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Biomedical subjects

A Bignami

Publications and source records attributed to A Bignami.

At least 55 records · Page 3Linked to original sources
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

Non-phosphorylated and phosphorylated neurofilaments in hypothyroid rat cerebellum.

Purkinje cell baskets in hypothyroid rat cerebellum were studied with antibodies reacting with phosphorylated or non-phosphorylated neurofilament epitopes. Compared to normal rats, Purkinje cell baskets were fewer in number and less developed in hypothyroid rat cerebellum. However, no differences were observed as to their immunoreactivity with monoclonal antibodies reacting with phosphorylated or non-phosphorylated neurofilament epitopes.

Animals↗
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↗
PubMed1986

Heterogeneity of desmin, the muscle-type intermediate filament protein, in blood vessels and astrocytes.

Monoclonal antibodies were isolated from mice immunized with chicken gizzard desmin. Antibodies reacting with desmin on immunoblots and selectively decorating chicken and rat intestinal smooth muscle as well as the Z-line in striated muscle, were selected for this study. Based on their staining pattern on cryostat sections of chicken and rat cerebellum, spleen, kidney, aorta and femoral artery, monoclonal supernatants could be divided in three groups: (i) antibodies decorating astrocytes and vascular smooth muscle; (ii) antibodies decorating only vascular smooth muscle; (iii) antibodies decorating only astrocytes. Antibodies in group (i) and (iii) also stained GFA-negative Bergmann glia in chicken cerebellum. It is proposed that desmin may vary depending on the histological localization.

Animals↗
PubMed1986

Brain-specific hyaluronate-binding protein. A product of white matter astrocytes?

The distribution of glial fibrillary acidic (GFA) protein and hyaluronectin, a hyaluronate-binding protein isolated from human brain, was compared in brain, spinal cord and optic nerves of pigs and dogs by indirect immunofluorescence with monoclonal antibodies. In spinal cord white matter the localization of the two proteins was similar, both antigens forming a mesh surrounding myelinated axons. A similar distribution of the two proteins was also observed in the periventricular glia as well as in the glia limitans of spinal cord and optic nerves. Cerebral white matter was hyaluronectin-positive, but the GFA-positive stellate astrocytes did not stain with hyaluronectin antibodies in this location. Hyaluronectin antibodies did not stain grey matter, the granular layer of the cerebellum excepted. The astrocytes identified with GFA antibodies in hyaluronectin-negative grey matter were: the fibrous astrocytes forming the glia limitans on the surface of the cerebral hemispheres; the protoplasmic astrocytes of cerebral isocortex and basal ganglia; the fibrous astrocytes of cerebral allocortex (hippocampus); Bergmann radial glia in the molecular layer of the cerebellar cortex; and fibrous astrocytes of spinal cord anterior and posterior horns. It is concluded that the hyaluronectin fraction reacting with the monoclonal antibodies is a brain-specific protein probably produced by white matter astrocytes. We propose to call this fraction brain-specific hyaluronectin, to be distinguished from other fractions reacting with polyclonal antibodies and with different localizations.

Animals↗
PubMed1986

Neurofilament phosphorylation in development. A sign of axonal maturation?

Monoclonal antibodies to the 200K neurofilament (NF) protein selectively decorated axons in tissue sections. Dilution of the antibodies in phosphate buffer and digestion with phosphatase abolished the stain. With conventional monoclonal and polyclonal NF antibodies, i.e. antibodies decorating NF regardless of their location (axons, perikarya and dendrites), the staining was not affected by this treatment. With all antibodies, axon-specific and conventional, the staining was abolished by trypsin digestion. Subsequent digestion with phosphatase did not restore the staining. Compared with conventional NF antibodies, staining with axon-specific anti-NF 200K was a late phenomenon in chick embryo development. NF 200K immunoreactivity was first observed in peripheral nerves and in the anterior columns of the spinal cord on day 10. Sensory ganglia and optic nerve fibers were negative. With conventional NF antibodies these structures were stained on days 4 and 5, respectively. In the following days of development the study was confined to the retina, optic nerves, cranial peripheral nerves and sensory ganglia. Up to day 16, bundles of thin peripheral nerve fibers, strongly decorated by conventional NF antibodies, did not stain with anti-NF 200K in double labelling experiments. Nerve bundles emerging from the ganglia were also negative, although some thick nerve fibers within the ganglia were stained. NF 200K immunoreactivity was first observed on day 17 in the optic nerve and in the layer of optic nerve fibers. At this time, staining was confined to the bundle emerging from the temporal side of the retina. In newborn chicken, only few fibers stained with anti-NF 200K in the nasal bundle, while the temporal bundle was well stained. It is suggested that the NF 200K antibodies reacted with a phosphorylated epitope in the axon, and that NF phosphorylation is a late event in ontogenesis probably related to axonal maturation.

Animals↗
PubMed1986

Brain-specific hyaluronate-binding protein: an immunohistological study with monoclonal antibodies of human and bovine central nervous system.

Hyaluronectin is a protein isolated from acid extracts of human brain by affinity chromatography on immobilized hyaluronate. With polyclonal antibodies, it was immunohistologically localized in the rat at the nodes of Ranvier of central and peripheral myelinated fibers and in mesenchymal tissues. Compared to adult rat, hyaluronectin-immunoreactive material was more abundant in embryonal rat brain and mesenchyma. We report a different localization in human and bovine tissues with monoclonal antibodies reacting with human hyaluronectin by NaDodSO4/PAGE and immunoblotting but not staining rat tissues by immunohistology. In human and calf the antigen reacting with hyaluronectin monoclonal antibodies was brain specific, while several peripheral tissues were stained by the polyclonal antibodies. In human and bovine central nervous system monoclonal antibodies stained white matter and tissues formed predominantly by glial fibers (e.g., subependymal glia). In white matter hyaluronectin-immunoreactive material formed a delicate mesh surrounding individual myelinated fibers, a pattern compatible with the distribution of fine astroglial processes in this location. Gray matter did not stain with monoclonal antibodies, the granular layer of the cerebellum excepted. The findings suggest that human hyaluronectin is heterogeneous and comprises at least two fractions. The main fraction is a brain-specific protein, probably produced by white matter astrocytes. Another fraction cross-reacting with rat is more abundant in embryonal tissues, including mesenchyma and brain.

Animals↗
PubMed1985

Glial fibrillary acidic (GFA) protein in vertebrates: immunofluorescence and immunoblotting study with monoclonal and polyclonal antibodies.

We report a comparative immunofluorescence and immunoblotting study of GFA protein, the subunit of glial filaments, in nonmammalian vertebrates. The study was conducted with polyclonal antibodies raised to human and shark antigen and with monoclonal antibodies isolated from mice immunized with chicken and bovine antigen. With the exception of cyclostomes, glial filaments appeared remarkably conserved in vertebrate phylogeny, both with respect to the molecular weight and immunoreactivity of their protein subunit. In most species, the antibodies decorated a single band in brain, spinal cord, and optic nerve extracts by the immunoblotting procedure. This band had the same molecular weight in the different CNS regions. With the exception of the turtle, species differences in the molecular weight of the band were not greater than those observed among mammalian vertebrates (human, bovine, and rat). However, there were some exceptional findings in fish. In goldfish and trout brain and spinal cord extracts, the antibodies decorated with the same intensity two bands. In accordance with previous immunofluorescence findings, goldfish optic nerve extracts were negative by the immunoblotting procedure. In four fishes (sea bass, tautog, trout, and scup), optic nerves reacted with the antibodies. However, the band decorated by the antibodies was higher in molecular weight than that obtained from brain and spinal cord extracts. Glial fibers were demonstrated by immunofluorescence in the brain, spinal cord, optic nerve, and retina of most species studied. In amphibia immunofluorescent structures were comparatively few, probably accounting for the negative results by immunoblotting. A comparative immunohistological study of the cerebellum showed the presence of perpendicular glial fibers in the molecular layer of most species examined. Birds and amphibia were different in this respect. Bergmann glia in chicken were GFA negative. In the frog and the toad, immunofluorescent fibers in the molecular layer of the cerebellum were haphazardly oriented. Ependymal radial glia was GFA-negative in the cerebellum of subavian vertebrates. Antisera raised in rabbit to shark GFA protein reacted with the same bovine GFA fragments recognized by polyclonal and monoclonal antibodies raised to human and bovine antigens, respectively, i.e., 30-kDa N-bromosuccinimide fragment (tryptophan cleavage); 35-kDa 2-nitro-5-thiocyanobenzoic acid fragment (cysteine cleavage); 18-kDa cyanogen bromide fragment (methionine cleavage). Conversely, the chicken GFA monoclonal antibodies selected for this study only reacted with noncleaved protein.

Animals↗
PubMed1985

Immunohistochemical demonstration of glial fibrillary acidic protein in normal rat Müller glia and retinal astrocytes.

The presence of glial fibrillary acidic protein (GFA)-positive Müller glia and retinal astrocytes were studied immunohistochemically in normal rat retina. Using GFA antiserum both Müller glia and separate star-shaped cells were observed in spread-preparations as well as cryostat sections. The retinal astrocytes were also visualized using two different monoclonal GFA antibodies. These cells were found to be located in the nerve fiber and ganglion cell layers. In contrast, Müller glia were not normally visualized with any of the monoclonal GFA antibodies but could be stained 4 days after an optic nerve crush. Our results demonstrate that normal rat Müller glia expresses GFA-like immunoreactivity.

Animals↗