Stimulation of some litter-decomposing basidiomycetes by shikimic acid

Shikimic acid, which constitutes 1.5-2.5% of the dry matter in needles of Scots pine, was found to stimulate the growth of various litter-decomposing basidiomycetes of the genera Marasmius, Mycena and Xeromphalina in a synthetic nutrient medium. Out of eighteen litter-decomposing species, ten were stimulated by shikimic acid, wheras the eight mycorrhizal and four wood-rotting species tested were not affected. Maximal effect was obtained at a concentration of ca 2 m M . Growth experiments at varying pH-values indicated active uptake of shikimic acid. Even in the presence of aromatic amino acids, shikimic acid stimulated the growth of the fungi. In certain species the strong inhibiting effect of phenylalanine, tyrosine and tryptophan, when added simultaneously, was reversed in the presence of shikimic acid. Fungi which were stimulated by shikimic acid were also able to use this compound as their sole carbon source. Maximal stimulating effect of shikimic acid occurred when glucose had been added at optimal concentration.     

Uptake of free amino acids by the diatom,Melosira mediocris

Individual ¹⁴C-labelled amino acids are rapidly removed from dilute solution in artificial sea water (0.2 mol 1^–1) by suspensions of Meliosira medocris. The rate of disappearance of radioactivity corresponds closely to removal of primary amines as determined by measurement of the rate of decrease of fluorescamine-positive material. Net removal of naturally occurring free amino acids from the sea water habitat from which the alga was isolated is demonstrated using high performance liquid chromatography. Removal of amino acids from natural sources makes a significant contribution to the carbon requirements of the alga as well as supplying significant amounts of amino nitrogen.     

Experimental Methyl Mercury Neurotoxicity: Locus of Mercurial Inhibition of Brain Protein Synthesis In Vivo and In Vitro

Brain cell-free protein synthesis is inhibited by methyl mercury chloride (MeHg) following in vivo or in vitro administration. In this report, we have identified the locus of mercurial inhibition of translation. Intraperitoneal injection of MeHg (40 nmol/g body wt) induced variable inhibition of amino acid incorporation into the post-mitochondrial supernatant (PMS) harvested from the brain of young (10-20-day-old) rats. No mercurial-induced disaggregation of brain polyribosomes nor change in the proportion of 80S monoribosomes was detected on sucrose density gradients. No difference in total RNA was found in the PMS. Initiation complex formation was stimulated by MeHg, as detected by radiolabelled methionine binding to 80S monoribosomes following continuous sucrose density gradient centrifugation. After micrococcal nuclease digestion of endogenous mRNA, both in vivo and in vitro MeHg inhibited polyuridylic acid-directed incorporation of [3H]phenylalanine. However, the in vivo inhibition was no longer observed when [3H]phenylalanyl-tRNAPhe replaced free [3H]phenylalanine in the incorporation assay. The formation of peptidyl[3H]puromycin revealed no difference from controls. There was significant mercurial inhibition of phenylalanyl-tRNA Phe synthetase activity in pH 5 enzyme fractions derived from brain PMS of MeHg-poisoned rats. These experiments revealed that the apparent MeHg inhibition of brain translation in vivo and in vitro is due primarily to perturbation in the aminoacylation of tRNA and is not associated with defective initiation, elongation, or ribosomal function.     

Concentration of Free Amino Acids in Primary Cultures of Neurones and Astrocytes

The cellular distribution of free amino acids was estimated in primary cultures (14 days in vitro) composed principally of cerebellar interneurones or cerebellar and forebrain astrocytes. In cultured neural cells, the overall concentration of amino acids resembled that found in brain at the corresponding age in vivo. In the two neural cell types, there were marked differences in the distribution of amino acids, in particular, those associated with the metabolic compartmentation of glutamate. In neuronal cell cultures, the concentrations of glutamate, aspartate, and gamma-aminobutyric acid were, respectively, about three, four, and seven times greater than in astrocytes. By contrast, the amount of glutamine was approximately 65% greater in astroglial cell cultures than in interneurone cultures. An unexpected finding was a very high concentration of glycine in astrocytes derived from 8-day-old cerebellum, but the concentrations of both serine and glycine were greater in nerve cell cultures than in forebrain astrocytes. The essential amino acids threonine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine were all present in the growth medium, and small cellular changes in the contents of some of these amino acids may relate to differences in their influx and efflux during culturing and washing procedures. The present results, together with our previous findings, provide further support for the model assigning the "small" compartment of glutamate to glial cells and the "large" compartment to neurones, and also underline the metabolic interaction between these two cell types in the brain.     

Purification and characterization of thermostable leucine dehydrogenase from Bacillus stearothermophilus

Leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) has been purified to homogeneity from a moderate thermophilic bacterium, Bacillus stearothermophilus. Am improved method of preparative slab gel electrophoresis was used effectively to purify it. The enzyme has a molecular mass of about 300,000 and consists of six subunits with identical molecular mass (Mr, 49,000). The enzyme does not lose its activity by heat treatment at 70° C for 20 min, and incubation in the pH range of 5.5–10.0 at 55° C for 5 min. It is stable in 10 mM phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol at over 1 month, and is resistant to detergent and ethanol treatment. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their keto analogs in the presence of NAD⁺ and NADH, respectively, as the coenzymes. The pH optima are 11 for the deamination of l-leucine, and 9.7 and 8.8 for the amination of -ketoisocaproate and -ketoisovalerate, respectively. The Michaelis constants were determined: 4.4 mM for l-leucine, 3.3 mM for l-valine, 1.4 mM for l-isoleucine and 0.49 mM for NAD⁺ in the oxidative deamination. The B. stearothermophilus enzyme shows similar catalytic properties, but higher activities than that from Bacillus sphaericus.Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

Functional groups and quaternary structure of chicken liver xanthine dehydrogenase

Xanthine dehydrogenase from chicken liver is a dimeric enzyme, each hemimolecule containing one FAD and two Fe/S groups. Determination of sulfhydryl groups with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuribenzoic acid (PMB) showed a variable number of sulfhydryl groups depending onpH, ionic strength, and nature of the reaction medium and buffer. The number of disulfide bonds was determined with DTNB and reducing conditions. Amino groups were determined with 2,4,6,-trinitrobencensulfonic acid (TNBS). At constant temperature andpH the reaction of DTNB and TNBS with native xanthine dehydrogenase showed an exponential dependence on time. From the obtained parameters the number of available sulfhydryl and amino groups at infinite concentration of enzyme and the rate constant of the equation were determined. The absorption spectrum of the enzyme changed with time when a chaotropic agent (1 M sodium nitrate) was added to the medium. This difference was detected by measuring the absorbance in the range 450–550 nm. The absorption spectrum (between 350 and 600 nm) also changed when a denaturating agent (sodium dodecyl sulfate) was added. This modification increased with time and depended on the medium.     

Development of a high-frequency transforming vector for Aspergillus nidulans

The pyr4 gene of Neurospora crassa, which codes for orotidine-5'-phosphate decarboxylase, is capable of transforming an Aspergillus nidulans pyrG mutant by chromosomal integration, despite low homology between the transforming DNA and the recipient genome. Integration of pFB6, a plasmid carrying pyr4 and capable of replication in Escherichia coli, was not observed at the pyrG locus. The efficiency of transformation was considerably enhanced (50-100 fold) by inclusion in the transforming vector of a 3.5-kb A.nidulans chromosomal sequence, ans1. Although this sequence was isolated on the basis of replicating activity in Saccharomyces cerevisiae, there was no evidence for such activity in A.nidulans. Part of the ans1 fragment appears to be reiterated in the A.nidulans genome, though it is not yet clear whether this is directly responsible for the high transformation frequency. The efficiency of transformation of A.nidulans by plasmids bearing ans1, using an improved protocol, was approx. 5 X 10(3) stable transformants per microgram of plasmid DNA.