Stage-dependent toxicity of N-acetyl-glucosamine to Plasmodium falciparum

The effects of N-acetyl-glucosamine on growth of synchronized cultures of Plasmodium falciparum were assessed by morphological observations and by measurement of parasite incorporation of 3H-hypoxanthine. Inhibition of 3H-hypoxanthine incorporation was more marked during the later stages of the erythrocytic cycle. At concentrations of the sugar below 20 mM, however, the deleterious effects were mainly a result of failure of released merozoites to invade erythrocytes, rather than a failure of schizonts to mature or release merozoites. These results are compatible with the hypothesis that a lectin-like substance on the merozoite interacts with a surface glycoprotein on the red cell and that sugar residues on this glycoprotein may be involved in this recognition.     

Incorporation of hypoxanthine by PHA-stimulated HPRT-deficient lymphocytes

Phytohemagglutinin (PHA) markedly stimulates ³H-hypoxanthine incorporation by lymphocytes of normal subjects as revealed by radioautography. There is no corresponding increase in activity of hypoxanthine phosphoribosyltransferase (HPRT) in lysates but the level of phosphoribosylpyrophosphate (PRPP), the cosubstrate for the reaction, is higher. Lymphocytes from a patient with partial HPRT deficiency responded to PHA as did the normals, whereas the response in Lesch-Nyhan (LN) subjects was variable. PHA-stimulated lymphocytes from two LN patients showed some increase in ³H-hypoxanthine incorporation, while two others failed to respond. The observations provide further evidence of genetic heterogeneity among LN patients.     

Eimeria tenella: Parasite-Specific Incorporation of 3H-Uracil as a Quantitative Measure of Intracellular Development1

ABSTRACT. An assay has been developed using parasite-specific incorporation of ³H-uracil to assess the intracellular growth of Eimeria tenella in vitro. As shown by both scintillation counts and autoradiography, ³H-uracil was incorporated specifically into intracellular parasites from the onset of infection and continued throughout development of the first generation schizonts. Mature schizonts and first generation merozoites did not continue to incorporate additional ³H-uracil, indicating that RNA synthesis had halted in these stages. Based on these findings, a semi-automated microscale uracil incorporation assay was developed to determine parasite viability. This method should be useful for biochemical studies with intracellular parasites and for screening compounds for anticoccidial activity. The ease, rapidity, and quantitative nature of this assay contrasts favorably with standard morphometric approaches of determining parasite development. In addition, parallel studies using host cell incorporation of ³H-uridine have been introduced as a method of determining whether antiparasitic activity is direct or indirect in relation to effects on the host cell.     

Adaptation in Lesch-Nyhan cells exposed to aminopterin

Fibroblasts from a particular patient with Lesch-Nyhan syndrome developed full resistance to aminopterin, in association with a progressive increase in ³H-hypoxanthine incorporation. This represented a gradual and generalized adaptation. When aminopterin treatment was stopped, the minimal ³H-hypoxanthine uptake of untreated cells was re-established. The hypoxanthine-guanine phosphoribosyltransferase activity of extracts of these cells was as low as in other Lesch-Nyhan cultures, but aminopterin-treated cells contained more than twice as much activity, suggesting that an increased amount of enzyme caused the adaptation. However, this conlusion can only be tentative in view of the complex and circular nature of the interconversions of hypoxanthine, inosine and inosinic acid in these extracts.     

Stage-Dependent Effects of Chloroquine on Plasmodium falciparum In Vitro1

The erythrocytic developmental cycle of Plasmodium falciparum can be conveniently divided into the ring, trophozoite, and schizont stages based on morphology and metabolism. Using highly synchronous cultures of P. falciparum, considerable variation was demonstrated among these stages in sensitivity to chloroquine. The effects of timed, sequential exposure to several clinically relevant concentrations of chloroquine were monitored by three techniques: morphological analysis, changes in the rate of glucose consumption, and changes in the incorporation of ³H-hypoxanthine into parasite nucleic acids. All three techniques gave essentially identical results. The trophozoite and schizont stages were considerably more sensitive to the drug than ring-stage parasites. Chloroquine sensitivity decreased as nuclear division neared completion. The increase in chloroquine sensitivity was coincident with a marked rise in the rate of glucose consumption and nucleic acid synthesis. The rate of nucleic acid synthesis decreased as schizogony progressed while glucose consumption continued at high rates during this process. The degree of chloroquine sensitivity was not highly correlated with either metabolic activity.     

Fine Structure of the Malaria Parasite Plasmodium lophurae Developing Extracellularly in vitro*

Duck malaria parasites (Plasmodium lophurae), synchronized at the uninucleate trophozoite stage, were freed from their host erythrocytes by immune lysis and cultured extracellularly in duck erythrocyte extract medium. At 0 time, 1, 2, and 3 days, samples were taken for light and electron microscopy and for measurement of incorporation of [¹⁴C]-methionine or [¹⁴C]-proline. For 2 days the parasites developed fairly normally, progressing from large trophozoites-early schizonts at 1 day to segmenters-forming merozoites at 2 days. However, the 3-day samples showed signs of deterioration: incorporation of amino acids dropped; the percentage degenerate cells rose; the progression of developmental stages slowed. At the fine structure level 2 abnormalities were observed which may indicate the limits of extracellular cultivation in vitro. Through 2 days of culture all parasites were surrounded by 2 membranes. The 3-day samples contained some organisms with only one membrane, which may have arisen from merozoites produced extracellularly. The 2nd alteration was in the food vacuoles, which were progressively fewer, smaller, and less dense in the cultured samples and may indicate an abnormality in the extracellular parasite's feeding mechanism.     

Does a Histidine-Rich Protein from Plasmodium lophurae Have a Function in Merozoite Penetration?*

SYNOPSIS. Autoradiography was used to localize the distribution of l -[³H]histidine incorporated in vitro by developing segmenters of Plasmodium lophurae. Under conditions used for the visualization of high specific activity loci, radioactivity appeared associated mainly with the histidine-rich protein of cytoplasmic granules as well as with rhoptries and micronemes of merozoites. The isolated histidine-rich protein caused agglutination of erythrocytes and increased their osmotic fragility. Based on the observed evidence suggesting the presence of this histidine-rich protein in the polar organelles of merozoites and its reactivity with erythrocyte membranes, the hypothesis that such a protein may have a function in the penetration of merozoites is proposed.     

Intercellular communication and tissue growth

Summary Three cancer cell strains that fail to make permeable membrane junctions were tested for ability to transfer an endogenous hypoxanthine derivative from cell to cell. The cells of these strains, loaded with³H-hypoxanthine, were grown in contact with cells from a mutant line incapable of incorporating exogenous hypoxanthine. The transfer of the³H-hypoxanthine derivative to the mutant cells was determined by radio-autography and, in the same preparations, the presence of permeable membrane junctions was determined by intercellular fluorescein tracer diffusion and electrical measurement. The cells of the three strains showed no transfer of hypoxanthine derivative to contiguous mutant cells; the cells that make permeable junctions did show such transfer, under the same conditions.In contrast to this contact-requiring mode of transfer, a contact-independent transfer phenomenon was observed with these three cancer cell strains.     

Biochemical and ultrastructural studies of ectoglycosyltransferase systems of murine L1210 leukemic cells

Intact murine L1210 leukemic cells incorporated significant quantities of [³H]-N-acetylneuraminic acid directly from CMP-N-acetylneuraminic acid. When pretreated with Vibrio cholerae neuraminidase, incorporation increased sixfold to tenfold. Biochemical studies comparing incorporation of N-acetyl-neuraminic acid from the nucleotide sugar with that from free sugar demonstrated that the relatively high levels of incorporation from CMP-N-acetyl-neuraminic acid could not be due to the incorporation of free sugar generated by extracellular degradation of the nucleotide sugar. Very little N-acetylneuraminic acid was taken up or incorporated by L 1210 cells from free sugar and this incorporation was not increased by neuraminidase pretreatment. Moreover, extracellular breakdown of CMP-N-acetylneuraminic acid during incubations with L 1210 cells was rather insignificant. Electron microscope autoradiography of cells incubated with CMP-N-acetylneuraminic acid demonstrated that greater than 84% of the incorporated radioactivity was associated with the plasma membrane and less than 1% with the Golgi apparatus. These findings are consistent with the conclusion that incroporation of N-acetylneuraminic acid from CMP-N-acetylneuraminic acid is the consequence of a cell surface sialytransferase system. Pretreatment of cells with the nonpenetrating reagent, diazonium salt of sulfonilic acid, significantly inhibited this ectoenzyme system while only marginally affecting galactose uptake and incorporation at the Golgi apparatus. Interestingly, incorporation from CMP-N-acetylneuraminic acid declined as the viability of the cell population declined. When taken together, the above evidence develops a rigorous argument for the presence of a sialyltransferase enzyme system at the cell surface of L 1210 cells. Studies directed towards the detection of a similar ectogalactosyltransferase system were also undertaken. Cells incubated in the presence of UDP-[³H]-galactose incorporated radioactivity into a macromolecular fraction. The presence of excess unlabeled galactose in the incubation medium significantly reduced this incorporation. Electron microscope autoradiographs of cells incubated with UDP-[³H]-galactose, demonstrated that incorporation occurred primarily at the Golgi apparatus. The grain distribution in these autoradiographs was similar to that for free galactose. Thus, the incorporation observed for L-1210 cells incubated in UDP-[³H]-galactose was due primarily to the intracellular utilization of free galactose generated by extracellular degradation of the nucleotide sugar. Inability t o demonstrate an ectogalacto-syltransferase system on L1210 cells does not rule out the possibility that the enzyme is present but undetectable due t o the absence of appropriate cell surface acceptor molecules.     

Ca2+‐mediated exocytosis of subtilisin‐like protease 1: a key step in egress of Plasmodium falciparum merozoites

Egress of Plasmodium falciparum merozoites from host erythrocytes is a critical step in multiplication of blood‐stage parasites. A cascade of proteolytic events plays a major role in degradation of membranes leading to egress of merozoites. However, the signals that regulate the temporal activation and/or secretion of proteases upon maturation of merozoites in intra‐erythrocytic schizonts remain unclear. Here, we have tested the role of intracellular Ca²⁺ in regulation of egress of P. falciparum merozoites from schizonts. A sharp rise in intracellular Ca²⁺ just before egress, observed by time‐lapse video microscopy, suggested a role for intracellular Ca²⁺ in this process. Chelation of intracellular Ca²⁺ with chelators such as BAPTA‐AM or inhibition of Ca²⁺ release from intracellular stores with a phospholipase C (PLC) inhibitor blocks merozoite egress. Interestingly, chelation of intracellular Ca²⁺ in schizonts was also found to block the discharge of a key protease PfSUB1 (subtilisin‐like protease 1) from exonemes of P. falciparum merozoites to parasitophorous vacuole (PV). This leads to inhibition of processing of PfSERA5 (serine repeat antigen 5) and a block in parasitophorous vacuolar membrane (PVM) rupture and merozoite egress. A complete understanding of the steps regulating egress of P. falciparum merozoites may provide novel targets for development of drugs that block egress and limit parasite growth.     

Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro

Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[³H]glucosamine, [³⁵S]sulfate, and L-[¹⁴C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-(3) and β-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[³H]glucosamine, but not of [³⁵S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[¹⁴C]leucine incorporation into total protein synthesis. These results suggest that analogs 3–5 exhibit an inhibitory effect on D-[³H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[³H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-α-(6) and β-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs, namely to ∼7% of controls, and a moderate inhibition of total protein synthesis, namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at 1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (∼40 kDa) than for control cultures (∼77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D -[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented.     

Inhibition by specific monosaccharides of interleukin 2-induced thymocyte proliferation

When rat thymocytes are cultured for 3 days in serum-free medium and are stimulated to divide by interleukin 2 (IL 2), concanavalin A, or sodium periodate oxidation, addition to the medium of 10–25 mMd-ribose, 2-deoxy-d-ribose, or N-acetyl-d-galactosamine inhibits by 40% or more the incorporation of [³H]thymidine. d-ribose and lectin-free IL 2 generated from sodium periodate oxidation of rat spleen cells were used to study the characteristics of this inhibition and to test possible mechanisms of inhibition. Viability of thymocytes cultured with d-ribose is similar to that of cells cultured without this sugar. In order to be inhibitory, d-ribose has to be added to the cultures within the first 24 hr, and the inhibition can be prevented if the sugar is removed 18–24 hr after the start of culture. d-Ribose does not block the absorption of IL 2 by unstimulated rat thymocytes or by concanavalin A-generated thymic or splenic blast cells. When thymocytes are cultured with d-ribose for 24 hr, inactivated with mitomycin C, and then cultured for 3 days with fresh mitogenically stimulated cells, [³H]thymidine incorporation into the latter is not altered. This suggests that the sugar does not generate suppressor cells or suppressor supernates. d-Ribose does not appear to be a general metabolic inhibitor since [³H]leucine incorporation into thymocyte proteins and the release of [³H]leucine into medium after a 2-hr. [³H]leucine pulse are not altered by d-ribose. Trivial or artifactual effects (nonspecific cytotoxicity, changes in thymidine transport, or changes in isotonicity of the culture medium) cannot explain the inhibition. A hypothetical mechanism of inhibition is discussed.     

Nutrient dynamics in a tritrophic system of ants,aphids and beans

The interactions between ants (Lasius niger), aphids (Aphis fabae) and plants (Phaseolus vulgaris) were studied in a laboratory experiment with the following treatments: application of N‐fertilizer for plants, supply of mealworms or sugar solution or their combination as alternative food sources for ants. Three main questions were studied: (1) Do ants reduce tending to honeydew‐producing aphids when an alternative sugar or protein resource was available? (2) Is aphid predation/protein consumption by ants higher when additional carbon is offered to maintain the carbon/protein balance? (3) Does fertilizer treatment propagate in the food web? For the experimental analysis stable isotope techniques were applied. δ¹⁵N served as a marker for the pathway from plants to higher trophic levels. Low δ¹⁵N‐value of fertilizer spread from plant shoots to aphids and ants. To trace which sugar‐/protein source was consumed by ants, the different ¹³C/¹²C‐ratios of C₃‐ and C₄‐plants were used with aphids feeding on C₃‐plant material, while mealworm food and sugar solution originated from C₄‐plant material. Fertilizer application had no effect on biomasses of plants, consumers or microflora. Ant biomass was significantly higher when additional sugar solution was offered. Higher contents of ¹³C indicated a high incorporation rate of additional sugar. Additional protein had no effect on colony biomass and no increasing predation on aphids could be observed when carbon was in excess. However, due to the lack of queens and newly produced larvae, protein requirements of experimental colonies were lower than in natural systems. Ants positively affected aphid populations, but reduced tending, whilst having access to an alternative sugar resource. When sugar/protein was offered to ants, the host plant had an increased root/shoot ratio. This indicates that decreasing aboveground activity of ants could lead to reduced plant growth of aphid‐infested plants, presumably due to higher fungal attack on shoots.     

Acute exposure to ozone inhibits rapid carbon translocation from source leaves of Pima cotton

Tropospheric ozone (O3 causes significant disruption of growth and yield in upland and Pima cottons. Pima cotton (Gossypium barbadense L.) was exposed to brief pulses (0.75 h) of a range of O3 concentrations (nominally 0.0, 0.2, 0.5, and 0.8 l l^-1) to investigate effects of phloem translocation of ¹⁴C-labelled recent photoassimilate. The initial phase of rapid efflux from source leaves was monitored with a Geiger-Muller Tube as activity remaining in the leaf as a function of time. Visual inspection of unprocessed efflux curves revealed disruption of efflux by O3. Single exponential decay functions were fitted to these efflux curves to extract first order rate constants for phloem leading and longitudinal transport of labelled carbohydrates. A single compartment model was applied, with and without an asymptote of non-transported carbohydrate, to calculate leaf sugar contents. The effect of O3 in retarding efflux of label, decreasing the rate constant, and increasing calculated soluble sugar pools, was consistent regardless of the method of analysis. Following incorporation of the asymptote, calculated rate constants and sugar pools were similar to values from the literature and to preliminary measurements of sugar contents in O3-treated cotton leaves. Total carbohydrate transported from source leaves was reduced both by O3 effects on assimilation (up to 205) and by O3 effects on efflux (up to 70%), but was clearly dominated by the impact on phloem translocation. These rapid efflux kinetics likely reflect oxidant damage at the plasmalemma or plasmodesmata of mesophyll or phloem companion cells. Evaluation of effects of O3 on tonoplast function and consequences for carbohydrate translocation await a more complete compartmental efflux analysis.     

Changes in Amino Acid Metabolism in vivo with Time after Feeding

Changes in the metabolism in vivo of amino acids with the lapse of time after feeding a diet were investigated by measuring the incorporation of ¹⁴C into some body components one hour after injection with ¹⁴C-amino acid mixture.

The incorporation of ¹⁴C into protein in the liver and carcass was rather constant, but that into blood sugar, liver glycogen, and lipids in the liver and carcass showed a change with the lapse of time after feeding a 25% casein diet or a protein-free diet. The incorporation of ¹⁴C into liver glycogen was stimulated shortly after feeding, but it was reduced at 7 hr, when a large amount of glycogen was still in the liver. On the contrary, the specific activity of blood sugar increased with the lapse of time after feeding. The conversion of ¹⁴C-amino acids into lipids in the liver and carcass was stimulated shortly after feeding.

The incorporation of ¹⁴C into protein was higher in the rats fed the protein-free diet than in those fed the 25% casein diet, and the higher incorporation was partly counterbalanced by the lower incorporation of ¹⁴C into lipids and glycogen in the rats fed the protein-free diet.     

The Suppression of Stareh Synthesis and the Accumulation of Uridine Diphosphoglucose in Cucumber Leaves due to Ammonium Toxicity

This work is a study of the metabolic disorder due to ammonium toxicity in cucumber (Cucumis sativus L. cv. suisei No. 2) leaves. The cucumber was cultured with 20 and 200 mg/l NH₃-N for 5 days. In the first half of this experiment, the plant leaves were photosynthesized for 3 hours to study the distribution of ¹⁴C in starch and other fractions. The incorporation of photosynthesized ¹⁴C into not only starch but also other higher polymers was suppressed by ammonium toxicity. On the other band, the rate of ¹⁴C incorporation in 80 % ethanol soluble fraction was higher in the treatment of 200 mg/l NH₃-N. In the latter half of experiment, phosphate esters in the loaves were analyzed by column chromatography. The opposing contents of uridine diphosphoglucose (UDPG) and the uridine compound containing sugar or sugar derivative (UDPX) were determined by different ammonia levels. Toxicity of ammonia resulted in an increase of UDPG and a decrease of UDPX. The level of ATP was not changed so much. The content of glucose-6-phosphate (G-6-P) in injured plants was lower than that of normal plants, while ghicose-1-phosphate (G-1-P) and fructose-6–phosphate (F-6-P) were higher in injured plants. These results further suggested the disorder of carbohydrate metabolism due to ammonium toxicity which was reported previously.     

In Vitro Cultivation of the Vascular Phase of Sarcocystis capracanis and Sarcocystis tenella1

ABSTRACT. Sporozoites of Sarcocystis capracanis and S. tenella (Apicomplexa) penetrated all four cell types tested (bovine monocytes, BM; bovine pulmonary artery endothelial cells, CPA; Madin-Darby bovine kidney; and ovine monocytes). Sporozoites of S. tenella developed to meronts in BM and CPA; those of S. capracanis developed to meronts in BM only. Both species of Sarcocystis developed to large first-generation meronts followed by small meronts. At 40 to 50 days after inoculation (DAI) of sporozoites, considerably more merozoites of S. tenella were harvested from CPA (24.9 × 10⁶ merozoites/75-cm² flask; n = 4) than from BM (1.9 × 10⁶ merozoites/75-cm² flask; n = 4). Merozoites of S. capracanis were most numerous in BM at 88 to 100 DAI during which time 2.1 × 10⁶ merozoites/75-cm² flask (n = 4) were harvested.     

Response of Chlorella to Scandium and Potassium at Various pH

The response of Chlorella pyrenoidosa cells to moderate potassium deficiency and to the addition of various scandium amounts in the nutrient solution were studied at different pH. The effects on growth, chlorophyll content, oxygen consumption, and the ¹⁴CO₂ incorporation in photosynthesizing cells were measured. Considerable metabolic changes inside the cells were registered under the given nutrient conditions. The separated ¹⁴C-labelled compounds showed, at acid and neutral pH in complete nutrient solution, an increase in the relative content of sugar phosphates and a decrease in the relative content of sucrose, caused by increasing scandium concentrations. Moderate potassium deficiency caused a diminution of the relative content of sugar phosphates. The relative content of amino acids was increased by scandium added to complete nutrient solution, but it decreased under potassium deficiency.     

Effetto Del Dinitrofenolo Sulla Conversione Del Glicerolo A Glucidi in Fettine Di Carote E in Internodi Di Pisello

Abstract

On the effect of dinitrophenol on carbohydrate activation in higher plant tissues. — Previous investigations on the effects of 2,4 dinitrophenol (DNP) on carbohydrate metabolism in isolated pea internodes and in yeast showed that the increased rate of glycolysis induced by the uncoupler corresponds to an increased rate of the conversion of free hexoses and polysaccarides to hexose phosphates. In yeast about 30% of the radioactivity supplied and taken up as ¹⁴C labelled glucose, and 20% of that supplied and taken up as glycerol is recovered as soluble sugar and glycogen; this phenomenon is almost completely suppressed by 10^-4M DNP.

This suggested that a mechanism involving kinase enzymes, on one hand, and phosphatases, on the other, is mediating the interconversion of phosphorylathed and free sugars, and that the apparent increase of hexose phosphorylation observed in the presence of DNP might depend on a decreased rate of phosphatase mediate reactions, consequent to the decrease of phosphorylated sugars level in the cell.

The experiments here reported were planned to test the validity of this hypothesis in the case of higher plant tissues.

Material used in these experiments were segments from the growing part of the third internode isolated from 7 day old, etiolated pea seedlings, and carrot root diks (0,7 mm thick, 7 mm diameter) preincubated for 24 hours in aerated distilled water. Both of these materials show an active, steady respiration and some growth activity, so that they may be taken as representing a condition close enough to that of the generally physiologically active higher plant tissues.

The reversibility of the hexose phosphate-free sugar interconversion process was tested by feeding 10^-3M 1-C¹⁴ labeled glycerol, and measuring after 150 minutes the amount of radioactivity incorporated into CO₂, soluble sugars, organic acids and proteins. The results of these experiments are summarized in table I and II.

Glycerol metabolism as well as its response to DNP appears very similar in the two material used. In both cases, glycerol uptake and incorporation into organic acids and amino acids is almost insensitive to DNP. In contrast large differences are observed for the free sugar fraction. In the absence of the uncoupler, a consistent amount of the radioactivity fed as glycerol is found in this fraction. It appears reasonable to assume that the glycerol-sugar interconversion comprehends, as intermediate steps, glycerol-P, fructose di-P (or sedoeptulose di-P) and hexose-6-P. If this is true, the observed data implicate that a continuous interconversion occurs, in the cell, between sugar phosphates and free sugars and vice-versa, one reaction direction involving the activity of phosphatases, and the other one that of kinases. The true rate of this interconversion process is probably much larger than indicated by the radioactivity found in free sugars: as a considerable part of the triose-P transormed into sugars must immediately re-enter the descending flux of glycolysis.

This view finds some support in the fact that DNP almost completely inhibits the incorporation of radioactivity in the free sugar fraction. It has been previously observed that DNP very markedly decreases the level of hexose mono- and di-phosphates and of triose-phosphates in the pea stem tissues. If phosphatases acting on fructose di-phosphate and on hexose-6-P are not saturated by their substrates, a decrease of the rate of free hexose synthesis from sugar phosphates should be expected.

The present results are thus consistent with the hypothesis that hexose phosphates and free sugars in the cell are continuously interconverted by the simultaneous action of phosphatases and kinases; and that the effect of DNP, and thus of any physiological conditions decreasing the ATP/ADP ratio in accelerating free hexose utilizations is at least in part due to a decreased rate of the reactions catalized by fructose diphosphate and hexose-6-P phosphatases. The reversibility of the kinase-phosphatase system would thus represent a crucial link in the mechanism by which the rate of carbohydrate activation and breackdown is controlled by the rate of utilization of high-energy phosphate bonds.     

Dual effect of adrenalin on sugar transport in rat diaphragm muscle

The effect of adrenalin on the membrane transport of the non-metabolized sugar, 3-methylglucose, was studied in isolated “intact” rat hemidiaphragms and related to simultaneously occurring changes in the internal levels of Na⁺, ATP, glucose-6-P, glycerol formation and ⁴⁵Ca uptake and loss. Basal sugar transport was inhibited by low (10⁻⁸−10⁻⁵ M) concentrations of adrenalin; this was antagonized by propranolol and practolol. High concentrations (10⁻⁴−10⁻³ M) stimulated sugar transport, and this was blocked by propranolol and butoxamine and was dependent on external Ca²⁺. These results suggest interaction with two different classes of adrenergic receptors, possibly of β₁ and β₂ types. Both low and high concentrations increased Na⁺ and K⁺ gradients by a practolol-sensitive effect. Isoproterenol behaved identically but phenylephrine had only the two practolol-sensitive effects on sugar and ion transport. Insulin did not interfere with inhibition of sugar transport and decrease in internal Na⁺ but prevented stimulation of sugar transport. Under anoxia adrenalin had no effect on sugar transport but led to greater Na⁺ gain by tissue. Addition of 3.0 mM palmitate decreased inhibition of sugar transport without changing receptor specificity. ATP was decreased and lipolysis enchanged by high adrenalin but glucose-6-P was increased by the low concentration as well. Influx of ⁴⁵Ca was decreased by low and increased by high adrenalin; ⁴⁵Ca efflux was also differentially affected. The results indicate that inhibition and stimulation of sugar transport depend on different receptors and that the latter response may override the former. The data are consistent with the earlier postulated regulatory role of sarcoplasmic Ca²⁺ on sugar transport in muscle, with adrenalin affecting Ca²⁺ fluxes and distribution both directly and indirectly.