, Cross-Linking Reagents metabolism, Enzyme-Linked Immunosorbent Assay, Immunoglobulin Fragments genetics, Immunoglobulin Fragments metabolism, Immunoglobulin Variable Region chemistry, Immunoglobulin Variable Region genetics, Luminescent Measurements, Models, Molecular, Molecular Sequence Data, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Terbium metabolism, Carrier Proteins chemical synthesis, Cross-Linking Reagents chemical synthesis, Immunoglobulin Fragments chemistry, Metals, Rare Earth metabolism, Protein Engineering methods, Recombinant Fusion Proteins chemical synthesis
- Abstract
The combination of an antibody fragment with a lanthanide chelating protein has desirable characteristics for fluorescence-based immunoassays and tumor radioimmunotherapy. As a model for this design, a fusion protein consisting of a single-chain antibody linked to an engineered version of oncomodulin, a protein with two Ca(2+)-binding motifs (the CD and EF loops), was produced by secretion from Escherichia coli in good yield. The single-chain antibody was specific for a Salmonella O-polysaccharide. The CD loop of oncomodulin had been redesigned to bind lanthanide ions with high affinity. The fusion protein was shown to have antigen-binding activity that was comparable to that of the unfused single-chain antibody, to bind Tb3+ with very high affinity and to give strong, sensitized Tb3+ luminescence via excitation of the tryptophan residue in the CD loop. A second fusion protein containing a 30-residue helix-loop-helix motif as the lanthanide-binding component was also prepared, but showed considerably lower solubility. Competition for Tb3+ binding by a series of metal chelators indicated that the affinities of the oncomodulin and 30 residue fusions for Tb3+ were approximately 10(11) M-1 and 10(7) M-1, respectively. Time-resolved lanthanide luminescence photography of electrophoresis gels demonstrated that the helix-loop-helix Ca(2+)-binding could be used to specifically visualize the scFv fragment.
- Published
- 1995
- Full Text
- View/download PDF
29. DNA fragmentation indicative of apoptosis following unilateral cerebral hypoxia-ischemia in the neonatal rat.
- Author
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Hill IE, MacManus JP, Rasquinha I, and Tuor UI
- Subjects
- Animals, Animals, Newborn, Brain Ischemia complications, Brain Ischemia metabolism, Hypoxia, Brain complications, Hypoxia, Brain metabolism, Neurons pathology, Rats, Rats, Wistar, Apoptosis genetics, Brain Ischemia pathology, DNA Damage, Functional Laterality physiology, Hypoxia, Brain pathology
- Abstract
DNA extracted from regional brain samples of hypoxic/ischemic neonatal rats showed internucleosomal cleavage indicative of apoptosis. Cells containing cleaved DNA were identified by in situ labelling in the cortex, hippocampus, striatum and thalamus of the ipsilateral hemisphere. When the effects of increasing the length of the hypoxia were examined, increases were seen in the amount of internucleosomally cleaved DNA and in the number of labelled cells.
- Published
- 1995
- Full Text
- View/download PDF
30. A protease assay using time-resolved lanthanide luminescence from an engineered calcium binding protein substrate.
- Author
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Clark ID, MacManus JP, and Szabo AG
- Subjects
- Animals, Cathepsin B analysis, Chymotrypsin analysis, HIV Protease analysis, Luminescent Measurements, Sensitivity and Specificity, Substrate Specificity, Subtilisins analysis, Time Factors, Calcium-Binding Proteins chemistry, Endopeptidases analysis, Metals, Rare Earth chemistry, Protein Engineering
- Abstract
Objectives: The objective of this work was to demonstrate the utility of luminescence from lanthanides bound to a mutant of the Ca2+ binding protein, oncomodulin, to monitor protease activity., Design and Methods: A mutant of oncomodulin with a cysteine residue at position 57 located in the CD binding loop was conjugated to a salicylic acid group. The luminescence of Tb3+ resulting from electronic energy transfer from the salicylic acid group was monitored using time resolved lanthanide luminescence in the presence of proteolytic enzymes., Results: Low detection limits for subtilisin (150 pg), chymotrypsin (2.5 ng), cathepsin B (3.5 ng), and HIV-1 protease (25 ng) were found., Conclusion: The simplicity of the assay coupled with its high level of sensitivity make it useful for the detection of protease at very low concentrations.
- Published
- 1995
- Full Text
- View/download PDF
31. An adherent cell model to study different stages of apoptosis.
- Author
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Desjardins LM and MacManus JP
- Subjects
- Adenocarcinoma, Cell Size, Colonic Neoplasms, DNA, Neoplasm metabolism, Humans, Teniposide pharmacology, Tumor Cells, Cultured, Apoptosis, Cell Adhesion drug effects
- Abstract
Apoptosis in the classical thymocyte model occurs very rapidly making it difficult to study the intermediate steps in the process. An alternative adherent cell model is characterized and proposed in this paper. HT29 cells treated with a teniposide were collected at various times for morphological and biochemical assessments. Large DNA breaks (450-500, 350-400, 100-200 kb) were observed in these cells between 6 and 24 h. The larger DNA breaks appeared initially and in progression such that the smaller DNA break of 100-200 kb became apparent by 24 h. These changes in DNA corresponded with an increase in cell diameter and a gradual rounding and detaching of cells from each other but not from the tissue culture plates. The smallest DNA break of 23-50 kb appeared at 48 h and persisted throughout the 96 h of incubation. DNA ladders of 180- to 200-bp oligomers were also observed between 48 and 96 h and these coincided with the presence of small floating cells. Changes in cell adherence after teniposide treatment have permitted the consistent isolation of cells in four distinct morphological and biochemical stages of apoptosis: (1) "preapoptotic," (2) "swelling," (3) "rounding," and (4) "floating." The main advantages of this adherent cell model are: (1) apoptosis occurs very slowly (minimum of 48 h) permitting the observation of progressive changes; (2) cells from four stages of apoptosis can be used to study the sequence of events of other biochemical and genetic factors involved in the process; and (3) extracellular matrix proteins are present in this model so their participation in apoptosis, if any, can be determined.
- Published
- 1995
- Full Text
- View/download PDF
32. Effects of metal ion binding on an oncomodulin mutant containing a novel calcium-binding loop.
- Author
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Clark ID, Bruckman AJ, Hogue CW, Macmanus JP, and Szabo AG
- Abstract
The Ca(2+)-binding protein oncomodulin was altered by cassette mutagenesis of the CD site (CDOM33) with a sequence that was derived by a consensus method using over 250 known Ca(2+)-binding loop sequences. This mutant was studied using time-resolved and steady-state fluorescence from the Trp residue included at position 7 of the loop (position 57 of the protein sequence). The fluorescence characteristics of this species in the absence and presence of metal ions were compared to those of a tetradecapeptide containing the loop and the single Trp mutant of oncomodulin, Y57W. The fluorescence properties of CDOM33 were quite different from the peptide, both in the apo form and in response to metal binding. The consensus CD loop in CDOM33 exhibited the characteristics of a Ca(2+)/Mg(2+) site in contrast to the Ca(2+) specificity of the wild-type CD loop. The Trp analogue, 5-hydroxytryptophan (5HW), was incorporated into both oncomodulin mutants to produce Y75(5HW) and 5HW-CDOM33. Results showed that this intrinsic probe was relatively insensitive to structural changes in the mutants upon metal binding compared to Trp itself.
- Published
- 1994
- Full Text
- View/download PDF
33. Detection of calcium binding proteins on polyacrylamide gels using time-resolved lanthanide luminescence photography.
- Author
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Hill IE, Hogue CW, Clark ID, MacManus JP, and Szabo AG
- Subjects
- Calcium-Binding Proteins genetics, Chelating Agents, Europium, Luminescent Measurements, Mutation genetics, Sensitivity and Specificity, Sodium Dodecyl Sulfate, Staining and Labeling methods, Terbium, Acrylic Resins, Calcium-Binding Proteins analysis, Metals, Rare Earth, Photography methods
- Abstract
Methods were developed for using the luminescent lanthanides Tb3+ and Eu3+ for the specific staining of calcium-binding proteins, as well as the nonspecific staining of proteins, on polyacrylamide gels. These methods involve equilibration of the gel after electrophoresis in solutions containing the appropriate lanthanide and a weak competitive chelating agent, such as N-(2-hydroxyethyl)iminodiacetic acid or nitrilotriacetic acid. This staining has the potential for complete reversibility using stronger chelating agents such as EDTA or diethylenetriaminepentaacetic acid, to allow for recovery of the protein. Specific staining produces an intense luminescent signal from those metal-binding proteins which have been modified either chemically or via site-directed mutagenesis. Gels were photographed using a time-resolved fluorescence camera system.
- Published
- 1994
- Full Text
- View/download PDF
34. DNA damage consistent with apoptosis in transient focal ischaemic neocortex.
- Author
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MacManus JP, Hill IE, Huang ZG, Rasquinha I, Xue D, and Buchan AM
- Subjects
- Animals, Male, Rats, Rats, Inbred SHR, Apoptosis genetics, Cerebral Cortex blood supply, DNA Damage, Ischemic Attack, Transient genetics
- Abstract
Transient focal ischaemia was produced in rat right neocortex by temporary middle cerebral artery occlusion. DNA damage was visualized in situ in cells of this right hemisphere but not in the contralateral hemisphere. The extracted damaged DNA exhibited laddered fragmentation which is indicative of apoptotic degradation. The amount of DNA damage was quantified by an end-labelling technique and shown to increase with the duration of the ischaemic insult. We conclude that the neurodegeneration resulting from focal ischaemia has an apoptotic component.
- Published
- 1994
- Full Text
- View/download PDF
35. Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain.
- Author
-
MacManus JP, Buchan AM, Hill IE, Rasquinha I, and Preston E
- Subjects
- Animals, Brain physiopathology, Brain Ischemia physiopathology, Ethidium, Histocytochemistry, Male, Rats, Rats, Sprague-Dawley, Apoptosis physiology, Brain cytology, Brain Ischemia metabolism, DNA Damage physiology
- Abstract
Laddered DNA indicative of apoptosis was observed in the CA1 layer of hippocampus and in dorsolateral striatum following a global cerebral ischemic insult produced by transient two vessel occlusion in rats. The extent of this DNA damage was proportional to the duration of the ischemic episode. Breaks in DNA were demonstrated in situ in sections from post-ischemic brain in neurons of the hippocampal CA1 which undergo selective neuronal death but not in other cell types. It is concluded that there is an apoptotic component to selective neuronal death following global ischemia in rat brain.
- Published
- 1993
- Full Text
- View/download PDF
36. A novel peptide designed for sensitization of terbium (III) luminescence.
- Author
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Clark ID, Hill I, Sikorska-Walker M, MacManus JP, and Szabo AG
- Subjects
- Amino Acid Sequence, Calcium-Binding Proteins chemical synthesis, Calcium-Binding Proteins metabolism, Iodoacetamide analogs & derivatives, Luminescence, Molecular Sequence Data, Peptide Fragments chemical synthesis, Peptide Fragments metabolism, Salicylates, Calcium-Binding Proteins chemistry, Peptide Fragments chemistry, Terbium metabolism
- Abstract
Several synthetic peptides, modelled from a Ca(2+)-binding loop of the EF-hand family of proteins, were prepared containing cysteine residues. The peptide, GDKNADGFICFEEL, was labelled covalently at the cysteine residue (loop position 9) with iodoacetamidosalicylic acid. This novel conjugate is a metal-binding loop containing a salicylic acid side chain that could not only chelate Tb3+ in conjunction with the other chelating groups in the sequence, but could also sensitize Tb3+ luminescence. The loop had a high Tb3+ affinity, with stoichiometric binding observed under experimental conditions. The luminescence from the Tb(3+)-peptide complex was more than 10-fold greater than the luminescence reported from a related peptide which contained Trp as the Tb3+ donor at loop position 7. This peptide has significant potential for use in lanthanide-based time-resolved luminescence immunoassays.
- Published
- 1993
- Full Text
- View/download PDF
37. Self-association of Ca(2+)-binding peptides induced by lanthanide ions: a fluorescence study.
- Author
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Clark ID, Brown CM, Sikorska-Walker M, MacManus JP, and Szabo AG
- Subjects
- Amino Acid Sequence, Calcium-Binding Proteins metabolism, Coumarins chemistry, Europium metabolism, Iodoacetamide analogs & derivatives, Iodoacetamide chemistry, Luminescent Measurements, Molecular Sequence Data, Pentetic Acid chemistry, Peptide Fragments chemistry, Peptide Fragments metabolism, Spectrometry, Fluorescence methods, Terbium metabolism, Calcium-Binding Proteins chemistry, Europium chemistry, Terbium chemistry
- Abstract
In this study a Ca(2+)-binding 14mer peptide was synthesized with the sequence GDKNADGCIEFEEL, allowing covalent attachment of sulfhydryl-reactive fluorescent molecules at position 7 of the 12-residue, metal-binding loop (underlined). This provided the opportunity to select donor molecules with suitable spectral characteristics for sensitized excitation of chelated terbium (Tb3+) or europium (Eu3+) ions. N-(1-Pyrene)-iodoacetamide and 7-diethylamino-3-((4'-iodoacetylamino)phenyl)-4-methylcoumarin were attached to the peptide and titrations carried out with terbium or europium stock solutions. It was possible to observe lanthanide ion binding to the loop in stoichiometric quantities, but maximal lanthanide luminescence was achieved with a large excess of lanthanide present, due to metal-induced peptide association. Obtaining maximal lanthanide luminescence is important in the development of systems for use in sensitive clinical diagnostic and time-resolved luminescence-based immunoassay applications.
- Published
- 1993
- Full Text
- View/download PDF
38. A study of sensitized lanthanide luminescence in an engineered calcium-binding protein.
- Author
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Clark ID, MacManus JP, Banville D, and Szabo AG
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Calcium-Binding Proteins genetics, Europium, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Engineering, Protein Structure, Secondary, Spectrophotometry, Terbium, Calcium-Binding Proteins chemistry, Luminescent Measurements, Metals, Rare Earth
- Abstract
In this study, the CD loop of the Ca(2+)-binding protein oncomodulin was replaced by a high-affinity, metal-binding sequence that was found to reverse the order of fill of the two sites in the protein. A cysteine was included at position 7 of this sequence, i.e., DKNADGCIEFEE. The cysteine allowed covalent attachment of chromophores to the loop that could subsequently be tested for their ability to sensitize the luminescence of Tb3+ or Eu3+ bound in the loop. 7-Diethylamino-3-((4'-iodoacetylamino)phenyl)-4-methylcoumarin was the most efficient Eu3+ sensitizer studied, consistent with a mechanism of energy transfer that involves the triplet state of the donor. 4-Iodoacetamidosalicylic acid was the most efficient Tb3+ donor tested. Levels of lanthanide ion and labeled C3 as low as 5 x 10(-10) mol/liter could be detected. This protein chelator system has potential to be a useful, flexible complement to the organic chelators currently used in lanthanide-based, time-resolved luminescence immunoassays.
- Published
- 1993
- Full Text
- View/download PDF
39. A new intrinsic fluorescent probe for proteins. Biosynthetic incorporation of 5-hydroxytryptophan into oncomodulin.
- Author
-
Hogue CW, Rasquinha I, Szabo AG, and MacManus JP
- Subjects
- Anisotropy, Calcium-Binding Proteins immunology, Epitopes, Macromolecular Substances, Recombinant Proteins biosynthesis, Spectrometry, Fluorescence, Spectrophotometry, Ultraviolet, 5-Hydroxytryptophan metabolism, Calcium-Binding Proteins metabolism, Fluorescent Dyes analysis, Fluorescent Dyes metabolism
- Abstract
The tryptophan analog, 5-hydroxytryptophan (5HW), has a significant absorbance between 310-320 nm, which allows it to act as an exclusive fluorescence probe in protein mixtures containing a large number of tryptophan residues. Here for the first time a method is reported for the biosynthetic incorporation of 5HW into an expressed protein, the Y57W mutant of the Ca2+ binding protein, oncomodulin. Fluorescence anisotropy and time-resolved fluorescence decay measurements of the interaction between anti-oncomodulin antibodies and the 5HW-incorporated oncomodulin conveniently provide evidence of complex formation and epitope identification that could not be obtained with the natural amino acid. This report demonstrates the significant potential for the use of 5HW as an intrinsic probe in the study of structure and dynamics of protein-protein interactions.
- Published
- 1992
- Full Text
- View/download PDF
40. Comparison of terbium (III) luminescence enhancement in mutants of EF hand calcium binding proteins.
- Author
-
Hogue CW, MacManus JP, Banville D, and Szabo AG
- Subjects
- Amino Acid Sequence, Calcium-Binding Proteins metabolism, Calmodulin metabolism, Cations, Luminescent Measurements, Molecular Sequence Data, Parvalbumins metabolism, Spectrometry, Fluorescence, Calcium-Binding Proteins genetics, Calmodulin genetics, Mutation, Parvalbumins genetics, Terbium chemistry
- Abstract
The luminescent isomorphous Ca2+ analogue, Tb3+, can be bound in the 12-amino acid metal binding sites of proteins of the EF hand family, and its luminescence can be enhanced by energy transfer from a nearby aromatic amino acid. Tb3+ can be used as a sensitive luminescent probe of the structure and function of these proteins. The effect of changing the molecular environment around Tb3+ on its luminescence was studied using native Cod III parvalbumin and site-directed mutants of both oncomodulin and calmodulin. Titrations of these proteins showed stoichiometries of fill corresponding to the number of Ca2+ binding loops present. Tryptophan in binding loop position 7 best enhanced Tb3+ luminescence in the oncomodulin mutant Y57W, as well as VU-9 (F99W) and VU-32 (T26W) calmodulin. Excitation spectra of Y57F, F102W, Y65W oncomodulin, and Cod III parvalbumin revealed that the principal Tb3+ luminescence donor residues were phenylalanine or tyrosine located in position 7 of a loop, despite the presence of other nearby donors, including tryptophan. Spectra also revealed conformational differences between the Ca2+- and Tb(3+)-bound forms. An alternate binding loop, based on Tb3+ binding to model peptides, was inserted into the CD loop of oncomodulin by cassette mutagenesis. The order of fill of Tb3+ in this protein reversed, with the mutated loop binding Tb3+ first. This indicates a much higher affinity for the consensus-based mutant loop. The mutant loop inserted into oncomodulin had 32 times more Tb3+ luminescence than the identical synthetic peptide, despite having the same donor tryptophan and metal binding ligands. In this paper, a ranking of sensitivity of luminescence of bound Tb3+ is made among this subset of calcium binding proteins. This ranking is interpreted in light of the structural differences affecting Tb3+ luminescence enhancement intensity. The mechanism of energy transfer from an aromatic amino acid to Tb3+ is consistent with a short-range process involving the donor triplet state as described by Dexter (Dexter, D. L. (1953) J. Chem. Phys. 21, 836). This cautions against the use of the Förster equation in approximating distances in these systems.
- Published
- 1992
41. Differentiation of HT-29 human colonic adenocarcinoma cells correlates with increased expression of mitochondrial RNA: effects of trehalose on cell growth and maturation.
- Author
-
Lu X, Walker T, MacManus JP, and Seligy VL
- Subjects
- Adenocarcinoma genetics, Base Sequence, Cell Differentiation, Cell Division drug effects, Colonic Neoplasms genetics, DNA, Mitochondrial analysis, Humans, Molecular Sequence Data, NADH Dehydrogenase genetics, RNA, Mitochondrial, Transcription, Genetic, Tumor Cells, Cultured, Adenocarcinoma pathology, Colonic Neoplasms pathology, RNA analysis, RNA, Neoplasm analysis, Trehalose pharmacology
- Abstract
The HT-29 human adenocarcinoma cell line has been used extensively in the study of colonic cell differentiation and colon cancer. We report here that substitution of glucose with trehalose (alpha-D-glucopyranosyl-alpha-D-glucopyranoside) depresses growth and promotes mucin-producing, goblet-like maturation of HT-29. An initial characterization of this process was made by analyzing several cDNA clones whose RNA templates were differentially expressed at elevated levels in cells grown in trehalose-containing medium. Seven of the 9 clones examined corresponded to 6 mitochondrial genes whose expression levels, relative to those from glucose-grown cells, ranged from approximately 3-fold for 16S rRNA to 8-23-fold for NADH dehydrogenase subunit 4. On the other hand, levels of mitochondrial DNA copy, measured by using NADH dehydrogenase subunit 4 cDNA as probe, were shown to be unaffected by trehalose treatment. Elevation of cellular NADH dehydrogenase subunit 4 RNA in HT-29 cultures grown in medium containing different components (sodium butyrate, galactose, no-sugar, glucose, cellobiose) generally correlated with depressed growth levels and specifically with increased numbers of mucin-producing cells present. Like butyrate, the sugar, trehalose, is an effective inducer of HT-29 differentiation, and may prove useful as a dietary therapeutic, and as a probe for elucidating mitochondrial involvement in colonic cell differentiation and transformation.
- Published
- 1992
42. Metal-induced changes in the fluorescence properties of tyrosine and tryptophan site-specific mutants of oncomodulin.
- Author
-
Hutnik CM, MacManus JP, Banville D, and Szabo AG
- Subjects
- Calcium metabolism, Calcium-Binding Proteins genetics, Calcium-Binding Proteins metabolism, Fluorescence, Magnesium metabolism, Metals chemistry, Models, Molecular, Mutagenesis, Site-Directed, Neoplasm Proteins genetics, Neoplasm Proteins metabolism, Phenylalanine chemistry, Protein Conformation, Spectrum Analysis, Calcium-Binding Proteins chemistry, Neoplasm Proteins chemistry, Tryptophan chemistry, Tyrosine chemistry
- Abstract
Oncomodulin is a 108-residue, oncodevelopmental protein containing two calcium-binding sites identified as the CD- and EF-loops. The protein contains no tryptophan and only two tyrosine residues, one which is a calcium ligand in the CD-loop (Tyr-57) and one which lies in the flanking D-helix of this loop (Tyr-65). Site-specific mutagenesis was performed to yield five mutants, two with phenylalanine substituted for tyrosine in positions 57 and 65 and three with tryptophan substituted into position 57 in the CD-loop, position 65 in the D-helix, and position 96 in the EF-loop. The single Tyr-containing mutants demonstrated that position 57 was perturbed to a significantly greater extent than position 65 upon calcium binding. Although both tyrosine residues responded to decalcification, the fluorescence intensity changes were in opposite directions, with the more dominant Tyr-57 accounting for the majority of the intrinsic fluorescence observed in native oncomodulin. The substitution of tryptophan for each tyrosyl residue revealed that in both positions the tryptophan resided in polar, conformationally heterogeneous environments. The environment of Trp-57 was affected by Ca2+ binding to a much greater extent compared to that of Trp-65. Only 1 equiv of Ca2+ was required to produce greater than 70% of the Trp fluorescence changes in positions 57 and 65, indicating that Ca2+ binding to the higher affinity EF-loop had a pronounced effect on the protein structure.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1991
- Full Text
- View/download PDF
43. Structure of oncomodulin refined at 1.85 A resolution. An example of extensive molecular aggregation via Ca2+.
- Author
-
Ahmed FR, Przybylska M, Rose DR, Birnbaum GI, Pippy ME, and MacManus JP
- Subjects
- Amino Acid Sequence, Animals, Calcium-Binding Proteins chemistry, Calcium-Binding Proteins genetics, Computer Graphics, Macromolecular Substances, Models, Molecular, Molecular Sequence Data, Parvalbumins chemistry, Parvalbumins genetics, Protein Conformation, Rats, Sequence Homology, Nucleic Acid, X-Ray Diffraction, Calcium metabolism, Calcium-Binding Proteins metabolism, Neoplasm Proteins metabolism
- Abstract
The crystal structure of oncomodulin, a 12,000 Mr protein isolated from rat tumours, has been determined by molecular replacement using the carp parvalbumin structure as a starting model. Refinement was performed by cycles of molecular fitting and restrained least-squares, using area-detector intensity data to 1.85 A resolution. For the 5770 reflections in the range 6.0 to 1.85 A, which were used in the refinement, the crystallographic R-factor is 0.166. The refined model includes residues 2 to 108, three Ca2+ and 87 water molecules per oncomodulin molecule. The oncomodulin backbone is closely related to that of parvalbumin; however, some differences are found after a least-squares fit of the two backbones, with root-mean-square (r.m.s.) deviations of 1 to 2 A in residues 2 to 6, 59 to 61 of the CD loop, 87, 90 and 108. The overall r.m.s. deviation of the backbone residues 5 to 108 is 0.62 A. Each of the two Ca2+ atoms that are bound to the CD and EF loops is co-ordinated to seven oxygen atoms, including one water molecule. The third Ca2+ is also seven-co-ordinated, to five oxygen atoms belonging to three different oncomodulin molecules and to two water molecules which form hydrogen bonds to a fourth oncomodulin; thus, this intermolecular Ca2+ and its equivalents interlink the molecules into zigzag layers normal to the b axis with a spacing of b/2 or 32.14 A. No such extensive molecular aggregation has been reported for any of the related Ca-binding regulatory proteins of the troponin-C family studied thus far. The Ca-O distances in all three polyhedra are in the range 2.07 A to 2.64 A, indicating tightly bound Ca polyhedra.
- Published
- 1990
- Full Text
- View/download PDF
44. A calcium-specific conformational response of parvalbumin.
- Author
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Hutnik CM, MacManus JP, and Szabo AG
- Subjects
- Amino Acid Sequence, Animals, Fishes, Kinetics, Models, Molecular, Molecular Sequence Data, Protein Conformation drug effects, Spectrometry, Fluorescence, Spectrophotometry, Atomic, Tryptophan, X-Ray Diffraction, Calcium pharmacology, Parvalbumins chemistry
- Abstract
The single tryptophan containing isotype III parvalbumin from codfish (Gadus callarius) was purified by a modified procedure and was shown to be homogeneous by a number of biochemical techniques. Sequence analysis established the location of the single tryptophan in position 102 of the 108 amino acid primary sequence. Atomic absorption spectroscopy showed that trichloroacetic acid (TCA) precipitation was more effective in parvalbumin decalcification compared to the more commonly used method of EGTA treatment. Magnesium induced steady-state fluorescence spectral changes of the EGTA-treated, but not the TCA-treated, parvalbumin. Steady-state fluorescence and circular dichroism spectra showed that calcium, but not magnesium, induced a conformational response in the TCA-treated protein. The fluorescence decay of the calcium-loaded native (holo) cod III parvalbumin was best described by two decay time components. By contrast, three lifetime components were necessary to describe the fluorescence decay of the metal-free (apo) protein. The decay-associated spectra of each temporal component were obtained. Collectively, these results demonstrate that it is possible for a parvalbumin to display a calcium-specific response.
- Published
- 1990
- Full Text
- View/download PDF
45. Comparison of metal ion-induced conformational changes in parvalbumin and oncomodulin as probed by the intrinsic fluorescence of tryptophan 102.
- Author
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Hutnik CM, MacManus JP, Banville D, and Szabo AG
- Subjects
- Amino Acid Sequence, Animals, Apoproteins metabolism, Calcium-Binding Proteins genetics, Fishes, Models, Molecular, Molecular Sequence Data, Mutation, Sequence Homology, Nucleic Acid, Spectrometry, Fluorescence methods, Calcium pharmacology, Calcium-Binding Proteins metabolism, Magnesium pharmacology, Muscle Proteins metabolism, Neoplasm Proteins metabolism, Parvalbumins metabolism, Tryptophan
- Abstract
The calcium-induced conformational changes of the 108-amino acid residue proteins, cod III parvalbumin and oncomodulin, were compared using tryptophan as a sensitive spectroscopic probe. As native oncomodulin is devoid of tryptophan, site-specific mutagenesis was performed to create a mutant protein in which tryptophan was placed in the identical position (residue 102) as the single tryptophan residue in cod III parvalbumin. The results showed that in the region probed by tryptophan-102, cod III parvalbumin experienced significantly greater changes in conformation upon decalcification compared to the oncomodulin mutant, F102W. Addition of 1 eq of Ca2+ produced greater than 90% of the total fluorescence response in F102W, while in cod III parvalbumin, only 74% of the total was observed. Cod III parvalbumin displayed a negligible response upon Mg2+ addition. In contrast, F102W did respond to Mg2+, but the response was considerably less when compared to Ca2+ addition. Time-resolved fluorescence showed that the tryptophan in both proteins existed in at least two conformational states in the presence of Ca2+ and at least three conformational states in its absence. Comparison with quantum yield measurements indicated that the local electronic environment of the tryptophan was significantly different in the two proteins. Collectively, these results demonstrate that both cod III parvalbumin and oncomodulin undergo Ca2(+)-specific conformational changes. However, oncomodulin is distinct from cod III parvalbumin in terms of the electronic environment of the hydrophobic core, the magnitude of the Ca2(+)-induced conformational changes, and the number of calcium ions required to modulate the major conformational changes.
- Published
- 1990
46. Terbium luminescence in synthetic peptide loops from calcium-binding proteins with different energy donors.
- Author
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MacManus JP, Hogue CW, Marsden BJ, Sikorska M, and Szabo AG
- Subjects
- Amino Acid Sequence, Calcium-Binding Proteins chemical synthesis, Energy Transfer, Luminescent Measurements, Molecular Sequence Data, Peptides isolation & purification, Spectrometry, Fluorescence, Structure-Activity Relationship, Terbium, Calcium-Binding Proteins metabolism, Peptides chemical synthesis
- Abstract
Fourteen 14-mer peptides corresponding to a consensus sequence of metal-binding loops from proteins of the calmodulin family were synthesized. The effect of varying both the position in the binding loop, and the type of aromatic side chains as energy donors for enhancement of terbium luminescence, was studied. It was concluded that tryptophan in loop position 7 gave optimal luminescence enhancement, and that the additional inclusion of a tyrosine in the loop at positions 2 or 4 could further boost emission from the bound terbium. In all further cases energy transfer from aromatic residues at positions other than 7 was markedly less efficient. These results suggest that the peptides assume a configuration which allows a hexadentate ligand structure around the bound terbium ion. This is consistent with a Dexter-type electron exchange model of energy transfer.
- Published
- 1990
47. Calcium- and magnesium-binding properties of oncomodulin. Direct binding studies and microcalorimetry.
- Author
-
Cox JA, Milos M, and MacManus JP
- Subjects
- Animals, Calcium-Binding Proteins genetics, Calorimetry, Dialysis, Edetic Acid pharmacology, Egtazic Acid pharmacology, Escherichia coli genetics, Kinetics, Parvalbumins metabolism, Protein Binding, Recombinant Proteins metabolism, Calcium metabolism, Calcium-Binding Proteins metabolism, Magnesium metabolism, Neoplasm Proteins metabolism
- Abstract
Ca2+ binding to the wild type recombinant oncomodulin was studied by equilibrium flow dialysis in the absence and presence of 1, 2, and 10 mM Mg2+. Direct Mg2(+)-binding experiments were carried out by the Hummel-Dryer gel filtration technique. These studies revealed that in the absence of Mg2+ oncomodulin binds two Ca2+ with KCa = 2.2 x 10(7) and 1.7 x 10(6) M-1, respectively. In the absence of Ca2+ the protein binds only one Mg2+ with KMg = 4.0 x 10(3) M-1.Mg2+ antagonizes Ca2+ binding at the high affinity site according to the rule of direct competition. Ca2+ binding to the low affinity site is only slightly affected by Mg2+, so that in the presence of 2-3 mM Mg2+ the two sites have apparently an equal affinity for Ca2+. Microcalorimetry showed that, in spite of the different affinities of the two Ca2(+)-binding sites, delta H0 for the binding of each Ca2+ is identical and exothermic for -18.9 kJ/site. It follows that the entropy gain upon binding of Ca2+ is +77.1 J K-1 site-1 for the high affinity Ca2(+)-Mg2+ site and +56.0 J K-1 site-1 for the low affinity Ca2(+)-specific site. Mg2+ binding is endothermic for +13 kJ/site with an entropy change of +111 J K-1 site-1. The thermodynamic characteristics of the Ca2(+)-Mg2+ site resemble most those of site II (the so-called EF domain) of toad alpha-parvalbumin. The characteristics of Ca2+ binding to the specific site (likely the CD domain) are different from those of the Ca2+ specific sites in troponin C and in calmodulin and suggest that in oncomodulin hydrophobic forces do not play a predominant role in the binding process at the specific site.
- Published
- 1990
48. Inhibition of glutathione reductase by oncomodulin.
- Author
-
Palmer EJ, MacManus JP, and Mutus B
- Subjects
- Animals, Calcium-Binding Proteins isolation & purification, Cattle, Cell Line, Chromatography, Affinity, Glutathione analogs & derivatives, Glutathione metabolism, Glutathione Disulfide, Glutathione Reductase isolation & purification, Intestinal Mucosa enzymology, Kinetics, Liver enzymology, Liver Neoplasms, Experimental, Rats, Calcium-Binding Proteins pharmacology, Glutathione Reductase antagonists & inhibitors, Neoplasm Proteins pharmacology
- Abstract
Evidence for a specific interaction between oncomodulin and glutathione reductase is presented. Glutathione reductase (EC 1.6.4.2) isolated from either the bovine intestinal mucosa or the rat liver was bound in a Ca2(+)-dependent manner to oncomodulin which was covalently attached to Sepharose. In addition, glutathione reductase was able to catalyze the reduction of the disulfide-linked dimer of oncomodulin. The interaction of these proteins could also be indirectly demonstrated by monitoring glutathione reductase activity since oncomodulin was shown to inhibit the enzyme in a dose-dependent manner with an apparent IC50 of approximately 5 microM. The kinetic analysis of the oncomodulin-dependent effects on glutathione reductase activity indicates that oncomodulin interacts at a site other than the active site as the oncomodulin-induced inhibition was of the noncompetitive type. The in vivo inhibition of glutathione reductase appears to be an oncomodulin-specific effect as closely related members of the troponin C superfamily such as rabbit (pI 5.5) or carp (pI 4.25) parvalbumins, as well as calmodulin, failed to affect the activity of this enzyme. The present in vitro study indicating that oncomodulin can regulate the activity of glutathione reductase could be very significant with respect to the elucidation of a physiological role for oncomodulin.
- Published
- 1990
- Full Text
- View/download PDF
49. Oncomodulin in normal and transformed cells.
- Author
-
MacManus JP, Brewer LM, and Banville D
- Subjects
- Animals, Calcium-Binding Proteins genetics, Cell Differentiation, Humans, RNA, Messenger genetics, Calcium-Binding Proteins metabolism, Cell Transformation, Neoplastic metabolism
- Published
- 1990
- Full Text
- View/download PDF
50. Stimulation of autophosphorylation of liver cell membrane proteins by calcium and partial hepatectomy.
- Author
-
MacManus JP and Whitfield JF
- Subjects
- Animals, Calmodulin pharmacology, Cell Division, Cyclic AMP pharmacology, Hepatectomy, Liver Neoplasms, Experimental metabolism, Phosphorylation, Protein Kinases metabolism, Rats, Calcium pharmacology, Liver metabolism, Liver Regeneration, Membrane Proteins metabolism
- Abstract
Partial hepatectomy in the rat stimulated the phosphorylation of three proteins (Mr 100,000, 48,000, and 35,000) in the plasma membranes of the proliferatively activated cells of the liver remnant. The autophosphorylation of these plasma membrane proteins began to rise about 8 hours after surgery, peaked at 14 hours, and then returned to the original low level by 24 hours. This increase in autophosphorylation was not evident in isolated plasma membranes from other proliferatively activated liver cells, such as those in fetal liver or hepatomas. The protein kinase responsible for the phosphorylation of the three membrane proteins was activated by calcium but appeared both cyclic-AMP- and calmodulin-independent.
- Published
- 1981
- Full Text
- View/download PDF
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