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R Bernhardt

Publications and source records attributed to R Bernhardt.

At least 91 records · Page 5Linked to original sources
PubMed1986

Map of retinal position onto the cross section of the optic pathway of goldfish.

The position of a retinal cell is defined by the two polar coordinates: r, the distance from the optic disc, and theta, the angular (or clock-face) position. Axons of similar theta value were labeled by the punctate application of horseradish peroxidase (HRP) to optic axons in the retina, and axons of similar r-value were labeled by the application of this same marker to a tectal fascicle. Labeled axons were traced in serial transverse sections of the optic pathway from the retina to the tectum to learn the map of the retinal surface onto the cross section of the pathway. Retinas were flat-mounted and treated for HRP to show the retinal origins of the labeled axons. Axons of similar r were clustered together, and the fraction of the pathway's cross-sectional area occupied by the cluster was about the same as the fraction of the retinal area occupied by the group of labeled somata. Axons of similar theta were also clustered, but the fraction of the cross-sectional area they occupied was larger than the fraction of retinal area occupied by their somata. The geometry of the clusters of labeled axons depended on the proximodistal location in the pathway. Near the retina both were strip-shaped, but the location and orientation of the strip varied. Both an r-strip and a theta-strip were labeled in some pathways by dual applications of HRP; the two strips were mutually orthogonal at all levels. Each of r and theta mapped onto a separate axis. The axons from most peripheral retina (largest r) were everywhere adjacent to the pia, and axons of progressively more central retina (smaller r) were progressively more separated from the pia (except in the nerve, where the secondary fasciculation complicates the geometry by wrapping old axons in new pia). The map of the circular variable, theta, onto a line, required a discontinuity, the location of which differed, depending on the proximodistal level. From the retina to the chiasm, the discontinuity was at the ventral retinal radius (i.e., the right retinal clock-face positions were ordered 6-9-12-3-6 o'clock across the line); just central to the chiasm, the fibers reordered to put the discontinuity at the nasal radius (clock-face positions ordered 3-6-9-12-3); at the brachial bifurcation, the 3-6-9 half turned dorsally, the 9-12-3 half, ventrally.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Microtubule-associated protein 2 and tubulin are differently distributed in the dendrites of developing neurons.

We have followed the appearance of two microtubule proteins, tubulin and microtubule-associated protein 2, in rat hippocampal neurons differentiating in cell culture. Double-label immunofluorescence staining showed that from day 1 in vitro onward tubulin appeared as filaments but that microtubule-associated protein 2 remained distributed throughout the cytoplasm. This difference persisted throughout development and was also detectable in cells that had reached morphological maturity. When cells were treated with the microtubule-depolymerizing agent nocodazole, the depolymerized tubulin became spread throughout the cytoplasm so that its distribution was then identical to microtubule associated protein 2. At the same time, multiple side branches began to emerge along the dendrites. When cells which had been exposed to nocodazole were allowed to recover before staining, the tubulin was again present as filaments but the microtubule-associated protein 2 remained distributed throughout the dendritic cytoplasm. Under these conditions the previously extended proximal side branches were resorbed into the main process. These results suggest that cellular microtubule-associated protein 2 is not necessarily exclusively associated with microtubules. Neuronal dendrites in particular appear to contain this protein at levels in excess of the capacity of microtubular microtubule-associated protein 2 binding sites. In view of the known effectiveness of microtubule-associated protein 2 as a promoter of tubulin polymerization, its abundance in dendrites suggests that it acts to ensure total polymerization of dendritic microtubules. In this way it would contribute both to the support of the growing process and the suppression of adventitious sidebranching.

Animals↗
PubMed1985

Differences in the developmental patterns of three microtubule-associated proteins in the rat cerebellum.

The developmental distribution patterns of microtubule-associated proteins (MAPs) 1, 2, and 3 were studied using three monoclonal antibodies. Immunochemical staining at the light and electron microscopic levels demonstrated the specific localization of each MAP in different cellular and subcellular compartments. MAP2, which is specifically associated with dendritic microtubules in the adult brain, is strictly associated with growing dendrites from the onset of their formation. MAP3, a recently described MAP of Mr = 180,000, which in the adult brain is associated with neurofilament-rich axons and glial processes, is associated with axons from the beginning of outgrowth. Although MAP3 is not detectable in granule cells and their parallel fiber axons in the mature cerebellum, it does appear transitorily in these axons during development. During neuronal differentiation, MAP1 is found first in axons and only later in dendrites where the highest concentrations are eventually to be found. These results indicate that the combined appearance of MAP1 and MAP2 (dendrites) or MAP1 and MAP3 (axons) correlates with the appearance of morphologically distinct microtubules and provide further evidence that specific MAPs are molecular determinants of dendritic and axonal formation.

Animals↗
PubMed1985

Chemical modification of cytochrome P-450 LM2 with N-acetylimidazole. Evidence for the functional involvement of tyrosyl residues.

Cytochrome P-450 LM2 has been reacted with N-acetylimidazole. About three tyrosyl residues of cytochrome P-450 LM2 are accessible to O-acetylation. The analysis of the spectral dissociation constants, substrate binding kinetics, reduction kinetics, N-demethylase activity and substrate conversion by artificial oxygen-donating agents of differently acetylated enzyme provides evidence for the existence of two groups of accessible tyrosines. One tyrosyl residue is located in the immediate environment of the heme iron and is involved in the binding of type II substrates. This tyrosine is not necessary for N-demethylation. Acetylation of two further tyrosyl residues, however, causes an almost complete inhibition of enzymatic activity. The results strongly suggest tyrosine(s) to be involved in NADPH-cytochrome P-450 reductase dependent N-demethylation.

Acetylation↗
PubMed1984

Light and electron microscopic studies of the distribution of microtubule-associated protein 2 in rat brain: a difference between dendritic and axonal cytoskeletons.

A specific antiserum was used to ascertain the distribution of microtubule-associated protein 2 (MAP2) in the rat brain at the light and electron microscope levels. Light microscopy showed MAP2 to be present only in neurons, and only in the dendrites and the perikaryon of each cell. This same polarized distribution pattern was found in the Purkinje, Golgi, basket, stellate, and granule cells of the cerebellum, and also in neurons of the hippocampus, the olfactory bulb, and the midbrain. While labelling of the dendritic arborization was extensive and intense, MAP2 density tended to decrease in the proximal dendritic trunk. Particularly in large neurons (e.g., Purkinje, Golgi, and pyramidal cells), staining was reproducibly weaker in the cell body than in the main dendrites. Dendritic contours generally appeared smooth, without any evidence of staining of dendritic spines. An electron microscope examination of the cerebellum confirmed the presence of MAP2 reactivity in neurons and its absence from axons and non-neuronal cells. MAP2 in dendrites was associated with microtubules, while MAP2 in neuronal perikarya was associated with polyribosomes. There was no evidence of specific staining in dendritic spines and in postsynaptic densities. MAP2 is a novel dendritic marker and labels part of a specific dendritic cytoskeleton, different from that in axons and non-neuronal cells.

Animals↗
PubMed1984

Chemical modification of cytochrome P-450 LM2. Characterization of tyrosine as axial heme iron ligand trans to thiolate.

Phenobarbital-inducible isozyme cytochrome P-450 LM2 (RH, reduced-flavoprotein:oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) from rabbit liver microsomes has been modified with N-acetylimidazole and tetranitromethane. Up to four tyrosine residues of cytochrome P-450 LM2 are accessible to O-acetylation and to nitration. N-Demethylase activity, spectral dissociation constants and substrate binding kinetics of differently acetylated enzyme indicate the existence of two groups of accessible tyrosines also differing in their reactivity towards N-acetylimidazole. The fast-reacting tyrosine residue representing the first group is involved in the binding of the type II substrate aniline and appears to be located near the heme as shown by the protecting effect of the inhibitor metyrapone against modification, but obviously is not necessary for N-demethylation. Acetylation of one further tyrosine residue, however, caused an almost complete inhibition of the enzyme, indicating its involvement in the catalytic mechanism at the active center. Nitration of two tyrosine residues inactivates to about 20%. Obviously the third and fourth tyrosine residue are without functional importance. The experiments evidencing two functionally linked tyrosines are in line with HPLC analyses of tryptic peptides of cytochrome P-450 LM2 nitrated in the presence of metyrapone which gave evidence for the location of two distinct tyrosine residues in the active center. Nitration of tyrosine residues results in the partial formation of a hyperporphyrin spectrum of cytochrome P-450 LM2. Its appearance is prevented in the presence of metyrapone and can be reversed by reduction of the nitrotyrosinate .

Acetylation↗
PubMed1984

Selective chemical modification of a functionally linked lysine in cytochrome P-450 LM2.

Fluorescein isothiocyanate (FITC) has been selectively bound to the epsilon-amino group of lysine-382 in cytochrome P-450 LM2 (RH, reduced-flavoprotein: oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) at pH 8.15. Benzphetamine N-demethylase activity of the reconstituted FITC-modified cytochrome P-450 LM2 was inhibited by 25%. This inhibition has been shown to be due to an impaired electron transfer from the NADPH-cytochrome P-450 reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) to the haemoprotein. The data indicate that cytochrome P-450 interacts with the flavoprotein via electrostatic interactions.

Animals↗
PubMed1984

Identification of lysine (384) in cytochrome P-450 LM2 as functionally linked residue.

Fluorescein isothiocyanate was selectively bound to the epsilon-amino group of a lysine residue of cytochrome P-450 LM2 at rho H 8.15. The decrease in the N-demethylase activity after modification evidences the functional importance of the modified group. After tryptic digestion the FITC-labeled peptide was isolated by means of HPLC and its amino acid composition determined. It was shown that the FITC-peptide can be attributed to the sequence Gly (379)- Arg (400) and that the label is selectively bound to Lys (384).

Amino Acid Sequence↗
PubMed1983

Modification of cytochrome P-450 with fluorescein isothiocyanate.

Fluorescein isothiocyanate (FITC) has been shown to be selectively attached to the N-terminus of cytochrome P-450 LM2. The N-demethylase activity of cytochrome P-450 LM2 reconstituted systems modified in this way was inhibited by 25%. As revealed by CD measurements the overall conformation as well as the immediate heme environment of cytochrome P-450 LM2 remained unchanged after attachment of the FITC molecule. The binding affinity of modified cytochrome P-450 LM2 toward benzphetamine and aniline and the cumene hydroperoxide- or H2O2-supported N-demethylation of benzphetamine are maintained. However, the introduction of the electron via NADPH-cytochrome P-450 reductase (EC 1.6.2.4) is impaired after modification of the alpha-amino group. The extent of reduced modified cytochrome P-450 LM2 in the cytochrome P-450 reductase-supported reduction reaction is diminished and the half-time of the reduction is increased. The diminished reducibility is ascribed to steric hindrance of groups directly involved in the interaction between cytochrome P-450 LM2 and NADPH-cytochrome P-450 reductase or to blocking of the charge-pair interactions between the alpha-amino group of P-450 LM2 and the respective negatively charged group of NADPH-cytochrome P-450 reductase. By energy-transfer measurements distances between the heme and the alpha-amino group of 2.65 and 3.97 nm for the oligomeric and the monomeric forms of P-450 LM2, respectively, have been determined.

Animals↗
PubMed1983

Fluorescent energy transfer measurements on fluorescein isothiocyanate modified cytochrome P-450 LM2.

The distance between the heme iron and the N-terminus of cytochrome P-450 LM2 was determined by fluorescence energy transfer measurements. Fluorescein isothiocyanate which was covalently bound to the N-terminal methionine was used as donor chromophor. The Ro value between fluorescein isothiocyanate and the heme was calculated to be 3.98 nm. The distance between the nitrogen of the N-terminal methionine and the heme was estimated with 2.84 +/- 0.23 nm excluding most likely the N-terminal amino acid of cytochrome P-450 LM2 to participate in the electron transfer to the heme iron. A cytochrome P-450 LM2 membrane model is proposed.

Animals↗
PubMed1982

Initial phase of dendrite growth: evidence for the involvement of high molecular weight microtubule-associated proteins (HMWP) before the appearance of tubulin.

It has recently been shown that high molecular weight microtubule-associated proteins (HMWP) in the brain are present in dendrites and are absent from axons (Matus et al., 1981, Proc. Natl. Acad. Sci. U. S. A. 78:3010-3014). In this study we followed the appearance of both HMWP and tubulin in the neonatal rat cerebellum by immunoperoxidase staining, concentrating particularly on comparing Purkinje cell dendrites with adjacent granule cell axons. In the axons both immunohistochemically demonstrable tubulin and structurally distinct microtubules are present at all stages of development. By contrast the Purkinje cell dendrites contain better neither tubulin nor microtubules at early stages of their growth. However, immunoperoxidase staining showed that these developing dendrites are rich in HMWP which are particularly concentrated in the dendritic distal regions. HMWP are also present as patches beneath the surface membrane of the cell body before the emergence of dendrites. Based on this data and the well-documented ability of HMWP to promote microtubule assembly, we propose the hypothesis that during the initial phase of Purkinje neuron differentiation HMWP form part of a specialized cytoskeletal structure which acts as a specifier for the development of dendrites as opposed to axons.

Axons↗
PubMed1981

High molecular weight microtubule-associated proteins are preferentially associated with dendritic microtubules in brain.

The distributions of tubulin and high molecular weight microtubule-associated proteins (HMWPs) in brain were determined by immunoperoxidase histochemistry with specific antisera. Tubulin was found in microtubules of both neurons and glia and both axons and dendrites. HMWPs were found only in neurons where, in all cases examined, they were associated with dendritic microtubules but not those in axons. Both tubulin and HMWPs were also found in postsynaptic densities. These results indicate that brain microtubules vary in chemical composition. The preferential association of HMWPs with dendritic microtubules suggests that they may play a role in distinguishing between dendritic and axonal export routes from the cell body.

Animals↗