Evidence that the decrease in liver glycogen is associated with the exercise-induced increase in IGFBP-1.

The purpose of the present study was to test the hypothesis that the exercise-induced increase in insulin-like growth factor binding protein (IGFBP)-1 is not always linked to a decrease in blood glucose level and to examine whether the decreasing levels of liver glycogen during exercise may be associated with the increase in IGFBP-1. Three groups of rats were submitted to a 70-min treadmill exercise. One group of rats was fed normally, and the two other groups had their food intake restricted by 50% (50% fast) the night before the experiment. One of these two 50% fasted groups of rats was infused (intravenously) with glucose throughout exercise to maintain euglycemia. Exercise in noninfused 50% fasted rats, compared with the normally fed rats, resulted in significantly lower blood glucose (minute 70) and insulin levels, significantly lower liver glycogen content, no change in IGF-I, and significantly higher increases in free fatty acid, glycerol, beta-hydroxybutyrate, and IGFBP-1. Maintenance of euglycemia during exercise in glucose-infused 50% fasted rats reduced to a large extent the decrease in insulin levels but only slightly attenuated the lipid response and the IGFBP-1 response seen in noninfused 50% fasted rats. Comparisons of all individual liver glycogen and IGFBP-1 values revealed that liver glycogen values were highly (P < 0.001) predictive of the IGFBP-1 response during exercise (R = 0.564). The present results indicate that the IGFBP-1 response during exercise is not always linked to a decrease in plasma glucose and suggest that the increase in IGFBP-1 during exercise may be related to the decrease in liver glycogen content.     

High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection

Endochitinases (E.C. 3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.     

The human DNA repair factor XPC-HR23B distinguishes stereoisomeric benzo[a]pyrenyl-DNA lesions

Benzo[a]pyrene (B[a]P), a known environmental pollutant and tobacco smoke carcinogen, is metabolically activated to highly tumorigenic B[a]P diol epoxide derivatives that predominantly form N(2)-guanine adducts in cellular DNA. Although nucleotide excision repair (NER) is an important cellular defense mechanism, the molecular basis of recognition of these bulky lesions is poorly understood. In order to investigate the effects of DNA adduct structure on NER, three stereoisomeric and conformationally different B[a]P-N(2)-dG lesions were site specifically incorporated into identical 135-mer duplexes and their response to purified NER factors was investigated. Using a permanganate footprinting assay, the NER lesion recognition factor XPC/HR23B exhibits, in each case, remarkably different patterns of helix opening that is also markedly distinct in the case of an intra-strand crosslinked cisplatin adduct. The different extents of helix distortions, as well as differences in the overall binding of XPC/HR23B to double-stranded DNA containing either of the three stereoisomeric B[a]P-N(2)-dG lesions, are correlated with dual incisions catalyzed by a reconstituted incision system of six purified NER factors, and by the full NER apparatus in cell-free nuclear extracts.     

SINE RNA induces severe developmental defects in Arabidopsis thaliana and interacts with HYL1 (DRB1), a key member of the DCL1 complex

The proper temporal and spatial expression of genes during plant development is governed, in part, by the regulatory activities of various types of small RNAs produced by the different RNAi pathways. Here we report that transgenic Arabidopsis plants constitutively expressing the rapeseed SB1 SINE retroposon exhibit developmental defects resembling those observed in some RNAi mutants. We show that SB1 RNA interacts with HYL1 (DRB1), a double-stranded RNA-binding protein (dsRBP) that associates with the Dicer homologue DCL1 to produce microRNAs. RNase V1 protection assays mapped the binding site of HYL1 to a SB1 region that mimics the hairpin structure of microRNA precursors. We also show that HYL1, upon binding to RNA substrates, induces conformational changes that force single-stranded RNA regions to adopt a structured helix-like conformation. Xenopus laevis ADAR1, but not Arabidopsis DRB4, binds SB1 RNA in the same region as HYL1, suggesting that SINE RNAs bind only a subset of dsRBPs. Consistently, DCL4-DRB4-dependent miRNA accumulation was unchanged in SB1 transgenic Arabidopsis, whereas DCL1-HYL1-dependent miRNA and DCL1-HYL1-DCL4-DRB4-dependent tasiRNA accumulation was decreased. We propose that SINE RNA can modulate the activity of the RNAi pathways in plants and possibly in other eukaryotes.     

Thermoluminescence and P700 redox kinetics as complementary tools to investigate the cyclic/chlororespiratory electron pathways in stress conditions in barley leaves

Cyclic electron flow around photosystem I drives additional proton pumping into the thylakoid lumen, which enhances the protective non-photochemical quenching and increases ATP synthesis. It involves several pathways activated independently. In whole barley leaves, P700 oxidation under far-red illumination and subsequent P700(+) dark reduction kinetics provide a major probe of the activation of cyclic pathways. Two 'intermediate' and 'slow' exponential reduction phases are always observed and they become faster after high light illumination, but dark inactivation of the Benson-Calvin cycle causes the emergence of both a transient in the P700 oxidation and a 'fast' phase in the P700(+) reduction. We investigate here the afterglow (AG) thermoluminescence emission as another tool to detect the activation of cyclic electron pathways from stroma reductants to the acceptor side of photosystem II. This transfer is activated by warming, yielding an AG band at about 45°C. However, treatments that accelerate the 'intermediate' and 'slow' P700(+) reduction phases (brief anoxia, hexose infiltration, fast dehydration of excised leaves) also produced a downshift of this AG band. This pathway ascribable to NADPH dehydrogenase (NDH) would be triggered by a deficit in ATP, while the 'fast' reduction phase corresponding to the ferredoxin plastoquinone reductase pathway is triggered by an overreduction of the photosystem I acceptor pool and is undetected in thermoluminescence. Contrastingly, slow dehydration of unwatered plants did not cause faster reduction of P700(+) nor temperature downshift of the AG band, that is no induction of the NDH pathway, whereas an increased intensity of the AG band indicated a strong NADPH + ATP assimilatory potential.     

Small efficient cell-penetrating peptides derived from scorpion toxin maurocalcine

Maurocalcine is the first demonstrated example of an animal toxin peptide with efficient cell penetration properties. Although it is a highly competitive cell-penetrating peptide (CPP), its relatively large size of 33 amino acids and the presence of three internal disulfide bridges may hamper its development for in vitro and in vivo applications. Here, we demonstrate that several efficient CPPs can be derived from maurocalcine by replacing Cys residues by isosteric 2-aminobutyric acid residues and sequence truncation down to peptides of up to 9 residues in length. A surprising finding is that all of the truncated maurocalcine analogues possessed cell penetration properties, indicating that the maurocalcine is a highly specialized CPP. Careful examination of the cell penetration properties of the truncated analogues indicates that several maurocalcine-derived peptides should be of great interest for cell delivery applications where peptide size matters.     

Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications

Yarrowia lipolytica requires the expression of a heterologous invertase to grow on a sucrose-based substrate. This work reports the construction of an optimized invertase expression cassette composed of Saccharomyces cerevisiae Suc2p secretion signal sequence followed by the SUC2 sequence and under the control of the strong Y. lipolytica pTEF promoter. This new construction allows a fast and optimal cleavage of sucrose into glucose and fructose and allows cells to reach the maximum growth rate. Contrary to pre-existing constructions, the expression of SUC2 is not sensitive to medium composition in this context. The strain JMY2593, expressing this new cassette with an optimized secretion signal sequence and a strong promoter, produces 4,519 U/l of extracellular invertase in bioreactor experiments compared to 597 U/l in a strain expressing the former invertase construction. The expression of this cassette strongly improved production of invertase and is suitable for simultaneously high production level of citric acid from sucrose-based media.