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1.
Water Sci Technol ; 54(2): 269-75, 2006.
Article in English | MEDLINE | ID: mdl-16939112

ABSTRACT

This paper presents the behaviour of a full-scale expanded bed reactor (160 m3) with overlaid anaerobic and aerobic zones used for municipal wastewater treatment. The research was carried out in two experimental steps: anaerobic and anaerobic-aerobic conditions, and the experimental results presented in this paper refer to four months of reactor operation. In the anaerobic condition, after inoculation and 60 days of operation, the reactor treating 3.40 kg CODm(-3)d(-1) for thetaH of 2.69 h, reached mean removal efficiencies of 76% for BOD, 72% for COD, and 80% for TSS, when the effluent presented mean values of 225 mg.L(-1) of COD, 98 mg.L(-1) of BOD and 35 mg.L(-1) of TSS. Under these conditions, for nitrogen loading of 0.27 kgN.m(-3)d(-1), the reactor generated an effluent with mean N-org. of 8 mg.L(-1) and N-ammon. of 37 mg.L(-1), demonstrating high potential of ammonification. For the anaerobic-aerobic condition (118th day) the system was operated with thetaH of 5.38 h presented mean removal efficiencies of 84% for BOD, 79% for COD, 76% for TSS, and 30% for TKN. The reactor's operation time was less than two months, which was not long enough to reach nitrification. Regarding the obtained results, this research confirmed that this reactor is configured as a flexible and adequate alternative for the treatment of sewage, requiring relatively small area and only thetaH of 10 h that can be adjusted to the local circumstances.


Subject(s)
Environmental Restoration and Remediation , Aerobiosis , Anaerobiosis
2.
Neuroscience ; 129(3): 615-28, 2004.
Article in English | MEDLINE | ID: mdl-15541883

ABSTRACT

Studies conducted in cell culture indicate that the gamma-secretase involved in amyloid beta-formation and Notch signaling is a multisubunit aspartic protease. Little is known, however, of the structure, function, or localization of gamma-secretase in the adult brain, or possible effects of familial Alzheimer's disease (FAD)-causing mutations on the brain protease. We report here that mouse brain contains a complex composed of gamma-secretase subunits presenilin-1 N-terminal fragment, presenilin-1 C-terminal fragment, Nicastrin, Aph-1a and Pen-2. A homozygous FAD-linked Presenilin-1 knock-in mutation does not alter relative subunit levels. Immunocytochemical localization of gamma-secretase subunits revealed overlapping but distinct regional and subcellular distributions. All subunits are expressed throughout the neuraxis predominantly in neurons, and are present in axons. Their distributions and levels of expression are unaffected by mutant presenilin-1. In a presenilin-1/amyloid precursor protein double knock-in mouse, subunits are associated with plaques, but are expressed at similar levels in amyloid-rich and -poor regions. gamma-Secretase subunits are distributed much more extensively than circumscribed amyloid deposits, suggesting the importance of other factors for localized amyloid deposition. These results indicate a widespread neuronal function for gamma-secretase in the adult brain, and suggest the pathogenic mechanism of FAD-linked mutations does not involve alterations in the composition, expression or brain distribution of the protease. The subcellular localization of gamma-secretase subunits is consistent with a nerve terminal source for amyloid aggregates.


Subject(s)
Brain/enzymology , Endopeptidases/metabolism , Membrane Proteins/metabolism , Amyloid Precursor Protein Secretases , Amyloid beta-Peptides/metabolism , Animals , Aspartic Acid Endopeptidases , Axons/metabolism , Blotting, Western/methods , Brain/cytology , Brain/metabolism , Endopeptidases/genetics , Humans , Immunohistochemistry/methods , Membrane Glycoproteins/metabolism , Membrane Proteins/genetics , Mice , Mice, Mutant Strains , Neurons/cytology , Neurons/metabolism , Peptide Hydrolases , Presenilin-1 , Protein Subunits/metabolism
3.
Water Sci Technol ; 49(11-12): 311-8, 2004.
Article in English | MEDLINE | ID: mdl-15303756

ABSTRACT

The effect of the filling stage on the behavior of a mechanically stirred anaerobic sequencing batch reactor containing biomass immobilized on 1 cm polyurethane foam cubes was investigated. The reactor was made of acrylic with a capacity of 6.3 L, treating per cycle 2.5 L synthetic low-strength wastewater with a concentration of 500 mgCOD/L, at 30+/-1 degrees C. Eight-hour cycles (tC) and agitation of 500 rpm were utilized. At the beginning of each cycle 60% of the wastewater volume was treated, sufficient to completely cover the bed. The remaining volume was added at different fill times (tF) of 10, 120, 240, 260 and 480 min. The results obtained showed that ratios of tF/tC < or = 0.5 enabled organic matter removal higher than 75% and 70% for filtered and non-filtered samples, respectively. Ratios of tF/tC > 0.5, despite operation stability, resulted in loss of efficiency and formation of viscous material, similar to extra-cellular polymeric substances.


Subject(s)
Bioreactors , Waste Disposal, Fluid/methods , Bacteria, Anaerobic/physiology , Biomass , Time Factors , Water Movements
4.
J Biol Chem ; 276(48): 44736-43, 2001 Nov 30.
Article in English | MEDLINE | ID: mdl-11574534

ABSTRACT

Endoplasmic reticulum (ER) stress elicits protective responses of chaperone induction and translational suppression and, when unimpeded, leads to caspase-mediated apoptosis. Alzheimer's disease-linked mutations in presenilin-1 (PS-1) reportedly impair ER stress-mediated protective responses and enhance vulnerability to degeneration. We used cleavage site-specific antibodies to characterize the cysteine protease activation responses of primary mouse cortical neurons to ER stress and evaluate the influence of a PS-1 knock-in mutation on these and other stress responses. Two different ER stressors lead to processing of the ER-resident protease procaspase-12, activation of calpain, caspase-3, and caspase-6, and degradation of ER and non-ER protein substrates. Immunocytochemical localization of activated caspase-3 and a cleaved substrate of caspase-6 confirms that caspase activation extends into the cytosol and nucleus. ER stress-induced proteolysis is unchanged in cortical neurons derived from the PS-1 P264L knock-in mouse. Furthermore, the PS-1 genotype does not influence stress-induced increases in chaperones Grp78/BiP and Grp94 or apoptotic neurodegeneration. A similar lack of effect of the PS-1 P264L mutation on the activation of caspases and induction of chaperones is observed in fibroblasts. Finally, the PS-1 knock-in mutation does not alter activation of the protein kinase PKR-like ER kinase (PERK), a trigger for stress-induced translational suppression. These data demonstrate that ER stress in cortical neurons leads to activation of several cysteine proteases within diverse neuronal compartments and indicate that Alzheimer's disease-linked PS-1 mutations do not invariably alter the proteolytic, chaperone induction, translational suppression, and apoptotic responses to ER stress.


Subject(s)
Cysteine Endopeptidases/metabolism , Endoplasmic Reticulum/enzymology , Heat-Shock Proteins , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutation , Alzheimer Disease/genetics , Animals , Apoptosis , Calpain/metabolism , Carrier Proteins/metabolism , Caspase 12 , Caspase 3 , Caspase 6 , Caspases/metabolism , Cells, Cultured , Cysteine Endopeptidases/genetics , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Chaperone BiP , Enzyme Activation , Glycosylation , HSP70 Heat-Shock Proteins/metabolism , Humans , Immunoblotting , Immunohistochemistry , Mice , Molecular Chaperones/metabolism , Neurons/metabolism , Presenilin-1 , Protein Biosynthesis , Protein Structure, Tertiary , Reverse Transcriptase Polymerase Chain Reaction , Stress, Physiological , Subcellular Fractions , eIF-2 Kinase/metabolism
5.
Exp Neurol ; 170(1): 27-35, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11421581

ABSTRACT

Activity of the Ca(2+)-dependent protease calpain is increased in neurons after global and focal brain ischemia, and may contribute to postischemic injury cascades. Understanding the time course and location of calpain activity in the post-ischemic brain is essential to establishing causality and optimizing therapeutic interventions. This study examined the temporal and spatial characteristics of brain calpain activity after transient forebrain ischemia (TFI) in rats. Male Long Evans rats underwent 10 min of normothermic TFI induced by bilateral carotid occlusion with hypovolemic hypotension (MABP 30 mm Hg). Brain calpain activity was examined between 1 and 72 h after reperfusion. Western blot analysis of regional brain homogenates demonstrated a bimodal pattern of calpain-mediated alpha-spectrin degradation in the hippocampus, cortex, and striatum with an initial increase at 1 h followed by a more prominent secondary increase at 36 h after reperfusion. Immunohistochemical analysis revealed that calpain activity was primarily localized to dendritic fields of selectively vulnerable neurons at one hour after reperfusion. Between 24 and 48 h after reperfusion neuronal calpain activity progressed from the dorsal to ventral striatum, medial to lateral CA1 hippocampus, and centripetally expanded from watershed foci in the cerebral cortex. This progression was associated with fragmentation of dendritic processes, calpain activation in the neuronal soma and subsequent neuronal degeneration. These observations demonstrate a clear association between calpain activation and subsequent delayed neuronal death and suggest broad therapeutic window for interventions aimed at preventing delayed intracellular Ca(2+) overload and pathologic calpain activation.


Subject(s)
Brain/enzymology , Calpain/metabolism , Ischemic Attack, Transient/enzymology , Animals , Blotting, Western , Brain/pathology , Cerebral Cortex/enzymology , Corpus Striatum/enzymology , Disease Models, Animal , Enzyme Activation , Hippocampus/enzymology , Immunohistochemistry , Ischemic Attack, Transient/pathology , Male , Neurons/pathology , Organ Specificity , Rats , Rats, Long-Evans , Spectrin/metabolism
6.
J Neurosci Res ; 64(4): 371-9, 2001 May 15.
Article in English | MEDLINE | ID: mdl-11340644

ABSTRACT

The myelin-deficient (MD) rat has a point mutation in its proteolipid protein (PLP) gene that causes severe dysmyelination and oligodendrocyte cell death. Using an in vitro model, we have shown that MD oligodendrocytes initially differentiate similarly to wild-type cells, expressing galactocerebroside, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin basic protein. However, at the time when PLP expression would normally begin, the MD oligodendrocytes die via an apoptotic pathway involving caspase activation. The active form of caspase-3 was detected, along with the cleavage products of poly-(ADP-ribose) polymerase (PARP) and spectrin, major targets of caspase-mediated proteolysis. A specific inhibitor of casapse-3, Ac-DEVD-CMK, reduced apoptosis in MD oligodendrocytes, but the rescued cells did not mature fully or express myelin-oligodendrocyte glycoprotein. These results suggest that mutant PLP affects not only cell death but also oligodendrocyte differentiation.


Subject(s)
Apoptosis/physiology , Caspases/metabolism , Myelin Proteolipid Protein/deficiency , Oligodendroglia/metabolism , Animals , Animals, Newborn , Apoptosis/drug effects , Caspase 3 , Caspase Inhibitors , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured , Culture Media/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Male , Myelin Proteolipid Protein/genetics , Oligodendroglia/drug effects , Point Mutation/genetics , Rats , Rats, Mutant Strains
7.
In Vivo ; 14(1): 265-8, 2000.
Article in English | MEDLINE | ID: mdl-10757085

ABSTRACT

The multicatalytic proteinase complex or proteasome possesses at least 4 distinct proteolytic activities. We have previously reported that the chymotrypsin-like activity of the rat natural killer cell proteasome may play a role in natural killer (NK) cell-mediated cytotoxicity or IL-2 activated NK (A-NK) cell-mediated cytotoxicity. Using a series of novel, Cephalon, Inc, synthetic proteasome inhibitors (CEP-1508, CEP-1612 and CEP-3117) which have been reported to be specific for the chymotrypsin-like activity of the proteasome, we have further investigated the possible role of the proteasome, with emphasis on the chymotryptic activity components, in cell-mediated cytotoxicity. We now report that these compounds can inhibit the rat NK proteasome in a dose dependent manner. Nevertheless, there is only a 50% inhibition of A-NK cell-mediated cytotoxicity. These results confirm and extend our previous results that the proteasome contributes, at least in part, to cell-mediated cytotoxicity. However, as anticipated, since multiple molecular pathways contribute to cell-mediated cytotoxicity, the proteasome contributes only partially to NK cell-mediated cytolytic reactivity. The exact role of the proteasome in NK cell-mediated killing, and whether single or multiple chymotryptic domains function directly or indirectly, remains to be fully determined.


Subject(s)
Cysteine Endopeptidases/drug effects , Lymphocytes/immunology , Multienzyme Complexes/drug effects , Animals , Binding Sites , Cell Survival/drug effects , Chymotrypsin/metabolism , Cysteine Endopeptidases/metabolism , Cytotoxicity, Immunologic/drug effects , Dipeptides/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Humans , Jurkat Cells , Killer Cells, Natural/cytology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Lymphocytes/cytology , Lymphocytes/drug effects , Male , Multienzyme Complexes/metabolism , Phthalimides/pharmacology , Proteasome Endopeptidase Complex , Rats , Rats, Inbred F344 , Tumor Cells, Cultured
8.
J Neurochem ; 74(4): 1646-55, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10737623

ABSTRACT

The pathophysiology of traumatic spinal cord injury (SCI) involves abnormal activation of the neutral cysteine protease calpain I (EC 3.4.22.17). In the present study we examined the effect of the calpain inhibitor CEP-4143 on cytoskeletal protection and neurological recovery after SCI in adult rats. Microinjection of 50 mM CEP-4143 into the T7 vertebral segment 10 min before a 35-g clip compression injury resulted in inhibition of calpain activation at 2 and 4 h postinjury, as determined by western blotting for calpain I-mediated spectrin degradation, and significantly attenuated the degradation of dephosphorylated NF200 neurofilament protein at 4 and 8 h postinjury. To examine the in vivo chronic neuroprotective effects of CEP-4143, animals underwent microinjection with saline or 50 mM CEP-4143 10 min before injury, followed by weekly blinded behavioral assessments for 6 weeks. Animals receiving CEP-4143 treatment showed significant improvement over saline-treated controls on the Basso Beattie Bresnahan locomotor rating scale (p < 0.02) and inclined plane test (p < 0.05). Counts of neurons in the red nucleus retrogradely labeled by fluoro-gold after introduction distal to the injury site were significantly higher in CEP-4143-treated animals. Finally, morphometric assessment of the injury site by computer-assisted image analysis revealed significant tissue preservation in CEP-4143-treated animals. We conclude that the calpain antagonist CEP-4143 exhibits biochemical, behavioral, and anatomical neuroprotection following traumatic SCI.


Subject(s)
Axons/metabolism , Calpain/antagonists & inhibitors , Cytoskeleton/enzymology , Enzyme Inhibitors/pharmacology , Spinal Cord Injuries/drug therapy , Stilbamidines , Animals , Axons/drug effects , Calpain/metabolism , Cell Count , Female , Fluorescent Dyes , Locomotion , Microinjections , Neurofilament Proteins/metabolism , Neurons/enzymology , Neurons/ultrastructure , Neuroprotective Agents/pharmacology , Phosphorylation , Rats , Rats, Wistar , Recovery of Function , Spinal Cord Injuries/enzymology , Spinal Cord Injuries/rehabilitation , Substrate Specificity
9.
J Neurosci ; 20(4): 1333-41, 2000 Feb 15.
Article in English | MEDLINE | ID: mdl-10662823

ABSTRACT

The selective degeneration of an axon, without the death of the parent neuron, can occur in response to injury, in a variety of metabolic, toxic, and inflammatory disorders, and during normal development. Recent evidence suggests that some forms of axon degeneration involve an active and regulated program of self-destruction rather than a passive "wasting away" and in this respect and others resemble apoptosis. Here we investigate whether selective axon degeneration depends on some of the molecular machinery that mediates apoptosis, namely, the caspase family of cysteine proteases. We focus on two models of selective axon degeneration: Wallerian degeneration of transected axons and localized axon degeneration induced by local deprivation of neurotrophin. We show that caspase-3 is not activated in the axon during either form of degeneration, although it is activated in the dying cell body of the same neurons. Moreover, caspase inhibitors do not inhibit or retard either form of axon degeneration, although they inhibit apoptosis of the same neurons. Finally, we cannot detect cleaved substrates of caspase-3 and its close relatives immunocytochemically or caspase activity biochemically in axons undergoing Wallerian degeneration. Our results suggest that a neuron contains at least two molecularly distinct self-destruction programs, one for caspase-dependent apoptosis and another for selective axon degeneration.


Subject(s)
Axons/physiology , Ganglia, Spinal/physiology , Nerve Degeneration/physiopathology , Nerve Growth Factors/physiology , Neurons/physiology , Optic Nerve/physiology , Retina/physiology , Sciatic Nerve/physiology , Wallerian Degeneration/physiopathology , Animals , Apoptosis , Axons/drug effects , Axons/pathology , Caspase 3 , Caspases/metabolism , Cycloheximide/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Enzyme Activation , Ganglia, Spinal/physiopathology , Mice , Mice, Inbred C57BL , Nerve Growth Factors/pharmacology , Optic Nerve/pathology , Optic Nerve/physiopathology , Organ Culture Techniques , Rats , Rats, Sprague-Dawley , Retina/drug effects , Sciatic Nerve/pathology , Sciatic Nerve/physiopathology
10.
J Neurosci ; 20(23): 8717-26, 2000 Dec 01.
Article in English | MEDLINE | ID: mdl-11102478

ABSTRACT

The pathogenic mechanism linking presenilin-1 (PS-1) gene mutations to familial Alzheimer's disease (FAD) is uncertain, but has been proposed to include increased neuronal sensitivity to degeneration and enhanced amyloidogenic processing of the beta-amyloid precursor protein (APP). We investigated this issue by using gene targeting with the Cre-lox system to introduce an FAD-linked P264L mutation into the endogenous mouse PS-1 gene, an approach that maintains normal regulatory controls over expression. Primary cortical neurons derived from PS-1 homozygous mutant knock-in mice exhibit basal neurodegeneration similar to their PS-1 wild-type counterparts. Staurosporine and Abeta1-42 induce apoptosis, and neither the dose dependence nor maximal extent of cell death is altered by the PS-1 knock-in mutation. Similarly, glutamate-induced neuronal necrosis is unaffected by the PS-1P264L mutation. The lack of effect of the PS-1P264L mutation is confirmed by measures of basal- and toxin-induced caspase and calpain activation, biochemical indices of apoptotic and necrotic signaling, respectively. To analyze the influence of the PS-1P264L knock-in mutation on APP processing and the development of AD-type neuropathology, we created mouse lines carrying mutations in both PS-1 and APP. In contrast to the lack of effect on neuronal vulnerability, cortical neurons cultured from PS-1P264L homozygous mutant mice secrete Abeta42 at an increased rate, whereas secretion of Abeta40 is reduced. Moreover, the PS-1 knock-in mutation selectively increases Abeta42 levels in the mouse brain and accelerates the onset of amyloid deposition and its attendant reactive gliosis, even as a single mutant allele. We conclude that expression of an FAD-linked mutant PS-1 at normal levels does not generally increase cortical neuronal sensitivity to degeneration. Instead, enhanced amyloidogenic processing of APP likely is critical to the pathogenesis of PS-1-linked FAD.


Subject(s)
Alzheimer Disease/genetics , Amyloid beta-Peptides/biosynthesis , Amyloid/metabolism , Membrane Proteins/genetics , Neurons/metabolism , Alzheimer Disease/etiology , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amino Acid Substitution , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/pharmacology , Amyloid beta-Protein Precursor/genetics , Animals , Apoptosis/genetics , Calpain/metabolism , Caspases/isolation & purification , Caspases/metabolism , Cell Survival/drug effects , Cell Survival/genetics , Cells, Cultured , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Disease Models, Animal , Gene Targeting , Glutamic Acid/pharmacology , Homozygote , Membrane Proteins/metabolism , Membrane Proteins/pharmacology , Mice , Mice, Mutant Strains , Mice, Transgenic , Neurons/drug effects , Neurons/pathology , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Presenilin-1 , Staurosporine/pharmacology
12.
Neuroscience ; 92(4): 1425-42, 1999.
Article in English | MEDLINE | ID: mdl-10426497

ABSTRACT

Activation of a member of the caspase family of cysteine proteases is thought to be required for the execution of apoptosis in neurons and other cell types. We describe here an antibody (Ab127) reactive with a neoantigenic site on caspase substrate proteins degraded during apoptosis, and its characterization as a biochemical and histochemical probe for apoptosis-associated proteolysis in growth factor-deprived neural cells in vitro and the developing postnatal rat brain. Neuronally differentiated PC12 cells became strongly Ab127 immunoreactive only during apoptosis following nerve growth factor withdrawal. Apoptosis-associated caspase proteolysis was detectable on western blots as markedly increased immunoreactivity of a approximately 46,000 mol. wt polypeptide, a product also generated by caspase-3 treatment of cell-free extracts. In the postnatal rat brain, intense immunoreactivity indicative of caspase activation was exhibited by small proportions of neurons and glia in distinct regional and temporal patterns. The degenerating nature of these cells was confirmed by their argyrophilia, cytoplasmic immunoreactivity for c-jun and fragmented processes. Combined immunofluorescence and Hoechst 33342 staining demonstrated that cells immunopositive for caspase activation have apoptotic nuclear morphologies. Caspase proteolysis was observed throughout the neuraxis in a minority of progenitor cells in germinal zones, postmitotic neurons in the parenchyma, and glia in the corpus callosum and other white matter tracts, but was observed rarely in the adult brain. These data characterize a new approach for evaluating apoptosis in physiological and pathological neurodegeneration, and demonstrate that caspase-associated apoptosis is a widespread mechanism for the programmed death of neurons and glia in the postnatal rat brain.


Subject(s)
Animals, Newborn/physiology , Apoptosis/physiology , Brain/cytology , Brain/enzymology , Caspases/metabolism , Caspases/physiology , Nerve Tissue Proteins/metabolism , Neuroglia/physiology , Neurons/physiology , Animals , Antibodies, Monoclonal , Cell Nucleus/immunology , Cell Nucleus/ultrastructure , Cells, Cultured , Fluorescent Antibody Technique, Direct , Immunoblotting , Immunohistochemistry , PC12 Cells , Proto-Oncogene Proteins c-jun/biosynthesis , Rats , Rats, Sprague-Dawley
13.
J Biol Chem ; 274(29): 20499-504, 1999 Jul 16.
Article in English | MEDLINE | ID: mdl-10400678

ABSTRACT

The Alzheimer's disease amyloid peptide Abeta has a heterogeneous COOH terminus, as variants 40 and 42 residues long are found in neuritic plaques and are secreted constitutively by cultured cells. The proteolytic activity that liberates the Abeta COOH terminus from the beta-amyloid precursor protein is called gamma-secretase. It could be one protease with dual specificity or two distinct enzymes. By using enzyme-linked immunosorbent assays selective for Abeta40 or Abeta42, we have measured Abeta secretion by a HeLa cell line, and we have examined the dose responses for a panel of five structurally diverse gamma-secretase inhibitors. The inhibitors lowered Abeta and p3 secretion and increased levels of the COOH-terminal 99-residue beta-amyloid precursor protein derivative that is the precursor for Abeta but did not alter secretion of beta-amyloid precursor protein derivatives generated by other secretases, indicating that the inhibitors blocked the gamma-secretase processing step. The dose-dependent inhibition of Abeta42 was unusual, as the compounds elevated Abeta42 secretion at sub-inhibitory doses and then inhibited secretion at higher doses. A compound was identified that elevated Abeta42 secretion at a low concentration without inhibiting Abeta42 or Abeta40 at high concentrations, demonstrating that these phenomena are separable pharmacologically. Using either of two methods, IC50 values for inhibition of Abeta42 and Abeta40 were found to have the same rank-order and fall on a trend line with near-unit slope. These results favor the hypothesis that Abeta variants ending at residue 40 or 42 are generated by a single gamma-secretase.


Subject(s)
Amyloid beta-Peptides/antagonists & inhibitors , Endopeptidases/metabolism , Protease Inhibitors/pharmacology , Amyloid Precursor Protein Secretases , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Aspartic Acid Endopeptidases , Dose-Response Relationship, Drug , Humans , Tumor Cells, Cultured
14.
J Neuropathol Exp Neurol ; 58(4): 365-75, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10218632

ABSTRACT

In animals and man, traumatic brain injury (TBI) results in axonal injury (AI) that contributes to morbidity and mortality. Such injured axons show progressive change leading to axonal disconnection. Although several theories implicate calcium in the pathogenesis of AI, experimental studies have failed to confirm its pivotal role. To explore the contribution of Ca2+-induced proteolysis to axonal injury, this study was undertaken in an animal model of TBI employing antibodies targeting both calpain-mediated spectrin proteolysis (CMSP) and focal neurofilament compaction (NFC), a marker of intra-axonal cytoskeletal perturbation, at 15-120 minutes (min) postinjury. Light microscopy (LM) revealed that TBI consistently evoked focal, intra-axonal CMSP that was spatially and temporally correlated with NFC. These changes were seen at 15 min postinjury with significantly increasing number of axons demonstrating CMSP immunoreactivity over time postinjury. Electron microscopy (EM) demonstrated that at 15 min postinjury CMSP was confined primarily to the subaxolemmal network. With increasing survival (30-120 min) CMSP filled the axoplasm proper. These findings provide the first direct evidence for focal CMSP in the pathogenesis of generalized/diffuse AI. Importantly, they also reveal an initial subaxolemmal involvement prior to induction of a more widespread axoplasmic change indicating a spatial-temporal compartmentalization of the calcium-induced proteolytic process that may be amenable to rapid therapeutic intervention.


Subject(s)
Axons/enzymology , Brain Injuries/metabolism , Calpain/metabolism , Spectrin/metabolism , Animals , Axons/chemistry , Axons/ultrastructure , Calpain/analysis , Cell Compartmentation/physiology , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , Disease Models, Animal , Microscopy, Electron , Neurofilament Proteins/analysis , Neurofilament Proteins/metabolism , Rats , Rats, Sprague-Dawley , Spectrin/analysis
15.
Neuroreport ; 10(2): 353-8, 1999 Feb 05.
Article in English | MEDLINE | ID: mdl-10203334

ABSTRACT

In traumatic axonal injury, Ca2+ influx across a focally damaged axolemma precipitates local mitochondrial failure, degradation of the subaxolemmal spectrin network and compaction of neurofilaments, which collectively contribute to axonal failure. In previous studies, cyclosporin A pretreatment preserved mitochondrial integrity and attenuated axonal failure following trauma. Here we investigate whether this CsA-linked protection was related to the concomitant blunting of intra-axonal, Ca2+-induced cytoskeletal changes in traumatic axonal injury, assessed with antibodies targeting spectrin proteolysis and neurofilament compaction. CsA pretreatment dramatically reduced Ca2+-induced cytoskeletal damage following injury; CsA-treated rats, compared with vehicle-treated rats, displayed a 70% decrease in immunoreactive/damaged profiles. We suggest that CsA-mediated preservation of mitochondrial integrity enables the restoration of ionic and metabolic homeostasis thereby short-circuiting Ca2+-induced proteolysis in injured axons.


Subject(s)
Axons/drug effects , Brain Injuries/pathology , Calcium/physiology , Cyclosporine/pharmacology , Animals , Axons/pathology , Biomarkers , Brain/pathology , Calpain/physiology , Image Processing, Computer-Assisted , Immunohistochemistry , Male , Peptide Hydrolases/metabolism , Pyramidal Tracts/metabolism , Rats , Rats, Sprague-Dawley , Spectrin/metabolism
16.
Brain Res ; 810(1-2): 181-99, 1998 Nov 09.
Article in English | MEDLINE | ID: mdl-9813316

ABSTRACT

Excitatory amino acids may promote microtubular proteolysis observed in ischemic neuronal degeneration by calcium-mediated activation of calpain, a neutral protease. We tested this hypothesis in an animal model of focal cerebral ischemia without reperfusion. Spontaneously hypertensive rats were treated with 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)quinoxaline (NBQX), a competitive antagonist of the neuronal receptor for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or cis-4-[phosphono-methyl]-2-piperidine carboxylic acid (CGS 19755), a competitive antagonist of the N-methyl-d-aspartate (NMDA) receptor. After treatment, all animals were subjected to permanent occlusion of the middle cerebral artery for 6 or 24 h. Infarct volumes measured in animals pretreated with CGS 19755 after 24 h of ischemia were significantly smaller than those quantified in ischemic controls. Rats pretreated with NBQX showed partial amelioration of cytoskeletal injury with preserved immunolabeling of microtubule-associated protein 2 (MAP 2) at 6 and 24 h and reduced accumulation of calpain-cleaved spectrin byproducts only at 6 h. Prevention of cytoskeletal damage was more effective after pretreatment with CGS 19755, as shown by retention of MAP 2 immunolabeling and significant restriction of calpain activity at both 6 and 24 h. Preserved immunolabeling of tau protein was observed at 6 and 24 h only in animals pretreated with CGS 19755. Western analysis performed on ischemic cortex taken from controls or rats pretreated with either NBQX or CGS 19755 suggested that loss of tau protein immunoreactivity was caused by dephosphorylation, rather than proteolysis. These results demonstrate a crucial link between excitotoxic neurotransmission, microtubular proteolysis, and neuronal degeneration in focal cerebral ischemia.


Subject(s)
Calpain/antagonists & inhibitors , Cytoskeleton/enzymology , Excitatory Amino Acid Antagonists/pharmacology , Ischemic Attack, Transient/enzymology , Receptors, Glutamate/drug effects , Animals , Blotting, Western , Cerebral Infarction/physiopathology , Cytoskeleton/drug effects , Immunohistochemistry , Male , Microtubule-Associated Proteins/metabolism , Pipecolic Acids/pharmacology , Quinoxalines/pharmacology , Rats , Rats, Inbred SHR , Spectrin/metabolism , Synaptic Transmission/drug effects , Synaptic Transmission/physiology , tau Proteins/metabolism
18.
Biochem Biophys Res Commun ; 248(2): 293-6, 1998 Jul 20.
Article in English | MEDLINE | ID: mdl-9675129

ABSTRACT

We have developed a sensitive and continuously recording fluorogenic assay for the thiol protease calpain. This assay uses the dipeptide substrate Suc-Leu-Tyr-4-Methoxy-2-Naphthylamine (Suc-LY-MNA) in Tris buffer (pH 7.5) in the presence of 0.1% CHAPS. The assay is linear over a wide range of enzyme concentration and is capable of detecting 10 picomolar calpain making it more sensitive than any previously published method. Moreover, this assay gives a rate that is linear for over ten minutes making it useful for mechanistic studies of inhibitors. This assay can be easily adapted to a 96-well plate format facilitating the large scale screening of inhibitors.


Subject(s)
Calpain/analysis , Erythrocytes/enzymology , Cholic Acids/pharmacology , Detergents/pharmacology , Dipeptides/metabolism , Enzyme Inhibitors/analysis , Fluorometry , Humans , Kinetics , Naphthalenes/metabolism , Recombinant Proteins/metabolism , Sensitivity and Specificity , Substrate Specificity
19.
Bioorg Med Chem ; 6(5): 509-22, 1998 May.
Article in English | MEDLINE | ID: mdl-9629465

ABSTRACT

Calpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration following an episode of cerebral ischemia. In this paper, we report on a series of peptidomimetic ketomethylene and carbamethylene inhibitors of recombinant human calpain I (rh calpain I). Our study reveals that the -NHCO-moiety (possible hydrogen-bonding site) at the P2-P3 region of a potent tripeptide or a dipeptide inhibitor of calpain I is not a strict requirement for enzyme recognition. Compounds 7d ((R)-2-isobutyl-4-oxo-4-(9-xanthenyl)butanoic acid ((S)-1-formyl-3-methyl)butyl amide), 31 ((R)-2-isobutyl-4-(2-sulfonylnaphthyl)butyric acid ((S)1-formyl-3-methyl)butyl amide) and 34 ((R)-2-isobutyl-4-(2-sulfoxylnaphthyl)butyric acid ((S)-1-formyl-3-methyl)butyl amide) which exhibited good activity in the enzyme assay, also inhibited calpain I in a human cell line.


Subject(s)
Calpain/antagonists & inhibitors , Cysteine Proteinase Inhibitors/chemistry , Dipeptides/chemistry , Oligopeptides/chemistry , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/pharmacology , Dipeptides/chemical synthesis , Dipeptides/pharmacology , Humans , Magnetic Resonance Spectroscopy , Oligopeptides/chemical synthesis , Oligopeptides/pharmacology , Protein Conformation , Recombinant Proteins/antagonists & inhibitors , Tumor Cells, Cultured
20.
J Biol Chem ; 273(26): 16576-82, 1998 Jun 26.
Article in English | MEDLINE | ID: mdl-9632729

ABSTRACT

The effects of dietary cholesterol on brain amyloid precursor protein (APP) processing were examined using an APP gene-targeted mouse, genetically humanized in the amyloid beta-peptide (Abeta) domain and expressing the Swedish familial Alzheimer's disease mutations. These mice express endogenous levels of APP holoprotein and abundant human Abeta. Increased dietary cholesterol led to significant reductions in brain levels of secreted APP derivatives, including sAPPalpha, sAPPbeta, Abeta1-40, and Abeta1-42, while having little to no effect on cell-associated species, including full-length APP and the COOH-terminal APP processing derivatives. The changes in levels of sAPP and Abeta in brain all were negatively correlated with serum cholesterol levels and levels of serum and brain apoE. These results demonstrate that secreted APP processing derivatives and Abeta can be modulated in the brain of an animal by diet and provide evidence that cholesterol plays a role in the modulation of APP processing in vivo. APP gene-targeted mice lacking apoE, also have high serum cholesterol levels but do not show alterations in APP processing, suggesting that effects of cholesterol on APP processing require the presence of apoE.


Subject(s)
Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Brain/metabolism , Cholesterol, Dietary/pharmacology , Alzheimer Disease/genetics , Animals , Apolipoproteins E/pharmacology , Cells, Cultured , Cholesterol/blood , Gene Targeting , Humans , Mice , Peptide Fragments/metabolism
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