Correlation of bacterial viability with uptake of [14C] acetate into phenolic glycolipid-1 ofMycobacterium leprae within Schwannoma cells

The viability ofMycobacterium leprae, maintained within 33B Schwannoma cells, was estimated in terms of incorporation of [¹⁴C] acetate into its specific phenolic glycolipid-1. This measure of viability was correlated with two other assays,viz., fluorescein diacetate/ethidium bromide staining and mouse footpad growth. Observation of a 2-fold increase in the number of intracellularMycobacterium leprae over an experimental period of 12 days also corroborated this contention. Furthermore, on addition of anti-leprosy drugs to these intracellularMycobacterium leprae there was significant decrease in phenolic glycolipid-1 synthesis indicative of loss of viability of the organisms. This study also established the importance of the host cell for active bacillary metabolism, asMycobacterium leprae maintained in cell-free conditions showed no incorporation into phenolic glycolipid-1. Moreover, compromising the host’s protein synthesis capacity with cycloheximide, also led to reduction in bacillary metabolism. As this system measures the metabolic synthesis of a uniqueMycobacterium leprae component, it would be useful for development and screening of compounds acting against specific bacillary targets.     

Cholesterol binding reserve of hyperlipemic rat serum lipoproteins in chronic ethanol administration

Chronic administration of ethanol in rats caused the reduction of serum cholesterol binding reserve. The very low density and high density lipoproteins, main serum cholesterol binding reserves, were slightly increased with corresponding increases in their lipid and protein components during initial stage of alcohol consumption. However, these capacities get deminished during reversal of hyperlipemia induced by prolonged action of ethanol. This situation may be an early indicator for the initiation of hepatic damage and a variety of secondary effects of ethanol.     

Carbonic anhydrase activity in acetate grown Methanosarcina barkeri

Cell extracts (27000xg supernatant) of acetate grown Methanosarcina barkeri were found to have carbonic anhydrase activity (0.41 U/mg protein), which was lost upon heating or incubation with proteinase K. The activity was inhibited by Diamox (apparent K _i=0.5 mM), by azide (apparent K _i=1 mM), and by cyanide (apparent K _i=0.02 mM). These and other properties indicate that the archaebacterium contains the enzyme carbonic anhydrase (EC 4.2.1.1). Evidence is presented that the protein is probably located in the cytoplasm. Methanol or H₂/CO₂ grown cells of M. barkeri showed no or only very little carbonic anhydrase activity. After transfer of these cells to acetate medium the activity was induced suggesting a function of this enzyme in acetate fermentation to CO₂ and CH₄. Interestingly, Desulfobacter postgatei and Desulfotomaculum acetoxidans, which oxidize acetate to 2 CO₂ with sulfate as electron acceptor, were also found to exhibit carbonic anhydrase activity (0.2 U/mg protein).     

N-15 in vivo NMR spectroscopic investigation of nitrogen deprived cell suspensions of the green alga Chlorella fusca

The possibility to apply N-15 in vivo NMR spectroscopy to study algal N-metabolism has been investigated. N-15 labelled cells of the green alga Chlorella fusca, subjected to nitrogen starvation and N-14 labelled cells supplied with K¹⁵NO₃ after prolonged nitrogen starvation were monitored by N-15 in vivo NMR spectroscopy at different times after the change in their nitrogen supply. During 20–40 min, necessary for the acquisition of 1 spectrum, the cells were under dark anaerobic conditions, but the relative amounts of the metabolites detected did not change. Signals from 2 acid amides, from the side chain nitrogens of arginine and lysine, from prolin as well as 4 signals from α amino groups of amino acids were detected. Besides two signals not yet reported in the literature were found. They may be due to amino compounds, but not to amino acids. The amount of free amino acids in the cells increases not only upon resupply of nitrogen starved cells with nitrate but also during the first hours after nitrate depletion. The spectra obtained from N-15 labelled autospores show that N-15 in vivo NMR spectroscopy can be applied to the investigation of N metabolism of the cells.     

4-aminobutyrate is not available to bacteroids of cowpea Rhizobium MNF2030 in snake bean nodules

Laboratory cultures of cowpea Rhizobium MNF2030 grew on 4-aminobutyrate (GABA) as sole source of carbon and nitrogen. GABA transport was active since it was inhibited by carbonyl cyanide mchlorophenyl hydrazone and 2,4-dinitrophenol and cells developed a 400-fold concentration gradient across the cell membrane. Arsenite treatment of GABA-grown cells revealed stoichiometric conversion of GABA to pyruvate, indicating that 2-oxoglutarate is not an intermediate in GABA catabolism. GABA catabolism by cells of strain MNF2030 grown on GABA appreared to involve GABA transaminase, succinic semialdehyde dehydrogenase and malic enzyme; the first two enzymes were specifically induced by growth on GABA. The deamination process and removal of NH₃ in cells catabolizing GABA involved GABA: 2-oxoglutarate transaminase; glutamate: oxaloacetate aminotransferase; asparate: pyruvate aminotransferase and alanine dehydrogenase.Isolated snakebean bacteroids of strain MNF2030 transported only small amounts of GABA and had uninduced levels of GABA catabolic enzymes, even though the nodules contained significant levels of GABA. The data suggest that GABA is not available to snakebean nodule bacteroids, presumably because of a control imposed by the peribacteroid membrane.Abbreviations CCCP Carbonyl cyanide m-chlorophenyl hydrazone - HEPES N-hydroxyethylpiperazine-N-2-ethanesulphonic acid - DTT dithiothreitol - SSAD succinic semialdehyde dehydrogenase - GABAT 4-aminobutyrate transaminase - GABA 4-aminobutyrate     

The phylogenic position of Peptococcus niger based on 16S rRNA sequence studies

A 1330 base-pair fragment of a 16S rRNA gene has been amplified, cloned and sequenced. Comparison to other 16S rRNA sequences of eubacteria showed that P. niger represents a deep branch within the subdivision "Gram-positive with Gram-negative cell walls". It is not related to peptostreptococci, representatives of this genus studied so far are more closely related to clostridia.     

Regional brain GABA metabolism and release during hepatic coma produced in rats chronically treated with carbon tetrachloride

Hepatic coma was induced in rats chronically treated with CCl₄, by means of a single injection of ammonium acetate. The activities of glutamate decarboxylase (GAD) and GABA transaminase (GABA-T), as well as the synaptosomal uptake and release of [³H]GABA, were measured in the following brain areas of the comatose rats: cortex, striatum, hypothalamus, hippocampus, midbrain and cerebellum. Hepatic coma was associated with a general decrease of GAD activity, whereas GABA-T activity was diminished only in the hypothalamus, striatum and midbrain. During hepatic coma, the K⁺-stimulated [³H]GABA release was notably diminished in the striatum and cerebellum, whereas a significant increase was observed in the hippocampus. [³H]GABA uptake increased in most regions after CCl₄ treatment, independently of the presence of coma. The results indicate that GABAergic transmission seems to be decreased in most cerebral regions during hepatic coma.     

Energy metabolism in glutamatergic neurons,GABAergic neurons and astrocytes in primary cultures

Several aspects of energy metabolism (glucose utilization, lactate production,¹⁴CO₂ production from labeled glucose, glutamate or pyruvate, oxygen consumption and contents of ATP and phosphocreatine) were measured in cerebellar granule cells (glutamatergic) in primary cultures and compared with corresponding data for cerebral cortical neurons (mainly GABA-ergic) and astrocytes. Cerebellar granule cells and astrocytes were metabolically more active than cerebral cortical neurons. Glutamate which is utilized as a major metabolic fuel as astrocytes and, to a lesser extent, in cerebral cortical neurons, was virtually not oxidized in cerebellar granule cells.Special Issue dedicated to Prof. Holger Hydén.     

Isolation of a cAMP-requiring mutant in Escherichia coli K-12: evidence of growth regulation via N-acetylglucosamine metabolism controlled by cAMP

Abstract An adenylate cyclase gene ( cya ) mutant was mutagenized and an adenosine 3,5-cyclic monophosphate (cAMP)-requiring mutant (KM8161) was obtained on Davis minimal medium containing glucose in the presence or absence of cAMP. KM8161 also required N -acetylglucosamine for its growth instead of cAMP. Furthermore, the mutant could use neither glucosamine nor N -acetylglucosamine as the carbon source. These results indicate that the cAMP-requiring property is due to multiple mutations of a few genes involved in amino sugar metabolism in addition to cya . By genetic analysis of KM8161, one gene, which was tentatively termed cidA and located near 2 min on the chromosomal map, proved to be defective. Reversion of cidA mutation in KM8161 resulted in recovery of not only the cAMP-requiring phenotype but also non-utilization of amino sugars. When both cAMP and N -acetylglucosamine or glucosamine were added to the culture medium for KM8161, only N -acetylglucosamine could be utilized as the carbon source. These studies s strongly suggest that the cidA or cya mutation in KM8161 causes deficiency in different stages of amino sugar metabolism and the regulatory circuit of growth by cAMP is mediated via control of N -acetylglucosamine metabolism.     

The effects of cultural conditions on growth and secondary metabolism in Streptomyces thermoviolaceus

Abstract Granaticin, an isochromate quinone antibiotic is synthesized as a secondary metabolite by Streptomyces thermoviolaceus . Antibiotic productivity was investigated under a variety of cultural conditions, including complex and defined media, mesophilic and thermophilic temperatures and a variety of sole carbon sources. In a defined medium growth was supported, to varying extents, by different carbon sources and in most cases granaticin production was observed. Highest biomass and granaticin yields were obtained when cultures were grown in the presence of xylan, fructose, glutamate or proline as carbon source. Changes in pH during growth affected both the timing and extent of granaticin production.