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Biomedical subjects

N G Cooper

Publications and source records attributed to N G Cooper.

At least 37 records · Page 2Linked to original sources
PubMed1995

Onset of expression of the alpha subunit of Ca2+/calmodulin-dependent protein kinase II and a novel related protein in the developing retina.

Calcium-calmodulin-dependent protein kinase II is an abundant protein in the nervous system and has been associated with many aspects of neuronal function, including events related to synaptic transmission. The purpose of this study is to correlate the onset of expression of this kinase with a specific developmental event in retinal morphogenesis using a monoclonal antibody to the 50-kDa alpha-subunit. Microscopy showed the antigen to be associated with the plexiform layers of the retina. Western blots demonstrated that the onset of expression of the alpha-subunit coincided in time with the initial formation of the plexiform layers. However, the onset of expression of the 50-kDa alpha-subunit was preceded by the earlier embryonic appearance of a related 82.5-kDa antigen that was recognized by the antibody. The amount of this latter protein declined as the amount of the alpha-subunit increased in retinal homogenates. Although this related 82.5 kDa protein disappeared from blots of retinal homogenates after embryonic d 14, it could be detected in concentrated supernatant fractions isolated from the retinae of hatched chicks. Microscopy showed that a subset of retinal cells and their processes contained this antigen in early embryonic chicks. Finally, the 50 kDa alpha-subunit of kinase II and the 82.5 kDa novel antigen were shown to be separable by differential centrifugation.

Animals↗
PubMed1995

Patterns of GFAP-immunoreactivity parallel the tonotopic axis in the developing dorsal cochlear nucleus.

The role of glia in the development of tonotopic and laminar organization in the auditory central nervous system has not been well characterized. In other systems, glia immunoreactive for glial fibrillary acidic protein (GFAP) appear to function in development of radial, laminar and topographic organization. Using a polyclonal antibody to GFAP, we have characterized the development of GFAP-immunoreactivity in the dorsal cochlear nucleus (DCN), a laminated and tonotopically organized central auditory system structure. Results suggest that in this nucleus, the GFAP-immunoreactive processes are not found within or between developing laminae, rather glial processes are observed parallel to presumptive isofrequency sheets before primary afferents have invaded the nucleus. Thus, GFAP-immunoreactive processes are positioned to play an early role in establishing the tonotopic axis of the DCN.

Animals↗
PubMed1994

Purification and characterization of the Ca2+/calmodulin-dependent protein kinase II from chicken forebrain.

CaM kinase II is known to be enriched in mammalian and avian brains. To determine the holoenzymic composition and functional characteristics of this kinase, a new approach for isolation was applied to isolate it from the chicken forebrain. Forebrains of hatched 45-d chicken were dissected, homogenized, and centrifuged. The supernatant was loaded onto a CaM-agarose affinity column and the calmodulin-binding proteins were eluted with EGTA. Selected eluates were loaded onto the antibody-agarose affinity column, which was prepared with monoclonal antibody (MAb) (6G9) to the CaM kinase II alpha subunit. Samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and either silver-stained or blotted onto a nitrocellulose membrane. The protein composition and the immunoreactivity of the antibody-agarose affinity eluate fractions were analyzed with a densitometric scanner. Silver staining of gels showed that the beta subunit doublet, the beta' subunit, and a putative substrate were coeluted with the alpha subunit from the antibody affinity column although only the alpha subunit bound the 6G9 antibody. Scintillation counting showed that the autophosphorylation of the kinase was significantly reduced in the eluate from the antibody affinity column. Whereas silver staining indicated an increase in the relative amount of alpha subunit had occurred during purification, phosphorylation assays indicated an increase in the relative amount of the alpha subunit after the last purification step. A possible reason for this is discussed. The presence of beta/beta' subunits in the antibody-agarose affinity eluate indicated the existence of an alpha beta/beta' heteropolymer. The phosphorylation assay was not a good indication of the amount of purification because of the loss of enzyme activity following purification. In contrast, the immunoassay indicated a 97-fold purification from the cytosolic fraction was achieved using the method. In conclusion, the data indicate the existence of the CaM kinase II alpha beta/beta' heteropolymer in the chicken forebrain.

Animals↗
PubMed1993

Phosphotyrosine-modified proteins are localized in Müller cells of the chick neural retina.

Phosphorylated protein substrates of tyrosine-specific protein kinases accumulate within areas of high density of neuronal and glial processes in the avian retina. To identify the cell type(s) containing elevated tyrosine kinase activity, phosphotyrosine-modified proteins were localized in the outer neural retina of the hatchling chick by immunoelectron microscopy using phosphotyrosine antibodies. In the outer retina, phosphotyrosine immunoreactivity was confined to the plasmalemma and cytoplasm of Müller cells. Prominent membrane labeling was associated with sites adjoining photoreceptor inner segments, cell bodies, and synaptic terminals. Immunoreactivity was not associated with pre- or post-synaptic membranes, synaptic ribbons, or synaptic vesicles. Immunoreactivity was also observed at sites of apposition between plasma membranes of adjacent Müller glial processes. Phosphotyrosine antibodies recognized nine principal proteins (106, 83, 68, 60, 52, 46, 44, 39, 38 kDa) in retinal extracts revealed by immunoblotting. The 60, 52, and 38 kDa phosphotyrosine-modified proteins were also prominent in extracts prepared from Müller glia-enriched cultures, but were absent or low in neuronal cultures; consistent with the higher immunolabeling of glia in retinal sections. Protein tyrosine kinases encoded by the fyn and yes proto-oncogenes were expressed in Müller glia-enriched cultures, and could contribute to the observed protein tyrosine phosphorylation. The localization of phosphotyrosine-modified proteins in the Müller glia leads us to suggest a possible role for protein tyrosine kinases in communication between Müller glia and neuronal cells.

Animals↗
PubMed1991

A mannose receptor is involved in retinal phagocytosis.

A 175-kD mannose-specific receptor has been described in macrophages which appears to mediate pinocytosis and phagocytosis. A mannose-specific receptor has also been found on retinal pigment epithelial cells (RPE). Its role was examined in the phagocytosis of photoreceptor outer segments (ROS) by the RPE. All testing was done on cultured RPE cells challenged with fluorescein isothiocyanate (FITC)-labeled ROS for 4 hr at 37 degrees C. Total (internalized and bound) and phagocytized (internalized) ROS were quantified, and the effects of several experimental conditions on ROS phagocytosis were examined. Incubation of RPE cells in the presence of rabbit anti-serum (1:100) raised against the rat alveolar macrophage mannose receptor (anti-Mr) showed a 80% reduction in ROS phagocytosis compared with RPE in the presence of ROS alone. Anti-Mr preabsorbed with purified human mannose receptor protein (4 micrograms) showed no reduction in ROS phagocytosis. Incubation of RPE with preimmune serum showed no reduction in ROS phagocytosis. When FITC-labeled ROS (1.8 X 10(7)) were preincubated with purified mannose receptor, there was a 93% reduction in phagocytosis. Immunoblots of solubilized rat RPE microvillus membranes stained with the anti-Mr showed a single stained band at the 175-kD region, and immunolocalization studies showed specific labeling along RPE microvilli. These results suggest that a mannose-specific receptor is involved in retinal phagocytosis.

Animals↗
PubMed1991

Glycoproteins in the retinal pigment epithelium of normal and dystrophic rats.

The apical membranes of retinal pigmented epithelium (RPE) were isolated from adult, normal (LE), and dystrophic (RCS) rats. The proteins of these RPE subfractions were separated through the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin-binding properties of glycoproteins were examined in western blots through the use of lectin-peroxidase conjugates. No differences were detected between RPE membrane proteins from normal and dystrophic rats in silver-stained gels. However, these two preparations showed significant differences with respect to their binding of the lectins, Lens culinaris (Lentil), Tetragonolobus purpurea (Lotus), and concanavalin A (Con A). In particular, a glycoprotein with a molecular weight of 86 kD in the RPE apical membrane from normal rats bound Lentil, Lotus, and Con A, but in the membrane from dystrophic rats these binding sites were absent or significantly reduced. Another glycoprotein with a molecular weight of 175 kD was recognized by Lotus in the normal membrane preparation but not in the dystrophic RPE membrane preparation. Developmental studies show that these lectin-binding anomalies appear after postnatal day 11 and are, therefore, most likely coincident with eye opening in RCS rats. These results demonstrate that the RPE glycoproteins (86 and 175 kD) are significantly modified in dystrophic rats. The data also confirm previous observations that differences in the oligosaccharide chains, but not the polypeptide chains, of RPE membrane glycoproteins can be detected between normal and dystrophic rats. To the authors' knowledge, this is the first study to correlate developmentally regulated alterations in specific membrane-associated molecules in the RPE of dystrophic rats with the breakdown in phagocytosis that occurs in these rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Early development of SI cortical barrel subfield representation of forelimb in normal and deafferented neonatal rat as delineated by peroxidase conjugated lectin, peanut agglutinin (PNA).

Development of the barrel field representation of the forelimb in the primary somatosensory cortex (SI) was studied in normal and deafferented neonatal rat pups by means of the peroxidase conjugated lectin peanut agglutinin (PNA), which most likely binds to radial glial cells within barrel boundaries. 1. Alterations in lectin binding were seen in animals sacrificed on postnatal day 8 (PND-8) if deafferentation took place on PND-1 (day of birth) through PND-6. 2. Deafferentation on PND-5 or on PND-6 had the least effect on lectin binding. In these animals, lectin binding was reduced, although the prospective representation was intact. 3. Deafferentation on PND-2, 3, and 4 had the greatest effect on lectin binding. In these animals, lectin binding was reduced and the prospective cortical representation was disrupted. 4. Deafferentation on PND-1 resulted in reduced lectin binding, however the prospective cortical representation was only slightly impaired compared to that in animals deafferented on PND-2, 3, and 4. 5. These results suggest that SI barrel field boundaries are important to plasticity and that a sensitive period for predevelopment of the forelimb barrels consists of postnatal days 1 through 6. Furthermore, the formation of normal SI barrel field boundaries requires an ongoing interaction between incoming afferents and radial glial cells.

Afferent Pathways↗
PubMed1990

Light and electron microscopic analysis of projection neurons retrogradely labeled with Fluoro-Gold: notes on the application of antibodies to Fluoro-Gold.

Fluoro-Gold has been a very popular fluorescent tracer used recently for retrogradely labeling projection neurons. In this study we described the advantages of using antibodies to Fluoro-Gold in conventional immunohistochemical reaction protocols to further extend the usefulness of this tracer for both light and electron microscopic neuroanatomical studies.

Animals↗
PubMed1989

Critical period-dependent alterations of the transient body image in the rodent cerebral cortex.

The present study demonstrates that the boundary patterns of cell surface-associated molecules detected with lectins in the barrel cortex of neonatal rodents are altered, as are the boundary patterns of cortical glia, following perturbation of large vibrissae in the contralateral mystacial face pad. The alterations in the transiently expressed molecular patterns of lectin-receptors provide data that are consistent with the idea that the periphery plays a prominent role in the establishment of functional cytoarchitecture in the developing cortex. The data are also consistent, however, with the notion that factors intrinsic to the cerebrum, such as the immature cortical glial cells, are of considerable importance in this respect and a direct or indirect interaction of thalamocortical afferents with glial cells in the somatosensory cortex of the neonate are indicated. It is suggested therefore that a critical period in early barrel development, a time in which the cortical neuronal architecture is malleable in response to altered afferent input, is directly related to the presence of these cellular and molecular boundaries. The transient barrel boundaries, it is argued, are the morphological and molecular substrates that form the physical basis of the critical period.

Afferent Pathways↗
PubMed1989

Early development of the representation of the body surface in SI cortex barrel field in neonatal rats as demonstrated with peanut agglutinin binding: evidence for differential development within the rattunculus.

Physiological studies have demonstrated a highly organized somatotopic representation of the body surface in SI cortex of rat. This representation is correlated morphologically with the presence of barrel-shaped structures in layer IV. Conventional staining techniques reveal barrels in the latter part of the first postnatal week. Recently, the peroxidase conjugates of lectins, which recognize glycosylated molecules, have been used to study barrel field formation. Con A, for example, has been shown to bind primarily to prospective barrel sides and septa as early as postnatal day 3 (PND-3) in mouse. To date, investigations of SI cortex using the lectin (Arachis hypogaea) peanut agglutinin (PNA) have been confined to the study of the barrel field representation of the face and mystacial vibrissae in the mouse. In the present study we extend these findings to the development of the representation of the entire body surface called the rattunculus. Rats ranging from PND-1 (first 24 h after birth) to PND-12 were anesthetized with Nembutal and perfused with 4% paraformaldehyde and 2% glutaraldehyde in 0.2 M sodium cacodylate buffer. Brains were removed, flattened tangentially, and sectioned on a vibratome at 30-120 microns. Sections were blocked in TRIS-buffered saline (TBS) plus 2% bovine serum albumin and incubated in peanut lectin at 4 degrees C. Following incubation, sections were washed with TBS and processed using peroxidase histochemistry. Lectin binding in the prospective forelimb representation was apparent by PND-5 whereas lectin binding to the prospective face-mystacial vibrissae representation occurred before PND-4. These results suggest that body part representations show individual variations during early pattern formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Boundaries defined by adhesion molecules during development of the cerebral cortex: the J1/tenascin glycoprotein in the mouse somatosensory cortical barrel field.

The distribution of the 200/220 KDa J1 glycoprotein (J1-200/220), within the developing vibrissae-related barrel field of the mouse somatosensory cortex, was studied by immunocytochemistry using a monoclonal antibody. J1-200/220, a member of the L2/HNK-1 family of adhesion molecules, also appears to be the mouse homologue of tenascin. J1/tenascin-positive barrel-like structures are visible in the somatosensory cortex between 24 and 48 hr after birth, with the molecule present in prospective barrel boundaries. Immunoelectronmicroscopy reveals labeling that is associated with glial and neuronal plasma membranes, as well as glial end-feet on blood vessels. A possible major source of J1/tenascin expression at this time is astrocyte precursor cells and radial glia. In the putative astrocyte precursor cells, immunolabeling was observed within organelles including the Golgi apparatus. At P6-7 J1/tenascin is most prevalent within prospective interbarrel septae. J1/tenascin-positive barrel boundaries are barely visible on P9 and not observed on P16. The findings indicate that J1/tenascin represents a major component of previously described "hidden" boundaries that we have seen during development using other methodologies. The expression of adhesion molecule-rich boundaries during the critical stages of barrel field formation indicates roles for such molecules during specific cerebral cortical pattern formation events.

Animals↗
PubMed1988

Glycoconjugate boundaries during early postnatal development of the neostriatal mosaic.

The dispositions of galactosyl-containing glycoconjugates were studied during postnatal development of the caudate putamen in mice. The binding of the lectin peanut agglutinin, which has an affinity for galactosyl B-1,3 N-acetylgalactosamine residues, was compared to acetylcholinesterase staining and tyrosine hydroxylase immunoreactivity in the immature and adult neostriatum. The binding of peanut agglutinin conjugated to horseradish peroxidase, in sections that were processed for peroxidase histochemistry, was extremely pronounced in the neostriatum through the first postnatal week and constituted ringlike or polygonally shaped structures, which, overall, produced a variegated mosaic. These structures consist of outer rims of dense lectin-associated reaction product surrounding lightly labeled centers. Lectin delineations of the neostriatal mosaic are no longer visible in the second postnatal week. When adjacent sections were processed for lectin binding or acetylcholinesterase histochemistry, the dense lectin binding sites represented borders of acetylcholinesterase-rich and -poor zones. The distribution of dense patches of tyrosine hydroxylase immunoreactive fibers and terminals also coincides with the acetylcholinesterase-rich zones during the same times, and thus the glycoconjugate-delineated boundaries can also be directly compared with the distribution of nigrostriatal dopaminergic projections. The findings presented here represent the first demonstration of a probe that recognizes apparent borders of neostriatal compartments during a limited period of development. They are consistent with previous observations made on transient glycoconjugate "hidden boundaries" during development of other central nervous system structures, including the somatosensory cortical barrel field, and thalamic and brainstem nuclei (Cooper and Steindler, '86a,b; Steindler and Cooper, in press). In those studies, glia were shown to be the major source of glycoconjugate-associated patterns, and thus, glia and glycoconjugates that they synthesize during pattern formation events may be involved in the formation and stabilization of neurochemically distinct components of the neostriatal mosaic.

Acetylcholinesterase↗
PubMed1987

Glial and glycoconjugate boundaries during postnatal development of the central nervous system.

The localization of glycosylated molecules and glia has been studied during early postnatal development in the mouse central nervous system (CNS) using autoradiographic detection of radiolabeled fucose incorporation, and in sections processed either for histochemistry or immunocytochemistry following binding of labeled lectins or an antibody to glial fibrillary acidic protein. Radiolabeled sugar incorporation, lectin binding of glycoconjugates, and glial labeling all reveal borders between nuclei within the diencephalon, midbrain, and brainstem through the first postnatal week. Glycoconjugate and glial boundaries exist throughout the CNS during pattern formation events, and they also are seen in relation to fine aspects of developing functional organization within individual structures (e.g. segmentation associated with the representation of mystacial vibrissae within the brainstem trigeminal complex). The observation that each of the probes employed in this study fails to label boundary organization during later postnatal times suggests that the distribution and chemistry of the glial/glycoconjugate network are dynamic, and they change in accordance with distinct maturational states of the nervous system.

Animals↗
PubMed1987

Abnormal glial and glycoconjugate dispositions in the somatosensory cortical barrel field of the early postnatal reeler mutant mouse.

During early postnatal development in reeler mutant mice, lectin binding delineates prospective abnormal barrels as they will appear in the adult mutant somatosensory cortex. Glial fibers also may be more condensed within fascicles in developing reeler barrels. These fibers also appear to be misaligned, coursing predominantly in the tangential plane within the abnormal reeler barrel sides as opposed to having a radial orientation as seen in normal mouse barrels. The thalamic barreloid complex, however, reveals a disposition of glycoconjugates that is completely normal in reeler. Thus, there are anomalies in glia and associated glycoconjugates during mainly cortical development in the reeler mutant mouse that might be related to the primary action of the abnormal gene.

Animals↗
PubMed1987

Lectin-affinity isolation of microvillous membranes from the pigmented epithelium of rat retina.

The pigmented epithelium of the vertebrate retina phagocytizes the discarded tips of photoreceptors and it is likely that a specific cellular recognition process is involved in this phenomenon. The apical surface of retinal pigmented epithelium (RPE) contains microvilli which interdigitate with the outer segment regions of photoreceptor cells and it is this apical microvillous surface that is of particular interest with respect to phagocytosis. The present study is a report of a method to isolate a fraction that is enriched in microvilli from the apical surface of this highly polarized epithelial cell. Wheat germ agglutinin (WGA) conjugated sepharose beads are used to remove the microvillous membranes which are then observed with scanning and transmission electron microscopy. The proteins of this RPE-subfraction are separated through use of SDS-polyacrylamide gel electrophoresis. The relative molecular weights (Mr) and lectin binding properties of glycoproteins are examined in Western blots through the use of lectin-peroxidase conjugates as probes for carbohydrate residues. A preliminary comparison of membranes isolated from Long Evans (normal) and Royal College of Surgeons (dystrophic) rat retina RPE shows that the glycoproteins in these two preparations are different with respect to the binding of Concanavalin-A (Con-A) and WGA. In particular a glycoprotein in the normal RPE preparation with a Mr of 175K binds Con-A and WGA, but in the dystrophic RPE preparation binds little or no WGA. A glycoprotein present in the normal RPE preparation with a Mr of 86K binds Con-A and WGA, but both lectins have reduced binding sites in the dystrophic preparation. Limax flavus agglutinin (specific for sialic acid residues) binds to a high molecular weight glycoprotein with a Mr of 195K-196K which is present in both normal and dystrophic RPE membrane preparations and which also binds Con-A and WGA.

Animals↗
PubMed1986

Monoclonal antibody to glial fibrillary acidic protein reveals a parcellation of individual barrels in the early postnatal mouse somatosensory cortex.

The relative dispositions of cells in immature and mature mouse barrel field cortices that bind antibody to glial fibrillary acidic protein (GFAP) were examined and photographed under the light microscope. Light micrographs demonstrate that radially oriented glial cells are present in the barrel field of postnatal day 6 cortices and that they are located predominantly within the presumptive barrel sides and/or septae, thus sharply delineating individual barrels from each other. The relative dispositions of radial glial fibers observed at this time implicate glia in development of topographic order during early postnatal development of the somatosensory cortex. In contrast, no such delineation could be detected in the cortices of more mature mice, because GFAP-positive astrocytes are present throughout the barrel field and are not confined to barrel sides. This ephemeral nature of the GFAP-delineated barrel field is of interest with respect to the recently reported ephemeral lectin-delineated barrel field.

Aging↗