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Biomedical subjects

M Bothwell

Publications and source records attributed to M Bothwell.

At least 73 records · Page 4Linked to original sources
PubMed1989

Nerve growth factor receptor immunoreactive profiles in the normal, aged human basal forebrain: colocalization with cholinergic neurons.

A monoclonal antibody raised against the receptor for nerve growth factor (NGF) has been used to map the distribution of NGF receptor-containing profiles within the human basal forebrain of four male and three female elderly patients without neurologic or psychiatric illness. Immunohistochemically processed tissue reveals a continuum of NGF receptor-positive neurons located within the medial septum, vertical and horizontal limb nuclei of the diagonal band, and nucleus basalis. NGF receptor-containing neurons are also found within the bed nucleus of the stria terminalis, the anterior commissure, the internal capsule, and the internal and external medullary laminae of the globus pallidus. Virtually all (greater than 95%) NGF receptor-containing neurons colocalize with the specific cholinergic marker choline acetyltransferase (ChAT) or the nonspecific marker acetylcholinesterase (AChE). Conversely, a few cholinergic perikarya are found which are not NGF receptor positive (and vice versa). These findings demonstrate that human basal forebrain neurons on which NGF receptor immunoreactivity is detected are primarily cholinergic and analogous to the nonhuman primate Ch1-Ch4 subgroups of Mesulam et al. (J. Comp. Neurol., 214:170-197, '83). NGF receptor-containing fiber tracts are observed emanating from the medial septum and vertical limb nucleus of the diagonal band coursing medially within the fornix. Another fascicle originating mainly from the nucleus basalis and travelling within the external capsule enroute to the cortex is observed innervating all cortical layers. Comparison of NGF receptor- and ChAT-containing neurons reveals cholinergic perikarya within the striatal complex, whereas virtually no NGF receptor-containing neurons are found in these structures. An occasional displaced NGF receptor-containing neurons is seen in the ventrolateral portion of the putamen and the white matter underlying the nucleus accumbens. These data are discussed in terms of the relationship of NGF receptor- and ChAT-containing neurons within the basal forebrain and in terms of the possible functional significance of NGF in normal and diseased brain.

Acetylcholinesterase↗
PubMed1989

Loss of nerve growth factor receptor-containing neurons in Alzheimer's disease: a quantitative analysis across subregions of the basal forebrain.

Magnocellular neurons comprising the Ch1-Ch4 regions of the basal forebrain provide topographic cholinergic innervation to the cerebral cortex, thalamus, and basolateral nucleus of the amygdala. Most quantitative studies analyzing the status of these neurons in Alzheimer's disease (AD) have employed Nissl-stained preparations. These studies principally analyzed large neurons of a prespecified cell diameter. Since basal forebrain neurons atrophy in Alzheimer's disease, an immunocytochemical marker for these neurons would appear to be a better alternative for determining whether there is regionally specific degeneration of cholinergic neurons across subregions of the basal forebrain. Brain sections from seven AD and five aged-matched control patients were immunocytochemically stained with a monoclonal antibody raised against the receptor for nerve growth factor (NGF), a probe which has previously been demonstrated to extensively and exclusively colocalize with cholinergic basal forebrain neurons in humans (17, 25, 35). NGF receptor-immunoreactive neurons within the hippocampal projecting nuclei of the medial septum (Ch1) and vertical limb of the diagonal band (Ch2) were minimally affected in AD as compared to control cases. In contrast, the Ch4 region demonstrated a significant loss of NGF receptor-immunoreactive neurons in AD that inversely correlated (-0.786) with the duration of the disease process. All four subregions of Ch4 were affected in the AD cases with the anterolateral (76.4%), intermediate (62.1%) and posterior divisions (76.5%) demonstrating the greatest reduction in NGF receptor-immunoreactive neurons. Nissl-counterstained sections failed to reveal magnocellular neurons which were not immunoreactive for the NGF receptor, suggesting that reductions in immunocytochemically stained neurons reflects neuron loss and not the failure of viable neurons to synthesize NGF receptors. These data indicate that cholinergic basal forebrain neurons which project to the amygdala, as well as to the temporal, frontobasal, and frontodorsal cortices, are most affected in AD.

Aged↗
PubMed1989

A monoclonal antibody against nerve growth factor receptor. Immunohistochemical analysis of normal and neoplastic human tissue.

The expression of human nerve growth factor (NGF) receptor in tumors and normal tissue was investigated with the use of a monoclonal antibody recently developed against that protein. This antibody, NGFR5, reacted strongly with 100% of 25 nerve sheath tumors. Eight of nine pheochromocytomas and three of three paragangliomas also had positive results, but the immunoreactivity was restricted to the sustentacular cell population. Within cells of melanocytic lineage, there was no immunostaining of melanocytes in normal epidermis, whereas 13 of 14 benign nevi had positive results, primarily involving spindled nevocytic structures within the dermis. NGF receptor was scarcely expressed in human melanoma; 9 of 19 melanomas had positive results, but immunoreactivity was generally restricted to rare cells within the larger tumor cell population. Among nonneurogenic mesenchymal tumors, results were generally negative: 0 of 5 chondrosarcomas, 0 of 6 malignant fibrous histiocytomas, 0 of 3 meningiomas, and 1 of 8 leiomyosarcomas were immunoreactive. Carcinomas were variable in immunoreactivity: 12 of 16 squamous cell carcinomas had positive results, whereas adenocarcinomas demonstrated focal, basal epithelial immunoreactivity and neuroendocrine tumors generally had negative results. Among normal tissues, in addition to expected neural immunostaining, NGFR 5 reacted positively with several nonneural cell types, including lymphoidal follicular dendritic cells, myoepithelial cells, vascular adventitia, and basal epithelium of oral mucosa and hair follicles. Antibodies to NGF receptor may play a role in the identification of benign and malignant soft tissue lesions.

Antibodies, Monoclonal↗
PubMed1988

Nerve growth factor receptor immunoreactivity in the nonhuman primate (Cebus apella): distribution, morphology, and colocalization with cholinergic enzymes.

A monoclonal antibody raised against the receptor for nerve growth factor (NGF) was used to examine the distribution and morphology of NGF receptor-containing neurons within the central nervous system of Cebus apella monkeys. Most somata demonstrating positive immunoreactivity were localized within the Ch1-4 regions of the basal forebrain. Neurons in the Ch1 region displayed morphological features typical of cholinergic medial septal neurons. These perikarya were primarily vertically oriented (40-50 micron along the vertical axis) with both apical and basal neuritic processes. Magnocellular (40-50 micron) neurons within the Ch2 (vertical limb of the diagonal band), Ch3 (horizontal limb of the diagonal band) and Ch4 (nucleus basalis of Meynert) regions were multipolar and had rounded perikarya that often displayed an eccentric nucleus. Fibers presumably originating from the Ch1-2 regions were observed throughout the fimbria-fornix system and were found to terminate preferentially within the CA1 and CA3 regions of the hippocampal formation and within the dentate gyrus of the hippocampus. An intense fiber network was also observed in the olfactory tubercle and other rhinencephalic structures, presumably originating from the Ch3 region of the basal forebrain. Beaded processes emanating from the Ch4 region primarily coursed within the external capsule and terminated preferentially within layers I, II, and IV of the cerebral cortex. In a pattern similar to that of cortical acetylcholinesterase (AChE) staining, NGF receptor immunopositive fibers were oriented in a tangential plane within the molecular layer of the cortex and in both a radial and tangential fashion within the cortical granular cell layers. In addition to neural innervation, there was an extensive vascular apposition by NGF receptor-containing neurites on both large caliber vessels and microcapillaries. NGF receptor immunoreactivity was extensively, but not exclusively, colocalized with choline acetyltransferase (ChAT) and AChE in the basal forebrain. A small population of cholinergic neurons were observed that were not NGF receptor-immunoreactive. Conversely, a few NGF receptor-containing neurons that were noncholinergic were also observed in this brain region. NGF receptor-containing somata were also identified in the putamen. The number of immunoreactive neurons observed in this structure, however, would not appear to be sufficient to account for the homologous NGF receptor binding densities described in rodents.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1988

Expression of NGF receptor in the developing and adult primate central nervous system.

Monoclonal antibodies against human NGF receptor have been used for immunocytochemical localization of NGF receptors in the CNS of macaques and baboons at various stages of development. In the adult, neurons in most brain regions are devoid of detectable NGF receptors. However, abundant NGF receptor immunoreactivity is present on a population of neurons in basal forebrain, which, on the basis of appearance and pattern of distribution, probably correspond, at least in part, to magnocellular cholinergic neurons of this region. NGF receptors were also associated with the vasculature in most brain regions. NGF receptor immunoreactivity is present on Mueller glia of neural retina. In macaque fetuses, approximately 1 month prenatally, retinal Mueller glia possess lower levels of receptor, while higher levels of receptor are present in the retinal nerve fiber layer. In fetal cerebellum, abundant receptor immunoreactivity is present on Purkinje cells, granule cells of the premigratory zone of the external granule layer, and neurons of the deep nuclei. Immunoreactivity decreases with subsequent development and is absent in the adult. In cerebellum, levels of NGF receptor assayed by affinity crosslinking to radioiodinated NGF, and levels of NGF receptor mRNA assayed by Northern blot analysis decrease dramatically during the last month of fetal life.

Animals↗
PubMed1987

Purification and amino terminal sequencing of human melanoma nerve growth factor receptor.

The nerve growth factor (NGF) receptor, solubilized with Triton X-100 detergent, has been purified from human melanoma cell line A875. Purification to near-homogeneity was achieved by chromatography on wheat germ agglutinin-agarose, followed by immunoaffinity chromatography on Sepharose columns coupled with anti-NGF receptor monoclonal antibody (MAb). The purified receptor, a 75,000-dalton protein, retains the capacity to bind NGF as well as anti-receptor MAbs. Final purification was achieved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of amino acid residues at the amino terminus has been determined. Possible sequence homology between the NGF receptor and several other proteins is discussed. Using the purified receptor as immunogen, new MAbs to the NGF receptor have been produced. The NGF receptor was visualized by immunoperoxidase staining in tissue sections of dorsal root ganglia from monkeys.

Amino Acid Sequence↗
PubMed1986

Expression and structure of the human NGF receptor.

The nucleotide sequence for the human nerve growth factor (NGF) receptor has been determined. The 3.8 kb receptor mRNA encodes a 427 amino acid protein containing a 28 amino acid signal peptide, an extracellular domain containing four 40 amino acid repeats with six cysteine residues at conserved positions followed by a serine/threonine-rich region, a single transmembrane domain, and a 155 amino acid cytoplasmic domain. The sequence of the extracellular domain of the NGF receptor predicts a highly ordered structure containing a negatively charged region that may serve as the ligand-binding site. This domain is conserved through evolution. Transfection of a full-length cDNA in mouse fibroblasts results in stable expression of NGF receptors that are recognized by monoclonal antibodies to the human NGF receptor and that bind [125I]NGF.

Amino Acid Sequence↗
PubMed1986

The nerve growth factor receptor gene is at human chromosome region 17q12-17q22, distal to the chromosome 17 breakpoint in acute leukemias.

Genomic and cDNA clones for the human nerve growth factor receptor have been used in conjunction with somatic cell hybrid analysis and in situ hybridization to localize the nerve growth factor receptor locus to human chromosome region 17q12-q22. Additionally, part, if not all, of the nerve growth factor receptor locus is present on the translocated portion of 17q (17q21-qter) from a poorly differentiated acute leukemia in which the chromosome 17 breakpoint was indistinguishable cytogenetically from the 17 breakpoint observed in the t(15;17)(q22;q21) translocation associated with acute promyelocytic leukemia. Thus the nerve growth factor receptor locus may be closely distal to the acute promyelocytic leukemia-associated chromosome 17 breakpoint at 17q21.

Chromosome Mapping↗
PubMed1985

Characterization of the human melanoma nerve growth factor receptor.

Monoclonal antibodies to the human nerve growth factor receptor have been used to biochemically characterize the receptor in the human melanoma cell line A875. Labeling of A875 cell proteins by culture with [35S]cysteine or labeling of cell surface proteins with 125I followed by immunoprecipitation with anti-nerve growth factor receptor antibody reveals a receptor protein with an apparent Mr of 70,000-75,000 and an isoelectric point of 4.9-5.2. Incorporation of [3H]glucosamine into this species indicates it is a glycoprotein. The receptor becomes phosphorylated on serine residues in intact cells and in isolated membranes incubated with [gamma-32P]ATP. The receptor appears to exist, at least partially, in the form of a disulfide-linked oligomer (probably a dimer) of Mr = 75,000 subunits. Kinetic [35S]cysteine labeling studies reveal an Mr = 59,000 core protein which is glycosylated via N-linked and probably also O-linked sugar moieties to produce the mature (Mr = 70,000-75,000) receptor.

Antibodies, Monoclonal↗
PubMed1985

Synergistic effects of cyclic AMP and nerve growth factor on neurite outgrowth and microtubule stability of PC12 cells.

The outgrowth of neurites from rat PC12 cells stimulated by combined treatment of nerve growth factor (NGF) with cAMP is significantly more rapid and extensive than the outgrowth induced by either factor alone. We have compared the responses of PC12 cells under three different growth conditions, NGF alone, cAMP alone, and combined treatment, with respect to surface morphology, rapidity of neurite outgrowth, and stability of neurite microtubules, to understand the synergistic action of NGF and cAMP on PC12. Surface events at early times in these growth conditions varied, suggesting divergent pathways of action of NGF and cAMP. This suggestion is strongly supported by the finding that cells exposed to saturating levels of dibutyryl cAMP without substantial neurite outgrowth initiated neurites within 5 min of NGF. This response has been adopted as a convenient assay for NGF. Neurites that regenerated in the three growth conditions showed marked differences in stability to treatments that depolymerize microtubules. The results indicate that microtubules in cells treated with both NGF and cAMP are significantly more stable than in either growth factor alone. We suggest that a shift of the assembly equilibrium favoring tubulin assembly is a necessary prerequisite for the initiation of neurites by PC12.

Animals↗
PubMed1984

Characterization of nerve growth factor receptor in neural crest tumors using monoclonal antibodies.

The nerve growth factor (NGF) receptor was characterized by using a new series of anti-receptor monoclonal antibodies (MAbs). These MAbs (i) showed significantly greater reactivity with a melanoma cell line expressing higher levels of NGF receptor, (ii) inhibited the binding of 125I-labeled NGF to its receptor, and (iii) immunoprecipitated both metabolically labeled and 125I-labeled NGF affinity-labeled receptor. These experiments defined the receptor as a 75-kDa cell-surface protein. The NGF receptor was visualized by immunoperoxidase staining in tissue sections of human nevi, melanomas, neurofibromas, a pheochromocytoma, and peripheral nerves. Uniform staining of the cytoplasm suggests that, in addition to cell-surface NGF receptors, there is a population of intracellular receptors.

Affinity Labels↗
PubMed1983

Use of iron- or selenium-coupled monoclonal antibodies to cell-surface antigens as a positive selection system for cells.

A system which confers selective growth advantage to cells expressing particular surface proteins would be extremely desirable, for such a technique would allow the study of receptors using somatic cell genetic techniques such as DNA-mediated cell transformation and selection of over-producing cell variants. Polypeptides bound to surface receptor, and antibodies bound to surface antigens generally are taken up efficiently by cells by endocytotic mechanisms. Several investigators have accordingly developed useful techniques for selection against cells expressing surface receptors and antigens, using hormones and antibodies conjugated to toxins. We reasoned that conjugation of nutrients to antibodies or hormones conversely might permit a positive selective pressure to be applied in appropriately constituted medium. We report here that monoclonal antibodies to cell-surface antigens will indeed deliver nutritional iron and selenium to cultured cells in an antigen-specific manner.

Animals↗
PubMed1983

The nerve growth factor receptor on PC12 cells: interconversion between two forms with different binding properties.

PC12 cells possess two classes of nerve growth factor (NGF) receptors on their surfaces which can be distinguished by kinetic criteria. The majority class binds and releases 125I-NGF at a relatively rapid rate and has been called fast. The second class of receptors has been called slow because of relatively slower rates of binding and release of 125I-NGF, and also may be distinguished from fast receptors by their cytoskeletal association and resistance to trypsin. PC12 cell plasma membranes were prepared and shown to have only the fast class of receptors. These membranes were fused to receptorless 3T3 cells with polyethylene glycol. The resultant fused cells were shown to possess NGF receptors, essentially all of which behave like slow receptors. Immunofluorescence microscopy was used to monitor the introduction of PC12 cell membrane and NGF receptors into 3T3 cells. Results obtained with C10-2, a monoclonal antibody specific for a major PC12 cell-surface antigen. show that up to 90% of 3T3 cells receive PC12 membrane and that the PC12 membrane becomes integrally incorporated into the 3T3 cell plasma membrane. It is suggested that an association of receptors with cytoskeleton may be involved in the conversion of fast to slow receptor behavior, and that the differing proportion of fast and slow NGF receptors in PC12 and 3T3 cells reflects the differing cytoskeletal organization of these cells.

Adrenal Gland Neoplasms↗
PubMed1980

Genetic analysis of epidermal growth factor action: assignment of human epidermal growth factor receptor gene to chromosome 7.

Purified murine epidermal growth factor (EGF) binds to mouse and human cells. Two mouse transformed cell lines of different origins, PG19 and B82, were found to lack EGF receptors (EGFR). The defect in each of these two cell lines seems to be identical because they fail to complement each other. Somatic cell hybrids between these EGFR-deficient mouse cells and human cells expressing EGFR were produced. Several of these hybrids bound labeled EGF. Detailed cytogenetic analysis of these cell hybrids, followed by correlation of EGFR expression with human chromosomes revealed that EGFR presence correlated with human chromosome 7. The results suggest that the structural gene or a gene necessary for expression of the human EGF receptor is located on human chromosome 7.

Animals↗