Effects of Osmotic Pressure on the Oxidative Metabolism of Leishmania major Promastigotes

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of ¹⁴C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H₂O to the cell suspension) caused an increase in the rates of ¹⁴CO₂ production from [6-¹⁴C]glucose and, to a lesser extent, from [1, (3)-¹⁴C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of ¹⁴CO₂ production from [1-: ¹⁴C]alanine, [1-¹⁴C]glutamate, and [1, (3)-¹⁴C]glycerol. The rates of ¹⁴CO₂ formation from [1-¹⁴C]laurate, [1-¹⁴C]acetate, and [2-¹⁴C]glucose (all of which form [1-¹⁴C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.     

METABOLISM OF GLUCOSE AND FREE AMINO ACIDS IN BRAIN, STUDIED WITH 14C-LABELLED GLUCOSE AND BUTYRATE IN RATS INTOXICATED WITH CARBON DISULPHIDE

Abstract— The effect of 15 h continuous exposure to CS₂ on the metaboliam of glucose and free amino acids in the brain of rats was studied. CS₂ caused a moderate hypoglycaemia. There were also changes in the amounts of some amino acids in the brain. Glutamate and γ-aminobutyrate were lower whereas glutamine was markedly increased. Comparative studies in vivo of the metabolism of [2-¹⁴C]glucose and [1-¹⁴C]butyrate indicated that CS₂ did not affect glycolysis or the incorporation of ¹⁴C from glucose into amino acids except into γ-aminobutyrate which was reduced. Contrary to the findings with [¹⁴C]glucose, CS₂ provoked distinct changes in the labelling of amino acids when [¹⁴C]butyrate was the precursor. The most notable change was a markedly increased incorporation of ¹⁴C into glutamine. Based on the two-compartment model of brain glutamate the experimental findings indicated that CS₂ affected metabolism associated with the 'small' pool of glutamate but had a minimal effect on metabolism associated with the 'large' glutamate pool. The possibility is suggested that the changes observed involved an increased rate of ammonia removal. The low incorporation of ¹⁴C into γ-aminobutyrate from either precursor is consistent with other evidence showing that CS₂ interferes with pyridoxal phosphate-dependent enzymes.     

INCORPORATION OF PHENYLALANINE AS A SINGLE UNIT INTO RAT BRAIN PROTEIN: RECIPROCAL INHIBITION BY PHENYLALANINE AND TYROSINE OF THEIR RESPECTIVE INCORPORATIONS

Abstract— Of seven amino acids studied, glutamic acid and phenylalanine were incorporated in highest amounts into the hot-TCA-insoluble material of the 100,000 g supernatant fraction of rat brain homogenate. The system for incorporation of phenylalanine was RNase-insensitive and required ATP (apparent K_m = 0.64 m m ), KC1 (apparent K_m = 14 m m ) and MgCl₂ (optimal concentration range 4-15 m m ). The apparent K_m for phenylalanine was 2.9 m m . [¹⁴C]Phenylalanine did not undergo modification before incorporation. Tyrosine and phenylalanine inhibited the incorporation, respectively, of [¹⁴C]phenylalanine and [¹⁴C]tyrosine when incubated simultaneously or successively. The K_m and K_t (3.3 m m ) values for phenylalanine in the incorporation reaction and as inhibitor of the incorporation of [¹⁴C]tyrosine were similar. We suggest that both the enzyme and the acceptor for the incorporation of these two amino acids are the same. [¹⁴C]Phenylalanine and [¹⁴C]tyrosine entered into COOH-terminal positions in the reactions described. Brain exhibited a 25- to 100-fold higher capacity to incorporate phenylalanine than that of liver, kidney or thyroid. The acceptor capacity in rat brain rapidly decreased from day 5 to day 15 of postnatal age and then slowly until age 150 days.     

Effects of In Vivo Hypoxia on Acetylcholine Synthesis by Rat Brain Synaptosomes

Abstract: Synaptosomes from normoxic and hypoxic rats were incubated aerobically in the presence and absence of veratridine. In the absence of veratridine, no significant difference was observed between the two types of preparation regarding either ATP/ADP ratio or ¹⁴CO₂ or [¹⁴C]acetylcholine synthesis from D-[U-¹⁴C]glucose. However, in the presence of veratridine, significant reductions in the output of ¹⁴CO₂ and [¹⁴C]acetylcholine by synaptosomes from hypoxic rats were apparent. It was concluded that irreversible metabolic lesions occur at the synapse as a result of hypoxia, which are apparent only when the metabolism of the preparation is accelerated to a level comparable with the maximal rate occurring in vivo. The presence of such lesions is further evidenced by the significant reductions in ATP/ADP ratio, ¹⁴CO₂ output, and [¹⁴C]acetylcholine synthesis that occur in synaptosomes from hypoxic rats made anoxic in vitro and permitted to recover. Such decreases are not seen when synaptosomes from normoxic rats are similarly treated.     

CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [¹⁴C]tyrosine into the C-terminal position of α-tubulin of rat brain cytosol was 10-fold higher for non-assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non-assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.
[¹⁴C]Tyrosine was released from assembled and non-assembled [¹⁴C]tyrosinated tubulin by the action of an endogenous carboxypeptidase. Release from non-assembled tubulin was shown by incubating a tubulinyl-[¹⁴C]tyrosine preparation in the presence of CaCl₂ at a concentration that abolished microtubule formation. Release from microtubules was inferred from the observation that the percentages of [¹⁴C]tyrosine released and the decrease of the specific radioactivity of the recovered microtubules were practically identical and did not change after a 10-fold dilution of the incubated microtubules.
[³H]Phenylalanine was released from a preparation of tubulinyl-[³H]phenylalanine also by an enzymatic activity.
The capacity of a tubulin preparation to incorporate tyrosine was increased 43% by pre-treatment with endogenous carboxypeptidase.
Tubulin tyrosinated in vitro was assembled to the same extent as native tubulin. After a mixture of tubulinyl-[¹⁴C]tyrosine and tubulinyl-[³H]phenylalanine was partially assembled, the ratio of ¹⁴C/³H found in the microtubules was the same as in the non-assembled tubulin fraction.     

THE RELEASE OF AMINO ACIDS FROM THE HEMISECTED SPINAL CORD DURING STIMULATION

Abstract— Isolated frog or toad hemicords were incubated for 40 min with either [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate. l -[¹⁴C]aspartate, l -[¹⁴C]serine, l [¹⁴C]threonine or l -[³H]leucine, and the release of these compounds from the cord was measured under resting conditions and during electrical stimulation. Stimulation of spinal roots produced no significant change in the efflux of any of the compounds tested. Direct stimulation of the rostral cord however, produced a large increase in the efflux of [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate and l -[¹⁴C]aspartate. These increased effluxes were calcium dependent, the effects of stimulation being reduced in a calcium-free, or magnesium-supplemented (10 mM) medium. Stimulation failed to produce an increase in the efflux of l -[¹⁴C]serine, l -[¹⁴C]threonine, l -[¹⁴H]leucine, [¹⁴C]mannitol or [¹⁴C]urea. These results are consistent with the suggestions that glycine, GABA, glutamate and aspartate may be synaptic transmitters in the spinal cord.     

Production of [14C]Acetylcholine and [14C]Carbon Dioxide from [U–14C]Glucose in Tissue Prisms from Aging Rat Brain

Abstract: Production of [¹⁴C]acetylcholine and ¹⁴CO₂ was examined by using tissue prisms from neocortex, hippocampus, and striatum from rats aged approximately 5 months, 13 months, and 27 months. [¹⁴C]Acetylcholine synthesis in the striatum showed highly significant decreases with age for measurements in the presence of both 5 m m - and 31 m m -K⁺, contrasting with the lack of significant change in ¹⁴CO₂ production in this region. The neocortex and hippocampus showed only small changes, especially when comparison was made between 13-month and senescent animals. Measurements of the release of [¹⁴C]acetylcholine and influence of atropine on this release confirmed the relative stability with age of the cholinergic system in the neocortex.     

Partitioning of CO2 Production Between Glucose and Lactate in Excised Sympathetic Ganglia, with Implications for Brain

Abstract: Chains of lumbar sympathetic ganglia from 15-day-old chicken embryos were incubated for 4 h at 36°C in a bicarbonate-buffered salt solution equilibrated with 5% CO₂-95% O₂. Glucose (1–10 m M ), lactate (1–10 m M ), [U-¹⁴C]glucose, [1-¹⁴C]glucose, [6-¹⁴C]glucose, and [U-¹⁴C]lactate were added as needed. ¹⁴CO₂ output was measured continuously by counting the radioactivity in gas that had passed through the incubation chamber. Lactate reduced the output of CO₂ from [U-¹⁴C]glucose, and glucose reduced that from [U-¹⁴C]lactate. When using uniformly labeled substrates in the presence of 5.5 m M glucose, the output of CO₂ from lactate exceeded that from glucose when the lactate concentration was >2 m M . The combined outputs at each concentration tested were greater than those from either substrate alone. The ¹⁴CO₂ output from [1-¹⁴C]glucose always exceeded that from [6-¹⁴C]glucose, indicating activity of the hexose monophosphate shunt. Lactate reduced both of these outputs, with the maximum difference between them during incubation remaining constant as the lactate concentration was increased, suggesting that lactate may not affect the shunt. Modeling revealed many details of lactate metabolism as a function of its concentration. Addition of a blood-brain barrier to the model suggested that lactate can be a significant metabolite for brain during hyperlactemia, especially at the high levels reached physiologically during exercise.     

Lumiflavin and Lumichrome Transport in the Central Nervous System

Abstract: The transport of the lipid-soluble sugarless flavins, [¹⁴C]lumiflavin and [¹⁴C]lumichrome, into and from the isolated choroid plexus and brain slices was studied in vitro. The isolated choroid plexus accumulated both [¹⁴C] flavins by a saturable, energy-requiring process that did not depend on binding or intracellular metabolism of the [¹⁴C] flavins. Both sugar-containing and sugarless flavins, as well as cyclic organic acids, significantly inhibited [¹⁴C]lumiflavin and [¹⁴C]Iumichrome uptake by the isolated choroid plexus. Within 2.5 min, 75% of the [¹⁴C]lumiflavin accumulated by the isolated choroid plexus was released into the medium. Brain slices accumulated [¹⁴C]lumiflavin by a saturable process that did not meet all the criteria for active transport. Ninety-five percent of the [¹⁴C]lumiflavin accumulated by brain slices was released into the medium within 7.5 min. In vivo , 2 h after the intraventricular injection of 6.5 nmol [¹⁴C]lumiflavin, almost all of the [¹⁴C]flavin was cleared from the CNS. Addition of 3.5 μmol FMN to the intraventricular injectate significantly decreased the clearance of [¹⁴C]lumiflavin from the CNS. These studies document that the sugarless flavins are transported by the flavin transport systems in the CNS.     

Trifluoperazine inhibits the incorporation of labelled precursors into lipids, proteins and DNA of Mycobacterium tuberculosis H37Rv

Abstract We have recently demonstrated that the calmodulin antagonist trifluoperazine has antitubercular activity in vitro against Mycobacterium tuberculosis H₃₇R_v susceptible and resistant to isoniazid. It is shown that trifluoperazine at a concentration of 50 μ g ml⁻¹ when added to the cells along with the labelled precursors inhibited the incorporation of [¹⁴C]acetate into lipids (63%) and uptake of [¹⁴C]glycine (74%) and [^3H]thymidine (52%) bu whole cells of M. tuberculosis H₃₇R_v by 6 h of exposure. After 48 h, the inhibition was 87%, 97% and 74%, respectively. However, when the drug was added to cells taking up and metabolizing the labelled precursors at a later point (3 h for [¹⁴C]acetate and [³H]thymidine and 12 h for [¹⁴C]glycine) it inhibited completely the uptake of all the precursors, at least up to 24 h. The onset of inhibitory action was very rapid, i.e. 3 h. It is suggested that trifluoperazine has multiple sites of action and acts probably by affecting the synthesis of lipids, proteins and DNA.     

THE EFFECT OF DIABETES, INSULIN AND WALLERIAN DEGENERATION ON LEUCINE METABOLISM OF ISOLATED RAT SCIATIC NERVES

Abstract– ¹⁴CO₂ production and ¹⁴C incorporation into proteins was studied in isolated rat sciatic nerves during incubation with 0.1 mM-[1-¹⁴C]leucine. Rats were made diabetic with streptozotocin. Nerves from diabetic rats incubated with glucose oxidized more [¹⁴C]leucine than controls. This difference was abolished in the presence of insulin (1 mU/ml). The effects of diabetes and insulin on leucine oxidation could not be demonstrated in the absence of glucose. Insulin stimulated the incorporation of [¹⁴C] from leucine into proteins by nerves from controls and diabetic rats.
Nerves undergoing Wallerian degeneration showed a marked increase in DNA content and stimulated incorporation of [¹⁴C]leucine into proteins. ¹⁴CO₂ production from leucine proceeded at 75% of the rate observed in intact nerves. Neither insulin nor diabetes affected leucine metabolism in degenerating nerves.
Neither the extracellular space nor the concentration of free amino acids were significantly different in nerves obtained from control and diabetic rats, except for lower glutamine content in the latter.
In vitro leucine metabolism of nerves is affected by diabetes, insulin and the integrity of the axon. The Schwann cell is suggested as a possible site of the observed changes in leucine metabolism.     

Isoprenoid biosynthesis in bacteria: Two different pathways?

Abstract The biosynthesis of isopentenylpyrophosphate, a central intermediate of isoprenoid formation, was investigated in six different bacterial organisms. Cell-free extracts of Myxococcus fulvus, Staphylococcus carnosus, Lactobacillus plantarum and Halobacterium cutirubrum converted [¹⁴C]acetyl-CoA or [¹⁴C]hydroxymethylglutaryl-CoA to [¹⁴C]mevalonic acid. Furthermore, [¹⁴C]mevalonic acid, [¹⁴C]mevalonate-5-phosphate and [¹⁴C]mevalonate-5-pyrophosphate were metabolized to [¹⁴C]isopentenylpyrophosphate in bacteria. In contrast, no intermediates of this reaction sequence could be detected using cell-free extracts of Zymomonas mobilis and Escherichia coli . These results indicate that at least two different pathways for the biosynthesis of isopentenylpyrophosphate are present in bacteria.     

UPTAKE OF [14C]ASPARTIC ACID AND [14C]GLUTAMIC ACID BY RETINAL SYNAPTOSOMAL FRACTIONS

Abstract— Uptake systems for [¹⁴C]aspartate and [¹⁴C]glutamate were characterized in two distinct synaptosomal fractions solated from rabbit retina. The P, synaptosomal fraction was highly enriched in large photoreceptor cell synaptosomes but contained very few conventional sized synaptosomes from amacrine, horizontal or bipolar cells. In contrast, the P₂ synaptosomal fraction contained numerous conventional sized synaptosomes and was virtually free of photoreceptor cell synaptosomes. Both synaptosomal fractions took up [¹⁴C]aspartate and [¹⁴C]glutamate with high affinity [ K _m= 1–2μM). Uptake characteristics were similar to those described for high affinity uptake systems in brain synaptosomes, i.e. saturation kinetics; temperature and Na⁺ dependence. Although the presence of a high affinity uptake system is not a definitive criterion for demonstration of functional neurotransmitter systems, it is an important and necessary prerequisite and can thus be considered as supportive evidence for the involvement of asparate and glutamate in neurotransmission in rabbit retina.     

SYNTHESIS AND RELEASE OF [14C]ACETYLCH0LINE IN SYNAPTOSOMES

Abstract— Synaptosomes took up [¹⁴C]choline, about half or more of which was converted to [^I4C]acetylcholine when incubated in an appropriate medium containing 1 to 5 μ M-[¹⁴C] choline and neostigmine. The amount of [¹⁴C]acetylcholine synthesized in synaptosomes increased in parallel with the increase of Na⁺ concentration in the incubation medium. The effect of Na⁺ on the uptake of [^I4C]choline into synaptosomes was dependent on the concentration of choline in the incubation medium.
About 25 per cent of [¹⁴C]acetylcholine synthesized in synaptosomes was released rapidly into the medium by increasing the K⁺ concentration in the medium from 5 m m to 35 m m . The change of Na⁺ concentration hardly affected the release of [¹⁴C]acetylcholine. The effect of K⁺ on the release of [¹⁴C]choline was rather small compared to that on [¹⁴C] acetylcholine. Ouabain promoted the release of [¹⁴C]acetylcholine.     

NEUROTRANSMITTER UPTAKE INTO SLICES OF RAT CEREBRAL CORTEX IN VITRO: EFFECT OF SLICE SIZE

Abstract— The uptake of [¹⁴C]GABA, [¹⁴C]taurine, [³H] β -alanine and [¹⁴C]dopamine was compared in slices of rat cerebral cortex of three different sizes (0.1 × 0.1 × 2 mm, 0.2 × 0.2 × 2 mm and 0.4 × 0.4 × 2 mm prepared with a mechanical tissue chopper). [¹⁴C]Taurine and [³H] β -alanine uptake increased whereas [¹⁴C]GABA uptake decreased with increasing slice size. [¹⁴C]Dopamine uptake was optimal in 0.2 × 0.2 × 2 mm slices. Increasing slice size was shown to decrease inhibition of [³H] β -alanine and [¹⁴C]GABA uptake by l -2,4-diaminobutyric acid. Lactate dehydrogenase activity increased with increasing slice size indicating decreased tissue damage or increased cellular integrity. The possibility that varying slice size can be used to distinguish between neuronal and glial uptake is discussed. It is suggested that taurine uptake in the cerebral cortex is predominantly glial.     

Confirmation of operative acetate, H2/CO2 and methanol methanogenic pathways in the solid-state refuse fermentation

By use of the radiolabelled substrates sodium [1–¹⁴C] acetate, sodium [2–¹⁴C] acetate, NaH¹⁴CO₃ and ¹⁴CH₃OH, three of the possible methanogenic pathways in fermenting refuse were confirmed. Due to the absence of a methanol pool, however, the relative contribution of each could not be determined. Circumstantial evidence for an operative trimethylamine pathway was gained but not confirmed whilst preliminary attempts to stimulate methanogenesis in refuse by supplementation with mono-and dimethylamine proved unsuccessful.     

IN VIVO FORMATION AND CATABOLISM OF [14C]HISTAMINE IN MOUSE BRAIN

Abstract— The formation of histamine in brain was studied in mice injected with l -[¹⁴C]-histidine (ring 2-¹⁴C) intravenously (i.v.) or intracerebrally; [¹⁴C]histamine appeared rapidly and exhibited a rapid rate of turnover. Drugs known to block various pathways of histamine catabolism were tested for effects on brain–[¹⁴C]histamine and [¹⁴C]-methyl-histamine in mice given (1) [¹⁴C]histamine i.v., (2) [¹⁴C]histamine intracerebrally, and (3) l -[¹⁴C]histidine i.v. Blood-borne histamine did not enter brain; brain histamine was formed locally by decarboxylation of histidine Methylhistamine did cross the blood-brain barrier. Methylation was the major route of histamine catabolism in mouse brain and some of the methylhistamine formed was destroyed by monoamine oxidase. No evidence for catabolism by the action of diamine oxidase was found.     

L-GLUTAMIC ACID DECARBOXYLASE IN NON-NEURAL TISSUES OF THE MOUSE

Abstract— Low levels of γ-aminobutyric acid (GABA) and of glutamic acid decarboxylase (GAD) activity have been detected in mouse kidney, liver, spleen and pancreas. Quantitation of both ¹⁴CO₂ and [¹⁴C]GABA produced in radiometric assays from [U-¹⁴CJglutamic acid has shown that measurement of ¹⁴CO₂ evolution alone is not, in all cases, a valid estimate of true GAD activity. As evidenced by increased ^,14CO₂ production upon addition of NAD and CoA to assay mixtures, radiometric assay of GAD activity in crude homogenates may yield ¹⁴CO₂ via the coupled reactions of glutamic acid dehydrogenase and a-ketoglutarate dehydrogenase. The addition of 1 mM aminooxyacetic acid (AOAA) to assays of kidney homogenates inhibited [^,14C]GABA production 92 per cent while ¹⁴CO₂ production was inhibited only 53 per cent. No evidence was found to confirm the reported existence of a second form of the enzyme, GAD II. previously described by Haber el al. (H aber B., K uriyama K. & R oberts E. (1970) Biochem. Pharmac. 19, 1119-1136). Based on sensitivity-to AOAA and chloride inhibition, the GAD activity in mouse kidney is. apparently, indistinguishable from that of neural origin.     

ACETYLCHOLINE METABOLISM AND CHOLINE UPTAKE IN CORTICAL SLICES

Abstract— The uptake of [¹⁴C]choline was studied in cortical slices from rat brain after their incubation in a Krebs-Henseleit medium containing either 4.7 m m -KCl (low K), 25 m m -KCl (high K) or 25 m m -KCl without calcium (Ca free, high K). With 0.84 μ m -[¹⁴C]choline in the medium the uptake per gram of tissue was 0.62 nmol after incubation in low K medium, 1.13 nmol after incubation in high K medium and 0.78 nmol after incubation in a Ca free, high K medium. The differences caused by potassium were greater in fraction P₂ than in fractions P₁ and S₂. With 17 and 50 μ m -[¹⁴C]choline in the medium greater amounts of [¹⁴C]choline were taken up, but the effect of potassium on the uptake almost disappeared. The amount of radioactive material in fraction P₂ followed Michaelis-Menten kinetics with K _m values of 2.1 and 2.3 μ m after incubation in low and high K medium, respectively. Hemicholinium-3 only slightly inhibited choline uptake from a medium with 0.84 μ m -[¹⁴C]choline, but it abolished the extra-uptake induced by high K medium. The radioactivity in the slices consisted mainly of unchanged choline and little ACh was formed after incubation in low K medium, but after incubation in high K medium 50% of the choline taken up was converted into ACh. The hemicholinium-3 sensitive uptake of choline, the conversion of choline into ACh and the synthesis of total ACh, were stimulated about 7–8-fold by potassium. It is concluded that in cortical slices from rat brain all choline used for the synthesis of ACh is supplied by the high-affinity uptake system, of which the activity is geared to the rate of ACh synthesis.     

Enrichment and characterization of a sulfate-reducing toluene-degrading microbial consortium by combining in situ microcosms and stable isotope probing techniques

A toluene-degrading microbial consortium was enriched directly in a BTEX-contaminated aquifer under sulfate-reducing conditions using in situ microcosms consisting of toluene-loaded activated carbon pellets. Degradation of toluene and concomitant sulfide production by the consortium was subsequently demonstrated in laboratory microcosms. The consortium was physiologically and phylogenetically characterized by isotope tracer experiments using nonlabeled toluene, [¹³C]-α-toluene or [¹³C₇]-toluene as growth substrates. Cells incubated with [¹³C]-α-toluene or [¹³C₇]-toluene incorporated 8–15 at.%¹³C and 51–57 at.%¹³C into total lipid fatty acids, respectively, indicating a lower specific incorporation of ¹³C from [¹³C₇]-toluene. In order to identify the toluene-assimilating bacteria, the incorporation of carbon from both [¹³C]-α-toluene and [¹³C₇]-toluene into rRNA was analyzed by stable isotope probing. Time and buoyant density-resolved 16S rRNA gene-based terminal restriction fragment length polymorphism profiles, combined with cloning and sequencing, revealed that an uncultured bacterium (99% sequence similarity) related to the genus Desulfocapsa was the main toluene-degrading organism in the consortium. The ratio of the respective terminal restriction fragments changed over time, indicating trophic interactions within this consortium.