Biomedical subjects

B W Festoff

Publications and source records attributed to B W Festoff.

At least 73 records · Page 4Linked to original sources

PubMed1994

[Serine proteases and their inhibitors: their role in the differentiation in neuromuscular system].

It is now well established that some serine proteases, such as plasminogen activators and thrombin, as well as their inhibitors, have roles in the development of both central and peripheral nervous systems. We have shown that muscle plasminogen activators, activated after denervation, were able to digest some components of the muscle basement membrane. We have also shown that several inhibitors of serine proteases were concentrated at the neuromuscular junction. These are protease nexin, I, also called glia-derived nexin, protease nexin II, a secreted form of the beta-amyloid precursor protein (APP), and alpha 1-antichymotrypsine (ACT). These results leads us to propose a model in which serine proteases would favor plasticity of motor nerve endings during neuromuscular development. On the contrary, the inhibitors of serine proteases would act to provide and secure maintenance of the synaptic contact. A dysequilibrium between serine proteases and their inhibitors might underlie one or more motor neuron diseases.

Amyloid beta-Protein Precursor↗

PubMed1993

Plasminogen activator inhibitor 1, the primary regulator of fibrinolysis, in normal human cerebrospinal fluid.

The occurrence of type-1 plasminogen activator inhibitor (PAI-1) in human cerebrospinal fluid (CSF) has not previously been reported. As a member of the serpin superfamily of serine protease inhibitors and an acute phase response component, PAI-1 has powerful potential roles in nervous system homeostasis. We have detected this serpin antigen using a polyclonal anti-PAI-1 antibody in normal human CSF. In Western blotting, PAI-1 in several CSF samples appears as a two-band antigen of Mr = 54 and 35 kDa, presumably the intact and proteolytic fragment, respectively. In vitro complex formation studies confirm that the 54 kDa form of PAI-1 interacts with 125I-urokinase after activation with SDS, but the 35 kDa form does not. Quantification of total PAI-1 antigen in 18 normal human CSF samples by ELISA reveals a mean value of 1.0 +/- 0.07 (SEM) micrograms/dL, indicating that a relatively low concentration of the inhibitor occurs in normal human CSF. This information should now allow comparison of PAI-1 levels and activity in various neurologic disorders.

Alzheimer Disease↗

PubMed1993

Recombinant growth hormone treatment of amyotrophic lateral sclerosis.

Based on the known trophic effects of growth hormone (GH) on nerve and muscle 75 patients with ALS were treated for up to 18 months with synthetic human growth hormone (Protropin) or a placebo. The course of ALS was assessed serially using a quantitative (TQNE) neuromuscular and manual exam (MRC) and laboratory chemistries. Average insulin-related growth factor (IGF-I) values increased from 1.2 to 2.3 U/mL in the treated group. Surprisingly, serum insulin levels did not increase. Hyperglycemia was noted in only 2 patients of the 38 patients receiving hGH, and this resolved with cessation of treatment. Over the 12 months of treatment there were 11 deaths (6 controls, 5 treated). Survival analysis, performed approximately 12 months following cessation of treatment, did not reveal a difference between the treatment and placebo group. The TQNE scores declined inexorably in both the control and treated group. Retrospective analysis of the TQNE data indicated a poor prognosis for patients who lost arm strength early. A correlation between the TQNE and MRC scores was evident at early stages of motor unit loss, less so when muscle weakness was advanced.

Adult↗

PubMed1993

Increased levels of plasminogen activator inhibitor-1 (PAI-1) in human brain tumors.

Considerable interest in the roles of serine proteases and serine protease inhibitors (serpins) in regulating physiologic and pathologic tissue remodeling has led to studies that indicate their critical participation in development and diseases of the brain. Plasminogen activator inhibitor-1 (PAI-1) is the most significant regulator of fibrinolysis in plasma, but little is known of the levels or activities of this important serpin in normal brain and brain tumors. For this reason, we estimated qualitative and quantitative levels of PAI-1 in normal human brain and various brain tumors. Western-blot results indicated that a 51 kDa band recognized with polyclonal anti-PAI-1 was more prominently in metastatic and glioblastoma than in meningiomas and low-grade gliomas; normal human brain lacked any detectable band. Reverse zymography also showed high levels of PAI-1 in malignant brain tumors. The complex formation with 125I-urokinase demonstrated that PAI-1 complex levels were increased in metastatic and glioblastoma when compared with low-grade gliomas and meningiomas. Since PAI-1 acts as a modulator of fibrinolysis, a better understanding of the balance between serine proteases and PAI-1 is likely to enhance our knowledge of brain tumor biology.

Blotting, Western↗

PubMed1993

A receptor for cathepsin G:alpha 1-antichymotrypsin complexes on mouse spinal cord astrocytes.

To determine whether there is a specific receptor for serpin:protease complexes on the astrocyte cell surface, we analyzed the cell-binding characteristics of an 125I-cathepsin G:alpha 1-antichymotrypsin complex. Complex formation is maximal at a 1:1 molar ratio of cathepsin G to alpha 1-antichymotrypsin (alpha 1-ACT) as revealed by sodium dodecyl sulfate-gel electrophoresis and autoradiography. Complex binding to mouse spinal cord astrocytes was inhibited by the presence of excess unlabeled complex, but not by the native protease, cathepsin G, or by the serpin, alpha 1-ACT. Scatchard analysis of the binding curve showed the Kd to be 8 x 10(-8) M. We estimated receptor numbers on astrocytes to be about 2.2 x 10(6) sites per cell. An alpha 1-ACT-derived pentapeptide, Phe-Leu-Met-Ile-Ile (FLMII), homologous to a well-conserved segment in the serpin superfamily, did not inhibit the binding of complexes to cells. These data indicate that a specific receptor for alpha 1-ACT:cathepsin G exists on the CNS glial cell surface. Study of this receptor in astrocytes should facilitate understanding of the serpin:protease balance in the brain.

Animals↗

PubMed1992

Plasminogen activators in the neuromuscular system of the wobbler mutant mouse.

Wobbler, the neurological mutant mouse, carries an autosomal recessive gene (wr) and has been characterized as a model of lower motoneuron disorders with associated muscle atrophy, denervation and reinnervation. During normal murine neuromuscular development a decrease in muscle plasminogen activator (PA) activity accompanies synapse maturation. In contrast, experimental denervation in adult mice leads to an increase in muscle PA activity. The purpose of the present study was to determine the possible involvement of PAs in the denervation/reinnervation phenomena and motoneuron degeneration that characterize the wobbler mutant mouse. We determined the degree of innervation and its characteristics in wobbler mice by measuring choline acetyltransferase (ChAT) activity. We measured ChAT in the spinal cord as well as in two different muscles known to be differentially affected, biceps brachii and gastrocnemius. We found a sharp decrease of ChAT activity in both muscles but not in spinal cord extracts. We estimated the extent of sprouting by the silver/cholinesterase stain. Motoneuron terminal sprouting, not detected in normal animals, was present in 40% of the neuromuscular junctions in wobbler mice. We estimated specific PA activities in biceps brachii and gastrocnemius muscle extracts, as well as spinal cord extracts, using both an amidolytic assay and fibrin zymography. Increased PA, predominantly urokinase-PA (uPA), was observed in wobbler mouse muscle. A greater uPA was detected in biceps brachii muscle than in gastrocnemius muscle, which is less impaired by the mutation. There was no change in spinal cord PA, although tissue type PA (tPA) is the predominant PA type there.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗

PubMed1992

Thrombospondin, a platelet alpha-granule and matrix glycoprotein, is increased in muscle basement membrane of patients with amyotrophic lateral sclerosis.

In an attempt to obtain a biological marker for the enigmatic and fatal neurologic disorder, amyotrophic lateral sclerosis (ALS), several laboratories have explored alterations in various extracellular matrix components in both skeletal muscle and skin. We have studied the distribution of fibronectin, laminin, heparan sulfate proteoglycan (HSPG) and collagen types I, III and IV, along with the platelet alpha-granule glycoprotein, thrombospondin (TSP), by immunofluorescence in frozen sections of muscle from control denervating conditions and ALS patients. In ALS and control muscle, types I and III collagen were localized to the endomysium and the perimysium. Type IV collagen and laminin precisely delineated each muscle fiber (endomysium or basement membrane) but did not stain the perimysium. We found no marked quantitative or qualitative differences in the distribution of collagen types I, III and IV, laminin, fibronectin or HSPG in ALS patients compared to controls. However, when polyclonal antisera for TSP was used we found a marked increase in the deposition of this multi-domain glycoprotein in ALS patients' muscle compared to control muscle. Quantitative analysis of soluble extracts from control and ALS patients' muscle by ELISA also indicated that TSP was increased in ALS. TSP is released from platelet alpha-granules in response to thrombin stimulation. TSP elevation implies coagulation activity via the extravascular thrombolytic system in ALS and may correlate with regeneration. Other studies have indicated decreased circulating protease inhibitors and increased serine proteases in this disorder.

Amyotrophic Lateral Sclerosis↗

PubMed1992

Characterization of the serpin, alpha 1-antichymotrypsin, in normal human cerebrospinal fluid.

Cerebrospinal fluid (CSF) from 20 male patients with nonneurologic disease (age 64.5 +/- 2.8 SEM) was analyzed for the presence of the serpin alpha 1-antichymotrypsin (alpha 1-ACT). A chymotrypsin-specific chromogenic substrate (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) was used to examine the CSF samples. All CSF samples showed inhibitory activity ranging from 45 to 80% inhibition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the samples revealed the presence of a 68-kDa protein migrating identical to authentic human plasma alpha 1-ACT. Complex formation was performed with iodinated bovine chymotrypsin for several representative CSF samples having the highest chymotrypsin inhibitory activity. Comparison was made with complex formation performed with commercially available authentic human plasma alpha 1-ACT. These studies showed the formation of complexes at 37 degrees C, regardless of whether the sample was subsequently boiled or not. In the case of CSF, two complex bands, mass smaller than with plasma alpha 1-ACT, were formed at the lower temperature whereas a single higher Mr band was formed when the samples were boiled. To determine whether cleavage of the serpin occurred, these studies were repeated using human neutrophil cathepsin G as target protease. A complex of approximately 90 kDa was formed with human alpha 1-ACT under these same conditions. alpha 1-ACT has been detected in senile amyloid plaques in brains of Alzheimer's disease patients, the only plasma serine protease inhibitor localized to these structures. Another serpin, protease nexin I, is also found in these plaques, but this inhibitor does not circulate in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1992

Protease nexin I, thrombin- and urokinase-inhibiting serpin, concentrated in normal human cerebrospinal fluid.

Protease nexin I (PNI), a 43,000- to 50,000-dalton glycoprotein, is a potent thrombin and urokinase inhibitor produced by many mammalian cells, including human glia, in tissue culture. PNI is a member of the growing superfamily of serine protease inhibitors now known as serpins, but, unlike many others of this family, it has not yet been detected in normal human plasma. Of interest to neurobiology and neurologic disease, PNI is identical to a glia-derived neurite-promoting factor, glia-derived nexin (GDN). Antibody to PNI stains the periphery of senile amyloid plaques in brain tissue from patients with Alzheimer's disease (AD), along with another serpin, alpha 1-antichymotrypsin (alpha 1-ACT). A soluble form of the beta-amyloid precursor protein (beta APP), containing a Kunitz-type trypsin inhibitor domain, the beta APP751 form, is identical to protease nexin II (PNII), a 100,000-dalton serine protease inhibitor present in a number of tissues besides the brain. PNII/beta APP is also found in normal and AD CSF. We found a 47,000-dalton PNI, a thrombin- and urokinase-inhibiting serpin, in normal human CSF by Western blotting using a monospecific antibody. We also demonstrated biologically active PNI capable of forming complexes with serine proteases 125I-urokinase or 125I-thrombin.

Amyloid beta-Protein Precursor↗

PubMed1991

Plasminogen activators and inhibitors in the neuromuscular system: III. The serpin protease nexin I is synthesized by muscle and localized at neuromuscular synapses.

Recent studies suggest that the nature of events leading to the formation, maintenance, and elimination of synapses may be regulated by cascade-type, locally expressed proteases and protease inhibitors acting on adhesive extracellular matrix components. We have identified a molecule in conditioned medium of murine skeletal muscle cells that in molecular weight, target protease inhibition, heparin-binding and cross-reactivity with authenic antisera is similar to the human serine proteinase inhibitor, protease nexin I. Protease nexin I is a 43-50 kDa glycoprotein of the serpin superfamily (arg-serpin class). Purified anti-protease nexin I antibody (anti-47 kDa) stains adult mouse skeletal muscle in discrete foci that precisely superimpose on synaptic neuromuscular junctions. Protease nexin I appears in patches on surfaces of cultured mouse skeletal myotubes, but not on myoblasts. These patches co-localize with acetylcholine receptor clusters and acetylcholinesterase staining during cellular maturation in culture. Evidence that protease nexin I is a synaptic, extracellular antigen is particularly intriguing since it has been shown to be identical, in structure and activity, with a factor released by glial cells, called glia-derived nexin that stimulates mouse neuroblastoma cell neurite outgrowth and inhibits granule cell migration. Protease nexin I inhibits both tumor cell and myoblast plasminogen activator-mediated destruction of extracellular matrix. Thus, such observations as presented in this report provide further evidence for involvement of cascade proteolytic systems, and their post-translational regulation by specific serpins, in the remodeling that occurs in synapse formation and elimination.

Amyloid beta-Protein Precursor↗

PubMed1991

Proteoglycan synthesis by clonal skeletal muscle cells during in vitro myogenesis: differences detected in the types and patterns from primary cultures.

Proteoglycan synthesis by two clonal murine skeletal muscle cell lines, G8-1 and C2, was examined. Cultures of skeletal muscle cells at both the myoblast and myotube stages were radiolabeled using [35S]sulfate as a precursor. The proteoglycans of the cell layer and medium were separately extracted and isolated by ion exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sepharose CL-2B. The cell layer proteoglycans eluted from Sepharose CL-20 as a single peak with a Kav of 0.66 and contained glycosaminoglycan chains with an average molecular weight of 20,000. The glycosaminoglycan chains were composed of nearly equal mixtures of chondroitin sulfate and heparan sulfate with the exception that C2 myoblast cultures contained larger amounts of heparan sulfate. Of interest, this line differentiates more rapidly in our laboratory than G8-1. The medium proteoglycans also eluted from Sepharose CL-2B as a single peak with a Kav of 0.66 but contained glycosaminoglycan chains with an average molecular weight of 32,000. Based upon enzymatic and chemical analysis, the medium glycosaminoglycan chains were composed of a mixture of chondroitin sulfate (71-80%) and heparin sulfate (19-22%). Following chondroitinase ABC digestion, the predominant disaccharide released from all glycosaminoglycan fractions was chondroitin-4-sulfate. When the extracted cell layer proteoglycans were chromatographed on Sepharose CL-28 in the absence of detergent, a small but consistent proportion (14-18%) eluted in the void volume, suggesting the association of at least a portion of this proteoglycan with cellular lipid. These differences distinguish proteoglycan metabolism in fusing clonal lines from primary muscle cell cultures suggesting their utility in evaluating the contribution of these macromolecules in myogenesis.

Animals↗

PubMed1991

Developmental appearance of thrombospondin in neonatal mouse skeletal muscle.

Immunocytochemical localization of the adhesive glycoprotein thrombospondin made in comparison with other components of extracellular matrices shows its sequential appearance in the mouse muscle endomysium during postnatal development. Thrombospondin, absent at birth, in contrast to laminin, type IV collagen and fibronectin, is progressively detected during the first month of neonatal life in the whole muscle extracellular matrix. Immunoblotting of thrombospondin showed the appearance 14 days after birth of a band migrating at 180 kDa corresponding to thrombospondin. A fragment of thrombospondin at 110 kDa was already present at birth, as was also a lower molecular mass band at 70 kDa. Another band at 50 kDa also appeared during development in muscle extracts. Clonal muscle cells in culture were able to synthesize thrombospondin but only at the myotube stage, since little thrombospondin was detected at the myoblast stage. These data show a development regulation of thrombospondin expression in muscle which correlates with muscle differentiation.

Animals↗

PubMed1990

Serpin inhibitors of urokinase and thrombin in normal rat brain and the 9L brain tumor: evidence for elevated expression of protease nexin I-like inhibitor and a novel sodium dodecyl sulfate-activated tumor antithrombin.

Increasing attention is being paid to alterations of the hemostatic balance in tumors, in general, and brain tumors, in particular. Apparently divergent results, showing excess fibrinolysis (i.e., increased plasminogen activator activity) or its inhibition (i.e., increased inhibitor activity), have been reported. The 9L rat brain tumor is a gliosarcoma and a model used to study treatment paradigms for human gliomas. To study the roles of fibrin and fibrinolysis in this brain tumor model, we used these features to investigate the nature of the plasminogen activator (PA) and thrombin inhibitors in normal rat brain and in the 9L rat brain tumor, growing both in vitro and in vivo in rat brain. The results indicate that cells cultured from the tumor in vitro express PA inhibitory activity which is both of the protease nexin I and PA inhibitor 1 types. However, the serpin PA inhibitory activity in extracts of both the normal brain and tumor is of the protease nexin I/PA inhibitor 3 type. This activity is higher in the tumor than in the surrounding "normal" tissue. In addition, we present evidence for a novel thrombin inhibitor which (a) is present only in the tumor growing in rat brain and undetectable either in the normal brain tissue or in vitro, (b) is in a latent, but sodium dodecyl sulfate-activatable, state, and (c) does not bind urokinase. In current studies, investigators are exploring the roles of these molecules and the target serine proteases they inhibit in the pathogenesis of gliomas.

Amyloid beta-Protein Precursor↗

PubMed1990

Plasminogen activators and their inhibitors in the neuromuscular system: I. Developmental regulation of plasminogen activator isoforms during in vitro myogenesis in two cell lines.

Plasminogen activators (PAs), were estimated qualitatively and quantitatively in two different clonal murine skeletal muscle cell lines. Both cell lines produced the two major types of PAs found in mammalian cells, urokinase-type (uPA) and tissue type (tPA). These two lines are models for the study of myogenesis in vitro, but differ in several growth and differentiation characteristics. Because of their possible involvement in these characteristics we assayed the expression of PAs in both cell systems during development in culture. Utilizing fibrin zymography two isoforms of tPA were detected. One co-migrated with human tPA at 75 kd and another may represent a tPA:inhibitor complex at 105 Kd. Several isoenzymes of uPA were detected and these changed depending on whether cell homogenates or conditioned medium was analyzed and whether myogenic cells were at single-cell myoblast or multi-nucleated myotube stage. Species-specific antisera to mouse uPA identified 4 uPA bands in muscle cell medium and 5 in cell layers. Antigenic uPA bands also varied depending on stage of myogenesis. Quantitative amidolytic studies using chromogenic substrates showed that maximal PA activity, both uPA and tPA, occurred at the time of myoblast fusion. Furthermore, uPA activity in membranes increased during myogenesis, while both uPA and tPA in medium decreased after fusion. These studies indicate that muscle PA expression is developmentally regulated and may correlate with growth and differentiation in skeletal muscle.

Animals↗

PubMed1990

Plasminogen activators and their inhibitors in the neuromuscular system: II. Serpins and serpin: protease complex receptors increase during in vitro myogenesis.

In the course of studies on the regulation of plasminogen activator-mediated extracellular matrix degradation in muscle we found the presence of a factor, a cellular inhibitor of serine proteases having features similar to the serpin protease nexin I (PNI). This factor was present in the medium and at maximum concentration following fusion of skeletal muscle cells in culture. The ability of the PNI homologue in mouse muscle to inhibit ECM degradation by urokinase in myoblast medium was compared to that of human PNI purified from human fibroblasts. Stable (to SDS) 1:1 molar ratio complex formation between PNI and proteases, the proposed means by which these enzymes are regulated and removed, was also detected. Cell surface receptors for protease:PNI complexes, the specific binding sites for inactive complex internalization, were found on multinucleated myotubes, while little or no receptor activity was detected on myoblasts. These data suggest that developmental regulation of a) increased PNI proteolytic inhibitory activity expression and b) the appearance of protease:inhibitor complex receptors on muscle cell surfaces during myogenesis may constitute important regulatory features of muscle surface proteolytic activity. They complement previous studies of proteoglycan metabolism in muscle, which itself contains molecules capable of regulating the activity of myotube surface proteases.

Amyloid beta-Protein Precursor↗

PubMed1990

Rapid neural regulation of muscle urokinase-like plasminogen activator as defined by nerve crush.

Muscle plasminogen activators (PAs), such as urokinase-like PA and, to a lesser extent, tissue PA, increase dramatically after denervation induced by axotomy. The PA/plasmin system has also been implicated in degradation of specific components of the muscle fiber basement membrane after local activation of plasminogen. These results suggest that neural regulation of muscle extracellular matrix metabolism accompanies or precedes regeneration after injury and is mediated by activation of PAs. In the present study, we have used nerve crush to explore the neural regulation of muscle PA activities directly after subtotal axon interruption and during the process of reinnervation. Muscle contraction after nerve stimulation and estimation of choline acetyltransferase activity were used to monitor reinnervation. Within 24 hr of nerve crush, muscle urokinase (but not tissue PA) activity rose in soluble and membrane-bound muscle fractions, as shown by an amidolytic assay and a fibrin zymography. Membrane-bound activity was 5-fold higher than cytosol activity, but there was no shift between cellular compartments during the time course of denervation. Coincident with the return of choline acetyltransferase activity and muscle contractility, muscle urokinase returned almost to baseline levels. These results show tight regulation of muscle urokinase levels by some neural influence.

Animals↗

PubMed1989

Monoclonal antibody detects embryonic epitope specific for nerve-derived transferrin.

Monoclonal antibodies were generated against transferrin purified from chick embryo extract by fusing spleen cells from BALB/c mice immunized against embryonic transferrin, with myeloma cells. Antibodies produced by the selected hybridoma clones were all type IgG. Twelve clones were selected for secretion of antibodies to the embryo extract-derived transferrin, and three clones were studied extensively. Immunoblotting was used to demonstrate antibody binding to several avian transferrin proteins derived from adult chicken serum, adult chicken peripheral nerves, and ovotransferrin. Screening and detailed epitope analysis were accomplished by solid-phase immunoassay. The results indicated that two clones, 2G9.1 and 2B11.1, recognized the embryonic and egg antigens in preference to the adult proteins. However, a third clone, 6H2.1, recognized the nerve-derived transferrin preferentially to both the embryonic and adult serum antigens. None of the clones recognized the serum-derived transferrin in preference to the other antigens. These results indicate that embryonic epitope(s) are conserved in the nerve- but not the serum-derived transferrin. They also show that the neural antigen has site(s) distinct from the embryonic proteins. No changes in displacement curves were observed after these proteins were digested with neuraminidase, indicating that the epitope differences discovered are not intimately related to sialic acid residues on the various transferrins.

Animals↗

PubMed1989

Protease nexin I, a serpin, inhibits plasminogen-dependent degradation of muscle extracellular matrix.

Clonal, fusing, mouse skeletal muscle cells (C2) were grown to the myotube stage (90% confluence) before they were subjected to isotope-containing serum-free media (3H-proline or 35S-methionine). C2 myotubes secrete and organize a biosynthetically labeled matrix which adheres to the plastic after removal of myotubes with detergent and ammonium hydroxide. When these homotypic-labeled myotube matrices were incubated with myoblast-conditioned media containing high specific activity urokinase-type plasminogen activator, slow, but clearly detectable, release of label occurred. However, degradation of matrix, with solubilization of label, was accelerated sixfold by addition of human plasminogen to diluted myoblast-conditioned media. If protease nexin I, a cellular serine protease inhibitor purified from human fibroblast-conditioned media, was added (0.2 microgram/ml) with plasminogen, inhibition of matrix hydrolysis by 52% occurred. Higher concentrations (0.8 microgram/ml or above) of protease nexin 1 completely inhibited the degradation of extracellular matrix components. A similar protease inhibitor was purified from C2 myotube-conditioned media, and this molecule also inhibited the plasminogen-dependent release of extracellular matrix. We propose that protease nexin 1 inhibits the destruction of myotube matrix by inactivating the plasmin/plasminogen activation system and may be the physiologic regulator of this system during muscle development in vivo.

Amyloid beta-Protein Precursor↗