Volker Neuhoff

Distribution of Gaba and Other Amino Acids In Different Tissues of The Gastropod Mollusc Helix Pomatia, Including In Vitro Experiments With ,4c Glucose and I4c Glutamic Acid

Microchromatography on 3 × 3 cm polyamide layers of dansyl-compounds was used to study the distribution of free amino acids, GABA, serotonin, 5-hydroxyindole and 5-hydroxytryptophan in the brain, optic tentacle, pharyngeal retractor muscle and heart of Helix pomatia. In addition the formation of GABA after incubation in U- 14C glucose or U- I4C glutamic acid was studied.Low amounts of GABA were for the first time shown to occur in all four tissues studied. In addition taurine, thought to be absent from terrestrial gastropods, was shown to occur in the snails tissues.In vitro incubation of brain and heart with 14C labelled glucose demonstrated the formation of 14C glutamic acid, 14 C glutamine, 14C alanine and 14C aspartic acid. These amino acids, in addition to 14C GABA, were formed when brain and heart were incubated with 14C labelled glutamic acid.The distribution of amino acids in all other tissues studied showed some similarities, with the exception of ornithine which is low in the brain compared wit...

Gas-phase sequencing after electroblotting on polyvinylidene difluoride membranes assigns correct molecular weights to myoglobin molecular weight markers

Commercially available polypeptide marker kits containing peptides generated by cyanogen bromide cleavage of either horse heart myoglobin or sperm whale myoglobin have been investigated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroblotting on polyvinylidene difluoride membranes, and gas-phase sequencing. It could be shown that the molecular weights assigned to the SDS-PAGE bands by the companies are incorrect. Arranged in descending order, the marker kits are composed of the following polypeptide fragments from myoglobin: positions 1-153, 1-131, 56-153, 56-131, 1-55, and 132-153. A polypeptide comprising residues 1-14 was not found. According to these results the log Mr versus Rf plot used for calibration must be revised. For the separation of low molecular weight polypeptides and peptides a new gel system based on the theory of multiphasic zone electrophoresis combined with a modified Coomassie staining procedure is reported.

Bulk Separation and Long‐Term Culture of Oligodendrocytes from Adult Pig Brain. I. Morphological Studies

Abstract: A method is described by which oligodendrocytes from adult pig brains can be isolated. It results in a cellular preparation suitable for long‐term culture. The entire procedure can be accomplished within 2–3 h. The purity of oligodendrocytes ranges between 80 and 95% depending on the Percoll gradient used and on the time in vitro. Yields between 2.5 and 4 × 107 cells per brain and plating efficiencies on the order of 60% make the system very useful for biochemical investigations. It was shown by immunocytochemical studies that oligodendrocytes produce extensive networks of processes, some of them having elaborate membranous expansions. Anti‐galactocerebroside (GC) antibodies as well as anti‐myelin basic protein (MBP), anti‐Wolfgram protein (WP), antiglial fibrillary acidic protein (GFAP), and monoclonal antibodies O1 and O4 are used to identify the cell types and to characterize the cellular composition of the cultures. Anti‐GC and O1 are suitable markers for these oligodendrocytes. Both antibodies label similar cells, and the staining intensities are equally strong. In the case of O4, variable staining intensities are observed, and a few additional cells are labeled that are anti‐GC−. After 31/2 weeks in culture, about 60% of the cells can be labeled by anti‐MBP. Here too differences in staining intensities are observed. The anti‐WP stain is too weak to be defined as positive. The percentage of GFAP+ cells lies in the range 15–20% at maximum. Cells were also mixed into collagen gels. This method appears to be more useful for outgrowth and branching of fibers than are monolayer systems. Drawbacks, however, include limited access for the antibodies and poor recovery of undamaged cells with their fibers.

Dopamine metabolism in characterised neurones ofPlanorbis corneus

A sensitive chromatographic procedure was used to study the metabolism of [14C]tyrosine, [3H]DOPA and [3H]dopamine in 3 defined cell-types situated in the nervous system of Planorbis corneus. One of the cell-types contains dopamine (GDC), the other serotonin (GSC) and the other neither amine (GC). The GDCs metabolise [14C]tyrosine to form DOPA and dopamine while the other two cells lack this ability. In contrast, the GDCs and the GSC, but not the GCs, metabolise [3H]DOPA to form dopamine. In addition the GDCs incorporate radioactivity from [3H]DOPA into DOPAC, homovanillic acid and methoxytyramine. After incubation of cells in [3H]dopamine, only the GDCs metabolise it to form DOPAC, homovanillic acid and methoxytyramine. In no instance did the GDCs form significant amounts of noradrenaline from the incorporated radioactive substances. These results, together with data on the amine histochemistry of the individual cell-types following pretretment of animals with drugs known to affect specific enzymes in the synthesis of amine transmitter substances, clearly demonstrate that the GDCs alone have the enzymes requisite for the biosynthesis and catabolism of dopamine, but not noradrenaline.

MEMBRANE PROTEINS OF RAT BRAIN: COMPOSITIONAL CHANGES DURING POSTNATAL DEVELOPMENT

Abstract— Membrane fractions from forebrain of rat were isolated at ages ranging from 5 to 93 days. Among these fractions were total membranes, three fractions isolated by density gradient centrifugation, and three subfractions which consisted of purified myelin and of two supernatant fractions. All membrane fractions showed an increase in protein content during the first postnatal month; however, only the myelin fraction and one of its supernatant fractions showed a prolonged accumulation. Myelin protein increased continually from 0.17 mg/g brain at 15 days to 8.3 mg/g brain at 93 days.

Isolation and cultivation of mature oligodendroglial cells

ConclusionThe isolation of mature ODC was improved appreciably during the past five years. It is now possible to maintain ODC cultures for several weeks. This should help to elucidate the functions of ODC in myelination and inflammation and their basic role within the neural network. However, several experimental strategies are needed to answer the manifold open questions addressed to ODC. Among others a very basic one is: what factors direct the glial cell lineage to become an oligodendrocyte or an astrocyte, if a common progenitor cell exists [76, 100].

A dot-blot assay for quantitation of nanogram amounts of protein in the presence of carrier ampholytes and other possibly interfering substances☆

A method for protein determination in one- and two-dimensional electrophoresis sample buffer is presented. Accurate quantitation of protein in two-dimensional electrophoresis sample buffer (9.5 M urea, 2% Nonidet P-40, 2% carrier ampholytes, and 5% 2-mercaptoethanol) required removal of carrier ampholytes prior to the assay. This was made possible by taking advantage of the mutual solubility/insolubility of carrier ampholytes/proteins in saturated ammonium sulfate solution. In addition, improvement of protein determination in denaturing electrophoresis sample buffer containing the anionic detergent sodium dodecyl sulfate and the reducing agent 2-mercaptoethanol was achieved. The assay covers a range of sensitivity from 40 ng to 20 micrograms of protein. The procedure is applicable to large numbers of samples.

Nervous system myelin in the electric ray, Torpedo marmorata: Morphological characterization of the membrane and biochemical analysis of its protein components

In Torpedo, PNS as well as CNS myelines are characterized by clearly separated double intraperiod lines. CNS myelin of Torpedo contains two glycosylated hydrophobic proteins labelled T1 (25,800 Da1) and T2 (29,700 Da1), and two basic proteins BP1 and BP2, migrating like mammalian large basic protein (BP2) and pre-small basic protein (BP1) (Barbarese et al., 1977). PNS myelin of Torpedo carries only BP1 and is characterized by a closely spaced doublet of the glycosylated hydrophobic proteins Con A+ (29,700 Da1) and Con A? (31,000 Da1); the latter does not bind Concanavalin A. These glycosylated proteins (T1, T2, Con A+, Con A?) contain mannose, N-acetylglucosamine and galactose, but lack fucose and sialic acids. They have isoleucine at their amino terminus. They bind anti-rat PNS myelin P(0) antibodies but do not react with anti-rat CNS myelin PLP antibodies. Limited proteolyses of isolated proteins suggest sequence homologies between T1 and T2, and possibly between Con A+ and Con A?. The two basic proteins BP1 and BP2 bind antibodies directed against human myelin basic protein. All Torpedo myelin proteins electrofocus in pH regions characteristic of their mammalian counterparts.

Amino acid and serotonin content in the nervous system, muscle and blood of the cockroach Periplaneta americana.

Abstract The levels of different free amino acids and related compounds in the nervous tissue, muscle and blood of the cockroach Periplaneta americana were measured using a micro dansyl procedure. More than 33 dansyl derivatives were identified in the various cockroach tissues. Of these only the nervous tissue contains serotonin but lacks an unknown substance (spot No. 24) which occurs in muscle and blood. The composition of the amino acid pool in the 3 tissue types shows a number of dissimilarities, although the predominant amino acids in all 3 tissue types include proline, alanine, glycine, glutamic acid and glutamine. The nervous tissue also contains a relatively high amount of GABA and aspartic acid. Studies on the accumulation of the 4 major nervous tissue amino acids (proline, GABA, glycine and glutamic acid) into the nervous tissue are also reported. When nervous tissue was incubated at 24 °C in a medium containing 10−5M of either [14C]-proline, [14C]GABA, [14C]glycine or [14C]glutamic acid, tissue-medium ratios of 10:1, 14:1, 8.5:1 and 13.5:1 respectively were obtained after 30 min incubation. The processes responsible for the accumulation of [14C]GABA and [14C]glutamic acid were most sensitive to temperature and were partly inhibited by 2,4-dinitrophenol and ouabain.

Determination of Amino Acids In Single Identd7iable Nerve Cells of Helix Pomatia

Microchromatography of dansylated compounds was used to study the distribution of free amino acids, GABA, serotonin and 5-hydroxyindole in the metacerebral serotonergic cell and one of the giant cells in the buccal ganglia of Helix pomaiia. The distribution of some of the substances in each of the cell types varied depending upon the isolation procedure. Metacerebral cells dissected in the presence of nialamide contained large amounts of serotonin. The same neurons isolated in the absence of nialamide, but dissected with methylene blue contained no serotonin. The distribution of ornithine, glycine, alanine, arginine, e-lysine, α-amino-histidine and cystine in each cell type varied depending upon the method of isolation.Generally the distribution of dansylated substances is similar; GABA is present in each cell type but in low concentrations. However, there are some exceptions. The serotonergic cell contains less ornithine and more glycine than do the buccal cells. In addition the metacerebral cells have h...