Uptake of l-Glutamate into Rat Brain Synaptic Vesicles: Effect of Inhibitors that Bind Specifically to the Glutamate Transporter

Abstract: In this study we have described a series of new and potent inhibitors of the vesicular uptake of glutamate. The two most efficient inhibitors were the dyes Evans blue and Chicago Skye Blue 6B, which are structurally related to glutamate and were competitive inhibitors in the nanomolar range. The anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (SITS) and the diuretics furosemide and bumetanide are inhibitors of chloride transport in other organs but were competitive inhibitors of glutamate and noncompetitive with respect to chloride ions. Evans blue, Chicago Skye Blue 6B, SITS, furosemide, and bumetanide are all large organic acids with two centers of negative charge and an electron-donating group at close vicinity of the negative charge at physiological pH. The inhibition of the glutamate uptake with these inhibitors was noncompetitive with respect to Cl⁻. The inhibitors, therefore, probably interact directly with the glutamate carrier. Bafilomycin A₁, which is a specific vacuolar ATPase inhibitor, was used as a control and inhibited the vesicular dopamine, glutamate, and GABA uptake to the same extent. None of the inhibitors had any effect on the plasma membrane carrier, which is therefore clearly different from the vesicular carrier.     

l-DOPA Is a Substrate for Tyrosine Hydroxylase

Abstract: In the presence of thiols, tyrosine hydroxylase (TH) oxidizes l -dihydroxyphenylalanine ( l -DOPA) with a specific activity of up to 140 nmol min⁻¹ mg⁻¹ at 37°C and pH 7.0, which is ∼12–50% of its TH activity under similar experimental conditions. Using assay conditions that are optimal for measuring TH activity, the specific DOPA oxidase activity of human TH is similar to that of mushroom tyrosinase, but the two enzymes are clearly different in terms of substrate specificities, cofactor dependencies, and selectivity with respect to the effects of metal chelators and other inhibitors. In the presence of an excess of dithiothreitol, 2-mercaptoethanol, cysteine, or reduced glutathione, the reaction products of the two enzymes are identical and have been identified tentatively as thioether derivatives of DOPA. Theoretically, the oxidation of l -DOPA by TH may contribute to the formation of neuromelanin (pheomelanin) in catecholaminergic neurons and in the metabolism of DOPA to reactive intermediates that can react with free thiol groups in cellular proteins. The DOPA oxidase activity of TH can lead to errors in the estimation of in vivo or in vitro TH activity, and currently used assay protocols may have to be modified to avoid interference from this activity.     

A family of modular type mannuronan C-5-epimerase genes controls alginate structure in Azotobacter vinelandii

The ToxR protein is a transmembrane protein that regulates the expression of several virulence factors of Vibrio cholerae. Previous analysis of fusion proteins between ToxR and alkaline phosphatase (ToxR-PhoA) suggested that ToxR was active as a dimer. In order to determine whether dimerization of the ToxR periplasmic domain was essential for activity, this domain was replaced by monomeric and dimeric protein domains. Surprisingly, PhoA (dimeric), β-lactamase (monomeric, ToxR–Bla), or the leucine zipper of GCN4 (dimeric, ToxR-GCN4-M) could substitute functionally for the ToxR periplasmic domain. ToxR-GCN4 fusion proteins, in which the ToxR trans-membrane domain was eliminated (ToxR-GCN4-C), were inactive, but an additional fusion protein that contained a heterologous membrane-spanning domain retained activity. Strains containing each of these ToxR fusion proteins were analysed for in vivo colonization properties and response to in vitro growth conditions that are known to affect expression of the ToxR regulon. Strains containing ToxR-GCN4-M and ToxR-Bla responded like wild-type strains to in vitro growth conditions. In the infant-mouse colonization model, strains containing ToxR fusion proteins were all deficient in colonization relative to strains containing wild-type ToxR, and strains containing monomeric ToxR-Bla were most severely outcompeted. These results suggest that, under in vitro conditions, ToxR does not require a dimerized periplasmic domain, but that, under in vivo conditions, the correct conformation of the ToxR periplasmic domain may be more important for function.     

Calcium and magnesium levels in isolated cardiac mitochondria from mice injected with isoproterenol

Summary Calcium (Ca) and Magnesium (Mg) are determined by atomic absorption flame spectrometry in isolated cardiac mitochondria from mice receiving subcutaneous injections of DL-isoproterenol HC1 (ISO), and in mitochondria of untreated controls. In the controls, mitochondria were isolated in the presence or absence of ruthenium red. On the absence of ruthenium red in the isolation medium, mitochondrial Ca levels increase by about 300%, while levels of Mg remain unchanged. Focal myocardial necrosis following a single ISO-injection is shown by electron microscopy. Ca and Mg levels are largely unaffected by a single dose of ISO until 24 h after the injection. A slight increase in Ca occurs in the 48 h samples. When multiple injections of ISO are given every 12th hour for 48 h, 72 h and 96 h, respectively, endogenous Ca and Mg increase significantly. It is suggested that this increase might be associated with ISO-induced cardiac hypertrophy rather than with the pharmacological effects of ISO per se.This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities     

Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation. Structure/activity studies and mechanistic implications.

Three isozymes of human tyrosine hydroxylase (hTH1, hTH2 and hTH4) were expressed in Escherichia coli and purified to homogeneity. Natural catecholamines and related synthetic compounds were found to be potent inhibitors, competitive to the tetrahydrobiopterin cofactor, of all the isozymes. Combining visible spectroscopy and equilibrium-binding studies, it was found that catecholamines bind to hTH1 and hTH2 with a stoichiometry of about 1.0 mol/mol enzyme subunit, interacting with the catalytic iron at the active site. All the isozymes tested were excellent substrates for cAMP-dependent protein kinase (Km = 5 microM, Vmax = 9.5 mumol.min-1.mg kinase-1). The incorporation of about 1.0 mol phosphate/subunit at Ser40 decreased the affinity of dopamine binding by a factor of 10. Conversely, the addition of stoichiometric amounts of Fe(II) and dopamine to the apoenzymes reduced both the affinity and stoichiometry of phosphorylation by cAMP-dependent protein kinase by 2-3-fold. These data provide evidence for a mutual interaction between the presumed regulatory and catalytic domains of hTH, and show that activation of the enzyme by phosphorylation and inactivation by binding of catecholamines are related events, which probably represent important mechanisms for the regulation of the enzyme activity in vivo.     

Oligotrophication as a result of planktivorous fish removal with rotenone in the small,eutrophic, Lake Mosvatn,Norway

In September 1987 the shallow, eutrophic, Lake Mosvatn was treated with rotenone to eliminate planktivorous fish (mainly whitefish,Coregonus lavaretus, L.), and the effects were studied. The first summer after treatment the zooplankton community changed markedly from rotifer dominance and few grazers, to a community with few rotifers and many grazers. Accordingly there was a fivefold increase in the biomass ofDaphnia galeata. Adult females ofD. galeata approximately doubled in weight. The decrease in rotifer biomass was probably mainly due to a loss of food by competition with the daphnids. The phytoplankton community was also markedly affected. Prior to treatment Secchi depth was 1.7 m and Chl-a 23μg l⁻¹ in the summer. After treatment there was an increase in the proportion of small and gelatinous algae and the mean chlorophyll concentration fell to 7μg Chl-a l⁻¹. Secchi depth increased to>2.3 m (bottom-sight most of the season). After the treatment there were also fewer cyanobacterial blooms. This seems to be related to oligotrophication caused indirectly by increased grazing by the zooplankton. Total nutrient concentrations were affected. Prior to treatment the mean summer concentration of total phosphate was 44μg P l⁻¹. This decreased to 29μg P l⁻¹ in the first summer and 23μg P l⁻¹ the second summer after the treatment. Total nitrogen decreased from 0.68 mg N l⁻¹ before treatment to 0.32 mg N l⁻¹ the first summer after the treatment. The phosphate loading was not reduced, therefor it can be concluded that the fish removal provided a biomanipulation which caused the more oligotrophic conditions.     

Amino acid control mechanism for bacitracin formation byBacillus licheniformis

Several amino acids affected the amount of bacitracin produced by Bacillus licheniformis ATCC 10716 and the high-yielding mutant B. licheniformis AL. The possibility of an amino acid control mechanism for bacitracin biosynthesis is discussed. The two strains showed a different response to the addition of some amino acids. This indicate that they may have differences in their amino acid control mechanism.     

Spruce sugars and poultry hydrolysate as growth medium in repeated fed-batch fermentation processes for production of yeast biomass

Bioprocess and Biosystems Engineering - The production of microbial protein in the form of yeast grown on lignocellulosic sugars and nitrogen-rich industrial residues is an attractive approach for...