Changes in orthophosphate, pyrophosphate and long-chain polyphosphate levels in Leishmania major promastigotes incubated with and without glucose

The intracellular levels of orthophosphate (Pi), pyrophosphate (PPi) and short- and long-chain polyphosphate (Poly P) were measured in Leishmania major promastigotes incubated in a phosphate-free medium. In the absence of exogenous substrate, the levels of both Pi and PPi increased during a 1 h incubation. The increase in both Pi and PPi was prevented when glucose was present, but glycerol prevented the rise in Pi only. A rise in Pi and PPi was also seen in cells incubated in the absence of exogenous substrate under anaerobic conditions. This was reversed upon addition of glucose plus oxygen. Polyphosphate, here shown to be present in L. major, was measured by means of a polyphosphate glucokinase assay. Short-chain Poly P content did not differ between cells incubated for 1 h in the absence of exogenous substrate or in the presence of glucose or glycerol. Long-chain Poly P content, however, was lower in cells incubated without glucose than in cells incubated with glucose and was also lower in cells incubated for 1 h with glycerol as compared with freshly washed cells. Up to 61% of the increase in Pi and PPi that occurred in promastigotes incubated in the absence of exogenous substrate could have arisen from the concomitant decrease in long-chain Poly P.     

Subcellular compartmentation of pyrophosphate and dark/light kinetics in comparison to fructose 2,6-bisphosphate

Subcellular compartmentation of pyrophosphate (PP₁) was determined by rapid membrane filtration of evaeuolated oat mesophyll protoplasts. By improving both the extraction procedure and its assay via bioluminescence, PP₁ recovery from samples was quantitative and linear down to below 200 fmol. Based on the content of the different fractions obtained after membrane filtration and compared to the respective pools of marker metabolites [cytosol, fructose 2,6-bisphosphate (F26BP); chloroplast stroma, ribulose bisphosphate] rather than enzymes, we found ca 2/3 of the total cellular content to be chloroplast-assotiated. Referred to compartmental volumes, cytosolic and stromal concentrations of PP₁ were nearly equal (70–100 μ M ). PP₁ was higher in evacuolated compared to racuotated protoplasts which indicates a possible role of the tonoplast-located H⁺ pumping PP₁ase in regulating the cellular pool size of PP₁. During dark-light-transition the pool sizes of PP₁ changed only marginally in both vacuolated and evacuolated protoplasts, while there were pronounced changes in those of F26BP, starch and sucrose. Thus our findings support the notion that the cellular pool size of PP₁ is kept rather constant. They are, however, in contrast to the assumption that appreciable PP₁ levels only exist in the cytosol.     

Muscarinic Cholinoceptor-Stimulated Synthesis and Degradation of Inositol 1,4,5-Trisphosphate in the Rat Cerebellar Granule Cell

Abstract: A detailed analysis of the generation and subsequent metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] following muscarinic cholinoceptor stimulation in primary cultures of rat cerebellar granule cells has been undertaken. Following incubation of cerebellar granule cell cultures with [³H]inositol for 48 h, labelling of the inositol phospholipid pool approached equilibrium. Significant basal labelling of inositol pentakisphosphate (InsP₅) and inositol hexakisphosphate (InsP₆), as well as inositol mono- to tetrakisphosphate, fractions was observed. Addition of carbachol (1 m M ) caused an immediate increase in level of Ins(1,4,5)P₃ (peak increase two-fold over basal by 60 s), which was well-maintained over the initial 300 s following agonist addition. In contrast, only a modest, more slowly developing, increase in inositol tetrakisphosphate accumulation was observed, whereas labelling of InsP₅ and InsP₆ was entirely unaffected by carbachol stimulation. Analysis of the products of Ins(1,4,5)P₃ and inositol 1,3,4,5-tetrakisphosphate metabolism in broken cell preparations strongly suggested that Ins(1,4,5)P₃ metabolism occurs predominantly via the inositol polyphosphate 5-phosphatase route, with metabolism via the Ins(1,4,5)P₃ 3-kinase being a relatively minor pathway. In view of the pattern of inositol (poly)phosphate metabolites observed on stimulation of the muscarinic receptor, it seems likely that, over the time course studied, the inositol polyphosphates are derived principally from phosphoinositide-specific phospholipase C hydrolysis of phosphatidylinositol 4,5-bisphosphate, although some hydrolysis of phosphatidylinositol 4-phosphate cannot be excluded.     

Irreversible blockade of sigma-1 receptors by haloperidol and its metabolites in guinea pig brain and SH-SY5Y human neuroblastoma cells

We evaluated the effect of haloperidol (HP) and its metabolites on [³H](+)-pentazocine binding to σ₁ receptors in SH-SY5Y human neuroblastoma cells and guinea pig brain P₁, P₂ and P₃ subcellular fractions. Three days after a single i.p. injection in guinea pigs of HP (but not of other σ₁ antagonists or (−)-sulpiride), [³H](+)-pentazocine binding to brain membranes was markedly decreased. Recovery of σ₁ receptor density to steady state after HP-induced inactivation required more than 30 days. HP-metabolite II (reduced HP, 4-(4-chlorophenyl)-α-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol), but not HP-metabolite I (4-(4-chlorophenyl)-4-hydroxypiperidine), irreversibly blocked σ₁ receptors in guinea pig brain homogenate and P₂ fraction in vitro . We found similar results in SH-SY5Y cells, which suggests that this process may also take place in humans. HP irreversibly inactivated σ₁ receptors when it was incubated with brain homogenate and SH-SY5Y cells, but not when incubated with P₂ fraction membranes, which suggests that HP is metabolized to inactivate σ₁ receptors. Menadione, an inhibitor of the ketone reductase activity that leads to the production of HP-metabolite II, completely prevented HP-induced inactivation of σ₁ receptors in brain homogenates. These results suggest that HP may irreversibly inactivate σ₁ receptors in guinea pig and human cells, probably after metabolism to reduced HP.     

Influence of P blood group phenotype on susceptibility to urinary tract infection

Abstract Bacterial attachment is an important event in the pathogenesis of urinary tract infection (UTI). Increased receptivity on the host cells has been suggested influence proneness to infection. The dual function of the globoseries of glycolipids both as receptors for attaching E. coli and as P blood group antigens lead us to examine the P blood group phenotype distribution in UTI prone patient populations. A correlation between the P₁ blood group phenotype and susceptibility to UTI was found. Patients with recurrent pyelonephritis had 74/79 (94%), P₁ compared to 75% in healthy controls. In contrast patients with asymptomatic bacteriuria (ABU) had a reduced frequency of P₁, 43/74 (58%). P₁ and P₂ individuals differ in amount and composition of the globoseries of glycolipids on their erythrocytes. A similar difference in other tissues, e.g. uroepithelial cells might explain the association of P₁ with UTI. There was, however, no significant difference in bacterial adherence to uroepithelial cells from P₁ and P₂ individuals. Other mechanisms explaining the increase in P₁ individuals in recurrent pyelonephritis are discussed.     

Identification of the Palmitoylation Site in Rat Myelin P0 Glycoprotein

Abstract: P₀ glycoprotein, the major protein of PNS myelin, contains approximately 1 mol of covalently bound long-chain fatty acids. To determine the chemical nature of the fatty acid-protein linkage, P₀ was labeled in rat sciatic nerve slices with [³H]palmitic acid and subsequently treated with various reagents. The protein-bound palmi-tate was released by incubation with the reducing agents dithiothreitol and 2-mercaptoethanol, and with 1 M hydrox-ylamine at pH 7.5. In addition, P₀ was deacylated by treatment with 10 m M NaBH₄ with the concomitant production of [³H]hexadecanol, indicating that the fatty acid is bound in a thioester linkage. This conclusion was supported further by the fact that deacylation with hydroxylamine generated free thiol groups, which were titrated with [¹⁴C]-iodoacetamide. To identify the cysteine residue involved in the thioester linkage, [¹⁴C]carboxyamidomethylated P₀was digested with trypsin and the resulting peptides analyzed by reversed-phase HPLC. Identification of the radioactive protein fragments by amino acid analysis and amino-terminal peptide sequencing revealed that Cys¹⁵³ in rat P₀ glycoprotein is the acylation site. The acylated cysteine is located at the junction of the putative transmem-brane and cytoplasmic domains. This residue is also present in the P₀ glycoprotein of other species, including human, bovine, mice, and chicken.     

Interactions between respiration and inorganic phosphate uptake in phosphate-limited cells of Chlamydomonas reinhardtii

Phosphate addition to P-limited cells of Chlamydomonas reinhardtii resulted in an immediate increase in the rate of respiratory O₂ consumption. The respiration rate continued to increase for several minutes after the addition of P₁. Similar patterns of P₁ stimulation of respiratory O₂ consumption were observed in the presence of cyanide (cytochrome oxidase inhibitor) and propyl gallate (alternative oxidase inhibitor). Stimulation of O₂ consumption was accompanied by rapid changes in levels of glycolytic intermediates. These changes were consistent with activation of ATP-dependent phosphofructokinase and pyruvate kinase. The adenylate pool exhibited only minor perturbations, P₁, uptake resulted in extracellular acidification, which continued for several minutes after the exhaustion of added P₁, whereas exhaustion of extracellular P₁ resulted in a rapid decline in the O₂ consumption rate. These results are consistent with control of respiration in P-limited cells occurring largely at the level of glycolysis.     

Kinetics of Entry of P0 Protein into Peripheral Nerve Myelin

Abstract: Sciatic nerves from 9-day-old rat pups were removed, sliced into 0.4-mm sections, and incubated with [³H]fucose or [¹⁴C]glycine precursors. The nerve slice system gave nearly linear incorporation of [³H]fucose as a function of time for 3 h, after an initial lag of ˜30 min for homogenate and ˜60 min for myelin. Incorporation of [³H]fucose at constant specific radioactivity was directly proportional to exogenous fucose levels over the range 3.0 × 10⁻⁸ m to 1.5 × 10⁻⁶ m . Analysis of labeled proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that greater than 50% of labeled glycoprotein was P₀, with no other major constituents. This system was used in fucose-chase experiments to determine that a period of ˜20 min elapses between fucosylation and assembly of P₀ into myelin. Cycloheximide inhibition of protein synthesis was used to determine that a period of ˜33 min elapses between protein synthesis and appearance of P₀ myelin.     

The possible role of fructose 2,6-bisphosphate in the cyanide-mediated removal of embryonic dormancy in apple

The effects of pretreatment with HCN on the level of fructose 2.6-bisphosphate (F-2,6-P₂). on activity of fructose 6-phosphate 2-kinase (EC 2.7.1.150; F-6-P. 2K. enzyme synthesizing F-2,6-P₂), as well as on activities of PP₁-dependent and ATP-dependent phosphofructokinases (EC 2.7.1.90. PP₁-PFK and EC 2.7.1.11, ATP-PFK) were studied in cultured, dormant embryos of apple ( Malus domestica Borb. cv. Antonówka). HCN increased the F-2.6-P₂ level and F-6-P, 2K activity in embryonal axes (3-fold), but had no effect in cotyledons. HCN pretreatment of embryos or the addition of F-2.6-P₂ to enzyme extract stimulated PP₁, -PFK activity, whereas the activity of ATP-PFK was slightly inhibited by HCN in axes and in cotyledons. Glycolysis is one of the first processes in the germination of apple embryos, and the stimulation of glycolysis by HCN may be the result of F-6-P, 2K activation in the axes. This will lead to accumulation of F-2,6-P₂, which, in turn, enhances glycolysis by activation of PP₁PKF.     

Changes of inositol phosphates during decapitation-induced lateral bud break of Malus domestica

An increase in phosphatidylcholine (PC), phosphatidyl-ethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI) occurred during bud break induced by decapitation. Inositol-1-phosphate [Ins(l)P₁], inositol-1,4-bisphosphate [Ins(1,4)P₂], and inositol-1,4,5-triphosphate [Ins(1,4,5)P₃] were found in apple buds and increased progressively following decapitation. Ins(1)P₁ and Ins(1,4)P₂ peaked 48 h after decapitation and Ins(1,4,5)P₃ peaked 72 h after decapitation during the metabolic transition when buds emerged from dormancy. Ins(1,4)P₂ and Ins(1,4,5)P₃ levels declined there after. The lateral buds on shoots with intact terminals and decapitated shoots treated with indole-3-acetic acid (IAA) in the terminals tip remained dormant and there were no significant changes in phospholipid and inositol phosphate contents.     

Glycoprotein Biosynthesis in Peripheral Nervous System Myelin: Effect of Tunicamycin

Abstract: The effect of an inhibitor of N -glycosylation of glycoproteins, tunicamycin, on synthesis of PNS myelin proteins was investigated in vitro by using chopped sciatic nerves or spinal roots of 21-day-old Wistar rats. Tunicamycin when incubated with these nerves in the presence of ³H-labeled fucose, mannose, or glucosamine inhibited the uptake of radioactivity into myelin proteins including some high-molecular-weight proteins, P₀, 23K protein, and 19K protein by amounts ranging from 42 to 79%. Uptake of ¹⁴Camino acid mixture was inhibited much less by tunicamycin, but a new radioactive protein peak appeared when the protein mixtures had been separated by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This protein ran directly in front of the P₀ peak, did not correspond to any bands stained by Fast green, and was not labeled by fucose. This peak appeared in increasing larger proportions with progressive time of incubation of nerves with ³H amino acids in the presence of tunicamycin. The new protein, which cross-reacts with P₀ antiserum, was tentatively identified as a nonglycosylated P₀ protein that appears to be almost as well incorporated as P₀ into the subcellular fraction containing myelin. At this time it is not possible to determine whether the unglycosylated P₀ is actually assembled into a site and configuration like that of P₀.     

Ultrastructural Localization of P2 Protein in Actively Myelinating Rat Schwann Cells

Abstract: The myelin specific protein, P₂, was localized immunocytochemically in electron micrographs of 4-day-old rat peripheral nerve by a preembedding technique. P₂ staining was restricted to Schwann cells that had established a one-to-one relationship with an axon. P₂ antiserum produced a diffuse staining throughout the entire cytosol of myelinating Schwann cells. In addition, the cytoplasmic side of Schwann cell plasma membranes and the membranes of cytoplasmic organelles that were exposed to cytosol were stained by P₂ antiserum. This cytoplasmic localization of P₂ protein is similar to that described for soluble or peripheral membrane proteins that are synthesized on free ribosomes. P₂ antiserum stained the cytoplasmic side of Schwann cell membranes that formed single or multiple loose myelin spirals around an axon. In the region of the outer mesaxon, P₂ antiserum stained the major dense line of compact myelin. These results demonstrate that P₂ protein is located on the cytoplasmic side of compact myelin membranes and are consistent with biochemical studies demonstrating P₂ to be a peripheral membrane protein.     

Formation of a Disulfide Bond in the Immunoglobulin Domain of the Myelin P0 Protein Is Essential for Its Adhesion

Abstract: It is widely accepted, although never demonstrated, that the formation of a disulfide bond in the majority of immunoglobulin (Ig)-like domains stabilizes their final conformation and thus is essential to their functioning as adhesion/recognition molecules. The myelin P₀ protein, which has been shown directly to behave as a homophilic adhesion molecule, contains a single Ig-like domain, stabilized by a putative Cys²¹-Cys⁹⁸ disulfide bond. To test if this bond is indeed necessary to the adhesive function of P₀, the nucleotides in the P₀ cDNA coding for Cys²¹ were altered to code for an alanine. The mutated P₀ cDNA was transfected into Chinese hamster ovary cells, expression of the mutated P₀ protein was characterized, and the adhesiveness of Cys²¹-mutated P₀-expressing cells and that of cells expressing equivalent surface amounts of the unmutated protein were compared. It was found, as we previously reported, that incubation of a single cell suspension of the unmutated P₀-expressing cells resulted in the rapid formation of large aggregates. In contrast, after a similar incubation the cells expressing the Cys²¹-mutated P₀ were still mostly single cells, a result indistinguishable from that observed with the control transfected cells. This suggests that the P₀ protein, when mutated at Cys²¹, does not behave as a homophilic adhesion molecule, which in turn implies that the formation of an Ig domain disulfide bond is essential to the functioning of this molecule.     

PROTEIN COMPOSITION OF MYELIN OF THE PERIPHERAL NERVOUS SYSTEM

Abstract— Myelin was purified from the peripheral nervous system (PNS) of several species. The protein composition of these preparations was examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Proteins characteristic of all samples include, in order of increasing mobility: a series of high molecular weight proteins, the major peripheral nerve protein (P₀), two uncharacterized proteins, and two basic proteins (P₁ and P₂). Quantitative results, obtained by densitometry of gels stained with Fast Green showed differences in protein distribution, both between species, and from different types of nerves obtained from the same animal. The relative amounts of P₁ and P₂ proteins were the most variable; e.g. myelin from guinea-pig sciatic nerve had little or no P₂ protein, whereas 15 per cent of the myelin protein of beef posterior intradural root was Pz protein. P₀, P₁ and P₂ proteins from rabbit sciatic nerve and P₀ and P₂ proteins from beef dorsal and ventral intradural roots were purified and their amino acid compositions were determined. Our results indicated that the P₁ protein is very similar in size and amino acid composition to the basic protein of central nervous system myelin, whereas the P₀ and P₂ proteins are unique to the PNS.     

Sphingosine-1-phosphate receptors mediate neuromodulatory functions in the CNS

Sphingosine-1-phosphate (S1P) is a ubiquitous, lipophilic cellular mediator that acts in part by activation of G-protein-coupled receptor. Modulation of S1P signaling is an emerging pharmacotherapeutic target for immunomodulatory drugs. Although multiple S1P receptor types exist in the CNS, little is known about their function. Here, we report that S1P stimulated G-protein activity in the CNS, and results from [³⁵S]GTPγS autoradiography using the S1P₁-selective agonist SEW2871 and the S1P_1/3-selective antagonist VPC44116 show that in several regions a majority of this activity is mediated by S1P₁ receptors. S1P receptor activation inhibited glutamatergic neurotransmission as determined by electrophysiological recordings in cortical neurons in vitro , and this effect was mimicked by SEW2871 and inhibited by VPC44116. Moreover, central administration of S1P produced in vivo effects resembling the actions of cannabinoids, including thermal antinociception, hypothermia, catalepsy and hypolocomotion, but these actions were independent of CB₁ receptors. At least one of the central effects of S1P, thermal antinociception, is also at least partly S1P₁ receptor mediated because it was produced by SEW2871 and attenuated by VPC44116. These results indicate that CNS S1P receptors are part of a physiologically relevant and widespread neuromodulatory system, and that the S1P₁ receptor contributes to S1P-mediated antinociception.     

Distribution of transition metal ions in multiple forms of Methanosarcina hydrogenase

There were significant levels of in vitro hydrogenase activity in Methanosarcina strains. The multiple forms of hydrogenase were observed in cell free extracts of cells grown on methanol. Strains having poor growth on H₂ : CO₂ had four forms while strains having normal growth on all substrates contained two forms of hydrogenase. These multiple forms differ in their charges as well as in their composition of transition metal ions. The strain having normal growth showed higher incorporation of ⁶³Ni²⁺ and ⁶⁵Zn²⁺. Both hydrogenases, A and D, of strain P₃ had methylviologen and F₄₂₀-reducing activity and contained Zn²⁺ and Co²⁺ respectively. Hydrogenases A and D of strains P₁ and P₄ also had similar characteristics whereas hydrogenases B and C had only methylviologen reducing activity.     

Lactate Is Released and Taken Up by Isolated Rabbit Vagus Nerve During Aerobic Metabolism

Abstract: To determine if lactate is produced during aerobic metabolism in peripheral nerve, we incubated pieces of rabbit vagus nerve in oxygenated solution containing d -[U-¹⁴C]glucose while stimulating electrically. After 30 min, nearly all the radioactivity in metabolites in the nerve was in lactate, glucose 6-phosphate, glutamate, and aspartate. Much lactate was released to the bath: 8.2 pmol (µg dry wt)⁻¹ from the exogenous glucose and 14.2 pmol (µg dry wt)⁻¹ from endogenous substrates. Lactate release was not increased when bath P o ₂ was decreased, indicating that it did not come from anoxic tissue. When the bath contained [U-¹⁴C]lactate at a total concentration of 2.13 m M and 1 m M glucose, ¹⁴C was incorporated in CO₂ and glutamate. The initial rate of formation of CO₂ from bath lactate was more rapid than its formation from bath glucose. The results are most readily explained by the hypothesis that has been proposed for brain tissue in which glial cells supply lactate to neurons.     

Effect of Hyper-Osmotic Stress On Alanine Content of Leishmania Major Promastigotes

ABSTRACT Earlier studies showed that Leishmania major promastigotes are sensitive to osmotic conditions. A reduction in osmolality caused the cells to shorten and to rapidly release most of their large internal pool of alanine. In this study some effects of hyper-osmotic stress were examined. an increase in osmolality of the culture medium from 308 to 625 mOsm/kg caused only a small decrease in growth rate. When cells grown in the usual culture medium (308 mOsm/kg) were washed, resuspended in iso-osmotic buffer, and subjected to acute hyper-osmotic stress by addition of mannitol, the alanine content increased even in the absence of exogenous substrate. Promastigotes, depleted of alanine by a 5-min exposure to hypo-osmotic conditions, also synthesized alanine when resuspended in iso-osmotic buffer. Washed cells resuspended in iso-osmotic buffer consume their internal pool of alanine under aerobic conditions, Rates of consumption decreased on addition of mannitol, becoming zero at about 440 mOsm/kg. At higher osmolalities, alanine synthesis occurred. to estimate whether proteolysis could account for alanine synthesis in the absence of exogenous substrate, cells that had been grown with [1-¹⁴C]leucine were washed and resuspended under hypo-, iso-, and hyper-osmotic conditions and the amounts of ¹⁴CO₂ and ¹⁴C-labelled peptides released in 1 h were measured. Little proteolysis occurred under these conditions, but the possibility that proteolysis was the source of the alanine increase, observed in response to hyper-osmotic stress, cannot be ruled out.     

Effects of Culture Age and Hexoses on Fatty Acid Oxidation by Leishmania major

ABSTRACT. The effect of culture age on the rate of oxidation of short-, medium-, and long-chain fatty acids by Leishmania major promastigotes was investigated. Promastigotes from 5-day stationary phase cultures oxidized several saturated fatty acids about 3-to-4-fold faster than cells from late log phase cultures, but [10⁻¹⁴C]oleate was oxidized 9-fold faster. The increase in rate of oxidation was partially reversed within 5 h and almost completely reversed within 30 h after resuspending cells from a 5-day stationary culture in fresh medium. Addition of acetate, leucine, or alanine caused moderate inhibitions of [1-¹⁴C]palmitate oxidation, while glycerol had little effect. Glucose, however, was a powerful inhibitor of the oxidation of [1-¹⁴C]palmitate and of [1-¹⁴C]octanoate. Mannose and fructose were also strong inhibitors of palmitate oxidation, but neither galactose, 2-deoxyglucose or 6-deoxyglucose caused appreciable inhibition. The extent of inhibition by acetate increased with increasing culture age, whereas inhibition by glucose decreased. In addition to demonstrating a reversible rise in β-oxidation capacity with culture age, these data also demonstrate a hitherto unrecognized strong and culture age-dependent inhibition of fatty acid oxidation by glucose.