Fructose-1,6-bisphosphate induces generation of reactive oxygen species and activation of p53-dependent cell death in human endometrial cancer cells.

Fructose-1,6-bisphosphate (F1,6BP), an intermediate of the glycolytic pathway, has been found to play a promising anticancer effect; nevertheless, the mechanisms involved remain poorly understood. The present study aimed to evaluate the effect and mechanisms of F1,6BP in a human endometrial cancer cell line (Ishikawa). F1,6BP showed an antiproliferative and non-cytotoxic effect on endometrial cancer cells. These effects are related to the increase in reactive oxygen species (ROS) levels and mitochondrial membrane potential (ΔΨm). These harmful stimuli trigger the upregulation of the expression of pro-apoptotic genes (p53 and Bax), leading to the reduction of cell proliferation through inducing programmed cell death by apoptosis. Furthermore, F1,6BP-treated cells had the formation of autophagosomes induced, as well as a decrease in their proliferative capacity after withdrawing the treatment. Our results demonstrate that F1,6BP acts as an anticancer agent through the generation of mitochondrial instability, loss of cell function, and p53-dependent cell death. Thus, F1,6BP proves to be a potential molecule for use in the treatment against endometrial cancer.

Phosphofructokinases A and B from Mycobacterium tuberculosis Display Different Catalytic Properties and Allosteric Regulation.

Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.

Mechanism and effects of fructose diphosphate on anti-hypoxia fatigue and learning memory ability.

This study aims to investigate the mechanisms through which fructose diphosphate (FDP) causes anti-hypoxia and anti-fatigue effects and improves learning and memory. Mice were divided into three groups: low-dose FDP (FDP-L), high-dose FDP (FDP-H), and a control group. Acute toxic hypoxia induced by carbon monoxide, sodium nitrite, and potassium cyanide and acute cerebral ischemic hypoxia were used to investigate the anti-hypoxia ability of FDP. The tests of rod-rotating, mouse tail suspension, and swimming endurance were used to explore the anti-fatigue effects of FDP. The Morris water maze experiment was used to determine the impact of FDP on learning and memory ability. Poisoning-induced hypoxic tests showed that mouse survival time was significantly prolonged in the FDP-L and FDP-H groups compared with the control group (p < 0.05). In the exhaustive swimming test, FDP significantly shortened struggling time and prolonged the time of mass-loaded swimming; the rod-rotating test showed that endurance time was significantly prolonged by using FDP (p < 0.05). FDP significantly decreased lactate and urea nitrogen levels and increased hepatic and muscle glycogen and glucose transporter-4 and Na+-K+-ATPase (p < 0.05). To conclude, FDP enhances hypoxia tolerance and fatigue resistance and improves learning and memory ability through regulating glucose and energy metabolism.

Beta-elemene inhibits breast cancer metastasis through blocking pyruvate kinase M2 dimerization and nuclear translocation.

Pyruvate kinase M2 (PKM2), playing a central role in regulating aerobic glycolysis, was considered as a promising target for cancer therapy. However, its role in cancer metastasis is rarely known. Here, we found a tight relationship between PKM2 and breast cancer metastasis, demonstrated by the findings that beta-elemene (ß-elemene), an approved drug for complementary cancer therapy, exerted distinct anti-metastatic activity dependent on PKM2. The results indicated that ß-elemene inhibited breast cancer cell migration, invasion in vitro as well as metastases in vivo. ß-Elemene further inhibited the process of aerobic glycolysis and decreased the utilization of glucose and the production of pyruvate and lactate through suppressing pyruvate kinase activity by modulating the transformation of dimeric and tetrameric forms of PKM2. Further analysis revealed that ß-elemene suppressed aerobic glycolysis by blocking PKM2 nuclear translocation and the expression of EGFR, GLUT1 and LDHA by influencing the expression of importin α5. Furthermore, the effect of ß-elemene on migration, invasion, PKM2 transformation, and nuclear translocation could be reversed in part by fructose-1,6-bisphosphate (FBP) and L-cysteine. Taken together, tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis, and ß-elemene inhibited breast cancer metastasis via blocking aerobic glycolysis mediated by dimeric PKM2 transformation and nuclear translocation, being a promising anti-metastatic agent from natural compounds.

Fructose 1,6-bisphosphate inhibits osteoclastogenesis by attenuating RANKL-induced NF-κB/NFATc-1.

BACKGROUND: Although some glycolytic intermediates have been shown to modulate several cell type formation and activation, the functional role of fructose 1,6-bisphosphate (FBP) on osteoclastogenesis is still unknown. METHODS: Osteoclastogenesis was evaluated on bone marrow preosteoclasts cultured with M-CSF - 30 ng/ml, RANKL - 10 ng/ml, and two concentrations of FBP (100 and 300 µM). TRAP-positive stained cells were counted, and osteoclastogenic marker genes expression were evaluated by qPCR. Osteoclasts resorption capacity was evaluated by the expression of specific enzymes and capacity to resorb a mineralized matrix. The NF-κB activation was detected using RAW 264.7, stably expressing luciferase on the NF-κB responsive promoter. RESULTS: We show that FBP, the product of the first stage of glycolysis, inhibited RANKL-induced osteoclasts differentiation and TRAP activity. The treatment of preosteoclasts with FBP attenuated osteoclast fusion and formation, without affecting cell viability. Moreover, the inhibition of several osteoclastogenic marker genes expression (TRAP, OSCAR, DC-STAMP, Integrin αv, NFATc1) by FBP correlates with a reduction of mineralized matrix resorption capacity. The mechanism underlying FBP-inhibition of osteoclastogenesis involves NF-κB/NFATc1 signaling pathway inhibition. CONCLUSION: Altogether these data show a protective role of a natural glycolytic intermediate in bone homeostasis that may have therapeutic benefit for osteolytic diseases.

Whole-protein alanine-scanning mutagenesis of allostery: A large percentage of a protein can contribute to mechanism.

Many studies of allosteric mechanisms use limited numbers of mutations to test whether residues play "key" roles. However, if a large percentage of the protein contributes to allosteric function, mutating any residue would have a high probability of modifying allostery. Thus, a predicted mechanism that is dependent on only a few residues could erroneously appear to be supported. We used whole-protein alanine-scanning mutagenesis to determine which amino acid sidechains of human liver pyruvate kinase (hL-PYK; approved symbol PKLR) contribute to regulation by fructose-1,6-bisphosphate (Fru-1,6-BP; activator) and alanine (inhibitor). Each nonalanine/nonglycine residue of hL-PYK was mutated to alanine to generate 431 mutant proteins. Allosteric functions in active proteins were quantified by following substrate affinity over a concentration range of effectors. Results show that different residues contribute to the two allosteric functions. Only a small fraction of mutated residues perturbed inhibition by alanine. In contrast, a large percentage of mutated residues influenced activation by Fru-1,6-BP; inhibition by alanine is not simply the reverse of activation by Fru-1,6-BP. Moreover, the results show that Fru-1,6-BP activation would be extremely difficult to elucidate using a limited number of mutations. Additionally, this large mutational data set will be useful to train and test computational algorithms aiming to predict allosteric mechanisms.

Inhibition of Recombinant Aldose-6-Phosphate Reductase from Peach Leaves by Hexose-Phosphates, Inorganic Phosphate and Oxidants.

Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves.

Fructose-1,6-bisphosphate reverts iron-induced phenotype of hepatic stellate cells by chelating ferrous ions.

Hepatic fibrosis is an extracellular matrix deposition by hepatic stellate cells (HSC). Fibrosis can be caused by iron, which will lead to hydroxyl radical production and cell damage. Fructose-1,6-bisphosphate (FBP) has been shown to deliver therapeutic effects in many pathological situations. In this work, we aimed to test the effects of FBP in HSC cell line, GRX, exposed to an excess of iron (Fe). The Fe-treatment increased cell proliferation and FBP reversed this effect, which was not due to increased necrosis, apoptosis or changes in cell cycle. Oil Red-O staining showed that FBP successfully increased lipid content and lead GRX cells to present characteristics of quiescent HSC. Fe-treatment decreased PPAR-γ expression and increased Col-1 expression. Both effects were reversed by FBP which also decreased TGF-ß1 levels in comparison to both control and Fe groups. FBP, also, did not present scavenger activity in the DPPH assay. The treatment with FBP resulted in decreased proliferation rate, Col-1 expression and TGF-ß1 release by HSC cells. Furthermore, activated PPAR-γ and increased lipid droplets induce cells to become quiescent, which is a key event to reversion of hepatic fibrosis. FBP also chelates iron showing potential to improve Cell redox state.

Synergistic Allosteric Mechanism of Fructose-1,6-bisphosphate and Serine for Pyruvate Kinase M2 via Dynamics Fluctuation Network Analysis.

Pyruvate kinase M2 (PKM2) plays a key role in tumor metabolism and regulates the rate-limiting final step of glycolysis. In tumor cells, there are two allosteric effectors for PKM2: fructose-1,6-bisphosphate (FBP) and serine. However, the relationship between FBP and serine for allosteric regulation of PKM2 is unknown. Here we constructed residue/residue fluctuation correlation network based on all-atom molecular dynamics simulations to reveal the regulation mechanism. The results suggest that the correlation network in bound PKM2 is distinctly different from that in the free state, FBP/PKM2, or Ser/PKM2. The community network analysis indicates that the information can freely transfer from the allosteric sites of FBP and serine to the substrate site in bound PKM2, while there exists a bottleneck for information transfer in the network of the free state. Furthermore, the binding free energy between the substrate and PKM2 for bound PKM2 is significantly lower than either of FBP/PKM2 or Ser/PKM2. Thus, a hypothesis of "synergistic allosteric mechanism" is proposed for the allosteric regulation of FBP and serine. This hypothesis was further confirmed by the perturbational and mutational analyses of community networks and binding free energies. Finally, two possible synergistic allosteric pathways of FBP-K433-T459-R461-A109-V71-R73-MG2-OXL and Ser-I47-C49-R73-MG2-OXL were identified based on the shortest path algorithm and were confirmed by the network perturbation analysis. Interestingly, no similar pathways could be found in the free state. The process targeting on the allosteric pathways can better regulate the glycolysis of PKM2 and significantly inhibit the progression of tumor.

Strontium fructose 1, 6-diphosphate alleviate cyclophosphamide-induced oligozoospermia by improving antioxidant and inhibiting testicular apoptosis via FAS/FASL pathway.

This study investigated the treatment effects of a new compound, strontium fructose 1, 6-diphosphate (FDP-Sr), in cyclophosphamide (CP)-induced oligozoospermia. FDP-Sr, with extra high-energy supply, could reverse male hypogonadism in the testis. Male Wistar rats were randomly divided into three groups: control group (vehicle treated), CP group and CP + FDP-Sr group. Both CP group and CP + FDP-Sr groups were orally administered CP (20 mg kg(-1) ) consecutively for the first 7 days to establish CP-induced testicular toxic models. Subsequently, CP group was given orally distilled water per day, whereas CP + FDP-Sr group was received FDP-Sr (200 mg kg(-1) ) for 49 days. Compared to the CP group, the FDP-Sr group showed significantly increased levels of serum testosterone, testis relative weights and epididymal sperm counts in rats. In addition, rats treated by FDP-Sr showed the recuperative activities of testicular marker enzymes and normalised levels of antioxidants in tissue. Testicular protection of FDP-Sr was further demonstrated by enhancing expression of P450scc, reducing ability of FAS/FASL and generating cytoprotection in the histopathological study. FDP-Sr appeared to possess an ability to attenuate CP-induced reproduction toxicity via the activation of antioxidants and steroidogenesis enzymes, and alleviate oligozoospermia via inhibition of testicular apoptosis by FAS/FASL pathway.

Regulation of the activity of lactate dehydrogenases from four lactic acid bacteria.

Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose 1,6-bisphosphate (FBP), phosphate (Pi), and ionic strength (NaCl concentration) on six LDHs from four LABs studied at pH 6 and pH 7. We found that 1) the extent of activation by FBP (Kact) differs. Lactobacillus plantarum LDH is not regulated by FBP, but the other LDHs are activated with increasing sensitivity in the following order: Enterococcus faecalis LDH2 ≤ Lactococcus lactis LDH2 < E. faecalis LDH1 < L. lactis LDH1 ≤ Streptococcus pyogenes LDH. This trend reflects the electrostatic properties in the allosteric binding site of the LDH enzymes. 2) For L. plantarum, S. pyogenes, and E. faecalis, the effects of Pi are distinguishable from the effect of changing ionic strength by adding NaCl. 3) Addition of Pi inhibits E. faecalis LDH2, whereas in the absence of FBP, Pi is an activator of S. pyogenes LDH, E. faecalis LDH1, and L. lactis LDH1 and LDH2 at pH 6. These effects can be interpreted by considering the computed binding affinities of Pi to the catalytic and allosteric binding sites of the enzymes modeled in protonation states corresponding to pH 6 and pH 7. Overall, the results show a subtle interplay among the effects of Pi, FBP, and pH that results in different regulatory effects on the LDHs of different LABs.

The study of inducing apoptosis effect of fructose 1,6-bisphosphate on the papillary thyroid carcinoma cell and its related mechanism.

This study aims to investigate the apoptosis-inducing effect of fructose 1,6-bisphosphate (F1,6BP) on the related mechanism of papillary thyroid carcinoma W3 and T cells. W3 cells were treated with F1,6BP alone or in combination with antioxidant catalase (CAT) or N-acetyl-L-cysteine (NAC). The changes of cell viability and cell nucleus morphology were examined by cell proliferation assay and Hoechst staining, and apoptosis levels of these cells were measured with flow cytometry. The changes of reactive oxygen species (ROS) level and the percentage of oxidized glutathione in total glutathione in W3 cells were detected by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining or colorimetry assay. At the same time, real-time fluorescence quantitative PCR was adopted to evaluate the expression levels of CAT and glutathione peroxidase (GSH-Px) mRNAs in W3 cells. F1,6BP inhibited the growth of W3 cells significantly, coupling with an increase in intracellular ROS level and the percentage of oxidized glutathione in total glutathione. Typical apoptotic morphological changes of the cell nucleus happened. The apoptosis rate and GSH-Px and CAT mRNAs expression levels were upregulated after F1,6BP treatment. The antitumor effect of F1,6BP was significantly decreased after W3 cells were pretreated with NAC and CAT. F1,6BP can induce the apoptosis of W3 cells through upregulating the generation of ROS, especially the production of H2O2.

Antitumor effect of fructose 1,6-bisphosphate and its mechanism in hepatocellular carcinoma cells.

We aimed to investigate the antitumor effect and mechanism of fructose 1,6-bisphosphate (F1,6BP) in a hepatocellular carcinoma cell line. HepG2 cells were treated with different concentrations of F1,6BP alone or in combination with antioxidant N-acetyl-L-cysteine (NAC) or catalase (CAT), and cell proliferation assays were performed. Nuclear morphology was observed by fluorescence microscopy after Hoechst staining, and apoptosis was measured with flow cytometry. Changes in reactive oxygen species (ROS) levels in HepG2 cells were detected by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. A colorimetric assay was adopted to determine the percentage of oxidized glutathione in these cells. CAT and glutathione peroxidase (GSH-Px) mRNA expression levels in HepG2 cells were measured by real-time fluorescence quantitative PCR. HepG2 cell proliferation was significantly inhibited by F1,6BP, accompanied by an increase in intracellular ROS levels and oxidized glutathione. Upregulated apoptosis and characteristic nuclear morphological changes were observed, and the expression of CAT and GSH-Px mRNA was increased after F1,6BP treatment. The antitumor effect of F1,6BP was significantly decreased after pretreatment with NAC and CAT in HepG2 cells. In conclusion, F1,6BP can induce the apoptosis of HepG2 cells. The mechanism involved may be associated with the generation of ROS, especially the production of H2O2.

Ultrasensitive regulation of anapleurosis via allosteric activation of PEP carboxylase.

Anapleurosis is the filling of the tricarboxylic acid cycle with four-carbon units. The common substrate for both anapleurosis and glucose phosphorylation in bacteria is the terminal glycolytic metabolite phosphoenolpyruvate (PEP). Here we show that Escherichia coli quickly and almost completely turns off PEP consumption upon glucose removal. The resulting buildup of PEP is used to quickly import glucose if it becomes available again. The switch-like termination of anapleurosis results from depletion of fructose-1,6-bisphosphate (FBP), an ultrasensitive allosteric activator of PEP carboxylase. E. coli expressing an FBP-insensitive point mutant of PEP carboxylase grow normally when glucose is steadily available. However, they fail to build up PEP upon glucose removal, grow poorly when glucose availability oscillates and suffer from futile cycling at the PEP node on gluconeogenic substrates. Thus, bacterial central carbon metabolism is intrinsically programmed with ultrasensitive allosteric regulation to enable rapid adaptation to changing environmental conditions.

Exogenous Glucose Interferes with Antimicrobial-Mediated ROS Accumulation and Bacterial Death.

Glucose is widely used in the reconstitution of intravenous medications, which often include antimicrobials. How glucose affects antimicrobial activity has not been comprehensively studied. The present work reports that glucose added to bacteria growing in a rich medium suppresses the bactericidal but not the bacteriostatic activity of several antimicrobial classes, thereby revealing a phenomenon called glucose-mediated antimicrobial tolerance. Glucose, at concentrations corresponding to blood-sugar levels of humans, increased survival of Escherichia coli treated with quinolones, aminoglycosides, and cephalosporins with little effect on minimal inhibitory concentration. Glucose suppressed a ROS surge stimulated by ciprofloxacin. Genes involved in phosphorylated fructose metabolism contributed to glucose-mediated tolerance, since a pfkA deficiency, which blocks the formation of fructose-1,6-bisphosphate, eliminated protection by glucose. Disrupting the pentose phosphate pathway or the TCA cycle failed to alter glucose-mediated tolerance, consistent with an upstream involvement of phosphorylated fructose. Exogenous sodium pyruvate or sodium citrate reversed glucose-mediated antimicrobial tolerance. Both metabolites bypass the effects of fructose-1,6-bisphosphate, a compound known to scavenge hydroxyl radical and chelate iron, activities that suppress ROS accumulation. Treatment with these two compounds constitutes a novel way to mitigate the glucose-mediated antimicrobial tolerance that may exist during intravenous antimicrobial therapy, especially for diabetes patients.

Fructose 1, 6-diphosphate regulates desmosomal proteins and collagen fibres in human skin equivalents.

We previously reported that fructose 1,6-diphosphate (FDP), a glycolytic metabolite, alleviates ultraviolet B-induced oxidative skin damage. Here, we further examined the effects of FDP on skin. FDP decreased the number of desmosomes, whereas it increased collagen fibres in skin equivalents (SEs). FDP significantly decreased the expression of corneodesmosomal components such as desmoglein 1 (DSG1), desmocollin 1 (DSC1) and corneodesmosin (CDSN), and desquamation-related proteases, kallikrein 5 (KLK 5) and kallikrein 7 (KLK7) in normal human epidermal keratinocytes (NHEKs). In addition, FDP treatment increased the phosphorylation of p38 MAPK, but the decreased expression of corneodesmosomal components is not recovered by the treatment of p38 MAPK inhibitors. Interestingly, FDP diminished the amplitude of Ca(2+) fluxes through down-regulation of SERCA2. Taken together, these results suggested that FDP induced a decrease in desmosomes and an increase in collagen fibres similar to the process of chemical peeling, the most common treatments for ageing skin.

Nitric oxide as a mediator of fructose 1,6-bisphosphate protection in galactosamine-induced hepatotoxicity in rats.

Fructose 1,6-bisphosphate (F1,6BP) has been widely used as a therapeutic agent for different harmful conditions in a variety of tissues. The hypothesis of the present work was that the increase in nitric oxide production and the prevention of oxidative stress induced by exogenous F1,6BP mediate its protective effect against the hepatotoxic action of GalN. Experimental groups used were sham, F1,6BP (2g/kg bw i.p.), GalN (0.4g/kg bw i.p), l-NAME (10mg/kg bw i.v.), F1,6BP+GalN, l-NAME+GalN and l-NAME+F1,6BP+GalN. Animals were killed after 24h of bolus administration. F1,6BP induced an increase in NO and the redox ratio (GSH/GSSG) in liver. Western blot assays pointed to overexpression of liver eNOS in F1,6BP-treated rats. The hepatic injury induced by GalN increased transaminases in plasma and decreased the reduced/oxidized glutathione ratio in liver. The concomitant administration of F1,6BP reversed this damage, while the addition of l-NAME worsened the liver injury. We provided evidence that this F1,6BP-induced protection may be related to the increase in NO production through the positive modulation of eNOS, and the increase in intracellular reduced glutathione, thus providing a higher reducing capacity.

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase is phosphorylated in wheat endosperm at serine-404 by an SNF1-related protein kinase allosterically inhibited by ribose-5-phosphate.

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) is a cytosolic unconventional glycolytic enzyme of plant cells regulated by phosphorylation in heterotrophic tissues. After interaction with 14-3-3 proteins, the phosphorylated enzyme becomes less active and more sensitive to regulation by adenylates and inorganic pyrophosphate. Here, we acknowledge that in wheat (Triticum aestivum), np-Ga3PDHase is specifically phosphorylated by the SnRK (SNF1-related) protein kinase family. Interestingly, only the kinase present in heterotrophic tissues (endosperm and shoots, but not in leaves) was found active. The specific SnRK partially purified from endosperm exhibited a requirement for Mg(2+) or Mn(2+) (being Ca(2+) independent), having a molecular mass of approximately 200 kD. The kinase also phosphorylated standard peptides SAMS, AMARA, and SP46, as well as endogenous sucrose synthase, results suggesting that it could be a member of the SnRK1 subfamily. Concurrently, the partially purified wheat SnRK was recognized by antibodies raised against a peptide conserved between SnRK1s from sorghum (Sorghum bicolor) and maize (Zea mays) developing seeds. The wheat kinase was allosterically inhibited by ribose-5-phosphate and, to a lesser extent, by fructose-1,6-bisphosphate and 3-phosphoglycerate, while glucose-6-phosphate (the main effector of spinach [Spinacia oleracea] leaves, SnRK1) and trehalose-6-phosphate produced little or no effect. Results support a distinctive allosteric regulation of SnRK1 present in photosynthetic or heterotrophic plant tissues. After in silico analysis, we constructed two np-Ga3PDHase mutants, S404A and S447A, identifying serine-404 as the target of phosphorylation. Results suggest that both np-Ga3PDHase and the specific kinase could be under control, critically affecting the metabolic scenario involving carbohydrates and reducing power partition and storage in heterotrophic plant cells.

Strontium fructose 1,6-diphosphate prevents bone loss in a rat model of postmenopausal osteoporosis via the OPG/RANKL/RANK pathway.

AIM: To evaluate the protective effects of strontium fructose 1,6-diphosphate (FDP-Sr), a novel strontium salt that combined fructose 1,6-diphosphate (FDP) with strontium, on bone in an ovariectomy-induced model of bone loss. METHODS: Eighty female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated. Three months later, the rats were assigned to six groups (10 for each): sham-operated, OVX control, OVX+FDP-Sr (110, 220, or 440 mg/kg), or OVX+strontium ranelate (SR, 180 mg/kg). Drugs were administered orally for 3 months. When the treatment was terminated, the following parameters were assessed: bone mineral density (BMD), the biomechanical properties of the femur and lumbar vertebrae, trabecular histomorphology, serum phosphorus, calcium, bone-specific alkaline phosphatase (B-ALP), tartrate-resistant acid phosphatase 5b (TRACP5b), N-telopeptide of type I collagen (NTx) and a series of markers for oxidative stress. Receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) levels in serum were measured using ELISA and their gene expression levels in the bone were measured using R-T PCR. RESULTS: Treatment with FDP-Sr (220 or 440 mg/kg) or SR (180 mg/kg) significantly increased the BMD and improved the bone microarchitecture and bone strength in OVX rats. The treatments also decreased in the levels of H(2)O(2) and MDA, restored the CAT level in serum and bone marrow, increased the serum B-ALP and decreased NTx and TRACP 5b in OVX rats. Treatment with FDP-Sr decreased the RANKL level, and increased the OPG level in serum in a dose-dependent manner. It also significantly down-regulated the RANKL expression and up-regulated OPG expression in bone marrow. CONCLUSION: FDP-Sr may be an effectve treatment for postmenopausal osteoporosis that acts, in part, via a decrease in osteoclastogenesis through the OPG\RANKL\RANK pathway.

The kinetics and regulation of phosphofructokinase from Teladorsagia circumcincta.

Phosphofructokinase (PFK-1) activity was examined in L(3) and adult Teladorsagia circumcincta, both of which exhibit oxygen consumption. Although activities were higher in the adult stage, the kinetic properties of the enzyme were similar in both life cycle stages. T. circumcincta PFK-1 was subject to allosteric inhibition by high ATP concentration, which increased both the Hill coefficient (from 1.4±0.2 to 1.7±0.2 in L(3)s and 2.0±0.3 to 2.4±0.4 in adults) and the K(½) for fructose 6 phosphate (from 0.35±0.02 to 0.75±0.05mM in L(3)s and 0.40±0.03 to 0.65±0.05mM in adults). The inhibitory effects of high ATP concentration could be reversed by fructose 2,6 bisphosphate and AMP, but glucose 1,6 bisphosphate had no effect on activity. Similarly, phosphoenolpyruvate had no effect on activity, while citrate, isocitrate and malate exerted mild inhibitory effects, but only at concentrations exceeding 2mM. The observed kinetic properties for T. circumcincta PFK-1 were very similar to those reported for purified Ascaris suum PFK-1, though slight differences in sensitivity to ATP concentration suggests there may be subtle variations at the active site. These results are consistent with the conservation of properties of PFK-1 amongst nematode species, despite between species variation in the ability to utilise oxygen.