Enhanced uptake and metabolism of riboflavin in erythrocytes infected with Plasmodium falciparum

Riboflavin deficiency inhibits the growth of malaria parasites both in vitro and in vivo in infected animals and humans. Although the precise mechanisms underlying this inhibition are unknown, they may involve enhanced requirements for riboflavin by parasites. To investigate this possibility, the rate of uptake of [14C]riboflavin and the biosynthesis of FMN and FAD from riboflavin were studied in infected (5-8% parasitemia) and uninfected human erythrocytes. All cells were incubated for 0-3 h at 37 degrees C in phosphate buffered saline containing MgCl2, glucose, and [14C]riboflavin (2.5-7.5 microM). At hourly intervals, samples were removed, centrifuged, washed twice with cold buffer, and lysed before counting the radioactivity. The rate of in vitro biosynthesis of FMN and FAD from riboflavin in erythrocytes was measured by ion exchange chromatography and reverse isotope dilution techniques. Results showed that the rate of riboflavin uptake and the biosynthesis of FMN and FAD were enhanced in erythrocytes with parasitemia as compared with results in unparasitized erythrocytes. Riboflavin uptake in erythrocytes was proportional to the extent of parasitemia and especially to percent of schizonts present in erythrocytes. These studies indicate that the requirement for riboflavin may be greater in the parasite than in the host erythrocyte. This increased riboflavin requirement may be due to rapid multiplication, higher metabolic rate, and extreme vulnerability to oxidative stress of malaria parasites compared with that of host erythrocytes. The differential requirement of riboflavin by the host and the malaria parasite may hold important potential for developing new strategies for malaria chemotherapy.     

Fractionation of δ15N and δ13C for Atlantic salmon and its intestinal cestode Eubothrium crassum

Stable isotopes of nitrogen (δ¹⁵N) and carbon (δ¹³C) were measured for Atlantic salmon Salmo salar and their intestinal cestode, Eubothrium crassum , sharing the same diet. Atlantic salmon muscle tissues were enriched in ¹⁵N and depleted in ¹³C compared to their prey (sprat Sprattus sprattus sprattus ) and their intestinal cestode. There was no significant difference in δ¹⁵N or δ¹³C between E. crassum and the sprat. Differences in nutrient uptake and intestine physiology between Atlantic salmon and E. crassum are discussed, as well as how these may give rise to different fractionations of stable isotopes between a host and its parasites. Furthermore, Atlantic salmon contained a significantly higher lipid content than their prey, which may partly explain differences in δ¹³C values between the host and its cestode. In addition, cestodes inhabiting lipid-rich hosts were also lipid rich. Larger Atlantic salmon were enriched in ¹⁵N compared to smaller fish. Cestodes inhabiting large hosts were also enriched in ¹⁵N compared to parasites living in smaller hosts. The last two results were explained by larger fish possibly feeding from a higher trophic level, or from larger and older prey, that resulted in both a higher lipid content and an enrichment in ¹⁵N.     

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.     

Carbon metabolism of Listeria monocytogenes growing inside macrophages

The intracellular metabolism of Listeria monocytogenes was studied by ¹³C-isotopologue profiling using murine J774A.1 macrophages as host cells. Six hours after infection, bacteria were separated from the macrophages and hydrolyzed. Amino acids were converted into tert-butyl-dimethylsilyl derivatives and subjected to gas chromatography/mass spectrometry. When the macrophages were supplied with [U-¹³C₆]glucose prior to infection, but not during infection, label was detected only in Ala, Asp and Glu of the macrophage and bacterial protein with equal isotope distribution. When [U-¹³C₆]glucose was provided during the infection period, ¹³C label was found again in Ala, Asp and Glu from host and bacterial protein, but also in Ser, Gly, Thr and Val from the bacterial fraction. Mutants of L. monocytogenes defective in the uptake and catabolism of the C₃-metabolites, glycerol and/or dihydroxyacetone, showed reduced incorporation of [U-¹³C₆]glucose into bacterial amino acids under the same experimental settings. The ¹³C pattern suggests that (i) significant fractions (50–100%) of bacterial amino acids were provided by the host cell, (ii) a C₃-metabolite can serve as carbon source for L. monocytogenes under intracellular conditions and (iii) bacterial biosynthesis of Asp, Thr and Glu proceeds via oxaloacetate by carboxylation of pyruvate.     

Effect of Tetanus Toxin on Transmitter Release from the Substantia Nigra and Striatum In Vitro

Abstract: The effect of tetanus toxin on the uptake and release of radiolabelled transmitters from slices prepared from substantia nigra (SN) and striatum of rats has been investigated. Tetanus toxin-500–750 mouse lethal doses (MLD)-injected into the SN 6 h before preparing the slices significantly reduced the calcium-dependent, potassium-evoked release of [³H]GABA. Endogenous GABA levels in the SN and [³H]GABA uptake by nigral slices were unaffected by pretreatment with the toxin. Injections of tetanus toxin (1000–2000 MLD) into the striatum significantly reduced the calcium-dependent, potassium-evoked release of [¹⁴C]GABA and also [³H]dopamine, but had no effect on the K⁺-evoked release of [³H]5-hydroxytryptamine or [¹⁴C]acetylcholine. It is concluded that tetanus toxin inhibits GABA release directly and not by interference with synthesis or inactivation processes.     

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.     

Potentiation of the antiviral activity of poly r(A-U) by riboflavin, FAD and FMN

The role of riboflavin (RFN), FAD or FMN in modulating the antiviral activity of poly r(A-U) was examined by the human foreskin fibroblast-vesicular stomatitis virus bioassay in which the concentrations of poly r(A-U) was fixed at 0.1 mM or 0.2 mM while the riboflavin, FAD or FMN concentration was varied to produce variable RFN (or FAD or FMN)/ribonucleotide ratios ranging from 1/16 to 2/1. Riboflavin, FAD and FMN tested individually did not exhibit any antiviral activity, while poly r(A-U) alone exhibited antiviral activity. When poly r(A-U) was combined with riboflavin, FAD or FMN, the antiviral activity was potentiated seven- to twelve-fold at RFN (or FAD or FMN)/ribonucleotide ratios in the region of 1/4.     

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.     

Glucose Metabolism in the Developing Cerebral Cortex as Detected by 1H{13C} Nuclear Magnetic Resonance Spectroscopy Ex Vivo

Abstract: Metabolism of [1-¹³C]glucose was monitored in superfused cerebral cortex slice preparations from 1-, 2-, and 5-week-old rats using ¹H-observed/¹³C-edited (¹H{¹³C}) NMR spectroscopy. The rate of label incorporation into glutamate C-4 did not differ among the three age groups: 0.52–0.67% of total ¹H NMR-detected glutamate/min. This was rather unexpected, as oxygen uptake proceeded at 1.1 ± 0.1, 1.9 ± 0.1, and 2.0 ± 0.1 µmol/min/g wet weight in brain slices prepared from 1-, 2-, and 5-week-old animals, respectively. Steady-state glutamate C-4 fractional enrichments in the slice preparations were ∼23% in all age groups. In the acid extracts of slices glutamate C-4 enrichments were smaller, however, in 1- and 2-week-old (17.8 ± 1.7 and 16.8 ± 0.8%, respectively) than in 5-week-old rats (22.7 ± 0.7%) after 75 min of incubation with 5 m M [1-¹³C]glucose. We add a new assignment to the ¹H{¹³C} NMR spectroscopy, as acetate C-2 was detected in slice preparations from 5-week-old animals. In the acid extracts of slice preparations acetate C-2 was labeled by ∼30% in 5-week-old rats but by 15% in both 1- and 2-week-old animals, showing that the turnover rate was increased in 5-week-old animals. In the extracts 3–4% of the C-6 of N -acetyl-aspartate (NAA; CH₃ of the acetyl group) contained label as determined by both NMR and mass spectrometry, which indicated that there was no significant labeling to other carbons in NAA. NAA accumulated label from [1-¹³C]glucose but not from [2-¹³C]acetate, and the rate of label incorporation increased by threefold on cerebral maturation.     

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.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of ¹⁴C into several subcellular fractions of adult rat brain was studied as a function of time, following intracerebral injection of [2-¹⁴C]mevalonic acid. As expected from previous studies, the microsomal fraction was indicated as the site of sterol biosynthesis. The myelin fraction showed a marked and early uptake of ^I4C-labelled, digitonin-precipitable material. This was assumed to be a non-enzymic uptake of sterol intermediates. The mitochondrial fraction exhibited a rapid uptake of ¹⁴C-labelled, nonsaponifiable material, but a very slow accumulation of ¹⁴C-labelled, digitonin-precipitable product. Examination of the nonsaponifiable ¹⁴C-fractions by TLC showed a rapid appearance of labelled 4-desmethyl sterols in the microsomal fraction. The myelin fraction selectively retained 4,4'-dimethyl sterol but seemed to release this with time, possibly to be further metabolized by the microsomes. Examination of [¹⁴C]digitonin-precipitable material by the dibromide method showed that although labelled 4-desmethyl sterol appeared quite early, cholesterol itself was formed slowly in all fractions.     

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.     

Distribution and metabolism of root-applied cytokinins in Pisum sativum

When N ⁶ [8–¹⁴C] furfuryladenine was applied to the intact root system of Pisum sativum L. cv. Meteor seedlings it was almost completely metabolised to other compounds within 24 h. Of the total activity recovered from the plants 94.5% was retained in the root system itself. ¹⁴C was recovered in a number of ethanol-soluble compounds and in ribonucleic acid, deoxyribonucleic acid and protein fractions of roots, stems, leaves and axillary buds. In rapidly growing axillary buds released from apical dominance by removal of the shoot apex the combined nucleic acid fractions accounted for 63.3% of the total ¹⁴C recovered from these organs. Xylem exudate collected from decapitated plants 0 to 12 h after supplying N ⁵[8–¹⁴C]furfuryladenine to the roots consistently contained a single major ¹⁴C-labelled compound which, in three different solvent systems, had the same Rf values as a major endogenous cytokinin isolated from the xylem of unlabelled plants. The content of N ⁶ [8–¹⁴C] furfuryladenine itself in the xylem exudate was always low and in some experiments it could not be detected.
It is suggested that part of the label from N ⁶ [8- ¹⁴CJfurfuryladenine taken up by the intact root system may have become incorporated in an endogenous cylokinin before export to the shoot.     

The riboflavin/FAD cycle in rat liver mitochondria.

Here we provide evidence that mitochondria isolated from rat liver can synthesize FAD from riboflavin that has been taken up and from endogenous ATP. Riboflavin uptake takes place via a carrier-mediated process, as shown by the inverse relationship between fold accumulation and riboflavin concentration, the saturation kinetics [riboflavin Km and Vmax values were 4.4+/-1.3 microM and 35+/-5 pmol x min(-1) (mg protein)(-1), respectively] and the inhibition shown by the thiol reagent mersalyl, which cannot enter the mitochondria. FAD synthesis is due to the existence of FAD synthetase (EC 2.7.7.2), localized in the matrix, which has as a substrate pair mitochondrial ATP and FMN synthesized from taken up riboflavin via the putative mitochondrial riboflavin kinase. In the light of certain features, including the protein thermal stability and molecular mass, mitochondrial FAD synthetase differs from the cytosolic isoenzyme. Apparent Km and apparent Vmax values for FMN were 5.4+/-0.9 microM and 22.9+/-1.4 pmol x min(-1) x (mg matrix protein)(-1), respectively. Newly synthesized FAD inside the mitochondria can be exported from the mitochondria in a manner sensitive to atractyloside but insensitive to mersalyl. The occurrence of the riboflavin/FAD cycle is proposed to account for riboflavin uptake in mitochondria biogenesis and riboflavin recovery in mitochondrial flavoprotein degradation; both are prerequisites for the synthesis of mitochondrial flavin cofactors.     

A GC-MS method for determination of amino acid uptake by plants

In this study, we present a rapid, robust and sensitive method for quantification of plant amino acid uptake using universally (U) (¹³C, ¹⁵N)-labelled amino acids and gas chromatography-mass spectrometry (GC-MS). Amino acids were analysed as their tert -butyldimethylsilyl (tBDMS) derivatives and displayed detection limits in the range 10–100 fmol on column, depending on the amino acid. The technique allows for simultaneous detection and quantification of both unlabelled and isotopically labelled species of amino acids. This makes simple quantification of plant amino acid uptake from an isotopically labelled source possible. The analytical variation was low, concerning total amino acid concentrations (relative standard deviation, rsd , less than 5.3%) as well as enrichment of U-¹³C, ¹⁵N-labelled glycine (Gly), arginine (Arg) and glutamic acid (Glu) ( rsd <2.1%). An application of the GC-MS method was conducted on non-mycorrhizal Pinus sylvestris roots supplied with U-¹³C, ¹⁵N-labelled amino acids. Intact, labelled amino acids were traced in root extracts. This provided conclusive evidence of plant root uptake of intact amino acids. Uptake rates of the three amino acids Gly, Glu and Arg in the range 0.5–37.9 μmol g⁻¹ dry weight h⁻¹ were recorded. These rates are comparable with those recorded in earlier studies of amino acid uptake, using other methods, as well as uptake rates measured for nitrate and ammonium.     

Axenic incorporation of [U-14C]palmitic acid into the phenolic glycolipid-I of Mycobacterium leprae

Abstract Incorporation of [U-¹⁴C]palmitic acid ([¹⁴C]PA) into the specific phenolic glycolipid-I (PGL-I) of freshly harvested, nude mouse-derived Mycobacterium leprae was investigated in an axenic modified Dubos medium. Incorporation was approximately linear for 10–14 days at pH 7.2, 33°C. No incorporation of radiolabeled phenol, acetate, tyrosine, phenylalanine, bicarbonate, proprionate or UDP-glucose was detected. Procedures known to remove residual host tissue did not diminish the rate of [¹⁴C]PA incorporation, indicating that bacterial metabolism was being measured. The antileprosy compounds, rifampicin and dapsone, significantly reduced incorporation of the label. The ability to quantitate PGL-I synthesis in the extracellular bacillus should facilitate a better understanding of the optimum conditions for metabolism in M. leprae .     

Biosynthesis of Polyamines in Mouse Brain: Effects of Methionine Sulfoximine and Adenosylhomocysteine

Abstract: This study examines the consequences on cerebral polyamine biosynthesis of increases and decreases in cerebral methylation. Increases were elicited by administering the convulsant agent methionine sulfoximine (MSO) and decreases by elevating in vivo the cerebral levels of the methylation inhibitor S -adenosyl-homocysteine. Following the intraventricular (i.vt.) administration of one of the two possible polyamine precursors, [1,4-¹⁴C]putrescine, the specific radioactivity (sra) of the newly formed [¹⁴C]spermidine remained unchanged. Conversely, after i.vt. l -[3,4-¹⁴C]methionine, the other polyamine precursor, significantly higher sra values for [¹⁴C]spermidine and [¹⁴C]spermine were recorded in the brains of the MSO-treated animals. [¹⁴C] S - adenosylmethionine in the brain of the MSO-treated animals was also more highly labeled following [1-¹⁴C]-methionine, indicating its accelerated formation relative to controls. We also investigated the effect of the administration of adenosine + homocysteine, a treatment that results in elevated brain adenosylhomocysteine levels, on polyamine biosynthesis from [3,4-¹⁴C]-methionine. The results of these experiments show both significantly lower sra values for [¹⁴C]spermidine and [¹⁴C]spermine and significantly higher than control endogenous methionine levels, a clear sign of the existence of a retardation in the conversion of methionine to polyamines under these conditions. In conclusion, the present study demonstrates that while interference with cerebral methylation results in significant alterations of the rate of formation of the methionine moiety of spermidine and spermine, it has no effect on the entry of the putrescine moiety into the two polyamine molecules.     

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.     

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.