Inactivation of Tyrosine Hydroxylase Activity by Ascorbate In Vitro and in Rat PC12 Cells

Abstract: Tyrosine hydroxylase activity is reversibly modulated by the actions of a number of protein kinases and phosphoprotein phosphatases. A previous report from this laboratory showed that low-molecular-weight substances present in striatal extracts lead to an irreversible loss of tyrosine hydroxylase activity under cyclic AMP-dependent phosphorylation conditions. We report here that ascorbate is one agent that inactivates striatal tyrosine hydroxylase activity with an EC₅₀ of 5.9 μM under phosphorylating conditions. Much higher concentrations (100 mM) fail to inactivate the enzyme under nonphosphorylating conditions. Isoascorbate (EC₅₀, 11 μM) and dehydroascorbate (EC₅₀, 970 μM) also inactivated tyrosine hydroxylase under phosphorylating but not under nonphosphorylating conditions. In contrast, ascorbate sulfate was inactive under phosphorylating conditions at concentrations up to 100 mM. Since the reduced compounds generate several reactive species in the presence of oxygen, the possible protecting effects of catalase, peroxidase, and superoxide dismutase were examined. None of these three enzymes, however, afforded any protection against inactivation. We also examined the effects of ascorbate and its congeners on the activity of tyrosine hydroxylase purified to near homogeneity from a rat pheochromocytoma. This purified enzyme was also inactivated by the same agents that inactivated the impure corpus striatal enzyme. Under conditions in which ascorbate almost completely abolished enzyme activity, we found no indication for significant prote-olysis of the purified enzyme as determined by sodium do-decyl sulfate-polyacrylamide gel electrophoresis. We also found that pretreatment of PC12 cells in culture for 4 h with 1 mM ascorbate, dehydroascorbate, or isoascorbate (but not ascorbate sulfate) also decreased tyrosine hydroxylase activity 25–50%. The inactivation seen under in vitro conditions appears to have a counterpart under more physiological conditions.     

Phosphorylation of tyrosine hydroxylase in the superior cervical ganglion

We studied the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion of the rat. Ganglia were preincubated with [³²P]P_i and were then incubated in non-radioactive medium containing a variety of agents that are known to activate tyrosine hydroxylase in this tissue. Tyrosine hydroxylase was isolated from homogenates of the ganglia by immunoprecipitation followed by polyacrylamide gel electrophoresis. ³²P-labelled tyrosine hydroxylase was visualized by radioautography, and the incorporation of ³²P into the enzyme was quantitated by densitometry of the autoradiograms. Veratridine produced a concentration-dependent increase in the incorporation of ³²P into tyrosine hydroxylase, with 50 μM veratridine producing a 5-fold increase in ³²P incorporation. The nicotinic agonist, dimethylphenylpiperazinium (100 μM), caused a 7-fold increase in the phosphorylation of tyrosine hydroxylase. The effect of dimethylphenylpiperazinium was maximal within 1 min and decreased upon continued exposure of the ganglia to this agent. The actions of dimethylphenylpiperazinium and of veratridine were dependent on extracellular Ca²⁺. Muscarine, 8-Br-cAMP, forskolin, vasoactive intestinal peptide, isoproterenol, deoxycholate and phospholipase C also stimulated the incorporation of ³²P into tyrosine hydroxylase. These data support the hypothesis that phosphorylation plays a role in activation of tyrosine hydroxylase produced by all of these agents.     

Measurement of tyrosine hydroxylase activity in serve terminals of rat hippocampus,hypothalamus, and striatum using an improved assay for tyrosine hydroxylase

The formation of ³H₂O from L-4-³H-phenylalanine is used as an index of tyrosine hydroxylase activity in synaptosomes from rat hippocampus, hypothalamus, and striatum. The reactions are linear with respect to time (up to 20 min) and with respect to protein concentration (up to 0.2 mg/ml). Formation of ³H₂O from L-4-³H-phenylalanine is inhibited by standard tyrosine hydroxylase inhibitors (α-methyl-p-tyrosine, L-3-iodotyrosine, dopamine, L-norepinephrine, and L-apomorphine) and by the tyrosine hydroxylase substrate L-tyrosine as well as by synaptosomal lysis. The blank ³H₂O produced from L-4-³H-phenylalanine (0.02% of total DPM) is 10-fold less than the blank ³H₂O produced from L-3,5-³H-tyrosine. The K_m values of tyrosine hydroxylase for phenylalanine determined by the production of ³H₂O from L-4-³H-phenylalanine are 3.1, 1.3, and 1.2 μm in hippocampal, hypothalamic and striatal synaptosomes respectively. The results indicate that analysis of ³H₂O formed from L-4-³H-phenylalanine is a sensitive and reliable method for quantitating synaptosomal tyrosine hydroxylase activity from tissues with low levels of tyrosine hydroxylase such as synaptosomes from hippocampus and hypothalamus.     

A denaturant-insoluble form of tyrosine hydroxylase in PC12 pheochromocytoma cells

A 6M urea-insoluble form of tyrosine hydroxylase (TH_i) was detected in PC12 pheochromocytoma cells by western blotting immunodetection methods, and the characteristics and mechanisms of formation of this insoluble species were investigated. TH_i accounts for about 4% of the immunodetectable tyrosine hydroxylase in exponentially dividing pheochromocytoma cells. It is unlikely that a subpopulation of dead or dying cells is the source of TH_i since essentially no changes in TH_i levels were detected when cell death was intentionally increased. To measure the kinetics of formation of cellular TH_i, exponentially dividing cells were metabolically labeled first with [³H]leucine and then with [¹⁴C]leucine, and though both³H and¹⁴C were incorporated into soluble tyrosine hydroxylase, the near absence of¹⁴C in TH_i demonstrated that a lag period of at least a day exists between biosynthesis of tyrosine hydroxylase and the accumulation of measurable TH_i. The cellular accumulation of TH_i can evidently be regulated by the cell, since upon nerve growth factor (NGF) treatment of cells the total content of tyrosine hydroxylase increased and the content of TH_i decreased to yield, overall, a fivefold lower proportion of TH_i after 4 days. A large increase in urea-insoluble enzyme was found upon sublethal exposure of cells to ferrous ion and hydrogen peroxide, indicating that oxidative damage via metal-ion-catalyzed formation of hydroxide free radical can yield an enzyme that is similar in its insolubility to TH_i.Abbreviations DOPA 3,4-dihydroxyphenylalanine - NGF nerve growth factor - TH_i denaturant-insoluble tyrosine hydroxylase - EDTA ethylene diamine tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - SDS sodium dodecyl sulfate - Tris Tris(hydroxymethyl)-aminomethane - LLPM low-leucine pulse medium - WS water-solubilized protein - US 6 M urea-solubilized protein - UI 6 M urea-insoluble protein     

Control of ketogenesis from amino acids: III. In vitro and in vivo studies on ketone body formation, lipogenesis and oxidation of tyrosine by rats

Ketone body formation from tyrosine was studied in rat liver in vitro with special references to the activities of tyrosine aminotransferase (EC 2.6.1.5) and p-hydroxyphenylpyruvate hydroxylase (EC 1.14.2.2). Liver was obtained from rats which had been given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase. The enzyme activities of the preparations were plotted against the amounts of ketone body formed from tyrosine. It was found that over a low range of tyrosine aminotransferase activities, activity was proportional to the amount of ketone body formed. However, above this range, ketone body formation ceased to increase and p-hydroxyphenylpyruvate started to accumulate. This inhibition of ketone body formation and accumulation of the p-hydroxyphenylpyruvate could be prevented by addition of ascorbate. These results suggest that the primary factor regulating metabolism of tyrosine in vitro is tyrosine aminotransferase and when the activity of this is high so that it is no longer rate limiting, p-hydroxyphenylpyruvate hydroxylase becomes the rate limiting step because its activity is inhibited by the accumulation of p-hydroxyphenylpyruvate.For in vivo studies rats were given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase and then injected with a tracer dose of [U- or 1-¹⁴ C]tyrosine. Then their respiratory ¹⁴CO₂ and the incorporation of ¹⁴C into total lipids of liver were measured. The amounts of radioactivity in CO₂ and lipids were found to be proportional to the tyrosine aminotransferase activity and were not affected by the free tyrosine concentration in the liver. After injection of [U-¹⁴C] acetate the radioactivities in CO₂ and lipids were not proportional to the tyrosine aminotransferase activity. These results indicate that the enzyme activity also regulates tyrosine metabolism in vivo. In vivo studies gave no evidence of the participation of p-hydroxyphenylpyruvate hydroxylase in regulation of tyrosine metabolism.     

Involvement of Intracellular or Extracellular Calcium in Activation of Tyrosine Hydroxylase Gene Expression in PC12 Cells

Abstract: The relationship between elevations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K⁺ concentration triggered rapid and sustained increases in [Ca²⁺]_i from a basal level of ～50 to 110–150 nM and three- to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5-trisphosphate (IP₃). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP₃ or inhibition of Ca²⁺-ATPase, rapidly elevated [Ca²⁺]_i to >200 nM and increased TH gene expression (three-to fivefold). Confocal imaging showed that the elevations in [Ca²⁺]_i in each case occurred throughout the cyto- and nucleoplasm. The initial rise in [Ca²⁺]_i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca²⁺]_i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca²⁺]_i, there are important differences among them in terms of the magnitude of elevated [Ca²⁺]_i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca²⁺]_i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.     

PROPERTIES OF TYROSINE HYDROXYLASE IN PERIPHERAL NERVES

Approximately 80 per cent of tyrosine hydroxylase activity in bovine mandibular nerve and rabbit sciatic nerve was soluble, and the rest of the activity was particle-bound. The soluble enzyme in bovine mandibular nerve was isolated by ammonium sulphate fractionation (25–35 per cent saturation). The enzyme had a pH optimum at 5·9 in Tris-acetate buffer, and at 6·5 in Tris-HCl or phosphate buffer. The enzyme required a tetrahydropteridine cofactor. K_m values toward various tetrahydropteridines such as l -erythro-tetrahydrobiopterin (a probable natural cofactor), 2-amino-4-hydroxy-6-methyltetrahydropteridine, and 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine were 2 × 10⁻⁵m , 5 × 10⁻⁵m and 4 × 10⁻⁴m , respectively. The K_m value for tyrosine at 1 × 10⁻³m -2-amino-4-hydroxy-6-methyltetrahydropteridine as a cofactor was 5 × 10⁻⁵m . The enzyme activity was markedly stimulated with Fe²⁺ or catalase, but Fe²⁺ gave higher activity. The activity was inhibited with α, α′-dipyridyl, l -α-methyl-p-tyrosine, and various catecholamines. Among catecholamines, dopamine was the most potent inhibitor. l -5-Hydroxytryptophan was an inhibitor as potent as dopamine. Neither d -5-hydroxytryptophan nor 5-hydroxytryptamine inhibited the enzyme. The inhibition by l -5-hydroxytryptophan was partially competitive with tetrahydrobiopterin at concentrations higher than 9 × 10⁻⁵m , and partially uncompetitive at concentrations lower than 9 × 10⁻⁵m . The addition of heparin or lysolecithin did not affect enzyme activity with tetrahydrobiopterin as cofactor.     

The effect of silver and other metal ions on the in vitro growth of root-rotting Phytophthora and other fungal species

A range of metal ions and the oxoanion WO₄^2-were toxic to zoospores of Phytophthora nicotianae parasitica in the order: Ag⁺ > Cu⁺⁺ > WO₄^2-> Ni⁺ > Co⁺⁺ > Zn⁺. The LD₅₀ for Ag⁺, 0.11 μM (11.4 ppb), compared with 1.84 μm (117 ppb) for Cu⁺⁺. Silver was similarly toxic to a range of pathogens including Pythium aphanidermatum, Thielaviopsis basicola and Fusarium oxy-sporum f.spp. Most zoospores of Phytophthora spp. were killed by Ag⁺ in the range 46–460 nM (5–50 ppb), bursting at the higher concentrations. A small sub-population of most propagules exhibited greater tolerance to silver than the whole. In 0.93 μM (100 ppb) Ag⁺ 1.4% of P. nicotianae parasitica zoospores survived but were all killed at 500 ppb. A population of P. cryptogea (1.9%) surviving 0.47 μm (50 ppb) were killed at 0.93 μM (100 ppb). Zoospore cysts and germlings showed the same sensitivity to silver. Oospores were mostly killed over the range 0.23–0.93 μm (25–100 ppb) Ag⁺, some surviving up to the lethal concentration of 9.26 μM (1000 ppb). Mycelium of P. cryptogea was generally less sensitive, with some growth occurring at 9.26 μm (100 ppb). Zoosporangiogenesis was unaffected over the range 0.47–4.65 μm (50–500 ppb). Toxicity increased with increased pH over the range 5.0–6.5. Ionic silver was lost from solution during a microscope slide bioassay by binding to the glass surface. In the presence of chloride ions, colloidal AgCl formed which was equally toxic to P. cryptogea. Silver and AgCl were further lost from solution by colloidal agglomeration - Ostwald ripening - and by AgCl adsorption to glass. Silver, < 90 nM (10 ppb) Ag⁺ as AgNO₃ and particles of silver chloride were both strongly attractive to zoospores of P. cryptogea. Spores burst or failed to germinate on entering lethal concentrations. The results are discussed in the context of the use of silver salts to control Phytophthora root-rot pathogens and the importance of ion availability in in vitro toxicity assays.     

Regulation of catecholamine synthesis in rat brain synaptosomes

Abstract— Catecholamine synthesis in synaptosomal preparations of rat striatum, cortex and brain stem was investigated. The striatum had much higher activity than either the cortex or brain stem. Equilibration of labelled tyrosine between tissue and incubation medium was completed within 2 min. The apparent K_m of tyrosine hydroxylase (EC 1.14.3a) and of the overall catecholamine synthetic pathway were both approximately 5 ± 10^?6m for tyrosine. The following amines were found to inhibit striatal dopamine synthesis: dopamine, 25% inhibition at 5 ± 10^?7m ; noradrenaline, 25% inhibition at 5 ± 10^?6m ;and serotonin, 30% inhibition at 10^?5m . The catecholamine-induced inhibition of synthesis was antagonized by pre-incubation with cocaine. Increasing the potassium concentration from 5 to 55 mm caused a release of amines into the medium which was accompanied by a 40% increase in dopamine synthesis, when synthesis was measured during the first 5 min of exposure to elevated potassium. These results indicate that synaptosomal catecholamine synthesis is inhibited by increases in intra-synaptosomal amine levels, and that short-term exposure to depolarizing concentrations of potassium can increase synthesis.     

A SOLUBLE PREPARATION FROM RAT BRAIN THAT INCORPORATES INTO ITS OWN PROTEINS [14C]ARGININE BY A RIBONUCLEASE-SENSITIVE SYSTEM AND [14C]TYROSINE BY A RIBONUCLEASE-INSENSITIVE SYSTEM

Abstract— A 100,000 g supernatant fraction from rat brain that was passed through a column of Sephadex G-25-40 was able, after addition of some factors, to incorporate [^I4C]arginine (apparent K_m= 5 μM) and [¹⁴C]tyrosine (apparent K_m= 20 μM) into its own proteins. The factors required for the incorporation of [¹⁴C]arginine were: ATP (optimal concentration = 0-25-2 μM) and Mg²⁺ (optimal concentration 5 mM). For the incorporation of [^I4C]tyrosine the required factors were: ATP (apparent K_m= 0-75 μM), Mg²⁺ (optimalconcentration 8-16 mM) and K⁺ (apparent K_m= 16 mM). Addition of 19 amino acids did not enhance these incorporations. Optimal pHs were: for [¹⁴C]arginine and [¹⁴C]tyrosine, respectively, 7-4 and 7-0 in phosphate buffer and 7–9 and 7-3-8-1 in tris-HCl buffer. Pancreatic ribonuclease abolished the incorporation of [¹⁴C]arginine but had practically no effect in the incorporation of [¹⁴C]tyrosine. Furthermore, [¹⁴C]arginyl-tRNA was a more effective donor of arginyl groups than [¹⁴C]arginine, whereas [¹⁴C]tyrosyl-tRNA was considerably less effective than [¹⁴C]tyrosine. The incorporations of [¹⁴C]arginine and [¹⁴C]tyrosine into brain proteins were from 25- to 2000-fold higher than for any other amino acid tested (12 in total). In brain [¹⁴C]arginine incorporation was higher than in liver and thyroid but somewhat lower than in kidney. In comparison to brain, the incorporation of [¹⁴C]tyrosine was negligible in liver, thyroid or kidney. Kinetic studies showed that the macromolecular factor in the brain preparation was complex. The protein nature of the products was inferred from their insolubilities in hot TCA and from the action of pronase that rendered them soluble. [¹⁴C]Arginine was bound so that its a-amino group remained free. Maximal incorporation of [¹⁴C]tyrosine in brain of 30-day-old rats was about one-third of that in the 5-day-old rat. The changes with postnatal age in the incorporation of [¹⁴C]arginine were not statistically significant.     

In situ phosphorylation of tyrosine hydroxylase in chromaffin cells: Localization to soluble compartments

The present studies investigated the subcellular distribution of acetylcholine's effects upon the phosphorylation of tyrosine hydroxylase in isolated purified bovine adrenal chromaffin cells. After labeling the intact chromaffin cells with ³²P_i, over 90% of the [³²P]tyrosine hydroxylase was found in soluble fractions. Stimulation of the cells with acetylcholine, the natural secretagogue of chromaffin cells, increased the phosphorylation of tyrosine hydroxylase and over 90% of the increase was associated with soluble tyrosine hydroxylase. Homogenates and subcellular fractions from chromaffin cells were also prepared and phosphorylated in vitro in an attempt to optimize detection of tyrosine hydroxylase phosphorylation. In chromaffin cell homogenates, both 8-bromo-cyclic AMP and calcium increased ³²P incorporation into tyrosine hydroxylase, and again over 90% of the increase was observed in soluble fractions. In the particulate fraction, phosphorylation of a band which comigrated with tyrosine hydroxylase in electrophoresis was occasionally detected but only with very long autoradiographic exposures.Tyrosine hydroxylase enzymatic activity in the isolated purified chromaffin cells was also found to be associated predominantly (approx 90%) with soluble fractions. In contrast, a large portion (40–50%) of the tyrosine hydroxylase activity from crude bovine adrenal medullae was associated with the particulate fraction.The data indicate that although tyrosine hydroxylase (and possibly kinases) can associate with particulate fractions when isolated from crude bovine adrenal medullae, the enzyme is predominantly soluble when isolated from the isolated cells. Further, the effects of acetylcholine on the isolated chromaffin cells are predominantly associated with this soluble tyrosine hydroxylase and its attendant kinases.     

PRIMARY PRODUCTION OF CRUSTOSE CORALLINE RED ALGAE IN A HIGH ARCTIC FJORD1

Crustose coralline algae occupied ～1%–2% (occasionally up to 7%) of the sea floor within their depth range of 15–50 m, and they were the dominant encrusting organisms and macroalgae beyond 20 m depth in Young Sound, NE Greenland. In the laboratory, oxygen microelectrodes were used to measure net photosynthesis (P) versus downwelling irradiance (E_d) and season for the two dominant corallines [Phymatolithon foecundum (Kjellman) Düwel et Wegeberg 1996 and Phymatolithon tenue (Rosenvinge) Düwel et Wegeberg 1996] representing> 90% of coralline cover. Differences in P‐E_d curves between the two species, the ice‐covered and open‐water seasons, or between specimens from 17 and 36 m depth were insignificant. The corallines were low light adapted, with compensation irradiances (E_c) averaging 0.7–1.8 μmol photons·m ^{? 2}·s ^{? 1} and light adaptation (E_k) indices averaging 7–17 μmol photons·m ^{? 2}·s ^{? 1}. Slight photoinhibition was evident in most plants at irradiances up to 160 μmol photons·m ^{? 2}·s ^{? 1}. Photosynthetic capacity (P_m) was low, averaging 43–67 mmol O₂·m ^{? 2} thallus·d ^{? 1} (～250–400 g C·m ^{? 2} thallus·yr ^{? 1}). Dark respiration rates averaged ～5 mmol O₂·m ^{? 2} thallus·d ^{? 1}. In ice covered periods, E_d at 20 m depth averaged ～1 μmol photons·m ^{? 2}·s ^{? 1}, with daily maxima of 2–3 μmol photons·m ^{? 2}·s ^{? 1}. During the open water season, E_d at 20 m depth averaged ～7 μmol photons·m ^{? 2}·s ^{? 1} with daily maxima of ～30 μmol photons·m ^{? 2}·s ^{? 1}. Significant net primary production of corallines was apparently limited to the 2–3 months with open water, and the small contribution of corallines to primary production seems due to low P_m values, low in situ irradiance, and their relatively low abundance in Young Sound.     

PARTIAL PURIFICATION AND PROPERTIES OF CHOLINE KINASE (EC 2.7.1.32) FROM RABBIT BRAIN: MEASUREMENT OF ACETYLCHOLINE

Choline kinase (EC 2.7.1.32; ATP: choline phosphotransferase) was purified 200-fold from an extract of acetone powder of rabbit brain by a combination of acid precipitation, ammonium sulphate precipitation, DEAE cellulose chromatography, and ultrafiltration. Maximal activity of 243 nmol of phosphorylcholine synthesized. min^?1 mg^?l of protein occurred at pH 9.5–10.0 in the presence of 10 mm MgS0₄, 10 mm choline and 0.005% (w/v) bovine serum albumin. 2-Aminoethanol, 2-methylaminoethanol, and 2-dimethylaminoethanol were also phosphorlyated by the enzyme preparation. The enzyme quantitatively converted low concentrations of choline (2.5–50 μm ) to phosphorylcholine [³²P] in the presence of ATP [y³²P], and may, therefore, be used to measure small amounts of choline acetylcholine. There were two K_m values for choline at pH 9.5; 32 μm and 0.31 mm . At pH 7.4, the higher K_m was not observed and enzyme activity was maximal with 0.1 mm choline. The K_m for ATP was 1.1 mm . Enzyme activity was inhibited by ATP (20 mm ), AMP, ADP, cytidine diphosphocholine (1 or 10 mm ), and activated by choline esters (1.0 mm ), NaCl or KCl(200 mm ).     

OBSERVATION OF SAND-DWELLING TOXIC DINOFLAGELLATES (DINOPHYCEAE) FROM WIDELY DIFFERING SITES, INCLUDING TWO NEW SPECIES

Dinoflagellate associations, including toxic and potentially toxic benthic species, were examined in sand from South Water Cay and Carrie Bow Cay, Belize. The inshore sand habitat in localized areas of warm shallow lagoonal waters supported blooms of toxic assemblages of dinoflagellates. In the sand, the dominant microalgae were dinoflagellates; cyanobacteria were a minor component and diatoms were absent. Ciliates and nematodes were present. Assemblages of microorganisms in colored sand were examined for 4 consecutive days after which a storm washed away the patch. The sand-dwelling dinoflagellate assemblage included 16 species where densities ranged from as low as 1.3% to 15% of total cell densities. The dominant species was Scrippsiella subsalsa, having 1.8 × 10⁵ to 2.6 × 10⁵ cells g^-1 sand. Toxic dinoflagellates identified in the sand were Gambierdiscus toxicus, Ostreopsis lenticularis, Prorocentrum lima, Prorocentrum mexicanum, and Amphidinium carteri. The potentially toxic Ostreopsis labens, Gambierdiscus belizeanussp. nov., and Coolia tropicalis sp. nov. were also identified. Toxic and potentially toxic species represented 36% to 60% of total microalgal cell assemblage. The morphology of a new sand-dwelling species, Gambierdiscus belizeanus sp. nov., was examined with the scanning electron microscope. The plate formula was P_o, 3′, 7″, 6c, s?, 5?, 1p, and 2″″.Dimensions of G. belizeanus cells were 53–67 pm long, 54–63 μm wide, and 92–98 μm in dorsoventral depth. Cells were deeply areolated, ellipsoid in apical view, and compressed anteroposteriorly. The cells of G. belizeanus were identified by the cell's long, narrow, pentagonal, posterior intercalary plate (1p) wedged between the wide postcingular plates 2″’and 4″; 1p occupied 20% of the width of the hypotheca. The plate formula for Coolia tropicalis sp. nov. was P_o, 3′, 7″, 7c, 8s?, 5″″, and 2″″, Cell size ranges were 23–40 μm long, 25–39 μm wide, and 35–65 μm in dorsoventral diameter. Cells were spherical, smooth, and covered with scattered round pores. The epitheca was smaller than the hypotheca. Precingular plates 1″ and 7″ were small and narrow, and the first apical plate 1″ and precingular plate 6″ were the largest plates on the epitheca. The apical pore was straight and 7 μm long, and was situated in the apical plate complex. Cells of C. tropicalis were distinguished from C. monotis by the wedge-shaped plate 1′, a four-sided 3’plate, and a short apical pore.     

In vivo evidence for the involvement of phospholipase A and protein kinase in the signal transduction pathway for auxin-induced corn coleoptile elongation

Auxin-induced elongation of com coleoptiles is accompanied by cell wall acidification, which depends upon H⁺-pump activity. We tested the hypothesis that phospholipase A and a protein kinase are involved in the pathway of auxin signal transduction leading to H⁺ secretion, and elongation of corn coleoptiles. Initially, the pH of the bath solution at 50–100 μm from the surface of a coleoptile segment (pH_o) ranged between 4.8 and 6.6 when measured with an H⁺-sensitive microelectrode. Twenty or 50 μM lysophosphatidylcholine, 50 μM linolenic acid or 50 μM arachidonic acid induced a decline in pH_o by 0.3 to 2.1 units. The effect was blocked by 1 mM vanadate, suggesting that lysophosphatidylcholine or linolenic acid induced acidification of the apoplast by activating the H⁺-pump. Lysophosphatidylcholine and linolenic acid also accelerated the elongation rate of the coleoptiles. While linolenic acid and arachidonic acid, highly unsaturated fatty acids, promoted pH_o decrease and coleoptile elongation, linoleic acid, oleic acid, and stearic acid, fatty acids with a lesser extent of unsaturation, had no such effects. The effects of lysophosphatidylcholine, linolenic acid, and arachidonic acid on H⁺ secretion were not additive to that of indoleacetic acid (IAA), suggesting that lysophospholipids, fatty acids and auxin use similar pathways for the activation of the H⁺-pump. The phospholipase A₂ inhibitors, aristolochic acid and manoalide, inhibited the IAA-induced pH_o decrease and coleoptile elongation. The general protein kinase inhibitors, H-7 or staurosporine, blocked the IAA- or lysophosphatidylcholine-induced decrease in pH_o. H-7 also inhibited the coleoptile elongation induced by IAA or lysophosphatidylcholine. These results support the hypothesis that phospholipase A is activated by auxin, and that the products of the enzyme, lysophospholipids and fatty acids, induce acidification of the apoplast by activating the H⁺-pump through a mechanism involving a protein kinase, which in turn promotes com coleoptile elongation.     

THE EFFECTS OF PHENYLKETONURIC AND OTHER METABOLITES ON SULFATED GALACTOCEREBROSIDE SYNTHESIS IN VIVO AND IN CULTURE

Abstract— Sulfated galactocerebroside synthesis was examined in vitro in mouse spinal cord cultures. This system permitted the study of the effects of phenylketonuric metabolites upon synthesis of a specific myelin component, sulfatide, formed early in postnatal development in mice. A significant reduction of Na₂³⁵SO₄ incorporation into myelin sulfatide was observed when spinal cord cultures were grown in the presence of 1000 μm -l -phenylalanine and 500 μm -phenylpyruvate (51 and 700%, respectively). No reduction was observed with β-phenyllactate (300 μm and) phenylacetate (250 μm ). Light microscopy indicated that the phenylpyruvate and phenylalanine treated cultures were less extensively myelinated compared to control and β-phenyllactate or phenylacetate treated cultures. The reduction of sulfatide synthesis by phenylpyruvate was shown to be reversible. Intracerebral bilateral injections (8 μg) of l -phenylalanine, phenylpyruvate, α-ketobutyrate, α-ketoisocaproate, α-ketoisovalerate, β-phenyllactate, and phenylacetate in mice 8–15 days old, followed by i.p. administration of radioactive sulfate, resulted in significantly reduced incorporation (all P < 0.05) of sulfate into brain sulfatides with all compounds tested with the exception of β-phenyllactate and phenylacetate. In adult mouse, phenylpyruvate treatment also resulted in a significant decrease in labelling of brain sulfatide. The effects of phenylpyruvate and other metabolites upon pyruvate oxidation in mouse brain homogenates were examined by measuring ¹⁴CO₂ release from [1-¹⁴C]pyruvate. Both phenylpyruvate and α-ketoisocaproate at 1 × 10^-3 resulted in a decrease in ¹⁴CO₂ produced, while phenylacetate and β-phenyllactate had no effect. Sulfate incorporation into sulfatide was reduced by α-ketoisocaproate and phenylpyruvate, and to a lesser extent by phenylalanine, α-ketobutyrate, and α-ketoisovalerate. Phenyllactate and phenylacetate had no effect, either in vivo, or in culture. This order of effectiveness may be related in part to the effects of these compounds on pyruvate oxidation.     

LIGHT AND ELECTRON MICROSCOPY OF GRAZING BY POTERIOOCHROMONAS MALHAMENSIS (CHRYSOPHYCEAE) ON A RANGE OF PHYTOPLANKTON TAXA1

Grazing of fluorescent latex beads, bacteria, and various species of phytoplankton by Poterioochromonas malhamensis (Pringsheim) Peterfi (about 8.0 μm in diameter) was surveyed. The alga ingested fluorescent beads and various live or killed and nomnotile or motile organisms including bacteria, blue-green algae, green algae, diatoms, and chrysomonads. The size range of grazed prey was from 0.1 to 6.0 μm for latex beads and from 1.0 μm (bacteria) to about 21 μm (Carteria inverse) for organisms. As many as 17 latex beads (2.0 μm) or more than 10 Microcystis cells (5–6 μm) were ingested by a single P. malhamensis cell. Following such grazing, the cell increased in volume by up to about 30-fold. The range of cell volume of ingested prey was from 0.52 μm³ (bacteria) to about 3178 μm³(Carteria inversa). This study demonstrates for the first time that P. malhamensis is capable of grazing algae 2–3 times larger in diameter than its own cell and of grazing intact motile algae. Poterioochromonas malhamensis is an omnivorous grazer. Food vacuole formation and digestion processes were examined. The membrane that was derived from the plasma membrane and surrounded the prey disappeared sometime after ingestion. The food vacuole was then formed by successive fusion of numerous homogeneous vesicles accumulated around the prey. The prey was enclosed in a single membrane-bound food vacuole and then digested.     

Effects of subchronic nicotine administration on central dopaminergic mechanisms in the rat

Nicotine was administered acutely and subchronically (14 days) to determine whether various synaptic mechanisms are selectively altered in the nigrostriatal and mesolimbic dopaminergic systems in the rat. When added to tissue preparations in vitro, nicotine had no effects on tyrosine hydroxylase, synaptosomal uptake of [³H]dopamine or binding of [³H]spiperone to D₂ receptors in either system. However, acute treatment in vivo stimulated tyrosine hydroxylase activity in the nucleus accumbens. This effect was prevented by pretreatment with a nicotinic antagonist, suggesting that it was mediated by nicotinic receptors. Since subchronic exposure to nicotine had no effect on tyrosine hydroxylase, it appears that tolerance develops to this action. In vivo treatment with nicotine did not alter dopamine uptake or receptor binding. The results suggest that, in doses which result in moderate plasma levels, nicotine has selective stimulant actions on nerve terminals of the mesolimbic system.     

Tyrosine hydroxylase of sheep brain: some catalytic and chemical properties of the detergent-solubilized, partially purified enzyme

Abstract– Detergent-solubilized tyrosine hydroxylase from the caudate nucleus of the sheep was purified 3-fold by affinity chromatography on 3-iodotyrosine modified agarose. Supplementation of the standard assay with 1 mM Fe²⁺ resulted in only slight stimulation of the enzymic activity. The enzyme was, however, markedly inhibited by certain complexing agents specific for either Fe²⁺ or Fe³⁺. Double reciprocal plots of the kinetic data for a representative complexing agent, bathophenanthroline, showed the inhibition to be competitive with O₂ (apparent K_m 0.15 mM) and noncompetitive with either l -tyrosine or the synthetic tetrahydropterin cofactor DMPH₄ (apparent K_m's 0.12 and 0.29 mM, respectively). The combined inhibition and kinetics studies strongly suggest that brain tyrosine hydroxylase is an iron enzyme. Furthermore, the prosthetic iron very likely participates directly in catalytic function, presumably by binding molecular oxygen. The activity of ammonium sulphate-precipitated enzyme was found to be stimulated 2-fold by Fe²⁺, catalase or peroxidase, while untreated enzyme was markedly less affected by these agents. Since the only ostensible difference between the two preparations was the extensive aggregation present in the former case, the change in physical state evoked by ammonium sulphate precipitation appeared to be somehow related to this peculiar property of the enzyme.