Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma

Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells.     

Jasmonate-dependent plant defense restricts thrips performance and preference

Background

Worldwide, diseases are important reducers of peanut (Arachis hypogaea) yield. Sources of resistance against many diseases are available in cultivated peanut genotypes, although often not in farmer preferred varieties. Wild species generally harbor greater levels of resistance and even apparent immunity, although the linkage of agronomically un-adapted wild alleles with wild disease resistance genes is inevitable. Marker-assisted selection has the potential to facilitate the combination of both cultivated and wild resistance loci with agronomically adapted alleles. However, in peanut there is an almost complete lack of knowledge of the regions of the Arachis genome that control disease resistance.

Results

In this work we identified candidate genome regions that control disease resistance. For this we placed candidate disease resistance genes and QTLs against late leaf spot disease on the genetic map of the A-genome of Arachis, which is based on microsatellite markers and legume anchor markers. These marker types are transferable within the genus Arachis and to other legumes respectively, enabling this map to be aligned to other Arachis maps and to maps of other legume crops including those with sequenced genomes. In total, 34 sequence-confirmed candidate disease resistance genes and five QTLs were mapped.

Conclusion

Candidate genes and QTLs were distributed on all linkage groups except for the smallest, but the distribution was not even. Groupings of candidate genes and QTLs for late leaf spot resistance were apparent on the upper region of linkage group 4 and the lower region of linkage group 2, indicating that these regions are likely to control disease resistance.     

Antibacterial halogenated metabolites from the Malaysian Laurencia species

Two halogenated C15 acetogenins, named lembyne-A and lembyne-B, have been isolated from an unrecorded Laurencia species collected off the Malaysian waters. Their structures were deduced on the basis of spectroscopic evidence. Previously known elatol and iso-obtusol showed potent antibacterial activity against some marine bacteria.     

Purification and properties of the manganese superoxide dismutase from the liver of bullfrog, Rana catesbeiana

A manganese superoxide dismutase (Mn-SOD) from the liver of bullfrog, Rana catesbeiana, was purified to electrophoretic homogeneity. The enzyme has a molecular weight of about 84,000 and is composed of four identical subunits, each containing one manganese atom. The amino acid composition of the enzyme is similar to that of Mn-SODs isolated from human and chicken livers, but differs considerably from that of the Escherichia coli enzyme (D. Barra et al. (1984) J. Biol. Chem. 259, 12595-12601; R. A. Weisiger and I. Fridovich (1973) J. Biol. Chem. 248, 3582-3592; H. M. Steinman (1978) J. Biol. Chem. 253, 8708-8720). The N-terminal amino acid is lysine. The sequence of 23 amino acid residues in the N-terminal region was determined. It shows excellent homologies with those of the human and chicken enzymes (H. M. Steinmam and R. L. Hill (1973) Proc. Natl. Acad. Sci. USA 70, 3725-3729; C. Ditlow et al. (1982) Carlsberg Res. Commun. 47, 81-91). The frog liver enzyme is also located exclusively in the mitochondrial matrix. Immunologically the same enzyme is also found in the tadpole liver, in an amount of about one-half of that in the adult bullfrog.     

Empirical evaluation of a dynamic experiment design method for prediction of MHC class I-binding peptides

The ability to predict MHC-binding peptides remains limited despite ever expanding demands for specific immunotherapy against cancers, infectious diseases, and autoimmune disorders. Previous analyses revealed position-specific preference of amino acids but failed to detect sequence patterns. Efforts to use computational analysis to identify sequence patterns have been hampered by the insufficiency of the number/quality of the peptide binding data. We propose here a dynamic experiment design to search for sequence patterns that are common to the MHC class I-binding peptides. The method is based on a committee-based framework of query learning using hidden Markov models as its component algorithm. It enables a comprehensive search of a large variety (20(9)) of peptides with a small number of experiments. The learning was conducted in seven rounds of feedback loops, in which our computational method was used to determine the next set of peptides to be analyzed based on the results of the earlier iterations. After these training cycles, the algorithm enabled a real number prediction of MHC binding peptides with an accuracy surpassing that of the hitherto best performing positional scanning method.     

Antioxidative galloyl esters as enzyme inhibitors of p-hydroxybenzoate hydroxylase

Gallic acid and its esters were evaluated as enzyme inhibitors of recombinant p-hydroxybenzoate hydroxylase (PHBH), a NADPH-dependent flavin monooxygenase from Pseudomonas aeruginosa. n-Dodecyl gallate (DG) (IC(50)=16 microM) and (-)-epigallocatechin-3-O-gallate (EGCG) (IC(50)=16 microM), a major component of green tea polyphenols, showed the most potent inhibition, while product-like gallic acid did not inhibit the enzyme significantly (IC(50)>250 microM). Inhibition kinetics revealed that both DG and EGCG inhibited PHBH in a non-competitive manner (K(I)=18.1 and 14.0 microM, respectively). The enzyme inhibition was caused by specific binding of the antioxidative gallate to the enzyme, and by scavenging reactive oxygen species required for the monooxygenase reaction. Molecular modeling predicted that EGCG binds to the enzyme in the proximity of the FAD binding site via formation of three hydrogen bonds.     

NTDB: Thermodynamic Database for Nucleic Acids,Version 2.0

The second release of Thermodynamic Database for Nucleic Acids, NTDB 2.0, includes more than 4600 entries (250% increase over release 1.0). It contains sequence types and details of several thermodynamic parameters (enthalpy, DeltaH; entropy, DeltaS; Gibbs free energy, DeltaG; melting temperature, T(m)), experimental models and methods for extracting thermodynamic parameters, buffer conditions as well as all relevant literature information. In addition, the database statistics and references related to NTDB are included. Information on normal and modified nucleobases and nucleosides are collected in a new section 'Nucleoside' whereby data collected thus far will be release in NTDB 2.0. The NTDB is freely available at http://ntdb.chem.cuhk.edu.hk.     

Plant regeneration from stem and petiole protoplasts of sweet potato (Ipomoea batatas) and its wild relative,I. lacunosa

A protoplast-to-plant regeneration system has been established for sweet potato (Ipomoea batatas (L.) Lam.) and its wild relative, I. lacunosa L. Viable protoplasts, isolated from preplasmolyzed stems and petioles of in vitro-grown plants, were cultured on liquid MS (Murashige & Skoog 1962) medium that supported cell division and colony formation. Embryogenic calli of sweet potato were induced on agar-solidified MS medium supplemented with 3% (w/v) sucrose, 50 mg l^-1 casamino acids, 0.2–0.5 mg l^-1 2,4-d, 1.0 mg l^-1 kinetin and 1.0 mg l^-1 ABA. On average, 3 plants were regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 3% (w/v) sucrose, 800 mg l^-1 glutamine, 2.0 mg l^-1 BA or 1.0 mg l^-1 kinetin and 1.0 mg l^-1 GA₃. Embryogenic calli of I. lacunosa L. were initiated on semi-solid MS medium containing 0.2–0.5 mg l^-1 IAA and 1.0–2.0 mg l^-1 BA. An average of 5 plants was regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 0.5 or 1.0 mg l^-1 GA₃.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole acetic acid - MES 2-(N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid     

Luteolin and Quercetin Affect the Cholesterol Absorption Mediated by Epithelial Cholesterol Transporter Niemann–Pick C1-Like 1 in Caco-2 Cells and Rats

Niemann–Pick C1-Like 1 (NPC1L1) mediates cholesterol absorption, and ezetimibe is a potent NPC1L1 inhibitor applicable for medication of hypercholesterolemia. Epidemiological studies demonstrated that consumption of polyphenols correlates with a decreased risk for atherosclerosis due to their antioxidant effect. This activity can hardly be attributable to the antioxidant activity only, and we hypothesized that polyphenols inhibit intestinal transport of cholesterol. We elucidated the kinetic parameters of intestinal cholesterol absorption, screened several polyphenols for their ability to specifically inhibit intestinal cholesterol absorption, and determined the inhibitory effects of selected flavonoids in vitro and in vivo. The concentration-dependent uptake of cholesterol by Caco-2 cells obeyed a monophasic saturation process. This indicates the involvement of an active-passive transport, i.e., NPC1L1. Parameters of cholesterol uptake by Caco-2 cells were as follows: J _max, K _t, and K _d were 6.89±2.96 19.03±11.58 µM, and 0.11±0.02 pmol/min/mg protein, respectively. Luteolin and quercetin inhibited cholesterol absorption by Caco-2 cells and human embryonic kidney 293T cells expressing NPC1L1. When preincubated Caco-2 cells with luteolin and quercetin before the assay, cholesterol uptake significantly decreased. The inhibitory effects of these flavonoids were maintained for up to 120 min. The level of inhibition and irreversible effects were similar to that of ezetimibe. Serum cholesterol levels significantly decreased more in rats fed both cholesterol and luteolin (or quercetin), than in those observed in the cholesterol feeding group. As quercetin induced a significant decrease in the levels of NPC1L1 mRNA in Caco-2 cells, the in vivo inhibitory effect may be due to the expression of NPC1L1. These results suggest that luteolin and quercetin reduce high blood cholesterol levels by specifically inhibiting intestinal cholesterol absorption mediated by NPC1L1.     

Identification by Suppression Subtractive Hybridization of Frankia Genes Induced under Nitrogen-Fixing Conditions

Frankia is an actinobacterium that fixes nitrogen under both symbiotic and free-living conditions. We identified genes upregulated in free-living nitrogen-fixing cells by using suppression subtractive hybridization. They included genes with predicted functions related to nitrogen fixation, as well as with unknown function. Their upregulation was a novel finding in Frankia.Frankia is a Gram-positive actinobacterium that establishes symbiosis with several angiosperms termed actinorhizal plants and forms nitrogen-fixing nodules on their roots (20). Frankia also fixes nitrogen in free-living culture under nitrogen-free conditions (19). Induction of the nitrogen-fixing ability is accompanied by differentiation of vesicles (19). Vesicles are spherical cells specialized to nitrogen fixation and are surrounded by multilayered lipid envelopes by which nitrogenase is protected from oxygen (3). Frankia plays an important role in the global nitrogen cycle, yet little is known about the genes involved in the induction of nitrogen-fixing activity. Recently, three Frankia genome sequences were determined (15), which facilitates the genetic dissection of Frankia biology. In this study, we identified Frankia genes induced in nitrogen-fixing cells under free-living conditions by using suppression subtractive hybridization (SSH) (4).