Membrane permeability of fructose-1,6-diphosphate in lipid vesicles and endothelial cells

Fructose-1,6-diphosphate (FDP) is a glycolytic intermediate which has been used an intervention in various ischemic conditions for two decades. Yet whether FDP can enter the cell is under constant debate. In this study we examined membrane permeability of FDP in artificial membrane bilayers and in endothelial cells. To examine passive diffusion of FDP through the membrane bilayer, L-a-phosphatidylcholine from egg yolk (Egg PC) (10 mM) multi-lamellar vesicles were created containing different external concentrations of FDP (0, 0.5, 5 and 50 mM). The passive diffusion of FDP into the vesicles was followed spectrophotometrically. The results indicate that FDP diffuses through the membrane bilayer in a dose-dependent fashion. The movement of FDP through Egg PC membrane bilayers was confirmed by measuring the conversion of FDP to dihydroxyacetone-phosphate and the formation of hydrozone. FDP (0, 0.5, 5 or 50 mM) was encapsulated in Egg PC multilamellar vesicles and placed in a solution containing aldolase. In the 5 and 50 mM FDP groups there was a significant increase in dihydroxyacetone/hydrazone indicating that FDP crossed the membrane bilayer intact. We theorized that the passive diffusion of FDP might be due to disruption of the membrane bilayer. To examine this hypothesis, small unilamellar vesicles composed of Egg PC were created in the presence of 60 mM carboxyfluorescein, and the leakage of the sequestered dye was followed upon addition of various concentrations of FDP, fructose, fructose-6-phosphate, or fructose-1-phosphate (0, 5 or 50 mM). These results indicate that increasing concentrations of FDP increase the leakage rate of carboxyfluorescein. In contrast, no concentration of fructose, fructose-6-phosphate, or fructose-1-phosphate resulted in any significant increase in membrane permeability to carboxyfluorescein. To examine whether FDP could pass through cellular membranes, we examined the uptake of ¹⁴C-FDP by endothelial cells cultured under hypoxia or normoxia for 4 or 16 h. The uptake of FDP was dose-dependent in both the normoxia and hypoxia treated cells, and was accompanied by no significant loss in endothelial cell viability. Our results demonstrate that FDP can diffuse through membrane bilayers in a dose-dependent manner.     

Fructose 1,6-diphosphate-activated L-lactate dehydrogenase from Streptococcus lactis: kinetic properties and factors affecting activation.

The L-(+)-lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) of Streptococcus lactis C10, like that of other streptococci, was activated by fructose 1,6-diphosphate (FDP). The enzyme showed some activity in the absence of FDP, with a pH optimum of 8.2; FDP decreased the Km for both pyruvate and reduced nicotinamide adenine dinucleotide (NADH) and shifted the pH optimum to 6.9. Enzyme activity showed a hyperbolic response to both NADH and pyruvate in all the buffers tried except phosphate buffer, in which the response to increasing NADH was sigmoidal. The FDP concentration required for half-maximal velocity (FDP0.5V) was markedly influenced by the nature of the assay buffer used. Thus the FDP0.5V was 0.002 mM in 90 mM triethanolamine buffer, 0.2 mM in 90 mM tris(hydroxymethyl)aminomethanemaleate buffer, and 4.4 mM in 90 mM phosphate buffer. Phosphate inhibition of FDP binding is not a general property of streptococcal lactate dehydrogenase, since the FDP0.5V value for S. faecalis 8043 lactate dehydrogenase was not increased by phosphate. The S. faecalis and S. lactis lactate dehydrogenases also differed in that Mn2+ enhanced FDP binding in S. faecalis but had no effect on the S. lactis dehydrogenase. The FDP concentration (12 to 15 mM) found in S. lactis cells during logarithmic growth on a high-carbohydrate (3% lactose) medium would be adequate to give almost complete activation of the lactate dehydrogenase even if the high FDP0.5V value found in 90 mM phosphate were similar to the FDP requirement in vivo.     

The characteristics of the cardioprotective action of fructose-1,6-diphosphate]

The cardioprotective effects of fructose-1,6-diphosphate (FDP) were investigated in infarcted rats and in conscious rabbits with myocardial ischemia. The influence of FDP on metabolic acidosis was studied in isolated hypoxic rat hearts. It was shown that FDP did not change the threshold of the initiation of ischemia in conscious rabbits, but decreased necrotic zone in infarcted rat hearts. After administration of FDP the myocardial contractility was prolonged significantly as compared with control under conditions of severe metabolic acidosis. However, FDP was not effective in hypoxic hearts with compensated metabolic acidosis. It was considered, that FDP influenced only ischemic myocytes with the changes in sarcolemmal permeability.     

Conventional detection method of fibrinogen and fibrin degradation products using latex piezoelectric immunoassay

We developed a conventional immunosensor for fibrinogen and fibrin degradation products (FDP) to combine a quartz crystal microbalance (QCM) with the agglutination reaction of immunized latex beads. FDP induced an immunoreaction due to anti-FDP antibody immobilized latex particles. We successfully measured FDP concentration of in human serum within 10 min by QCM method. The detection range of QCM immunosensor is covered with screening concentration of FDP in serum (<10 microg/ml of FDP). The time course of latex agglutination obtained from QCM immunosensor is synchronized to that of latex photometric immunoassay. SEM was used to observe the surface of QCM that applied FDP serum. The size of latex particles agglutinated on the QCM electrode increased concomitant with FDP concentration. Frequency shift on immunoreaction explains the increased adsorption amount of agglutinated latex on QCM.     

血浆中D- 二聚体、C- 反应蛋白及FDP 比值在急性脑梗死中 应用价值研究

目的:探讨血浆中D-二聚体、C-反应蛋白及FDP比值在急性脑梗死中应用价值,为临床诊断提供指导。方法:抽取我院于2010年1月至2011年1月收治的30名急性脑梗死患者编为实验组。30名陈旧性脑梗死患者编为对照组。检测血浆中的D-二聚体、C-反应蛋白、FDP及其相互之间比值。结果:两组相比,实验组血浆D-二聚体、C-反应蛋白、FDP均与对照组差异无统计学意义(P>0.05);两组相比,实验组D-二聚体/FDP,C-反应蛋白/FDP均高于对照组,差异有统计学意义(P<0.05)。结论:急性和陈旧性脑梗死患者血浆中单独比较D-二聚体、C-反应蛋白、FDP差异均不明显,但D-二聚体/FDP,C-反应蛋白/FDP的阳性检出率较高,对临床有一定的指导意义。     

Kringle domains and plasmin denaturation.

The rate of plasmin denaturation was in the order of Lys-plasmin greater than miniplasmin greater than microplasmin. Fibrinogen degradation products (FDP) dose dependently increased the denaturation rate of Lys-plasmin and mini-plasmin with a maximal rate constant at the FDP/plasmin ratio of about 0.5. The denaturation rate constant of microplasmin was not affected. FDP increased the rate of plasmin denaturation was in parallel with its effect on the interaction among kringle domains. Without FDP only trace amounts of plasminogen dimer could be detected by cross-linking with bis-(sulfo-succinimidyl)-suberate followed by SDS gel electrophoresis. In the low concentration of FDP significant amounts of oligomers of Glu-, mini-plasminogens, kringle 1-3 and kringle 1-5 were observed. High concentration of FDP, however, decreased plasminogen oligomer.     

Properties of a fructose-1,6-diphosphate-activated lactate dehydrogenase from Acholeplasma laidlawii type A

Acholeplasma laidlawii A possesses a nicotinamide adenine dinucleotide (NAD)-dependent l(+)-lactate dehydrogenase (LDH) which is activated specifically by low concentrations of fructose-1, 6-diphosphate (FDP). Studies with partially purified enzyme show that the kinetic response to FDP is hyperbolic. The enzyme is inhibited by inorganic phosphate, adenosine triphosphate, and high concentrations of reduced NAD (NADH). Low activity is demonstrable in the absence of FDP at pH 6.0 to 7.2, but FDP is absolutely required in the region of pH 8. FDP causes an upward shift in the optimum pH of the enzyme, which is near 7.2 in tris (hydroxymethyl)aminomethane buffer. Activation of the enzyme by FDP is markedly affected by substrate concentration; FDP lowers the apparent K(m) for pyruvate and NADH. The affinity of the enzyme for pyruvate is also influenced by H(+) concentration. The pyruvate analogue alpha-ketobutyrate serves as an effective substrate for the enzyme; when it is utilized, the enzyme is still activated by FDP. Reversal of the pyruvate reduction reaction catalyzed by the enzyme can be demonstrated with the 3-acetylpyridine analogue of NAD. The catalytic properties of the A. laidlawii enzyme and the known FDP-activated LDHs which occur among lactic acid bacteria are discussed.     

The effect of fructose 1,6 diphosphate on pyruvate kinase from the liver of the flounder (Platichthys flesus L.)

1. Pyruvate kinase purified from flounder liver in two forms, i.e. PKI and PKII, is activated by fructose 1,6 diphosphate. 2. Two or more binding sites for FDP are demonstrated for PKII, the binding to which is influenced by the levels of substrates. 3. FDP reduces or abolishes the cooperative effect of PEP. 4. FDP increases the maximal activity. 5. The inhibition observed at higher levels of ADP is not abolished by FDP.     

Modification of the regulatory properties of pyruvate kinase of Neurospora by growth at elevated temperatures.

Pyruvate kinase (EC 2.7.1.40) was isolated from Neurospora crassa mycelium grown at 28 degrees C (PK-28) and at 42 degrees C (PK-42). The regulatory properties, particularly the response towards the allosteric effector fructose 1,6-diphosphate (FDP), was different in the two enzymes. PK-28 showed an activation by FDP but PK-42, under comparable conditions, appeared to be activated by low concentrations of FDP and inhibited by higher ones. For PK-28, complex formation with FDP results in a lowering of the isoelectric point from 6.40 to 5.50, representing the pI of the unliganded enzyme and that of the complex, respectively. In contrast to this, PK-42 exhibits a weak binding to FDP as suggested by a lack of decrease in the isoelectric point on treatment with comparable concentrations of FDP. Studies with quenching of aromatic residue fluorescence of PK-28 and PK-42, following binding of FDP, indicate that although this ligand binds to both types of enzymes the affinity for the two is vastly different. Dissociation constants of 9.3 muM and 0.1 mM were calculated for the binding of FDP to PK-28 and PK-42, respectively. It is concluded that growth at elevated temperatures induced a conformational change in the pyruvate kinase leading to partial desensitization of the allosteric site. The nature of the factor(s) responsible for this change is not understood at present.     

Some effects of fructose-1,6-diphosphate on rat myocardial tissue related to a membrane-stabilizing action

This study aims at elucidating the mechanism of action of extracellular fructose-1,6-diphosphate (FDP). FDP is able to inhibit Ca++ entry into the myocardial tissue with an IC50 value of 11.5 mM and in addition, it is bound by rat heart slices, the binding being activated by Zn and conditions of chemical hypoxia induced by KCN and iodoacetate. The overall effect of extracellular FDP includes an increase of frequency and amplitude of contraction of perfused heart at concentration below 1 mM, and, in general, a stimulation of the oxygen consumption of the tissue. The antihaemolytic effect of FDP suggests its action as a membrane stabilizer. The effects of extracellular FDP on the myocardial cell can be interpreted both on the basis of a limited permeability of the cell membrane to it and as a purely extracellular effect transduced through the cell membrane with a final response consisting of an increase in the intracellular FDP.     

A statistical evaluation of the fixed dose procedure

The Fixed Dose Procedure (FDP) was first proposed in 1984 by the British Toxicology Society, as an alternative to the conventional LD50 test (OECD Test Guideline 401), for determining acute oral toxicity. The FDP used fewer animals and caused less suffering than the LD50 test, and provided information on acute toxicity which allowed substances to be classified according to the European Union hazard classification system. In 1992, the FDP was introduced as OECD Test Guideline 420. In 1999, as part of an initiative to phase out Test Guideline 401, a review of the FDP was undertaken. The aim of the review was to provide further reductions and refinements, and classification according to the criteria of the Globally Harmonised Hazard Classification and Labelling Scheme. The revised FDP was adopted by the OECD in 2001. This article concerns the development and revision of the FDP. It illustrates how statistical modelling and simulation can be used to increase the efficiency of a test procedure and reduce the number of animals needed for an in vivo validation of the procedure.     

Differential effect of glycolytic intermediaries upon cyclic ADP-ribose-, inositol 1',4',5'-trisphosphate-, and nicotinate adenine dinucleotide phosphate-induced Ca(2+) release systems.

We investigated the effect of glycolytic pathway intermediaries upon Ca(2+) release induced by cyclic ADP-ribose (cADPR), inositol 1',4', 5-trisphosphate (IP(3)), and nicotinate adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenate. Fructose 1,6, -diphosphate (FDP), at concentrations up to 8 mM, did not induce Ca(2+) release by itself in sea urchin egg homogenate. However, FDP potentiates Ca(2+) release mediated by agonists of the ryanodine channel, such as ryanodine, caffeine, and palmitoyl-CoA. Furthermore, glucose 6-phosphate had similar effects. FDP also potentiates activation of the ryanodine channel mediated by the endogenous nucleotide cADPR. The half-maximal concentration for cADPR-induced Ca(2+) release was decreased approximately 3.5 times by addition of 4 mM FDP. The reverse was also true: addition of subthreshold concentrations of cADPR sensitized the homogenates to FDP. The Ca(2+) release mediated by FDP in the presence of subthreshold concentrations of cADPR was inhibited by antagonists of the ryanodine channel, such as ruthenium red, and by the cADPR inhibitor 8-Br-cADPR. However, inhibition of Ca(2+) release induced by IP(3) or NAADP had no effect upon Ca(2+) release induced by FDP in the presence of low concentrations of cADPR. Furthermore, FDP had inhibitory effects upon Ca(2+) release induced by both IP(3) and NAADP. We propose that the state of cellular intermediary metabolism may regulate cellular Ca(2+) homeostases by switching preferential effects from one intracellular Ca(2+) release channel to another.     

Interacting coexistence mechanisms in annual plant communities: Frequency-dependent predation and the storage effect

We study frequency-dependent seed predation (FDP) in a model of competing annual plant species in a variable environment. The combination of a variable environment and competition leads to the storage-effect coexistence mechanism (SE), which is a leading hypothesis for coexistence of desert annual plants. However, seed predation in such systems demands attention to coexistence mechanisms associated with predation. FDP is one such mechanism, which promotes coexistence by shifting predation to more abundant plant species, facilitating the recovery of species perturbed to low density. When present together, FDP and SE interact, undermining each other’s effects. Predation weakens competition, and therefore weakens mechanisms associated with competition: here SE. However, the direct effect of FDP in promoting coexistence can compensate or more than compensate for this weakening of SE. On the other hand, the environmental variation necessary for SE weakens FDP. With high survival of dormant seeds, SE can be strong enough to compensate, or overcompensate, for the decline in FDP, provided predation is not too strong. Although FDP and SE may simultaneously contribute to coexistence, their combined effect is less than the sum of their separate effects, and is often less than the effect of the stronger mechanism when present alone.     

Fructose-1,6-diphosphate as an in vitro and in vivo anti-alcohol agent in the rat

Fructose-1, 6-diphosphate (FDP) decreases the effect of ethanol on Ca++ entry and inhibits the ethanol-stimulated phosphate efflux in rat heart slices. FDP also inhibits the ethanol-stimulated [36Cl-]-uptake by rat brain microvesicles and affects the isolated GABA-receptor in a way opposite to that of ethanol. The in vivo effects of FDP include a dose-dependent decrease in ethanol-induced gastric ulcers and a decrease in the serum transaminase levels raised by chronic ethanol administration. Other central actions of ethanol such as diuresis, narcosis, dependence and withdrawal symptoms are also counteracted by FDP.     

Cooperative atp inhibition and fructose-1,6-biphosphate activation of rat liver pyruvate kinase

• 1.1. For the determination of relationship between FDP and ATP in the rat liver pyruvate kinase regulation, kinelic studies have been carried out at several ATP and FDP concentrations.
• 2.2. The results obtained on FDP activation show a great cooperativity for FDP saturation with a Hill coefficient of h = 2.79.
• 3.3. Kinetic studies on ATP inhibition also show a great cooperativity for ATP saturation (h = 2.84) at high FDP concentrations.
• 4.4. These results may contribute to explain the regulation of rat liver pyruvate kinase accounting for the activity of this enzyme at high FDP concentrations modulated by small changes in ATP concentrations.

     

Expression of the farnesyldiphosphate synthase gene of Saccharomyces cerevisiae in tobacco

The coding region of the farnesyldiphosphate synthase (FDP synthase) gene from Saccharomyces cerevisiae has been inserted into a pBin19 vector, downstream of the cauliflower mosaic virus (CaMV) 35S promoter, in order to allow its expression in the genome of a higher plant, Nicotiana tabacum. We have produced transgenic tobacco in which the expression of the foreign gene leads to functional FDP synthase activity. In these transgenic plants, total FDP synthase-specific activity is increased 12-fold compared to controls. This increase of FDP synthase activity has been correlated to a clear increase of both sterol and carotenoid biosynthesis. This heterologous expression is also related to an increased resistance of transformed plants to R172117, a specific inhibitor of FDP synthase, and to sterol biosynthesis inhibitors such as flusilazol and fenpropimorph.Abbreviations: AP, Annick Petit; BAP, benzylaminopurine; CaMV, cauliflower mosaic virus; CTAB, cetyltrimethylammonium bromide; DMAEDP, dimethylamino ethyl diphosphate; DMADP, dimethylallyl diphosphate; DTT, dithiothreitol; ERG12, mevalonate kinase yeast gene; ERG20, FDP synthase yeast gene; FDP, farnesyl diphosphate; GGDP, geranylgeranyl diphosphate; GDP, geranyl diphosphate; IDP, isopentenyl diphosphate; LB, Luria Bertani; MS, Murashige and Skoog; NAA, Naphtaleneacetic acid; PVP, polyvinyl pyrrolidone; SBI, sterol biosynthesis inhibitor.     

Factors affecting the activity of the lactate dehydrognease of Streptococcus cremoris

Studies with partially purified extracts of the nicotinamide adenine dinucleotide-linked l(+)-lactate dehydrogenase of Streptococcus cremoris US3 showed that fructose-1,6-diphosphate (FDP) was essential for the catalytic reduction of pyruvate in the pH range 5.0 to 7.0, outside of which the organism does not grow. In the absence of FDP, enzyme activity was observed only in the region of pH 8.0. The optimal pH for the oxidation of lactate was approximately 8.0 in the presence and absence of FDP. The FDP-activated enzyme was markedly inhibited by inorganic phosphate. The enzyme lost activity on standing at 5 C in alkaline triethanolamine, was quite stable at pH 6.0 to 6.5, and underwent irreversible denaturation below pH 5.0. Inorganic phosphate or FDP increased the stability of the enzyme in alkaline buffers. Some distinguishing properties of individual lactate dehydrogenases, activated by FDP, are discussed.     

Properties of fructose-1,6-diphosphate phosphatase and fructose-1,6-diphosphate aldolase fromPseudomonas putida

The fructose-1,6-P₂ (FDP) phosphatase, (FDPase) and FDP aldolase fromPseudomonas putida were partially purified by a combination of (NH₄)₂SO₄ fractionation and DEAE-Sephadex column chromatography. Michaelis-Menten kinetics were observed with, respect to FDP in both FDPase and FDP aldolase. TheK _m for FDP at pH 8.0 was 1.2×10⁻⁵M for FDPase and 3.0×10⁻⁵M for FDP aldolase. The specific activities of these two enzymes (assayed under optimal conditions in cell-free extracts ofP. putida grown ond-fructose), as well as their kinetic properties, are consistent with the suggestion that during growth ond-fructose most, of the FDP generated is converted to fructose-6-P (F-6-P), which is subsequently utilized via the Entner-Doudoroff pathway (EDP).     

Fructose, 1,6-diphosphate mitigates CCl4 suppressed nitric oxide synthase activity in rats

Effect of fructose 1,6-diphosphate (FDP) and carbon tetrachloride (CCl4) were studied individually and in combination on rat endothelial (ET) and smooth muscle cell (SMC) nitric oxide synthase (NOS) activities in vivo, inhibition of ET and SMC NOS activity in CCl4 treated rats was reversed in FDP + CCl4 treated animals. Cellular based NOS activity was significantly increased in FDP treated group of rats when compared to non treated controls. The results suggest a significant increase in NOS in rats treated with a combination of FDP + CCl4 thus overcoming the suppression of NOS exposed to CCl4 alone.     

Effect of two strains of Flavescence dorée phytoplasma on the survival and fecundity of the experimental leafhopper vector Euscelidius variegatus Kirschbaum

We have investigated the influence on longevity and fecundity of Flavescence dorée phytoplasma (FDP), the agent of a grapevine yellows disease, in the experimental vector Euscelidius variegatus Kirschbaum. Late instar nymphs were exposed to one or the other of two strains of FDP (FD92 and FD2000) by feeding on infected broad bean (Vicia faba L.) or on healthy broad bean or maize (Zea mays L.) for an acquisition access period of 13 days. Detection of FDP in individual insects was done with PCR assays and revealed that almost all exposed leafhoppers had acquired FDP, for both FD92 and FD2000 strains. FDP infection significantly reduced the life span of males and females (ANOVA of the quartiles of survival distribution and Weibull scale parameter). FDP-exposed females produced significantly fewer nymphs. The two FDP strains had similar effects on reduction of survival and fecundity of leafhoppers. There was no significant differences in longevity of E. variegatus males exposed to FD broad bean than held on healthy broad bean or maize, but female survival and fecundity were reduced when they fed on maize versus healthy broad bean.