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Biomedical subjects

D Dahl

Publications and source records attributed to D Dahl.

At least 127 records · Page 7Linked to original sources

The 200- and 150-kDa neurofilament proteins react with IgG autoantibodies from patients with kuru, Creutzfeldt-Jakob disease, and other neurologic diseases.

Sera from 65 patients with spongiform virus encephalopathies (29 with kuru, 36 with Creutzfeldt-Jakob disease), 79 with other neurologic diseases, and 65 control subjects were examined for reactivity in immunoblots of preparations of myelinated axons and neurofilaments from mouse brain. The sera reacted most frequently with the 200-kDa and 150-kDa neurofilament proteins and less frequently with the 70-kDa neurofilament protein and a 62-kDa neurofilament-associated protein. The sera reacted with the same proteins as those which reacted with rabbit and mouse polyclonal antibodies and mouse monoclonal antibody to neurofilament proteins. Serum reactions were also seen with Trixon X-100 extracts of chimpanzee brain and bovine spinal cord but not with Triton extracts of liver, kidney, and muscle.

Alzheimer Disease↗

The 200- and 150-kDa neurofilament proteins react with IgG autoantibodies from chimpanzees with kuru or Creutzfeldt-Jakob disease; a 62-kDa neurofilament-associated protein reacts with sera from sheep with natural scrapie.

Sera from 46 chimpanzees with spongiform encephalopathy (18 kuru, 28 Creutzfeldt-Jakob disease) and sera from 12 sheep with natural scrapie were tested for reactivity with immunoblots of neurofilament preparations obtained from mouse brain. The sera from the chimpanzees reacted mainly with the 200- and 150-kDa proteins of the neurofilament triplet and less frequently with the 70-kDa component of the triplet and with a 62-kDa neurofilament-associated protein. In contrast, the sera of sheep with natural scrapie reacted exclusively against the 62-kDa protein. The specificity of the reactions was established by comparison of sera reactivities with those of rabbit and mouse polyclonal antibodies and mouse monoclonal antibody to neurofilament proteins.

Animals↗

Primary neuroendocrine (Merkel cell?) carcinoma of the skin. II. An immunocytochemical study of 21 cases.

Twenty-one examples of neuroendocrine carcinoma of the skin were examined by the unlabeled antibody enzyme method for several neural hormones and peptides, carcinoembryonic antigen, S-100 protein, neuron-specific enolase, and three intermediate filaments: neurofilament, glial fibrillary acidic protein, and cytokeratin. Vasoactive intestinal polypeptide from two sources reacted with the neoplastic cells of four (18%) and seven (32%) of the cases, and pancreatic polypeptide reacted with scattered cells of one case. Neuron-specific enolase reactivity occurred in 50% of the cases. Neurofilament (70, 150, 200 kilodaltons) was strongly positive in 40% of the tumors whereas neurofilament (200 kilodaltons) was negative. Two monoclonal anticytokeratin antibodies of 54 kilodaltons and 44-54 kilodaltons reacted in 77% and 64% of the cases, respectively, in a distribution similar to the neurofilament. Sections reacted with antisera against cytokeratins of higher molecular weight were negative. The demonstration of vasoactive intestinal polypeptide, pancreatic polypeptide, neurofilament, and neuron-specific enolase is evidence of the neuroendocrine nature of this neoplasm.

Carcinoma↗

Intermediate filaments in cultured human pleomorphic adenomas. An immunohistochemical study.

Primary cultures of 9 benign human pleomorphic adenomas were analyzed by immunofluorescence and antibodies against the different intermediate filaments and microfilaments. The cultured cells were also cytogenetically characterized by G- and C-banding techniques. The adenoma cells expressed at least three different classes of intermediate filaments, viz. prekeratin, vimentin and glial fibrillary acidic protein (GFA). No correlation between chromosomal pattern and intermediate filament expression was found. The frequency of cells stained for each of the different antibodies varied considerably among the tumours. Prolonged culturing appeared to induce a reduction of cells positive for prekeratin. This was probably a true loss of antigens and not an effect of overgrowth of stromal cells. It is concluded that the cells studied, on the basis of several criteria, are of neoplastic origin. This in vitro system appears to be well suited for further histogenetic studies on pleomorphic adenomas.

Actin Cytoskeleton↗

Varying expression of cytokeratin and neurofilaments in neuroendocrine tumors of human gastrointestinal tract.

Twelve cases of gastrointestinal neuroendocrine tumors, including eight carcinoids and four pancreatic islet cell tumors or their metastases, were immunohistochemically analyzed for the expression of different types of intermediate filament proteins. All of the tumors showed cytokeratin positivity in immunostaining, and the Western blotting technique revealed 45- and 52-kilodalton cytokeratins in carcinoid tumors. Three of the islet cell tumors, but none of the carcinoid tumors, showed, in addition, varying numbers of neurofilament-positive tumor cells when evaluated with rabbit and mouse monoclonal antineurofilament antibodies. The presence of only the 70-kilodalton neurofilament and cytokeratin polypeptides in an islet cell tumor was revealed also by using the Western blotting technique. On the other hand, both fetal and adult pancreatic islet cells showed only cytokeratin positivity. Neurofilament-positive epithelial cells were not found in normal small intestines either. The results show epithelial characteristics in normal gastrointestinal neuroendocrine cells and neuroendocrine tumors by their expression of cytokeratin. In addition, some islet cell tumors display the 70-kilodalton neurofilament protein which suggests the acquisition of a new type of intermediate filament during the neoplastic change.

Adenoma, Islet Cell↗

The relationship of bovine intermediate filament proteins. A comparative analysis of glial fibrillary acidic protein, desmin and the neurofilament 70 kDa protein.

Three bovine intermediate filament proteins, glial fibrillary acidic protein, desmin and the 70 kDa component of the neurofilament are compared by cleavage at cysteine and tryptophan. The results of these experiments show that the difference in molecular weight between the glial fibrillary acidic protein and desmin is due to a longer portion of the desmin amino terminal to the tryptophan. On the other hand, the 70 kDa protein contains a carboxy terminal addition. The tryptophan and cysteine contents of these proteins are also determined by amino-acid analysis. Differences in the apparent amount of cysteine determined by these methods in the glial fibrillary acidic protein and 70 kDa proteins are discussed. Interchain disulfide bonds result in the formation of dimers in glial fibrillary acidic protein. The bovine 70 kDa neurofilament protein and desmin also form dimers under nonreducing conditions. This emphasizes the structural similarity of these intermediate filament proteins.

Animals↗

Bronchial carcinoid cells contain neural-type intermediate filaments.

Monospecific antibodies and indirect immunofluorescent microscopic examination, combined with immunochemical analysis, were used to examine intermediate filaments in four cases of bronchial carcinoid tumors. The results show that carcinoid cells express intermediate filaments of neural type (neurofilaments) but are negative for intermediate filaments of mesenchymal type (vimentin), epithelial type (keratin), muscle type (desmin), and glial type (glial fibrillary acidic protein). Since the expression of intermediate filaments shows a high degree of tissue specificity, the results suggest either derivation of bronchial carcinoid cells from maternal cells displaying neural characteristics or from cells with the capacity to acquire neural properties on neoplastic growth. It is also suggested that antineurofilament antibodies can be used as a useful aid in differential diagnosis of bronchial carcinoids from other pulmonary tumors.

Adolescent↗

Expression of the 70 kdalton neurofilament protein in clonal lines of mouse neuroblastoma.

The expression of the neurofilament (NF) protein triplet (70, 150 and 200 k daltons (K)) was studied by immunofluorescence in two clones of murine C-1300 neuroblastoma. Clone 1 formed clumps of round cells with few processes, while in clone 2 the cells were more dispersed and extended many processes. In both clones selective decoration of tight bundles of filaments was only observed with anti-NF 70 K. Round cells contained curvilinear bundles with the appearance of whorls and ringlets , while in cells extending processes the bundles were more rectilinear in shape. Ringlets were the most prominent feature in large clumps of cells. Antisera to vimentin, the mesenchymal-type intermediate filament (IF) protein, decorated the neuroblastoma cells with a different pattern. Treatment of the two clones with dibutyryl cAMP, a neurite-inducing agent, did not result in the expression of NF 150 K and NF 200 K, although many cells had extended processes and the shape of the NF bundles had changed accordingly.

Animals↗

Appearance and distribution of neurofilament immunoreactivity in iris nerves.

We have used antiserum raised against neurofilament (NF) protein and indirect immunofluorescence techniques to visualize neuronal structures in rodent, cat, and cow irides. In the adult rat iris a large population of nerve fibers with a nonautonomic distribution show NF-like immunoreactivity. In whole mounts, smooth fluorescent fibers were seen in a fine-meshed plexus from the sphincter margin to the ciliary processes. Superimposed, a sparse pattern of thick meandering axon bundles were seen. Electroblotting and peroxidase immunochemical staining techniques unequivocally showed the presence of all three NF polypeptides in the adult rat iris. Adult mouse irides showed a somewhat sparser pattern of NF-positive nerves than that of the rat. Adult guinea pig irides contained irregular NF-positives fibers and few axon bundles. In cryostat-sectioned cat iris numerous irregularly distributed individual fibers were found, whereas in similarly sectioned cow iris thick NF-positive axon bundles were more numerous. By embryonic day 18 numerous sparse NF-positive axons were seen, and the subsequent gradual increase in both axons in bundles and fine-meshed plexuses of individual fibers produced an appearance similar to that in the adult by 6 days of postnatal age. One week after grafting of irides to the anterior eye chamber, most NF-positive nerves had disappeared from the iris grafts. Sympathetic and parasympathetic denervation of the irides did not influence the distribution of the NF-positive iris nerves. Five days after electrothermal lesion of the trigeminal nerve just distal to its ganglion a large proportion of the NF-positive nerves had disappeared from the iris. All perikarya in the parasympathetic ciliary and most perikarya in the superior cervical sympathetic and in the trigeminal sensory ganglion showed NF immunoreactivity. The present report shows a way to visualize nonautonomic nerve populations in stretch-prepared as well as sectioned irides by immunofluorescence techniques using an antiserum to neurofilament protein.

Animals↗

Laminin is induced in astrocytes of adult brain by injury.

Laminin is a high mol. wt. non-collagenous matrix glycoprotein, confined in adult tissues to basement membranes. In normal rat brain we found laminin mainly in vessel walls but, after injury, induced by stereotaxic injection of a neurotoxin, laminin immunoreactivity appeared also in reactive astrocytes, which are characteristically positive for the glial fibrillary acidic protein (GFAP). Laminin was first detected in GFAP-immunoreactive glial cells 24 h after injury. Four days later the majority of reactive astrocytes in the gray matter were positive for laminin and the laminin immunoreactivity, but not that of GFAP, gradually subsided within a month. Fibronectin, the other major matrix glycoprotein, was found only in capillary structures both in normal and lesioned brain tissue. The results indicate that mature astrocytes have the potential to produce laminin and suggest a role for this glycoprotein in brain regeneration.

Animals↗

Formation of 10-nanometer filaments from the 150K-dalton neurofilament protein in vitro.

In the present study we report self-assembly of individual neurofilament (NF) triplet proteins (70K, 150K, and 200K daltons) isolated by anion exchange chromatography from bovine spinal cord. Formation of smooth 10-nm filaments by both NF 150K and NF 70K is shown. Optimal conditions for NK 150K filament formation were incubation in 100 mM MES, 0.2 M NaCl, 1 mM DTT, 0.5 mM EGTA, pH 6.5, at 37 degrees C for 24 hr. Under the same assembly conditions, NF 200K formed 7-nm coiled structures. These thin filaments were similar to those formed by NF 70K and 150K under less than optimal conditions. Our results indicate that NF 150K is an integral part of the filament (self-assembly of NF 70K was previously demonstrated by others). We suggest that the optimal conditions resulting in the formation of a 10-nm 200K homopolymer remain to be determined and that the thin coiled structures formed by all three NF proteins are protofilaments that coalesce to form a double helical 10-nm filament.

Animals↗

Neurons cultured from developing rat brain attach and spread preferentially to laminin.

Neurons, mechanically dissociated from newborn rat brain and identified by immunostaining for neurofilaments, attached preferentially to laminin-coated coverslips without need of an underlying glial monolayer. The most extensive neurite outgrowth was seen when 20-30 micrograms/ml of laminin was used to coat the coverslips. Higher concentrations of laminin (greater than 30 micrograms/ml) supported single neurons to spread on the coverslips. Fibronectin coating of the coverslips allowed glial cells to attach more rapidly than on uncoated surfaces, but it did not support neuronal spreading or neurite outgrowth. Spreading of neurons and neurite outgrowth were completely inhibited by preincubation of laminin-coated coverslips with laminin antibodies but were unaffected by fibronectin antibodies. These results indicate that laminin is an attachment and spreading factor for central neurons in culture and suggest the presence of a laminin receptor on the neuronal cell surfaces.

Animals↗

Intracranial cerebellar grafts: intermediate filament immunohistochemistry and electrophysiology.

Pieces of the developing cerebellar anlage were prepared from 13-15 day old rat embryos and transplanted to the cerebellar region of 5-7 and 13-14 day old rat pups. Approximately two months later, sections showed most grafts to consist of both cerebellar cortex, with a typical trilaminar organization, and white matter areas containing large neuronal perikarya. The astrocytic populations were studied using immunohistochemistry with antisera raised against the intermediate filaments, glial fibrillary acidic protein (GFA), and vimentin. The GFA-antiserum revealed a glial interface along most of the border between host brain and graft. Both antisera stained long, slender, although slightly distorted Bergmann fibers spanning the molecular layer. Using GFA-antiserum, star-shaped fluorescent astrocytes were seen in the granular layer and in the white matter. Only in the white matter did the amount of GFA-like immunoreactivity suggest an astrocytic gliosis. With vimentin antiserum fluorescent astrocytes in the white matter were seen. There were no signs of increased amounts of vimentin-like immunoreactivity. Taken together, the amount and distribution of GFA- and vimentin-like immunoreactivity suggests a rather normal astrocytic development in the cerebellar grafts. Using an antiserum against the neurofilament (NF) triplet, delicate immunoreactive fibres were seen in both the molecular and the granular layer. No positive cell bodies could be visualized in the cortical areas. Although the Purkinje cells themselves were negative, fibre baskets around them were intensely stained. In the white matter a high density of NF-positive fibres and some positive perikarya were visualized. Thus the distribution of NF-like immunoreactivity in the grafts corresponded well to the normal NF distribution. The functional maturation of the cerebellar grafts was studied electrophysiologically. A spontaneous mean discharge rate of 19.3 + 1.7 Hz was recorded from the Purkinje cells. This compares with a discharge rate of 26.8 + 1.0 Hz for Purkinje neurons in situ. The difference was at least partly ascribable to the absence of climbing fibre bursts in the grafts. Local stimulation of the graft surface caused both decreased and increased Purkinje cell discharge. In conclusion, these experiments suggest that grafts of fetal cerebellar buds to the young cerebellum develop into cerebellar tissue having both morphological and electrophysiological characteristics quite similar to the normal cerebellum.

Animals↗

Masking of epitopes in tissue sections. A study of glial fibrillary acidic (GFA) protein with antisera and monoclonal antibodies.

Antisera to chicken brain antigen (CBA) isolated by hydroxyapatite chromatography from 8 M urea extracts following repeated extractions with phosphate buffer selectively decorate neurofilaments (NF) in neuronal perikarya, dendrites and axons. The antisera also reacted with GFA protein, the astrocyte-specific intermediate filament protein, as indicated by the adsorption of NF immunoreactivity following passage of the antisera through columns prepared with purified GFA protein. Moreover, the antisera stained the polypeptides of the NF triplet (70 kd, 150 kd, 200 kd) and GFA protein by the immunoblotting procedure. Monoclonal antibodies selectively decorating NF in tissue sections were isolated from a fusion of mouse myeloma cells with spleen cells of mice immunized with CBA. By the immunoblotting procedure the antibodies decorated the 150 kd NF polypeptide and GFA protein. No staining of glial filaments or any other structure on tissue sections was also observed with antibodies derived from another fusion strongly reacting with GFA protein on immunoblots. All antibodies (monoclonal and polyclonal) appeared to react with the same region of the GFA polypeptide as indicated by immunoblots of cleavage products.

Animals↗

Distribution of neurofilament-immunoreactive nerve fibers in human skin.

Neurofilament immunoreactive nerve fibers were demonstrated in human skin using indirect immunohistochemical technique with antibodies to neurofilament polypeptides. Neurofilament-positive fibers were seen as free nerve endings in the epidermis and in dermal papilla, in Meissner's corpuscles and as fibers crossing in the dermis. Strongly fluorescent nerve fibers were also seen around hair follicles, sweat gland ducts and sometimes in relation to blood vessels. From the distribution pattern it was concluded that predominantly sensory nerve fibers were labelled and that this technique may be used to study reinnervation of cutaneous sensory nerves following traumatic injuries and surgical procedures.

Fluorescent Antibody Technique↗

Amyloid-like (Congophilic) neurofibrillary tangles do not react with neurofilament antisera in Alzheimer's cerebral cortex.

The immunohistological properties of Alzheimer's neurofibrillary tangles (NFT) were studied by immunofluorescence with neurofilament (NF) antisera and with antiserum raised to paired helical filaments (PHF) in NFT preparations, brain smears, and cryostat sections. NFT decorated by NF antisera were Congo red-negative. Conversely, PHF antisera stained Congo red-positive NFT but failed to decorate NF-positive NFT. It is concluded that NF do not cross react with typical NFT, i.e., NFT displaying amyloid-like birefringence, under the conditions reported in this study.

Alzheimer Disease↗

Neuronal and glial markers in tumours of neuroblastic origin.

The presence and distribution of different neural markers in 30 neuroblastic tumours (neuroblastomas, ganglioneuroblastomas) and 6 non-neuroblastic tumours were investigated by immunocytochemistry. Neuron-specific enolase (NSE), S-100 protein, tyrosine hydroxylase, neurofilaments and glial fibrillary acidic protein (GFAP) were localised in 3 undifferentiated neuroblastic tumours (group A), 12 poorly differentiated tumours (group B) and 15 well differentiated neuroblastic tumours (group C). Non-neuroblastic tumours (3 lymphomas and 3 Ewing sarcomas) showed no immunoreactivity. Tyrosine hydroxylase and, in particular, NSE were found in mature ganglion cells and developing neuroblasts of poorly and well differentiated tumours (groups B and C). S-100 was localised in neuroblasts with slender cytoplasmic processes in the same groups. Neurofilaments were detected in ganglion cells and differentiated neuroblasts (groups B and C) while GFAP was localised in immature neuroblasts of undifferentiated and poorly differentiated tumours (groups A and B). Thus, there are differences in the neural proteins found in neuroblastic tumours and a wide panel of antibodies against neural markers may be a useful tool in the histological assessment of nervous system neoplasms.

Adolescent↗