PubMed Health⌕ Search

Biomedical subjects

A Bignami

Publications and source records attributed to A Bignami.

At least 37 records · Page 2Linked to original sources
PubMed1989

Isolation and partial characterization of a glial hyaluronate-binding protein.

A glial hyaluronate-binding protein (GHAP) with an isoelectric point of 4.3-4.4 was isolated from human brain white matter. The 60-kDa glycoprotein appeared to be quite resistant to proteolysis, and comparison with GHAP from a viable glioma removed at surgery showed that the protein isolated from autopsy material was not a degradation product resulting from postmortem autolysis. The protein was localized immunohistochemically with mouse monoclonal and rabbit polyclonal antibodies in cerebral white matter. Only small amounts could be found in the gray matter. After enzymatic deglycosylation, an immunoreactive 47-kDa polypeptide was obtained. Two amino acid sequences of GHAP showed a striking similarity (up to 89%) with a highly conserved region of cartilage proteins (bovine nasal cartilage proteoglycan and rat and chicken link protein). However, the amino acid composition and other amino acid sequences suggested that there are also differences between brain-specific GHAP and cartilage proteins.

Amino Acid Sequence↗
PubMed1989

Glial hyaluronate-binding protein in Wallerian degeneration of dog spinal cord.

Wallerian degeneration of spinal cord dorsal columns was produced in three dogs by unilateral extradural dorsal rhizotomy at the lower thoracic level. The spinal cord was studied 1 month, 2 months, and 3 months after surgery. Transverse cryostat sections at the site rhizotomy and at the mid-thoracic level were stained by indirect immunofluorescence with antibodies to the glial fibrillary acidic protein (GFAP) and to the glial hyaluronate-binding protein (GHAP). GHAP immunoreactivity was almost unchanged in the degenerated dorsal column 1 month after rhizotomy. After 2 and 3 months, staining with GHAP antibodies was markedly decreased in the gliosed dorsal column at the site of rhizotomy, but it still persisted at the mid-thoracic level. It is concluded that GHAP persists for long periods of time in dorsal columns undergoing Wallerian degeneration, a finding consistent with its putative role as a nonpermissive substrate preventing nerve regeneration in CNS white matter.

Animals↗
PubMed1989

Axonal regeneration in old multiple sclerosis plaques. Immunohistochemical study with monoclonal antibodies to phosphorylated and non-phosphorylated neurofilament proteins.

Cryostat sections of two old plaques removed at autopsy from the spinal cord of a 62-year-old man with multiple sclerosis of 24-year duration were studied by indirect immunofluorescence with antibodies to neurofilament proteins, glial fibrillary acidic protein (GFAP), glial hyaluronate-binding protein (GHAP), vimentin and laminin. The neurofilament monoclonal antibodies used in this study reacted with phosphorylated epitopes of the two large polypeptides of the neurofilament triplet (NF 150K, NF 200K). As previously reported [Dahl D, Labkovsky B, Bignami A (1989) Brain Res Bull 22:225-232], the neurofilament antibodies either stained axons in the distal stump of transected sciatic nerve in the early stages of regeneration or late in the process, i.e., after regenerating axons had reached the distal stump of the transected sciatic nerve. Both multiple sclerosis plaques were positive for GFAP and vimentin, but negative for GHAP, while astrocytes in myelinated spinal cord white matter stained with both GFAP and GHAP antibodies. Laminin immunoreactivity in the plaques and normal spinal cord was confined to blood vessels. One plaque was almost devoid of axons as evidenced by indirect immunofluorescence with neurofilament antibodies. Another plaque was packed with bundles of thin axons running an irregular course in the densely gliosed tissue. Axons in the plaque only stained with neurofilament antibodies reacting with sciatic nerve in the early stages of regeneration while axons in the surrounding myelinated white matter were decorated by all neurofilament antibodies, regardless of the time of appearance of immunoreactivity in crushed sciatic nerve. It is concluded that reactive astrocytes forming glial scars do not constitute a non-permissible substrate for axonal growth.

Antibodies, Monoclonal↗
PubMed1989

Glial hyaluronate-binding protein in dysmyelinating mice mutants: jimpy, quaking and shiverer.

Cryostat sections of cerebral hemispheres, cerebellum and spinal cord from dysmyelinating mice mutants (quaking, jimpy and shiverer) and littermate controls were stained by indirect immunofluorescence with polyclonal antibodies to the glial hyaluronate-binding protein (GHAP), a brain-specific extracellular matrix glycoprotein produced by astrocytes. In normal mice, the distribution of GHAP was similar to that previously reported in human, calf, pig and dog. The antigen was mainly localized in white matter, the granular layer of the cerebellum being the main exception. No differences were observed between mutants and littermate controls, except that with both GHAP and glial fibrillary acidic protein antibodies the glial framework was denser in the mutants, probably due to the reduction in myelin. The findings suggest that GHAP expression by astrocytes is not induced by myelination and that white matter astrocytes constitute a distinct glial population.

Animals↗
PubMed1989

Intermediate filament proteins immunologically related to desmin in astrocytes: a study of chicken spinal cord by two-dimensional gel electrophoresis and immunoblotting.

Co-migration experiments by two-dimensional SDS-PAGE using chicken spinal cord extracts and desmin purified from chicken gizzard showed that desmin is not present in spinal cord. However, by the immunoblotting procedure, desmin antibodies recognized 3 spinal cord antigens with different molecular weights and isoelectric points than desmin and the glial fibrillary acidic (GFA) protein. These antigens which also reacted with GFA protein antibodies were not identified in chicken gizzard extracts. The reactivity of the antigens with a monoclonal antibody recognizing an epitope common to most intermediate filament proteins (1) suggests that immunostaining of astrocytes with desmin antibodies (2, 3) is due to the presence of new intermediate filament proteins immunologically related to desmin.

Animals↗
PubMed1989

Early and late appearance of neurofilament phosphorylation events in nerve regeneration.

Neurofilament phosphorylation in regenerating rat sciatic nerve was studied by indirect immunofluorescence with monoclonal antibodies reacting with phosphorylated epitopes of the 2 large polypeptides of the neurofilament protein triplet (NF 150K, NF 200K). One group of antibodies decorated axons early in the process. In fact, no differences were seen in double labeled sections between these antibodies and polyclonal neurofilament antibodies as to their reactivity with the distal stump of transected sciatic nerves. Another group stained axons after they had completed their elongation, i.e., after they had reached the distal part of the denervated sciatic nerve. In general, the epitopes recognized by antibodies in this group appeared more sensitive to phosphatase digestion as compared to the first group. Furthermore, there was a good correlation between the thickness of the regenerated axons and staining with these monoclonal antibodies. Thick axons (like those observed in normal nerves) were stained, while bundles of thin axons remained unstained. Monoclonal II32 stained regenerated axons in a remarkable segmental pattern. With this antibody, continuous decoration of the axons was still not observed 7 weeks after transection, the longest follow-up period in this study. We suggest that some neurofilament phosphorylation events may contribute to the stabilization of the axonal cytoskeleton and that abnormalities persist in regenerated axons as to the extent of neurofilament phosphorylation.

Animals↗
PubMed1989

Structural similarity of hyaluronate binding proteins in brain and cartilage.

A glial hyaluronate-binding protein (GHAP) was isolated from human brain white matter by affinity chromatography on immobilized hyaluronate. The 60 kDa protein appeared remarkably homogeneous by reversed-phase high pressure liquid chromatography analysis. Four cyanogen bromide peptides and 10 tryptic peptides were characterized by amino acid sequence, a total of 12 sequences since overlaps were found between 2 cyanogen bromide and 2 tryptic peptide sequences. Two sequences of brain GHAP had similarity with rat link protein, a hyaluronate binding protein in cartilage. The region of similarity was contained in the evolutionary conserved COOH-terminal half of link protein which is involved in the binding of hyaluronate. The remaining 10 amino acid sequences of brain GHAP had no similarity with link protein, nor with previously reported protein sequences. The findings suggest that the hyaluronate binding domains of such diverse proteins as brain GHAP and cartilage link protein are similar, probably due to the fact that hyaluronic acid is highly conserved in evolution.

Amino Acid Sequence↗
PubMed1989

Vimentin-GFAP transition in primary dissociated cultures of rat embryo spinal cord.

Primary dissociated cultures derived from 15-day-old rat embryo spinal cord with or without dorsal root ganglia (DRG) were grown on polylysine, Primaria and laminin substrates. On polylysine and Primaria substrates, spinal cord neurons formed aggregates connected by bundles of neurites in a distinctive pattern similar to that observed in cultures derived from embryonal rat brain and neonatal rat cerebellum. After 2 days in culture, the number of cells stained with GFAP antibodies progressively increased within the vimentin-positive monolayer surrounding the neuronal aggregates. These astrocytes had the typical appearance of astrocytes in primary dissociated cultures derived from late fetal or early neonatal murine brain, i.e. large flat or stellate cells with thick processes staining equally well with GFAP and vimentin antibodies. Astrocytes found within the neuronal aggregates in 4-5 day cultures were markedly different, i.e. small stellate cells with slender processes forming a delicate mesh throughout the aggregate. These GFAP-positive cells stained only weakly with vimentin antibodies. Spinal cord neurons formed aggregates on laminin substrates but failed to extend neurites and rapidly degenerated. The large flat cells in the surrounding monolayer gradually invaded the aggregates. These cells stained with both GFAP and vimentin antibodies. DRG neurons developed equally well on Primaria and laminin substrates, extending their neurites on the vimentin-positive flat cells forming the monolayer regardless of their reactivity with GFAP antibodies.

Animals↗
PubMed1989

A case of post-traumatic syringomyelia. Neuropathological findings after 1 year of cystic drainage.

A 58-year-old man sustained C4-C5 post-traumatic myelopathy with C3-C4 subluxation, slight compression of C3 vertebral body, C4 spinous process fracture and C6 compression fracture. He subsequently developed syringomyelia from C4 to C6, which was shunted into the subarachnoid space. Postoperatively, there was some but insignificant improvement of his symptoms although a postoperative metrizamide spinal computerised tomography showed complete drainage of the cyst. This patient died 1 year later. Autopsy examination of the spinal cord showed extensive damage of the posterior half of the cord at C3-C4 but the damage was much less extensive from C4 to C6 (where the syrinx was located), affecting mainly the right dorsal column at C4 and the right dorsal column and right anterior horn at C5-C6. In this patient, the syrinx developed in the partially damaged segments of the cord at the level of the spinal fractures and complete drainage of the cyst was not followed by satisfactory relief of his symptoms.

Humans↗
PubMed1989

Glial hyaluronate-binding protein in polar spongioblastoma.

Glial hyaluronate-binding (GHA) protein is a 60 kDa glycoprotein isolated from human white matter by affinity chromatography on immobilized hyaluronate. It is localized in white matter astrocytes by immunofluorescence with monoclonal antibodies. Amino acid sequences have not revealed similarities with other proteins except cartilage extracellular matrix proteins, the region of similarity being located within the hyaluronate-binding region. Cryostat sections of 13 intracranial neoplasms removed at surgery were tested for the presence of GHA protein by indirect immunofluorescence with monoclonal antibodies. These included seven astrocytomas, one oligodendroglioma, one medulloblastoma and one spinal cord ependymoma. All tumors were negative with the exception of one astrocytoma in which the GHA protein-positive areas had the typical appearance of polar spongioblastoma, i.e. small cells palisading around blood vessels and very delicate glial fibrillary acidic (GFA) protein-positive fibrils. Conversely, neoplastic as well as reactive GFA protein-positive astrocytes were GHA protein-negative. We suggest that polar spongioblastoma derives from a GHA protein-positive glial precursor and pertinent to this suggestion is the observation that the periventricular germinal layer was found GHA protein-positive in a 22-week human fetus.

Antibodies, Monoclonal↗
PubMed1988

Astroglia-induced detachment of central neurons but astroglia-dependent growth of peripheral neurons in rat embryonic spinal cord primary cultures.

In mixed primary cultures, intrinsic neurons from embryonic mammalian brains degenerate secondary to their detachment from the substratum and this is caused by the under-growing co-cultured astroglia. In the present study we sought to find out whether or not peripheral neurons, sensory and motor neurons which reside and/or only project outside the CNS respectively, interact with astroglia similarly as their central counterparts do. Mixed primary cultures prepared from dissociated embryonic rat spinal cord and dorsal root ganglia were examined by phase and immunofluorescence microscopy after labeling with antibodies to neurofilaments (neuronal markers) and to glial fibrillary acidic protein and vimentin (astroglia markers). Acetylcholinesterase staining served as a marker for motor neurons. In this system astroglia grew exclusively under intrinsic neurons of the spinal cord and with time (about 8 days) all these neurons detached and disappeared. In contrast, astroglia were intimately associated with perikarya of peripheral neurons, sometimes growing over them. Furthermore, the neuritic processes of these neurons were attached to the undergrowing astroglia. Central neurons could be rescued by treatment of cultures with the antimitotic drug cytosine arabinoside which led to the elimination of astroglia. However, this treatment resulted in death of all peripheral neurons. We conclude: (1) survival of intrinsic CNS neurons in culture is independent of astroglia; (2) astroglia are responsible for the detachment of these neurons from their growth substratum; (3) survival of peripheral sensory and motoneurons is dependent on co-cultured astroglia and (4) the differences in neuron-astroglia interactions between central and peripheral neurons are membrane-associated and probably independent of soluble factors.

Animals↗
PubMed1988

Neurofilament phosphorylation in axons and perikarya: immunofluorescence study of the rat spinal cord and dorsal root ganglia with monoclonal antibodies.

Rat dorsal root ganglia and spinal cord were stained with 12 monoclonal antibodies reacting with phosphorylated epitopes of two neurofilament proteins (NF 150K and NF 200K). Three monoclonal antibodies were axon-specific in both locations; neuronal perikarya were not stained. Nine monoclonal antibodies stained a subpopulation of neurofilament-positive sensory neurons, as indicated by double labeling experiments with polyclonal antibodies reacting with phosphorylated and dephosphorylated forms of the neurofilament protein triplet. Of these nine antibodies, two stained motor neuron perikarya in the spinal cord, while the remaining seven antibodies were axon-specific in this location. Subpopulations of stained and unstained motor neurons were not observed. With all 12 antibodies, the staining pattern in the lumbar dorsal root ganglia and spinal cord remained unchanged following sciatic nerve crush and ligature. The findings suggest that, in the neurofilament, some phosphorylated epitopes are axon specific, while other phosphorylated epitopes are present in both axons and perikarya. Furthermore, they suggest that differences exist between neuronal populations as to the presence of phosphorylated epitopes in perikaryal neurofilaments. It remains to be seen whether phosphorylation events in perikarya and axons have similar or different effects on neurofilament structure and function.

Antibodies, Monoclonal↗
PubMed1988

Expression of brain-specific hyaluronectin (BHN), a hyaluronate-binding protein, in dog postnatal development.

Monoclonal antibodies reacting with the brain-specific form of hyaluronectin, a hyaluronate-binding protein, were used in conjunction with antibodies to the glial fibrillary acidic protein (GFAP), the subunit of astrocyte-specific intermediate filaments, to study the postnatal development of spinal cord and cerebral white matter in the dog. As previously reported, the distributions of brain-specific hyaluronectin (BHN) and GFAP in adult dog spinal cord white matter were similar. Both antigens formed a mesh surrounding individual myelinated axons. Furthermore, the glia limitans on the surface of the spinal cord and the glial septa were stained by both antibodies. In newborn dog spinal cord, hyaluronectin immunoreactivity was confined to the glia limitans on the surface. At this time GFAP-positive fibers formed a dense mesh throughout the myelinated white matter. Staining of spinal cord white matter with BHN antibodies first appeared on day 15 and reached its mature appearance in the fully myelinated spinal cord on day 21. In cerebral white matter BHN immunoreactivity was first observed on day 21. With GFAP antibodies astrocytes were extremely few in the nonmyelinated cerebral white matter of 1- and 3-day-old dogs. GFAP-positive astrocytes in cerebral white matter had markedly increased on day 9 before the onset of myelination on day 15. On day 21, myelination was confined to deep cerebral white matter and myelin sheaths were still very few in subcortical white matter. We conclude that BHN expression by white matter astrocytes is an extremely late event in brain development, first occurring after the onset of myelination.

Aging↗
PubMed1987

Axonal maturation in development--II. Immunofluorescence study of rat spinal cord and cerebellum with axon-specific neurofilament antibodies.

Neurofilament monoclonal antibodies derived from mice immunized with chicken brain antigen or purified bovine NF 150K and NF 200K either stained only axons or they stained neuronal perikarya, dendrites and axons. Antibodies in the second group were called conventional because they decorated tissue sections like the neurofibrillary methods of traditional histology. Axon-specific antibodies either reacted with phosphorylated epitopes or they were phosphate/phosphatase insensitive thus suggesting reactivity with post-translational modifications other than phosphorylation. Another possibility was reactivity with phosphorylated epitopes inaccessible to exogenous phosphatases. Conventional neurofilament antibodies stained motor and sensory neurons in day 12 and day 13 rat embryos, respectively, as previously reported with neurofilament antisera. Immunoreactivity with axon-specific antibodies first appeared in motor and sensory axons at different times in development: day 13-14 (3 monoclonals); day 17 (6 monoclonals); day 21 (1 monoclonal); postnatal day 2 (1 monoclonal). There were no major differences between conventional and axon-specific antibodies as to the time of appearance of Purkinje cell baskets in postnatal rat cerebellum. With two exceptions all monoclonals first stained thin baskets on day 11. Immunoreactivity of Purkinje cell baskets with two monoclonals reacting with phosphorylated NF 200K first appeared on days 14 and 20. It is suggested that post-translational modifications may stabilize the neurofilaments, thus accounting for their late appearance by electron microscopy in development.

Animals↗
PubMed1986

Neurofilament proteins in fish: a study with monoclonal antibodies reacting with mammalian NF 150K and NF 200K.

Monoclonal antibodies were obtained upon immunization of mice with chicken brain antigen and with the two high molecular weight neurofilament proteins (NF 150K and NF 200K) isolated from bovine spinal cord by anion exchange chromatography. By the immunoblotting procedure, the antibodies selected for this study reacted with bovine NF 150K and NF 200K. By the same procedure the antibodies reacted with sea raven, goldfish, sea bass, shark, and trout spinal cord extracts. In goldfish and sea raven the antibodies stained a single band at approximately 150 kDa and 200 kDa, respectively. Two bands were stained in the shark, sea bass, and trout. In the shark and sea bass these bands were in the molecular weight range of mammalian NF 150K and NF 200K. In the trout the upper band was approximately 150 kDa and the lower band 130 kDa. Our findings suggest an early origin of NF 150K and NF 200K in vertebrate phylogeny as well as considerable divergence in several species.

Animals↗
PubMed1986

Neurofilament phosphorylation in peripheral nerve regeneration.

A monoclonal antibody to the 200 kdalton neurofilament (NF) polypeptide selectively decorated axons in tissue sections. Neuronal perikarya and dendrites, including motor and sensory neurons reacting to axotomy, were not stained. Axonal staining was abolished by dilution of the monoclonal supernatants with phosphate buffer and by digestion of tissue sections with phosphatase, thus suggesting that the antibody reacted with a phosphorylated epitope. Conventional monoclonal and polyclonal antibodies, i.e. antibodies decorating NF regardless of their location (axons, perikarya and dendrites) were not affected by these procedures. Compared to conventional NF antibodies, staining with the axon-specific monoclonal antibody was a late event in peripheral nerve regeneration. One week after operation, the whole distal stump of crushed rat sciatic nerve was invaded by bundles of axons strongly reacting with conventional NF antibodies. Axon-specific NF immunoreactivity was confined to the proximal segment of the stump at this time and progressively extended distally in the following week. Furthermore, NF phosphorylation appeared to coincide with the return of a normal nerve structure as evidenced by the distribution of laminin immunoreactivity. Bundles of axons growing within columns of laminin-positive Schwann cells did not stain with the axon-specific NF antibody. Immunoreactivity with this antibody coincided with the return of a normal laminin pattern, i.e. selective decoration of the endoneurial basal membranes surrounding the axons.

Animals↗
PubMed1986

Delayed phosphorylation of the largest neurofilament protein in rat optic nerve development.

Monoclonal antibodies selectively reacting with the high molecular weight neurofilament proteins (NF 150K and NF 200K) on immunoblots of bovine spinal cord extracts were obtained upon immunization of mice with chicken brain antigen and with highly purified NF 150K or NF 200K isolated from bovine spinal cord by anion exchange chromatography. Antibodies reacting with NF 200K or with both NF 150K and NF 200K were selected for this study. The antibodies were screened on immunoblots for reactivity with phosphorylated epitopes by dilution of the supernatants in sodium potassium phosphate as well as by treatment of nitrocellulose transfers with alkaline phosphatase. Abolishment of staining under these conditions was taken as evidence of reactivity with phosphorylated epitopes. With phosphate/phosphatase-sensitive antibodies, NF 200K immunoreactivity was a late event in rat optic nerve development. It was first observed at day 18 on immunoblots of sodium dodecyl sulfate extracts analyzed by gel electrophoresis. Conversely, with phosphate/phosphatase-insensitive antibodies, NF 200K immunoreactivity was already present on day 10, the earliest age in this study. With one monoclonal reacting with phosphorylated NF 150K and NF 200K, NF 150K immunoreactivity was already present on day 10. It is proposed that NF 200K expression precedes NF 200K phosphorylation in development.

Aging↗