Kinetic properties of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from spinach leaves.

A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. The soluble protein had a molecular mass of 320 kDa, estimated by gel filtration chromatography, and a subunit size of 90.8 kDa. The purified protein had activity of both 6-phosphofructo-2-kinase specific activity 10.4-15.9 nmol min(-1) x mg protein (-1) and fructose-2,6-bisphosphatase (specific activity 1.65-1.75 nmol x mol(-1) mg protein(-1). The 6-phosphofructo-2-kinase activity was activated by inorganic phosphate, and inhibited by 3-carbon phosphorylated metabolites and pyrophosphate. In the presence of phosphate, 3-phosphoglycerate was a mixed inhibitor with respect to both fructose 6-phosphate and ATP. Fructose-2,6-bisphosphatase activity was sensitive to product inhibition; inhibition by inorganic phosphate was uncompetitive, whereas inhibition by fructose 6-phosphate was mixed. These kinetic properties support the view that the level of fructose 2,6-bisphosphate in leaves is determined by the relative concentrations of hexose phosphates, three-carbon phosphate esters and inorganic phosphate in the cytosol through reciprocal modulation of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities of the bifunctional enzyme.     

Post heroin dose tissue distribution of 6-monoacetylmorphine (6-MAM) with MALDI imaging

Heroin is an illicit opioid drug which is commonly abused and leads to dependence and addiction. Heroin is considered a pro-drug and is rapidly converted to its major active metabolite 6-monoacetylmorphine (6-MAM) which mediates euphoria and reward through the stimulation of opioid receptors in the brain. The aim of this study was to investigate the distribution and localization of 6-MAM in the healthy Sprague Dawley rat brain following intraperitoneal (i.p) administration of heroin (10 mg/kg), using matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI), in combination with quantification via liquid chromatography mass spectrometry (LC–MS/MS). These findings revealed that 6-MAM is present both in plasma and brain tissue with a T_max of 5 min (2.8 µg/mL) and 15 min (1.1 µg/mL), respectively. MSI analysis of the brain showed high intensities of 6-MAM in the thalamus-hypothalamus and mesocorticolimbic system including areas of the cortex, caudate putamen, and ventral pallidum regions. This finding correlates with the distribution of opioid receptors in the brain, according to literature. In addition, we report a time-dependent distribution in the levels of 6-MAM, from 1 min with the highest intensity of the drug observed at 15 min, with sparse distribution at 45 min before decreasing at 60 min. This is the first study to use MSI as a brain imaging technique to detect a morphine’s distribution over time in the brain.     

Homocysteine modulates 5‐lipoxygenase expression level via DNA methylation

Elevated levels of homocysteinemia (Hcy), a risk factor for late‐onset Alzheimer's disease (AD), have been associated with changes in cell methylation. Alzheimer's disease is characterized by an upregulation of the 5‐lipoxygenase (5LO), whose promoter is regulated by methylation. However, whether Hcy activates 5LO enzymatic pathway by influencing the methylation status of its promoter remains unknown. Brains from mice with high Hcy were assessed for the 5LO pathway and neuronal cells exposed to Hcy implemented to study the mechanism(s) regulating 5LO expression levels and the effect on amyloid β formation. Diet‐ and genetically induced high Hcy resulted in 5LO protein and mRNA upregulation, which was associated with a significant increase of the S‐adenosylhomocysteine (SAH)/S‐adenosylmethionine ratio, and reduced DNA methyltrasferases and hypomethylation of 5‐lipoxygenase DNA. In vitro studies confirmed these results and demonstrated that the mechanism involved in the Hcy‐dependent 5LO activation and amyloid β formation is DNA hypomethylation secondary to the elevated levels of SAH. Taken together these findings represent the first demonstration that Hcy directly influences 5LO expression levels and establish a previously unknown cross talk between these two pathways, which is highly relevant for AD pathogenesis. The discovery of such a novel link not only provides new mechanistic insights in the neurobiology of Hcy, but most importantly new therapeutic opportunities for the individuals bearing this risk factor for the disease.     

High-lysine corn produced by the combination of enhanced lysine biosynthesis and reduced zein accumulation

Corn is one of the major crops in the world, but its low lysine content is often problematic for animal consumption. While exogenous lysine supplementation is still the most common solution for today's feed corn, high-lysine corn has been developed through genetic research and biotechnology. Reducing the lysine-poor seed storage proteins, zeins, or expressing a deregulated lysine biosynthetic enzyme, CordapA, has shown increased total lysine or free lysine content in the grains of modified corn plants, respectively. Here, by combining these two approaches through genetic crosses, the total lysine content has more than doubled in F1 progeny. We also observe a synergy between the transgenic zein reduction and the enhanced lysine biosynthesis by CordapA expression. The zein reduction plants are found to accumulate higher levels of aspartate, asparagine and glutamate, and therefore, provide excess precursors for the enhanced lysine biosynthesis.     

Stimulation of prostaglandin E2 (PGE2) production by arachidonic acid, oestrogen and parathyroid hormone in MG-63 and MC3T3-E1 osteoblast-like cells

Polyunsaturated fatty acids (PUFAs) as well as oestrogen (E2) and parathyroid hormone (PTH) affect bone cells. The aim of the study was to determine whether arachidonic acid (AA), E2, and PTH increase prostaglandin E₂ (PGE₂) synthesis in MG-63 and MC3T3-E1 osteoblastic cells and the level of mediation by COX-1 and COX-2. PGE₂ levels were determined in the conditioned culture media of MG-63 and MC3T3-E1 osteoblasts after exposure to AA, PTH and E2. Cells were pre-incubated in some experiments with the unselective COX inhibitor indomethacin or the COX-2 specific blocker NS-398. Indirect immunofluorescence was performed on MG-63 cells to detect the presence and location of the two enzymes involved. AA increased PGE₂ secretion in both cell lines; production by MC3T3-E1 cells, however, was significantly higher than that of MG-63 cells. This could be due to autoamplification via the EP₁ subtype of PGE receptors in mouse MC3T3-E1 osteoblasts. Both COX-1 and COX-2 affected the regulation of PGE₂ synthesis in MG-63 cells. E2 had no effect on PGE₂ secretion in both cell lines, while PTH caused a slight increase in PGE₂ synthesis in the MG-63 cell line.     

Activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase by fructose 2,6-bisphosphate stimulates conversion of hexose phosphates to triose phosphates but does not influence accumulation of carbohydrates in phosphate-deficient tobacco cells

The aim of this work was to investigate the contribution of fructose 2,6-bisphosphate to the regulation of carbohydrate metabolism under phosphate stress. The study exploited heterotrophic tobacco callus lines expressing a modified mammalian 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase that increased the fructose 2,6-bisphosphate content of the tissue. The phosphate status of two transgenic and one untransformed cell line was perturbed by incubation with 2-deoxyglucose, a phosphate-sequestering agent, and by growth of callus on phosphate-depleted media. 31P-NMR spectroscopy confirmed that both treatments decreased cellular levels of inorganic phosphate and phosphorylated metabolites. Despite large decreases in the amounts of phosphate esters, UDPglucose and adenylates in response to phosphate deficiency, the fructose 2,6-bisphosphate content of each line was unaffected by 2-deoxyglucose and increased during growth on phosphate-limited media. Short-term treatment of callus with 2-deoxyglucose had only minor effects on the carbohydrate status of each line, whereas long-term phosphate deficiency caused an increase in starch and a decrease in soluble sugar content in both transgenic and control lines. There were no consistent differences between the three callus lines in metabolism of [U-14C]glucose in response to incubation with 2-deoxyglucose. In contrast, there was a decrease in partitioning of label into glycolytic products (particularly organic acids) in untransformed callus during growth on phosphate-depleted medium. This decrease was greatly attenuated in the transgenic lines with increased fructose 2,6-bisphosphate content. This suggests that the conversion of hexose phosphates to triose phosphates is constrained under phosphate-deficient conditions, and that this restriction can be relieved by activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase. However, since the transgenic and control lines did not differ in the extent to which the carbohydrate content changed in response to growth on phosphate-depleted media, it is concluded that an increase in flux through pyrophosphate:fructose-6-phosphate 1-phosphotransferase is not a major component of the metabolic response of heterotrophic tobacco cells to phosphate deficiency.     

Monitoring flux through the oxidative pentose phosphate pathway using [1-14C]gluconate

The aim of this work was to examine the metabolism of exogenous gluconate by a 4-day-old cell suspension culture of Arabidopsis thaliana (L.) Heynh. Release of (14)CO(2) from [1-(14)C]gluconate was dependent on the concentration in the medium and could be resolved into a substrate-saturable component (apparent K(m) of approximately 0.4 mM) and an unsaturable component. At an external concentration of 0.3 mM, the rate of decarboxylation of applied gluconate was 0.2% of the rate of oxygen consumption by the cells. There was no effect of 0.3 mM gluconate on the rate of oxygen consumption, or on the rate of (14)CO(2) release from either [1-(14)C]glucose or [6-(14)C]glucose by the culture. The following observations argue that gluconate taken up by the cells is metabolised by direct phosphorylation to 6-phosphogluconate and subsequent decarboxylation through 6-phosphogluconate dehydrogenase. First, more than 95% of the label released from [1-(14)C]gluconate during metabolism by the cell culture was recovered as (14)CO(2). Secondly, inhibition of the oxidative pentose phosphate pathway (OPPP) by treatment with 6-aminonicotinamide preferentially inhibited release of (14)CO(2) from [1-(14)C]gluconate relative to that from [1-(14)C]glucose. Thirdly, perturbation of glucose metabolism by glucosamine did not affect (14)CO(2) from [1-(14)C]gluconate. Fourth, stimulation of the OPPP by phenazine methosulphate stimulated release of (14)CO(2) from [1-(14)C]gluconate to a far greater extent than that from [1-(14)C]glucose. It is proposed that measurement of (14)CO(2) from [1-(14)C]gluconate provides a simple and sensitive technique for monitoring flux through the OPPP pathway in plants.     

Brain repair by endogenous progenitors

Stem cells within the adult brain can be stimulated by injury and growth factor treatment to replace damaged neurons, even neurons that are not normally generated in adults. Coupled with recent insights into the mechanism by which Nogo inhibits axonal regeneration, this discovery may inspire new treatments for central nervous system injuries and neurodegenerative diseases.