Regulation of nitric oxide production in limb and ventilatory muscles during chronic exercise training

In this study, we evaluated the differential influence of chronic treadmill training (30 m/min, 15% incline, 1 h/day, 5 days/wk) on nitric oxide (NO) production and NO synthase (NOS) isoform expression as well as 3-nitrotyrosine formation (footprint of peroxynitrite) both in limb (gastrocnemius) and ventilatory (diaphragm) muscles. A group of exercise-trained rats and a control group (no training) were examined after a 4-wk experimental period. Exercise training elicited an approximate fourfold rise in gastrocnemius NOS activity and augmented protein expression of the endothelial (eNOS) and neuronal (nNOS) isoforms of NOS to approximately 480% and 240%, respectively. Qualitatively similar but quantitatively smaller elevations in NOS activity and eNOS and nNOS expression were observed in the diaphragm. No detectable inducible NOS (iNOS) protein expression was found in any of the muscle samples. Training increased the intensity of 3-nitrotyrosine only in the gastrocnemius muscle. We conclude that whole body exercise training enhances both limb and ventilatory muscle NO production and that constitutive and not iNOS isoforms are responsible for increased protein tyrosine nitration in trained limb muscles.     

Tree genetic engineering and applications to sustainable forestry and biomass production

Forest trees provide raw materials, help to maintain biodiversity and mitigate the effects of climate change. Certain tree species can also be used as feedstocks for bioenergy production. Achieving these goals may require the introduction or modified expression of genes to enhance biomass production in a sustainable and environmentally responsible manner. Tree genetic engineering has advanced to the point at which genes for desirable traits can now be introduced and expressed efficiently; examples include biotic and abiotic stress tolerance, improved wood properties, root formation and phytoremediation. Transgene confinement, including flowering control, may be needed to avoid ecological risks and satisfy regulatory requirements. This and stable expression are key issues that need to be resolved before transgenic trees can be used commercially.     

Estradiol-dependent regulation of angiopoietin expression in breast cancer cells

Angiopoietin-1 (Ang-1) is a ligand for Tie-2 receptors and a promoter of angiogenesis. Angiogenesis plays an important role in breast cancer, as it is one of the critical events required for tumors to grow and metastasize. In this study, we investigated the influence of estradiol (E2) on the expression of angiopoietins in breast cancer cell lines. Ang-1 mRNA and protein expressions were significantly higher in estrogen receptor-negative (ERα-) breast cancer cells than in estrogen receptor-positive (ERα+) cells. Exposure of ERα+ cells to E2 resulted in further reductions of Ang-1 levels. In mouse mammary pads inoculated with breast cancer cells, both tumor size and Ang-1 production were significantly lower in ERα+ cell-derived xenografts, as compared to those derived from ERα- cells. Reduction of circulating levels of E2 by ovariectomy eliminated this response. Overall, these results indicate that Ang-1 mRNA and protein expressions: (1) negatively correlate with the level of ERα in breast cancer cell lines; (2) are downregulated by E2 in an ERα dependent manner; and (3) positively correlate with the degree of angiogenesis in vivo. We conclude that Ang-1 is an important modulator of growth and progression of ERα- breast cancers.     

Rapid,High Quality DNA Isolation from Cypress (Cupressus sempervirens L.) Needles and Optimization of the RAPD Marker Technique

A protocol for extracting high quality DNA from cypress (Cupressus sempervirens L.), a gymnosperm of economic and ecological importance to the Mediterranean, is presented using the commercially supplied DNeasy^TM Plant Mini Kit (QIAGEN GmbH, Germany). Additional steps were introduced in the QIAGEN protocol to significantly enhance DNA quantity and quality. For one sample, the procedure can be completed in less than one hour, and more than 10 samples can be processed in a day. DNA yield and purity were monitored by gel electrophoresis and by determining absorbance at UV (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀). Both ratios were between 1.7 and 2.0, indicating that the presence of contaminating metabolites was minimal. The average DNA yield obtained from 100 mg starting material was around 22 g, which compares very favorably with numbers indicated by the manufacturer. Additionally, restriction and PCR analyses of the extracted DNA showed its compatibility with downstream applications. Using this DNA, the parameters for the randomly amplified polymorphic DNA (RAPD) protocol were optimized based on the use of (1) an AmpliTaq^® DNA Polymerase, Stoffel fragment (Perkin-Elmer), and (2) a high initial denaturation temperature (97.5 °C). Reproducible amplification products were achieved in all PCR reactions. Seven to eight bands per primer were obtained with most individuals. This represents a number at the high end of published results with other plant species.     

Roles of iNOS and nNOS in sepsis-induced pulmonary apoptosis

Apoptosis(programmed cell death) is induced in pulmonary cells and contributes to the pathogenesis of acute lung injury in septic humans. Previous studies have shown that nitric oxide (NO) is an important modulator of apoptosis; however, the functional role of NO derived from inducible NO synthase (iNOS) in sepsis-induced pulmonary apoptosis remains unknown. We measured pulmonary apoptosis in a rat model of Escherichia coli lipopolysaccharide (LPS)-induced sepsis in the absence and presence of the selective iNOS inhibitor 1400W. Four groups were studied 24 h after saline (control) or LPS injection in the absence and presence of 1400W pretreatment. Apoptosis was evaluated using DNA fragmentation, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and caspase activation. LPS administration significantly augmented pulmonary cell apoptosis and caspase-3 activity in airway and alveolar epithelial cells. Pretreatment with 1400W significantly enhanced LPS-induced pulmonary apoptosis and increased caspase-3 and -7 activation. The antiapoptotic effect of iNOS was confirmed in iNOS-/- mice, which developed a greater degree of pulmonary apoptosis both under control conditions and in response to LPS compared with wild-type mice. By comparison, genetic deletion of the neuronal NOS had no effect on LPS-induced pulmonary apoptosis. We conclude that NO derived from iNOS plays an important protective role against sepsis-induced pulmonary apoptosis.     

Accelerating the domestication of forest trees in a changing world

In light of impending water and arable land shortages, population growth and climate change, it is more important than ever to examine how forest tree domestication can be accelerated to sustainably meet future demands for wood, biomass, paper, fuel and biomaterials. Because of long breeding cycles, tree domestication cannot be rapidly achieved through traditional genetic improvement methods alone. Integrating modern genetic and genomic techniques with conventional breeding will expedite tree domestication. Breeders will only embrace these technologies if they are cost-effective and readily accessible, and forest landowners will only adopt end-products that meet with regulatory approval and public acceptance. All parties involved must work together to achieve these objectives for the benefit of society.