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Biomedical subjects

D Monard

Publications and source records attributed to D Monard.

At least 37 records · Page 2Linked to original sources
PubMed1995

Regulation of protease nexin-1 and angiotensin II receptor subtype 1 expression: inverse relationship in experimental models of nerve injury.

The up-regulation of PN-1 following nerve lesion has been investigated in vitro in cultures of dorsal root ganglion (DRG) explants, sciatic nerve segments, and isolated Schwann cells. In the first culture model, Schwann cells associated with neuronal processes synthesized small amounts of PN-1. Injury of the neurites emerging from the DRGs led to enhanced levels of PN-1 in Schwann cells located distal to the lesion site where degeneration of neuronal processes took place. In cultured sciatic nerve segments, PN-1 synthesis increased with a time-course comparable to that in ganglion explants following lesion. In the third model, PN-1 levels gradually rose in isolated Schwann cells during the first 3-8 days in culture. Dissociation of Schwann cells from the sciatic nerve therefore causes an effect similar to nerve damage. Impairment of Schwann cells-neuron interactions was followed by a reduction in the expression levels of the angiotensin II (Ang II) receptor subtype AT1 in all three systems studied. Since the neuropeptide Ang II is able to repress PN-1 synthesis in cultured Schwann cells, loss of neuronal contact might decrease their responsiveness to Ang II, thus resulting in PN-1 up-regulation by default.

Amyloid beta-Protein Precursor↗
PubMed1995

Inhibition of acrosin by serpins. A suicide substrate mechanism.

The serpins antithrombin, protease nexin 1, and alpha 1-antitrypsin with a reactive-center arginine (Arg-alpha 1-antitrypsin) were found to inhibit the sperm protease acrosin with varying efficiency. The serpins were titrated against acrosin to determine their specific activity with respect to this enzyme. While antithrombin was fully active against acrosin, more than one molecule of Arg-alpha 1-antitrypsin and protease nexin 1 was required to inhibit one molecule of acrosin. In particular, only 2.7% of protease nexin 1 molecules interacting with acrosin formed stable complexes with the enzyme at 37 degrees C and this value decreased to 0.03% at 12 degrees C. N-terminal sequence analysis indicated that acrosin had cleaved protease nexin 1 at its reactive-center Arg-Ser bond. The results could be interpreted in terms of protease nexin 1 acting as a suicide substrate for acrosin; after the formation of an initial complex, the serpin partitioned between pathways yielding either inactivated (cleaved) serpin or a stable serpin-enzyme complex. The association rate constant (k(ass)) and inhibition constant (Ki) for the stable complexes were determined for each of the serpins by using slow-binding kinetics. The values of k(ass) were 2 x 10(5), 4 x 10(4), and 5 x 10(3) M-1 s-1 for Arg-alpha 1-antitrypsin, antithrombin, and protease nexin 1, respectively. The Ki values for the serpins were 1 nM or less. Heparin markedly accelerated the inhibition of acrosin by antithrombin and protease nexin 1; at the optimal concentration, the degree of heparin acceleration of the inhibition rate was 250- and 500-fold for antithrombin and protease nexin 1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosin↗
PubMed1995

Inactivation of protease nexin-1 by xanthine oxidase-derived free radicals.

Neuronal viability is affected by reactive oxygen species. Lipid peroxidation is often defined as a major reason for cellular breakdown. Additionally, certain indispensable proteins are possible targets for excessively formed reactive oxygen species. Evidence is given here that protease nexin-1 (PN-1), an endogenous thrombin inhibitor and neurite outgrowth promoter, is inactivated by xanthine oxidase-derived free radicals. Varying protection by superoxide dismutase and catalase was observed, depending on the reaction conditions. The water-soluble alpha-tocopherol analogues MDL 74,406 (R(+)-3,4-dihydro-6-hydroxy-N,N,N-2,5,7,8-heptamethyl-2H-1-benzopy ran-2- ethanaminium 4-methylbenzenesulfonate), MDL 74,180DA (2,3-dihydro-2,2,4,6,7-pentamethyl-3-(4-methyl-piperazino)-1-benzo furan-5-ol dihydro-chloride) and trolox also protected PN-1. Neurodegeneration may be triggered by oxidative inactivation of protease inhibitors such as PN-1. Protection of PN-1 in Alzheimer's or Parkinson's diseases, could be a possible target for a therapeutic function of antioxidants in these diseases.

Amyloid beta-Protein Precursor↗
PubMed1995

Regulation of protease nexin-1 expression in cultured Schwann cells is mediated by angiotensin II receptors.

Protease nexin-1 (PN-1) is a potent inhibitor of serine proteases, such as thrombin and plasminogen activators, which is secreted into the extracellular space. Since PN-1 is induced following lesion of the sciatic nerve, the effect of substances known to accumulate at the site of injury was examined in primary cultures of Schwann cells. Among the cytokines, growth factors, mitogens, neurotrophins, and neuroactive peptides analyzed, only angiotensin II (Ang II), calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP) were found to regulate the expression of PN-1 on Schwann cells. While Ang II and CGRP caused downregulation, VIP acted as a positive modulator of PN-1. Displacement of Ang II binding using the selective ligands losartan and CGP 42112 led to a severalfold increase of PN-1 protein and mRNA over basal levels, indicating that the observed effect was mediated by specific binding sites. Indeed, the presence of AT1 and AT2 angiotensin receptor subtypes was demonstrated in cultured Schwann cells as well as in the rat sciatic nerve. Moreover, the detection of angiotensinogen- and renin-mRNA in these cultures suggested an endogenous production of Ang II. This data identified one of the mechanisms regulating PN-1 synthesis. Altogether our results indicate that neuropeptides can differentially control the proteolytic activity of the microenvironment, providing new aspects of neuron-glia interactions in the intact tissue and following nerve injury.

Amyloid beta-Protein Precursor↗
PubMed1994

Granzyme A released upon stimulation of cytotoxic T lymphocytes activates the thrombin receptor on neuronal cells and astrocytes.

Granzymes are a family of serine proteases that are harbored in cytoplasmic granules of activated T lymphocytes and are released upon target cell interaction. Immediate and complete neurite retraction was induced in a mouse neuronal cell line when total extracts of granule proteins were added. This activity was isolated and identified as granzyme A. This protease not only induced neurite retraction at nanomolar concentrations but also reversed the stellation of astrocytes. Both effects were critically dependent on the esterolytic activity of granzyme A. As neurite retraction is known to be induced by thrombin, possible cleavage and activation of the thrombin receptor were investigated. A synthetic peptide spanning the N-terminal thrombin receptor activation sequence was cleaved by granzyme A at the authentic thrombin cleavage site Leu-Asp-Pro-Arg-Ser. Antibodies to the thrombin receptor inhibited both thrombin and granzyme A-mediated neurite retraction. Thus, T-cell-released granzyme A induces cellular responses by activation of the thrombin receptor. As brain-infiltrating CD4+ lymphocytes are the effector cells in experimental allergic encephalomyelitis, granzyme A released in the brain may contribute to the etiology of autoimmune disorders in the nervous system.

Amino Acid Sequence↗
PubMed1994

Localization of the heparin-binding site of glia-derived nexin/protease nexin-1 by site-directed mutagenesis.

Recombinant rat glia-derived nexin was expressed in insect cells using the baculovirus system. The kinetics for the inhibition of thrombin by this recombinant material were indistinguishable from those observed with natural glia-derived nexin and recombinant nexin expressed in yeast. In addition, the dependence of the rate of inactivation on the concentration of heparin was similar for the three preparations. At the optimal heparin concentration, the association rate constant was 330-fold higher than that observed in the absence of heparin. A putative heparin-binding site is found in glia-derived nexin between residues 71 and 86; heparin-binding sites are found in homologous regions of antithrombin III and heparin cofactor II. Lysines in this region were mutated to glutamates, and the kinetics for the inhibition of thrombin by mutant proteins were determined. Concurrent mutation of all seven lysines in this region (residues 71, 74, 75, 78, 83, 84, and 86) did not affect the rate constant for the association of glia-derived nexin with thrombin in the absence of heparin, but it resulted in complete loss of the heparin acceleration of the rate of association. Mutations of residues 83, 84, and 86 together also caused a marked decrease in the acceleration by heparin of the reaction between glia-derived nexin and thrombin. These results support the hypothesis that the heparin-binding sites of glia-derived nexin, antithrombin III, and heparin cofactor II are found in homologous regions of the molecules. Heparin was also found to potentiate the ability of wild-type glia-derived nexin to inhibit the thrombin-induced retraction of neurites from neuroblastoma NB2a cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Re-expression of glia-derived nexin/protease nexin 1 depends on mode of lesion-induction or terminal degeneration: observations after excitotoxin or 6-hydroxydopamine lesions of rat substantia nigra.

The serine protease inhibitor and neurite outgrowth promoter glia derived nexin (GDN) is expressed in the rat CNS during embryogenesis and persists in the olfactory system of the adult where receptor neurons are replaced throughout life. We investigated whether GDN-immunoreactivity also appears in the adult at sites of synaptic rearrangement following nerve cell death and anterograde terminal degeneration in experimental models for Parkinson's disease. Rat substantia nigra was unilaterally lesioned by stereotaxic application of different toxins: 6-hydroxydopamine, which selectively destroys dopaminergic neurons, the excitotoxic glutamate analog ibotenic acid, or the glutamate receptor agonists N-methyl-D-aspartate and quisqualate, which cause circumscript lesions of the whole substantia nigra. Nerve cell death and astroglial reactivity were monitored by parallel cresyl staining and immunocytochemistry for glial fibrillary acidic protein, at survival times ranging from 2 to 100 days. Sustained de novo synthesis of GDN occurred in the dopamine depleted caudate putamen following excitotoxin or 6-hydroxydopamine induced degeneration of the substantia nigra and of the nigrostriatal pathway provided that the lesions were nearly complete. This is consistent with compensatory changes occurring in deafferented caudate putamen and suggests a permissive role of GDN in neuronal plasticity. In the substantia nigra astroglia exhibited GDN-immunoreactivity following excitotoxin injection but not after application of 6-hydroxydopamine. Thus differences in action mechanisms of neurotoxins may have distinct consequences on the astrocyte mediated response of the same affected brain region.

Amyloid beta-Protein Precursor↗
PubMed1994

Glia-derived nexin/protease nexin-1 is expressed by a subset of neurons in the rat brain.

Glia-derived nexin/protease nexin-1 (GDN/PN-1) is a serine protease inhibitor that is secreted by glial cells and fibroblasts in culture. In the adult mammalian nervous system it has been shown to be expressed in the olfactory system and by some glial cells in response to neuronal injury. In situ hybridization and immunocytochemical studies were performed to identify the structures expressing GDN/PN-1 in the developing and adult rat brain. In contrast to a transient widespread expression during pre- and postnatal development, some brain structures constitutively express GDN/PN-1. These include the olfactory nerve layer of the olfactory bulb, basal forebrain, striatum, pyramidal neurons of layer V in the cortex, thalamic nuclei, pars compacta of the substantia nigra, inferior and superior colliculi, and deep cerebellar nuclei. All of these parts, excluding the olfactory nerve layer, are characterized by a high neuronal cell density. Neurons in these regions were immunoreactive for GDN/PN-1. Furthermore GDN/PN-1 expression in cell lines showed that the active protein was synthesized and secreted from B104 but not from NB2a neuroblastoma cells. Although GDN/PN-1 has only been reported to be synthesized by glia, the results presented here demonstrate that in addition, a subset of neurons express this protease inhibitor.

Amyloid beta-Protein Precursor↗
PubMed1994

Expression of the thrombin receptor mRNA in rat brain.

Thrombin activates its receptor in a number of cultured cells of neural origin, but the functional significance of this activation in the nervous system is unknown. It is also not known which cells in brain express the thrombin receptor and whether the level of its expression is developmentally regulated. In the present study, Northern blot analysis showed that thrombin receptor mRNA was expressed at higher levels in brain compared to some other tissues, such as skeletal muscle, liver or kidney. The level of expression is substantially higher in the brain of newborn rats compared to that of postnatal day 28 (P28). At embryonic day 18, thrombin receptor mRNA is present throughout in the brain and in dorsal root ganglia as detected by in situ hybridization. The regions of the P28 brain in which the thrombin receptor mRNA was present include the substantia nigra and the ventral tegmental area, the pretectal area, some hypothalamic nuclei and some cells of the cerebral cortex. These results represent one of the first steps needed to understand the role played by the thrombin receptor in the development and function of the nervous system.

Animals↗
PubMed1993

Decreased levels of glia-derived nexin/protease nexin I in irradiated rat spinal cord in vivo.

The pathophysiology of the cellular basis of radiation induced demyelination and white matter necrosis of the Central Nervous System (CNS) is poorly understood. There have been no previous studies that have shown the effect of irradiation on glia-derived nexin. In this study, rats were given cervical spinal cord irradiation, and glial derived nexin or protease nexin I (GDN/PNI) was measured on days 2, 7, 30, 60, 120, 130, and 145 after irradiation. The level of GDN/PNI significantly decreased after irradiation compared to levels in control spinal cord, and there was no detectable levels of GDN/PNI by the time paralysis developed. This study adds considerable weight to the proposal that GDN/PNI has an important role in the pathogenesis of CNS radiation damage.

Amyloid beta-Protein Precursor↗
PubMed1993

The glia-derived protease nexin 1 persists for over 1 year in rat brain areas selectively lesioned by transient global ischaemia.

The re-expression of the developmentally regulated serine protease inhibitor glia-derived nexin (GDN) was investigated 1 year after transient global ischaemia induced by the four-vessel occlusion technique in rats. The CA1 sector of the hippocampus was severely shrunken due to the absence of pyramidal cells, but still clearly discernible due to the continued presence of the parvalbumin-containing GABAergic neurons. In this partially neuron-depleted hippocampus, GDN immunoreactivity was found in reactive astrocytes containing glial fibrillary acidic protein. GDN-positive astrocytes were also found in other lesioned areas, the reticular thalamic nucleus and the cerebellar cortex. Thus, the re-expression of GDN in the adult excitotoxically lesioned brain described previously in the gerbil model of ischaemia persists. The continued presence of the protease inhibitor might disturb the proteolytic balance and lead to the deposition of pathological breakdown products of proteins, e.g. beta-amyloid.

Amyloid beta-Protein Precursor↗
PubMed1993

Protease-nexin I as an androgen-dependent secretory product of the murine seminal vesicle.

A search for inhibitors of urokinase-type plasminogen activator (uPA) in the male and female murine genital tracts revealed high levels of a uPA ligand in the seminal vesicle. This ligand is functionally, biochemically and immunologically indistinguishable from protease-nexin I (PN-I), a serpin ligand of thrombin and uPA previously detected only in mesenchymal cells and astrocytes. A survey of murine tissues indicates that PN-I mRNA is most abundant in seminal vesicles, where it represents 0.2-0.4% of the mRNAs. PN-I is synthesized in the epithelium of the seminal vesicle, as determined by in situ hybridization, and is secreted in the lumen of the gland. PN-I levels are much lower in immature animals, and strongly decreased upon castration. Testosterone treatment of castrated males rapidly restores PN-I mRNA levels, indicating that PN-I gene expression is under androgen control.

Amyloid beta-Protein Precursor↗
PubMed1993

Variable and multiple expression of Protease Nexin-1 during mouse organogenesis and nervous system development.

Protease Nexin-1 (PN-1) also known as Glia-Derived Nexin (GDN) inhibits the activity of several serine proteases including thrombin, tissue (tPA)- and urokinase (uPA)-type plasminogen activators. These and other serine proteases seem to play roles in development and tissue homeostasis. To gain insight into where and when PN-1 might counteract serine protease activities in vivo, we examined its mRNA and protein expression in the mouse embryo, postnatal developing nervous system and adult tissues. These analyses revealed distinct temporal and spatial PN-1 expression patterns in developing cartilage, lung, skin, urogenital tract, and central and peripheral nervous system. In the embryonic spinal cord, PN-1 expression occurs in cells lining the neural canal that are different from the cells previously shown to express tPA. In the developing postnatal brain, PN-1 expression appears transiently in many neuronal cell populations. These findings suggest a role for PN-1 in the maturation of the central nervous system, a phase that is accompanied by the appearance of different forms of PN-1. In adults, few distinct neuronal cell populations like pyramidal cells of the layer V in the neocortex retained detectable levels of PN-1 expression. Also, mRNA and protein levels did not correspond in adult spleen and muscle tissues. The widespread and complex regulation of PN-1 expression during embryonic development and, in particular, in the early postnatal nervous system as well as in adult tissues suggests multiple roles for this serine protease inhibitor in organogenesis and tissue homeostasis.

Amyloid beta-Protein Precursor↗
PubMed1993

Tinkering with certain blood components can engender distinct functions in the nervous system.

A 43 kd protein called glia-derived nexin or protease nexin-1 (GDN/PN-1) with both neurite-promoting and serine protease inhibitor activity is developmentally regulated during the differentiation of the nervous system. The synthesis of GDN/PN-1 remains high in structures such as the olfactory system where degeneration and regeneration take place throughout life. It is also up-regulated following injury both in the peripheral and the central nervous systems. Together with hirudin (a protease inhibitor from the leech), GDN/PN-1 is the most potent thrombin inhibitor known today. The surprising discovery of this potent thrombin inhibitor in the nervous system led to demonstration that mRNAs coding for prothrombin and the thrombin receptor are detected in neural tissue. Neuronal cells can cleave the inactive prothrombin into the active thrombin, which, in turn, specifically cleaves its own receptor to trigger a metabolic cascade causing sudden neurite retraction. Other macromolecules, such as vitronectin and thrombospondin, also found in the blood, can stimulate neurite outgrowth. Altogether, the data available today indicate that many molecules considered until now to be components of the hematopoietic system could perform distinct tasks and specific functions in the nervous system. Some of the experimental facts still required for demonstration of this hypothesis are discussed.

Amyloid beta-Protein Precursor↗
PubMed1993

Molecular organization of the rat glia-derived nexin/protease nexin-1 promoter.

The first three exons and the promoter of rat glia-derived nexin, also called protease nexin-1 (GDN/PN-1), have been identified through analysis of rat genomic clones. A 1.6 kilobase (kb) fragment containing 105 base pairs of the first exon and 5'-flanking sequences was sequenced. The 5'-flanking sequence and the first exon were found to be GC-rich, indicating that the 5' region of the rat GDN/PN-1 gene resides within a CpG island. A TATA box-like sequence, but no CAAT box, was found. The rat GDN/PN-1 promoter contains five SP1 consensus sites, four consensus sites for the MyoD1 transcription factor, and one binding site for the transcription factors NGFI-A, NGFI-C, Krox-20, and Wilms tumor factor. The presence of these consensus sequences is consistent with the known expression pattern of GDN/PN-1. Primer extension and RNase protection assays identified one transcriptional start site. The 1.6 kb promoter fragment cloned in a reporter plasmid was found to induce firefly luciferase expression in a cell-specific manner. A positive regulatory element is localized in the region -1545 to -389. In vitro CpG methylation blocked transcription from the GDN/PN-1 promoter in rat hepatoma cells but not in C6 rat glioma cells.

Amyloid beta-Protein Precursor↗
PubMed1992

Characterization of the heparin-binding site of glia-derived nexin/protease nexin-1.

The interaction of heparin with glia-derived nexin (GDN) has been characterized and compared to that observed between heparin and antithrombin III (ATIII). Heparin was fractionated according to its affinity for immobilized GDN, and the ability of various fractions to accelerate the inhibition rate of thrombin by either GDN or ATIII was examined. Fractions with different affinities for GDN accelerated the thrombin-GDN reaction to a similar extent; heparin with a high affinity for immobilized GDN stimulated the reaction only about 30% more than the fraction that did not bind to immobilized GDN. Slightly greater differences were observed for the effect of these fractions on the thrombin-ATIII reaction; heparin that did not bind to the GDN affinity column was about 60% more effective than heparin with a high affinity for GDN in accelerating the inhibition of thrombin by ATIII. The CNBr fragment of GDN between residues 63 and 144 was able to reduce the heparin-accelerated rate of inhibition of thrombin by GDN indicating that this region of GDN was able to bind the heparin molecules responsible for the acceleration. Shorter synthetic peptides within this sequence did not significantly reduce the rate, suggesting that the heparin-binding activity of fragment 63-144 depends on a specific conformation of the polypeptide chain. Fragment 63-144 was less effective in decreasing the heparin-accelerated rate of inhibition of thrombin by ATIII. The results are discussed in terms of the heparin species that are responsible for the acceleration of the GDN- and ATIII-thrombin reactions and the heparin-binding sites of GDN and ATIII.

Amino Acid Sequence↗