The origin of indoleacetic acid and indolepropionic acid in rat and human cerebrospinal fluid

Abstract: Using a new high performance liquid chromatographic method we have measured tryptophan, 5-hydroxyindoleacetic acid (5HIAA), indoleacetic acid (IAA), and indolepropionic acid (IPA) in rat and human CSF. Experiments on rats indicate that IPA in CSF is not derived from the CNS but from bacterial metabolism in the intestine. However, IAA in CSF is derived from CNS tryptamine metabolism. Some tryptamine that is formed peripherally diffuses across the blood-brain barrier and augments the tryptamine formed within the CNS. We have concluded from our data that (i) measurements on CSF are a useful way of studying trace amine metabolism in human CNS, but it is essential to establish the anatomical and metabolic origin of any metabolite found in the CSF; and (ii) tryptamine metabolism is more important in man than in the rat.     

The organization and structure of nerve and muscle in the jellyfish Cyanea capillata (coelenterata; scyphozoa)

The perirhopalial tissue and swimming muscle of Cyanea were examined with light microscopical and electron microscopical techniques. The perirhopalial tissue is a thin, triangular septum found on the subumbrellar surface of the animal. It separates part of the gastric canal system from the surrounding seawater, and is bound on two sides by radial muscle bands and on the third, the shorter side, by a rhopalium and the margin of the bell. The ectoderm of the perirhopalial tissue is composed of large, somewhat cuboidal, vacuolated, myoepithelial cells. The muscle tails of these cells form a single layer of radial, smooth muscle. Neurons of the “giant fiber nerve net” (GFNN), which form an extensive net over the perirhopalial tissue, lie at the base of the vacuolated portion of the myoepithelial cells. These neurons are visible in living tissue. The morphology of individual GFNN neurons was examined following intracellular injection of the fluorescent dye Lucifer Yellow. The neurons are usually bipolar and free of branches. At the electron microscope level, one usually finds that the GFNN neurons contain large vacuoles. The other characteristic feature of these cells is that they form symmetrical, or nonpolarized, synapses; that is, synaptic vesicles are found on both sides of the synapse. The swimming muscle is striated and composed of myoepithelial cells. Each myoepithelial cell has several muscle tails, and those of adjacent cells are linked to gether by desmosomes. The endoderm of the perirhopalial tissue also was examined. This investigation of the organization and ultrastructure of the perirhopalial tissue and surrounding muscle was undertaken to provide essential background information for an ongoing physiological study of the GFNN neurons and their synapses.     

Mechanistic investigations of Escherichia coli cytidine-5'-triphosphate synthetase. Detection of an intermediate by positional isotope exchange experiments

CTP synthetase from Escherichia coli catalyzes exchange of 18O from the beta gamma-bridge position of [gamma-18O4] ATP into the beta-nonbridge position. This positional isotope exchange occurs in the presence of UTP and MgCl2 but in the absence of NH3. The enzyme also has an ATPase activity in the presence of UTP that occurs under conditions that are identical to those used in the positional isotope exchange experiments. These data provide evidence for the stepwise nature of the reactions catalyzed by CTP synthetase with the initial step involving phosphorylation of UTP by ATP. The relative rate of the isotope exchange reaction is approximately 3 times faster than the ATPase reaction, but the isotope exchange rate is approximately 3% of the overall rate in the presence of NH3. These results are consistent with the ATPase reaction involving attack of water on the phosphorylated intermediate (4-phospho-UTP). The positional isotope exchange reaction is independent of the UTP concentration above saturating levels of UTP demonstrating that the order of addition of substrates is UTP followed by ATP and then NH3.     

Characterization of the isolated rat flexor digitorum brevis for the study of skeletal muscle phosphorylase kinase phosphorylation

The flexor digitorum brevis skeletal muscle, a nearly homogeneous fast-twitch oxidative glycolytic fiber type, has been examined for its suitability to explore the regulation of phosphorylase kinase by multisite phosphorylation. A characterization of the adrenergic response of glycogenolytic enzymes, together with the previous data on contractile properties (Carlsen, R. C., Larson, D. B., and Walsh, D. A. (1985) Can. J. Physiol. Pharm. 63, 958-965), has demonstrated that this muscle is stably maintained for the several hours necessary for phosphorylation studies. The phosphorylase kinase in this muscle is primarily the alpha' isozyme, suggesting that the alpha versus alpha' isozyme distribution in muscle is related more to oxidative capacity than to fiber contractile characteristics. Using this muscle system, beta-adrenergic activation of phosphorylase kinase was observed to occur with concomitant phosphorylation of both the alpha' and beta subunits, with the total in the alpha' subunit being approximately 3-fold greater. Similarly, deactivation, following initial adrenergic activation, occurred concomitantly with the dephosphorylation of the two subunits. These results are compatible with the conclusions drawn from previous studies of the isolated enzyme and of the enzyme in perfused rat cardiac muscle, that both alpha' (or alpha) and beta subunit phosphorylation regulate phosphorylase kinase activity.     

Involvement of the carboxy-terminal residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site that involves His-15, which is phosphorylated to form a N delta 1-P-histidine, Arg-17, and the carboxy-terminal residue Glu-85. Mutant HPrs with alterations to the three C-terminal residues, Glu-85, Leu-84, and Glu-83, were produced by site-directed mutagenesis. The properties of these mutants were assessed by kinetic analysis of enzyme I, enzyme IImannose, enzyme IIN-acetylglucosamine, and enzyme IImannitol, and the phosphohydrolysis properties of the HPr mutants. The results show that it is the C-terminal alpha-carboxyl of Glu-85 that is involved in the active site, and this involvement may be restricted to the phosphoryl donor action of HPr. The contribution of this alpha-carboxyl group is modest as the deletion of Glu-85 resulted in the reduction of the enzyme II activity (kcat/Km) to about 33%. Removal of both Glu-85 and Leu-84 yields an HPr that is an impaired substrate of both the enzyme I and enzyme II reactions. Glu-83 appears to have no role in the active site.     

Effects of urea, trimethylamine oxide, and osmolality on respiration and citrulline synthesis by isolated hepatic mitochondria from Squalus acanthias

The effects of osmolality and of urea and trimethylamine oxide on citrulline synthesis and respiration by isolated hepatic mitochondria from Squalus acanthias (spiny dogfish) were determined. Optimal rates of citrulline synthesis and of respiration, as well as maximal respiratory control ratios, were obtained when the osmolality was about 300 mOsm when physiological concentrations of urea and trimethylamine oxide were present, analogous to mammalian mitochondria. Urea and trimethylamine oxide do not act as osmolytes and do not significantly affect these rates. When glutamate is not saturating, increasing concentrations of urea over the range of concentrations of physiological significance (e.g. 0.1-0.5 M) significantly inhibit citrulline synthesis, but not respiration. This response to urea concentration may reflect a specific homeostatic mechanism for maintaining physiological concentrations of urea.