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P Seubert

Publications and source records attributed to P Seubert.

52 records · Page 3Linked to original sources
PubMed1991

Increased spectrin proteolysis in fibroblasts from aged and Alzheimer donors.

Since calcium homeostasis is altered in cultured skin fibroblasts from aged and Alzheimer donors, the present study examined the degradation of spectrin, a substrate of the calcium dependent protease calpain. Spectrin proteolysis was estimated as the percentage of spectrin breakdown products (e.g., 150 + 155 kDa bands) per total spectrin immunoreactivity. In the baseline condition (e.g., unstimulated fibroblasts), spectrin breakdown was 53% greater in cells from aged donors when compared to cells from either young or Alzheimer donors. Compared to unstimulated cells, serum increased spectrin breakdown in cells from aged (22.4%) or Alzheimer (92.1%) donors but was ineffective in cells from young donors. Thus, when compared to young donors (100%), serum stimulation increased spectrin proteolysis by 183.9% (aged) or 231.7% (Alzheimer) after serum stimulation. Treatment of unstimulated cells with carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP), an uncoupler of mitochondrial function, increased spectrin degradation by 360.6% (young), 242.4% (aged) or 239.7% (Alzheimer) when compared to unstimulated cells of the same group. The combination of FCCP and serum stimulation enhanced spectrin breakdown in cells from aged (123.6%) and Alzheimer (154.0%) donors when compared to young cells (100%). Thus, changes in the regulation of calcium dependent proteases may contribute to decreased cell spreading and may play a role in the altered cytoskeletal dynamics characteristic of Alzheimer's disease.

Aged↗
PubMed1990

The protease inhibitory properties of the Alzheimer's beta-amyloid precursor protein.

We have expressed the 57-amino acid Kunitz domain of the Alzheimer's beta-amyloid precursor protein (APP751) as a bacterial fusion protein. The protease inhibitory properties of the purified fusion protein, BX9, were virtually identical in all respects tested to those of purified secreted APP751. Both proteins strongly inhibited pancreatic trypsin (Kis = 0.2 and 0.3 nM) and less well epidermal growth factor-binding protein (Kis = 1 and 3.5 nM), alpha-chymotrypsin (Kis = 3 and 6 nM), and the gamma-subunit of nerve growth factor (Kis = 8 and 9 M). Neither protein appreciably inhibited plasma and pancreatic kallikreins, thrombin, lung tryptase, neutrophil elastase, or cathepsin G. The remarkable similarity of the protease inhibitory profile of BX9 to that of secreted APP751 suggests that proper intramolecular disulfide bond formation has occurred in the bacterial fusion protein and leads to the conclusion that the amyloid precursor protein Kunitz domain is a relatively specific inhibitor of only a few trypsin-like arginine esterases.

Amino Acid Sequence↗
PubMed1990

Increased spectrin proteolysis in the brindled mouse brain.

Proteolytically generated fragments of the microfilament anchoring protein brain spectrin were found to accumulate in brindled mouse brain. Proteolysis was most extensive in brain regions possessing high concentrations of N-methyl-D-aspartate (NMDA) receptors (e.g. cortex, striatum, hippocampus). The brindle mutation affects copper homeostasis and thus a variety of copper-dependent enzymes needed in intermediary metabolism. The altered mitochondria of these mice are suggested to less efficiently buffer NMDA receptor-gated calcium fluxes, thus promoting activation of calcium-activated proteases and subsequent degradation of the spectrin meshwork.

Aging↗
PubMed1989

Proteolytic conversion of xanthine dehydrogenase to xanthine oxidase: evidence against a role for calcium-activated protease (calpain).

The present study tested the hypothesis that calpain is responsible for the limited proteolytic conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO). We compared the effects of various proteases on the activity and molecular weight of a purified preparation of xanthine dehydrogenase from rat liver. In agreement with previous reports, trypsin treatment produced a complete conversion of XD to XO accompanied by a limited proteolysis of XDH from an Mr of 140 kD to an Mr of 90 kD. Treatment with calpain I or calpain II did not produce a conversion from XD to XO nor did it result in partial proteolysis of the enzyme. Similarly, trypsin treatment partially degraded a reversibly oxidized form of xanthine dehydrogenase while calpain I or calpain II were ineffective. The possibility that an endogenous inhibitor prevented the proteolysis of XDH by calpain I or II was excluded by verifying that brain spectrin, a known calpain substrate, was degraded under the same incubation conditions. The results indicate that calpain is not likely to be responsible for the in vivo conversion of XD to XO under pathological conditions.

Animals↗
PubMed1989

Ischemia triggers NMDA receptor-linked cytoskeletal proteolysis in hippocampus.

Transient forebrain ischemia is followed within minutes by accelerated proteolysis of the cytoskeletal protein, spectrin. This effect is most pronounced in the selectively vulnerable CA1 region of hippocampus which also experiences a second proteolytic phase during the terminal stages of neuronal degeneration. Both proteolytic phases are suppressed by MK-801, an NMDA receptor antagonist. Cytoskeletal disruption, via NMDA receptor-linked proteolytic events, is suggested to predispose vulnerable neurons to delayed cell death.

Animals↗
PubMed1989

Intrahippocampal colchicine injection results in spectrin proteolysis.

Neurons in the hippocampal formation vary markedly in their susceptibility to colchicine toxicity. The present study was directed at evaluating the effects of colchicine on the proteolytic breakdown of the cytoskeletal protein spectrin within the hippocampus in the rat. Quantified by immunoblot analysis of spectrin breakdown products, the extent of proteolysis was found to correlate with the relative vulnerability of different hippocampal subfields to colchicine toxicity. Levels of breakdown products increased dramatically between 1 and 2 days after colchicine injection, peaked between 2 and 4 days, and remained detectably elevated for at least 35 days. Two days after colchicine injection, the spectrin breakdown products were significantly more concentrated in the molecular layer than in the granule cell/hilar region of the dentate gyrus. The colchicine-induced increase in spectrin breakdown products was significantly reduced by pretreatment with the protease inhibitor leupeptin and was significantly elevated by pretreatment with the lysosomal inhibitor chloroquine. Immunohistochemical analyses of the hippocampus at various times after colchicine injection revealed changes in the distribution of spectrin-like immunoreactivity that paralleled the changes observed by Western blot analysis. Thus increased staining was observed in the molecular layer of the dentate gyrus at 2 and 4 days after the injection, while staining in CA3 was only slightly increased. In addition, abnormal staining of reactive astrocytes was prominent at 2 days. The mechanism whereby colchicine results in neuronal death is as yet unknown. However, the results presented here demonstrate that extensive proteolysis of a cytoskeletal protein occurs in response to the drug, suggesting a plausible mechanism for its neurotoxicity. The protease responsible for the effect is likely to be calpain since the process is non-lysosomal, leupeptin-sensitive and produces spectrin breakdown products indistinguishable from those generated by calpain treatment in vitro. These data support the hypothesis that calpain-mediated degradation of cytoskeletal elements is a common and early response to neurodegenerative events and serves as a trigger in the development of various neuropathologies.

Animals↗
PubMed1988

Stimulation of NMDA receptors induces proteolysis of spectrin in hippocampus.

Stimulation of N-methyl-D-aspartate (NMDA) receptors was found to induce proteolysis of brain spectrin in hippocampal slices. The effect was dependent upon extracellular calcium, blocked by the antagonist 2-amino-5-phosphonovalerate (AP5), and was not reproduced by potassium-induced depolarization. These results are consistent with the hypothesis that the involvement of NMDA receptors in plasticity and excitotoxicity is at least partially mediated by calcium-activated proteolysis of cytoskeletal proteins.

2-Amino-5-phosphonovalerate↗
PubMed1988

Lesions of entorhinal cortex produce a calpain-mediated degradation of brain spectrin in dentate gyrus. I. Biochemical studies.

Lesions of the rat entorhinal cortex cause extensive synaptic restructuring and perturbation of calcium regulation in the dentate gyrus of hippocampus. Calpain is a calcium-activated protease which has been implicated in degenerative phenomena in muscles and in peripheral nerves. In addition, calpain degrades several major structural neuronal proteins and has been proposed to play a critical role in the morphological changes observed following deafferentation. In this report we present evidence that lesions of the entorhinal cortex produce a marked increase in the breakdown of brain spectrin, a substrate for calpain, in the dentate gyrus. Two lines of evidence indicate that this effect is due to calpain activation: (i) the spectrin breakdown products observed following the lesion are indistinguishable from calpain-generated spectrin fragments in vitro; and (ii) their appearance can be reduced by prior intraventricular in fusion of leupeptin, a calpain inhibitor. Levels of spectrin breakdown products are increased as early as 4 h post-lesion, reach maximal values at 2 days, and remain above normal to some degree for at least 27 days. In addition, a small but significant increase in spectrin proteolysis is also observed in the hippocampus contralateral to the lesioned side in the first week postlesion. At 2 days postlesion the total spectrin immunoreactivity (native polypeptide plus breakdown products) increases by 40%, suggesting that denervation of the dentate gyrus produces not only an increased rate of spectrin degradation but also an increased rate of spectrin synthesis. These results indicate that calpain activation and spectrin degradation are early biochemical events following deafferentation and might well participate in the remodelling of postsynaptic structures. Finally, the magnitude of the observed effects as well as the stable nature of the breakdown products provide a sensitive assay for neuronal pathology.

Animals↗
PubMed1988

Lesions of entorhinal cortex produce a calpain-mediated degradation of brain spectrin in dentate gyrus. II. Anatomical studies.

Lesions of the various afferents to the hippocampus have been widely used to investigate the mechanisms underlying growth and degeneration in adult mammalian CNS. It has been proposed that disturbances in intracellular calcium and activation of calcium-dependent proteases represent key steps in producing come of the consequences of the lesions. In this study, we show that lesions of the entorhinal cortex or of the commissural pathway result in profound changes in the distribution of brain spectrin. At 2 days after lesions of the entorhinal cortex, immunoreactivity to spectrin is markedly increased in the outer molecular layer (OML) of the dentate gyrus; conversely at 2 days after commissural lesions, immunoreactivity to the same antigen is increased in the inner molecular layer. The increase in immunoreactivity to spectrin varies with survival time after lesions of the entorhinal cortex. By 24 h post lesion, the increase is homogeneous across the OML, and becomes more intense by 48 h. Between 1 and 3 weeks the increase is much less than at 48 h and is concentrated at the inner border of the OML. Pretreatment of the animals with the calpain inhibitor leupeptin reduces the increase in spectrin immunoreactivity normally seen 48 h after the lesion of the entorhinal cortex. Changes in the pattern of immunoreactivity to GFAP are very different to that seen with spectrin antibodies and are consistent with the known modifications in astrocytes that follow lesions of hippocampal afferents.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

The ultrastructural localization of calcium-activated protease "calpain" in rat brain.

Calpain I, a calcium-activated neutral protease which degrades a number of cytoskeletal proteins, has been implicated in the rapid turnover of structural proteins that may participate in synaptic plasticity. In the present study, an antibody raised against purified erythrocyte calpain I was biochemically characterized and demonstrated to specifically bind the Mr = 80,000 subunit of both rat erythrocyte and brain calpain I. This antibody was used to examine the cellular distribution of calpain I at the electron microscopic level in rat brain and spinal cord using the avidin-biotin immunocytochemical technique. Reaction product was observed throughout neuronal perikarya, within both axonal and dendritic processes, and within spine heads and necks. Postsynaptic densities in both shaft and spine synapses were also immunoreactive. Glial cell bodies and processes were densely stained. In both neurons and glia, the reaction product was deposited along cytoskeletal elements. The localization of calpain I immunoreactivity to glial processes suggests this degradative enzyme may play a role in the glial hypertrophy and process retraction seen in brain. The presence of the enzyme in spines and postsynaptic densities is consistent with the hypothesis that it is involved in the turnover of synaptic cytoskeleton, thus providing a means through which transient physiological events effect lasting changes in the chemistry and morphology of spines.

Animals↗
PubMed1988

Long-term potentiation: persisting problems and recent results.

In this paper we discuss recent experimental results pertinent to three unresolved issues regarding the long-term potentiation (LTP) effect: the nature of its enduring substrates, the biochemical mechanisms that produce it, and its potential role in memory. LTP appears to be triggered by a postsynaptic influx of calcium and is associated with alterations in the shape of dendritic spines and probably the formation of new synapses. We discuss the possibility that morphological reorganization also modifies membrane surface chemistry of synaptic elements. Evidence is presented that LTP is not associated with changes in presynaptic calcium currents. Activation of protein kinase C is shown to be insufficient for the induction of LTP, although it may play a modulatory role. The hypothesis that activation of a calcium-sensitive protease (calpain) is pivotal to the establishment of LTP is supported by experiments showing that a calpain inhibitor, leupeptin, blocks LTP. Furthermore, activation of NMDA receptors, an event implicated in LTP induction, is accompanied by calcium-sensitive proteolysis of spectrin, a major dendritic cytoskeletal protein. The finding that stimulation patterns designed to mimic naturally-occurring cell discharge patterns are highly effective for LTP induction greatly strengthens the hypothesis that LTP actually occurs during the encoding of information in cortical systems. Potential contributions of LTP to learning are explored using computer simulations of a simple cortical network.

Animals↗
PubMed1987

Calmodulin stimulates the degradation of brain spectrin by calpain.

Brain spectrin has been shown to be a preferential substrate of calcium-dependent proteases (Baudry, Bundman, Smith, and Lynch: Science 212:937-938, 1981) and a major calmodulin-binding protein (Kakiuchi, Sobue, and Fujita: FEBS Lett. 132:144-148, 1981). Since calmodulin, spectrin, and a proteolytically derived spectrin fragment are all components of isolated postsynaptic density preparations (Grab, Berzins, Cohen, and Siekevitz: J. Biol. Chem. 254:8690-8696, 1979; Carlin, Bartelt, and Siekevitz: J. Cell Biol. 96:443-448, 1983), we investigated the functional role of calmodulin binding to brain spectrin with respect to its susceptibility to digestion by proteases. We report that calmodulin's interaction with brain spectrin results in a marked acceleration of the rate of spectrin degradation by calcium-dependent proteases (calpains I and II), but not by chymotrypsin. The cleavage of erythrocyte spectrin (which lacks a high-affinity calmodulin binding site) by calpain I is unaffected by the presence of calmodulin. The stimulatory effect of calmodulin is blocked by trifluoperazine, a calmodulin antagonist, which by itself does not modify brain spectrin proteolysis by calcium-dependent proteases. These results suggest a novel role for calmodulin in neuronal function--namely, a synergistic interaction with calcium-dependent proteases in the regulation of cytoskeletal integrity.

Animals↗
PubMedDate not supplied

[Experimental and clinical studies on the relevance of proton spin tomography in patients with coxarthrosis].

We examined 2 in formalin fixed hip specimens from a corpse, 27 patients with coxarthrosis and 3 healthy young volunteers with MRT concerning the illustration of the hip joint and pathological changes by coxarthrosis. Specimen of the femoral head after endoprosthetic operation could be achieved in 18 cases for comparison with MRT. All pathological alterations as cartilage alterations, sclerosis, cysts, effusion, edema, fibrosis were figured with high sensitivity. Cartilage was figured much better by gradient echo sequence with short echo time than by spin echo sequence. Gradient echo sequence made it possible to figure and quantify thinning of cartilage with high sensitivity. Both sequences showed artefacts because of the chemical shift in the area of the cartilage-bone boundary, which impaired especially the judgement of cartilage by spin echo sequence. Gradient echo sequence allowed because of the phase-contrast effect the distinction of hematopoietic and fatty bone marrow and possibly the delineation of bone marrow edema.

Adult↗