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R L Beach

Publications and source records attributed to R L Beach.

28 records · Page 2Linked to original sources
PubMed1982

Identification of cell types in rat hippocampal slices maintained in organotypic cultures.

We have cultured transverse slices of the hippocampal formation from neonatal rats and have identified the cell types which appear in the outgrowth with cell type specific markers. Tetanus toxin and anti-tetanus toxoid, as well as antisera to neurofilaments and 14-3-2 protein, were used to identify neurons. Astrocytes were identified with antisera to glial fibrillary acidic protein and were the predominant non-neural cell type. Fibroblastic cells were labeled with antisera to fibronectin and to myosin and oligodendroglia were identified with antisera to galactocerebroside. The hippocampal neurons could be classified as 1 or the 3 types present in vivo (pyramidal cells, granule cells, or GABAergic interneurons) on the basis of their size, shape, location, or reaction with antisera to glutamic acid decarboxylase. Outgrowth of glial cells and neurites occurred within hours of explantation. Within a few days granule cell neurons migrated onto the glial cell layer from the explant. Their movement is probably related to their migration during in vivo development of the granule cell layer. Synapse formation was observed by electron microscopic analysis beginning about 3-5 days in vitro and areas of neuropil containing many synapses were observed after 3-4 weeks. This culture system should be useful for further studies on the cellular processes which occur during hippocampal development and plasticity.

Animals↗
PubMed1981

Identification of myosin in isolated synaptic junctions.

A monospecific antibody prepared against chicken gizzard myosin reacted with only one peptide corresponding to myosin heavy chain (Mr = 200,000) in gels of synaptic plasma membranes (SPM) and synaptic junctions (SJ) prepared from several species. Preadsorption of antisera with purified brain myosin eliminated antibody reactivity to SPMs and SJs. SJs were found to contain approximately 3 times the concentration of myosin found in SPMs when assayed by an indirect immunoradiometric assay. Postsynaptic density and myelin fractions contained no myosin detectable by immunoradiometric assay, antibody binding to gels, or Coomassie blue staining. The band identified as myosin in SJ fraction yielded peptide fingerprints indistinguishable from fingerprints of purified brain myosin but distinct from fingerprints of purified smooth and skeletal muscle myosins. The distribution of exogenous [125I]myosin during subcellular fractionation indicated that myosin in isolated synaptic junction could not have resulted from artifactual re-distribution of soluble myosin. Together these results show that a non-muscle myosin is an endogenous component of CNS asymmetric synapses.

Animals↗
PubMed1977

Identification of D-threo-alpha-methylisocitrate as stereochemically specific substrate for bovine heart aconitase and inhibitor of TPN-linked isocitrate dehydrogenase.

DL-threo-alpha-Methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) is a substrate for bovine heart aconitase and an inhibitor of TPN-linked isocitrate dehydrogenase from liver and heart. The isomer of alpha-methylisocitrate formed from alpha-methyl-cis-aconitate (cis-2-butane-1,2,3-tricarboxylate) by aconitase inhibits TPN-linked isocitrate dehydrogenase and has been identified as D-threo-alpha-methylisocitrate (2S,3R)-3-hydroxy-1,2,3-butanetricarboxylate) by optical rotation and circular dichroism studies. Mitochondrial bovine heart aconitase catalyzes a reversible reaction between D-threo-alpha-methylisocitrate (Km, 0.2 mM) and alpha-methyl-cis-aconitate (Km, 0.05 mM) at pH 7.4. However, formation of methylcitrate (2-hydroxy-1,2,3-butanetricarboxylate) from these substrates or utilization of synthetic methylcitrate for formation of these products could not be demonstrated with bovine heart aconitase. DL-threo-alpha-Methylisocitrate is also a substrate for aconitase from rat liver cytosol (Km, 0.1 mM); Vmax with citrate is approximately 1.4 times that with DL-threo-alpha-methylisocitrate. The ratio of activities for these substrates observed with the bovine heart enzyme is about 5. Formation of alpha-methyl-cis-aconitate from synthetic methylcitrate could not be detected spectrophotometrically with the liver aconitase; if it occurs with either the liver or the heart enzyme, the rate would be less than 0.1% that obtained with DL-threo-alpha-methylisocitrate. A new synthesis of methylcitric acid in good yields from diethyl alpha-methyl-beta-ketoglutarate (diethyl 2-methyl-3-oxoglutarate) and cyanide has been described. NMR spectroscopy indicates that this synthetic methylcitric acid contains the two racemic pairs of diastereoisomers.

Aconitate Hydratase↗
PubMed1975

Alpha-methylisocitrate. A selective inhibitor of TPN-linked isocitrate dehydrogenase from bovine heart and rat liver.

Alpha-Methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) is a potent inhibitor, competitive with isocitrate (1-hydroxy-1,2,3-propanetricarboxylate), of the TPN-linked isocitrate dehydrogenase from bovine heart and rat liver; it does not inhibit the DPN-specific enzyme from these tissues. In the presence of magnesium ion, values of Kis for DL-alpha-methylisocitrate for purified bovine heart enzyme, rat liver cytosol, and rat liver mitochondrial extract were in the range of 0.1 muM to 0.3 muM. This compared to values of apparent Km for DL-isocitrate for the same tissue preparations of 14 muM to 20 muM. One of the DL isomer pairs of alpha-methylisocitrate was inactive; the observations suggest that it is threo-alpha-methylisocitrate which inhibits TPN-linked isocitrate dehydrogenase. A method of synthesis of DL-threo-alpha-methylisocitric lactone (2-methyl-5-oxo-2,3-furandicarboxylic acid) from dimethyl trans-epoxymethylsuccinate and dimethylmalonate is described.

Animals↗
PubMed1975

Substrate activity of structural analogs of isocitrate for isocitrate dehydrogenases from bovine heart.

D-Garcinia acid (D-threo-1,2-dihydroxy-1,2,3-propanetricarboxylate), like D-isocitrate, has an alpha-DS-hydroxyl group and a beta-LS configuration of the second carboxyl group. The maximal velocity of pyridine nucleotide reduction with D-garcinia acid is 8 and 21% of D-threo-isocitrate with the DPN-linked and TPN-linked isocitrate dehydrogenase from bovine heart, respectively. The other stereoisomers of hydroxycitrate [L-garcinia acid, D- and L-hibiscus acid (D- and L-erythro-1,2-dihydroxy-1,2,3-propanetricarboxylate)] are inactive. DL-threo-Homoisocitrate (DL-threo-1-hydroxy-1,2,4-butanetricarboxylate) supports DPN+ reduction at 10-15% of the rate observed for isocitrate with the DPN-specific enzyme, but is not a substrate for TPN-linked isocitrate dehydrogenase. The values of apparent S0.5 for total isocitrate and total garcinia acid are similar with both enzymes; the apparent S0.5 of total homoisocitrate is two- to threefold higher than that of total isocitrate with the DPN-linked enzyme. Enzymatic oxidative decarboxylation of garcinia acid and homoisocitrate leads to formation of alpha-keto-beta-hydroxyglutarate and alpha-ketoadipate, respectively. DL-Methylmalate (DL-1-hydroxy-2-methylsuccinate) is inactive as a substrate for either dehydrogenase as are the newly synthesized compounds: DL-threo-gamma-isocitrate amide (DL-threo-1-hydroxy-3-carbamy01,2-propanedicarboxylate), beta-methyl-DL-isocitrate (DL-1-hydroxy-2-methyl-1,2,3-propanetricarboxylate), beta-methyl-DL-garcinia acid (DL-threo-1-hydroxyl-2-methoxy-1,2,3-propanetricarboxylate), DL-1-hydroxyl-1,2,2-ethanetricarboxylate, and DL-1,4-dihydroxy-1,2-butanedicarboxylate.

2,6-Dichloroindophenol↗