Daily fluctuations of titratable acidity, content of organic acids (malate and citrate) and soluble sugars of varieties and wild relatives of Ananas comosus L. growing under natural tropical conditions

The genus Ananas has its centre of origin in northern South America. In this area, several varieties of Ananas comosus are widely cultivated, and a number of wild species are found growing under variable conditions of light intensity, soil fertility and water availability. Here we report detailed daily courses of titratable acidity, and malate, citrate and free-sugars content of several cultivated varieties of A. comosus and of A. ananassoides, a closely related species growing on granitic rock-outcrops in southern Venezuela. Day-night oscillations of both malate and citrate were detected in plants growing under full sun, but malate was by far the most important organic anion associated with CAM performance in ail populations sampled. Fructose was the dominant compound in the neutral fraction, but only sucrose showed a consistent inverse relation with the cycle of titratable acidity. The diel oscillations of free sugars measured were not always enough to account for the amount of organic anions accumulated during the night. Plants cultivated under shady conditions always showed a lower night-time increase in titratable acidity and organic acids, and also smaller oscillations in the amount of free sugars than sun exposed plants. In all populations growing under full sun, osmolality increased during the night, but it was not always possible to explain these changes on the basis of variations in molar concentrations of organic acids and sugars. Besides, no diel variations in the cations K⁺, Ca²⁺ and Mg²⁺ were detected. K⁺ was always the dominant cation (K/Ca ratios ～ 19), while Mg²⁺ was always higher than Ca²⁺ (Mg/Ca ～ 2).     

Growth and maintenance respiration in leaves of northern red oak seedlings and mature trees after 3 years of ozone exposure

A two-component model of growth and maintenance respiration is used to study the response of northern red oak (Quercus rubra L.) seedlings and 32-year-old trees to sub-ambient (10 μmol h; cumulative dose based on 7 h daily mean), ambient (43 μmol h), and twice-ambient (85 μmolh) ozone. The relative growth rates (RGR) of leaves sampled from seedlings and trees were similar across treatments, as were specific leaf respiration rates (SRR). Growth coefficients estimated from the SRR versus RGR relationship averaged 25-3 mol CO2 kg^?1 leaf dry mass produced for seedlings and 21-5 mol kg^?1 for trees. Maintenance coefficients ranged from 0-89 to 1-07 mol CO₂ kg^?1 leaf dry mass d^?1 for seedlings and from 0-64 to 0-84 mol kg-1 d^?1 for trees. Neither coefficient was affected by ozone. Leaves sampled throughout the growing season also showed little response of respiration to ozone. This occurred despite a 30% reduction in net photosynthesis for trees grown at twice-ambient ozone. These results suggest that growth and maintenance respiration in young northern red oak leaves are not affected by ozone and that in older leaves injury can occur without a parallel increase in so-called ‘maintenance’ respiration.     

Sodium Fluoride Mimics the Effect of Prostaglandin E2 on Catecholamine Release from Bovine Adrenal Chromaffin Cells

We have reported recently that prostaglandin E2 (PGE2) stimulated phosphoinositide metabolism in bovine adrenal chromaffin cells and that PGE2 and ouabain, an inhibitor of Na+, K(+)-ATPase, synergistically induced a gradual secretion of catecholamines from the cells. Here we examined the involvement of a GTP-binding protein(s) in PGE receptor-induced responses by using NaF. In the presence of Ca2+ in the medium, NaF stimulated the formation of all three inositol phosphates, i.e., inositol monophosphate, bisphosphate, and trisphosphate, linearly over 30 min in a dose-dependent manner (15-30 mM). This effect on phosphoinositide metabolism was accompanied by an increase in cytosolic free Ca2+. NaF also induced catecholamine release from chromaffin cells, and the dependency of stimulation of the release on NaF concentration was well correlated with those of NaF-enhanced inositol phosphate formation and increase in cytosolic free Ca2+. Although the effect of NaF on PGE2-induced catecholamine release in the presence of ouabain was additive at concentrations below 20 mM, there was no additive effect at 25 mM NaF. Furthermore, the time course of catecholamine release stimulated by 20 mM NaF in the presence of ouabain was quite similar to that by 1 microM PGE2, and both stimulations were markedly inhibited by amiloride, with half-maximal inhibition at 10 microM. Pretreatment of the cells with pertussis toxin did not prevent, but rather enhanced, PGE2-induced catecholamine release over the range of concentrations examined. These results demonstrate that NaF mimics the effect of PGE2 on catecholamine release from chromaffin cells and suggest that PGE2-evoked catecholamine release may be mediated by the stimulation of phosphoinositide metabolism through a putative GTP-binding protein insensitive to pertussis toxin.     

Activation of OR1A1 suppresses PPAR-γ expression by inducing HES-1 in cultured hepatocytes

Olfactory receptors (ORs) comprise the largest G protein-coupled receptor gene superfamily. Recent studies indicate that ORs are also expressed in non-olfactory organs, including metabolically active tissues, although their biological functions in these tissues are largely unknown. In this study, OR1A1 expression was detected in HepG2 liver cells. OR1A1 activation by (−)-carvone, a known OR1A1 ligand, increased the cyclic adenosine monophosphate (cAMP), but not intracellular Ca²⁺ concentration, thereby inducing protein kinase A (PKA) activity with subsequent phosphorylation of cAMP response element-binding protein (CREB) and upregulation of the CREB-responsive gene hairy and enhancer of split (HES)-1, a corepressor of peroxisome proliferator-activated receptor-γ (PPAR-γ) in hepatocytes. In (−)-carvone-stimulated cells, the repression of PPAR-γ reduced the expression of the target gene, mitochondrial glycerol-3-phosphate acyltransferase, which encodes a key enzyme involved in triglyceride synthesis. Intracellular triglyceride level and lipid accumulation were reduced in cells stimulated with (−)-carvone, effects that were diminished following the loss of OR1A1 function. These results indicate that OR1A1 may function as a non-redundant receptor in hepatocytes that regulates the PKA-CREB-HES-1 signaling axis and thereby modulates hepatic triglyceride metabolism.     

Establishing a novel biosynthetic pathway for the production of 3,4-dihydroxybutyric acid from xylose in Escherichia coli

3-Hydroxy-γ-butyrolactone (3HBL) is an attractive building block owing to its broad applications in pharmaceutical industry. Currently, 3HBL is commercially produced by chemical routes using petro-derived carbohydrates, which involves hazardous materials and harsh processing conditions. Only one biosynthetic pathway has been reported for synthesis of 3HBL and its hydrolyzed form 3,4-dihydroxybutyric acid (3,4-DHBA) using glucose and glycolic acid as the substrates and coenzyme A as the activator, which involves multiple steps (>10 steps) and suffers from low productivity and yield. Here we established a novel five-step biosynthetic pathway for 3,4-DHBA generation from D-xylose based on the non-phosphorylative D-xylose metabolism, which led to efficient production of 3,4-DHBA in Escherichia coli. Pathway optimization by incorporation of efficient enzymes for each step and host strain engineering by knocking out competing pathways enabled 1.27 g/L 3,4-DHBA produced in shake flasks, which is the highest titer reported so far. The novel pathway established in engineered E. coli strain demonstrates a new route for 3,4-DHBA biosynthesis from xylose, and this engineered pathway has great potential for industrial biomanufacturing of 3,4-DHBA and 3HBL.     

Inhibition of Mitochondrial Complex II by the Anticancer Agent Lonidamine

The antitumor agent lonidamine (LND; 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid) is known to interfere with energy-yielding processes in cancer cells. However, the effect of LND on central energy metabolism has never been fully characterized. In this study, we report that a significant amount of succinate is accumulated in LND-treated cells. LND inhibits the formation of fumarate and malate and suppresses succinate-induced respiration of isolated mitochondria. Utilizing biochemical assays, we determined that LND inhibits the succinate-ubiquinone reductase activity of respiratory complex II without fully blocking succinate dehydrogenase activity. LND also induces cellular reactive oxygen species through complex II, which reduced the viability of the DB-1 melanoma cell line. The ability of LND to promote cell death was potentiated by its suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation. Using stable isotope tracers in combination with isotopologue analysis, we showed that LND increased glutaminolysis but decreased reductive carboxylation of glutamine-derived α-ketoglutarate. Our findings on the previously uncharacterized effects of LND may provide potential combinational therapeutic approaches for targeting cancer metabolism.     

Regulation of the phosphate (Pi) concentration in UMR 106 osteoblast-like cells: Effect of Pi,Na+ and K+

Osteoblast-like cells possess Na-dependent transporters which accumulate orthophosphate (Pi) from the extracellular medium. This may be important in bone formation. Here we describe parallel measurements of Pi uptake and cellular [Pi] in such cells from the rat (UMR 106–01 and UMR 106–06) and human (OB), and in non-osteoblastic human fibroblasts (Detroit 532 (DET)). In UMR 106–01, cellular [Pi] was weakly dependent on extracellular [Pi] and higher than expected from passive transport alone. [³²Pi]-uptake was inhibited by Na deprivation, but paradoxically increased on K deprivation. With Na, 87 per cent of cellular ³²P was found in organic phosphorus pools after only 5 min. Na deprivation also decreased cellular [Pi], in both UMR 106–01 and DET, but the decrease was smaller than that in [³²Pi]-uptake. Ouabain decreased [³²Pi]-uptake and cellular [Pi] in DET, but not in UMR 106–01. Regulation of cellular [Pi] is therefore at least partly dependent on Na/Pi co-transport, but this does not seem to be an exclusive property of osteoblasts.