Measurement of exhaled hydrogen peroxide from rabbit lungs

Exhaled H2O2 is considered an indicator of lung inflammatory and oxidative stress. Moreover, H2O2 may be involved in signal transduction processes. It is not fully elucidated to what extent (i) H2O2 escapes from the intravascular compartment, and (ii) pulmonary H2O2 generation and nasopharyngeal H2O2 generation contribute to exhaled H2O2. We investigated H2O2 concentrations in breath condensate from isolated buffer-perfused and ventilated rabbit lungs, and from both intubated and spontaneously breathing rabbits with a horseradish peroxidase/2',7'dichlorofluorescin assay. For the perfused lungs, a H2O2 concentration of 58 +/- 19 nM was found. Addition of H2O2 to the buffer fluid resulted in only minute appearance in the exhaled air (<0.001%). Levels of exhaled H2O2 in intubated rabbits and perfused lungs were virtually identical. Nearly ten-fold higher levels were detected in spontaneously breathing rabbits. Decreasing the inspired oxygen concentration from 21% to 1% resulted in a tendency toward decreased H2O2 exhalation in perfused lungs. In contrast, phorbol-12-myristate-13-acetate (PMA) prompted a approximately 4-fold increase in H2O2 exhalation. We conclude that the horseradish peroxidase/2',7'dichlorofluorescin assay is a feasible technique to measure H2O2 in exhaled breath condensate in rabbits. When collecting exhaled air via the tracheal tube, the signal represents pulmonary H2O2 generation with the contribution of the remaining body being negligible.     

Activation of the human neutrophil by calcium-mobilizing ligands. II. Correlation of calcium, diacyl glycerol, and phosphatidic acid generation with superoxide anion generation

Calcium and protein kinase C (Ca2+/phospholipid-dependent enzyme) have been proposed to act as signals in triggering superoxide anion (O2-) generation by neutrophils. We have probed the adequacy and necessity of calcium and diacylglycerol (DG), activators of protein kinase C, in eliciting O2- generation and degranulation. Activation of neutrophils by the ligand 10(-7) M fMet-Leu-Phe triggered elevation of cytosolic calcium (fura-2) and a rapid, biphasic increase in labeled DG in [14C]glycerol and [3H]arachidonate prelabeled cells. Buffering of the fMet-Leu-Phe-induced elevation of cytosolic calcium with MAPTAM (a cell permeant EGTA analogue) inhibited O2- generation by 90% and degranulation by 50%, concordant with a role of calcium in signaling. However, buffering the increase in calcium also decreased DG. Since phosphatidylinositol 4,5-bisphosphate breakdown in response to fMet-Leu-Phe was not inhibited and phosphatidic acid levels were enhanced in MAPTAM pretreated cells, the removal of calcium may enhance further DG metabolism. Thus, a requirement for calcium could not be differentiated from a requirement for DG, and the profound inhibition of O2- generation in the presence of MAPTAM may reflect removal of DG. Four stimuli, fMet-Leu-Phe, 10(-7) M leukotriene B4, 100 micrograms/ml concanavalin A, and 200 nM ionomycin elevated cytosolic calcium and triggered release of specific granules, but only fMet-Leu-Phe and concanavalin A triggered substantial O2- generation. Nevertheless, all four stimuli significantly increased labeled DG. Therefore, elevated DG and elevated calcium may be necessary but do not appear adequate to elicit O2- generation. Only fMet-Leu-Phe and concanavalin A triggered generation of phosphatidic acid (PA) together with DG. Correlation of O2- generation with PA may reflect a requirement for PA per se or for a specific pool of DG that can be further metabolized to PA.     

Functional and Muscular Adaptations in an Experimental Model for Isometric Strength Training in Mice

Exercise training induces muscular adaptations that are highly specific to the type of exercise. For a systematic study of the differentiated exercise adaptations on a molecular level mouse models have been used successfully. The aim of the current study was to develop a suitable mouse model of isometric strength exercise training characterized by specific adaptations known from strength training. C57BL/6 mice performed an isometric strength training (ST) for 10 weeks 5 days/week. Additionally, either a sedentary control group (CT) or a regular endurance training group (ET) groups were used as controls. Performance capacity was determined by maximum holding time (MHT) and treadmill spirometry, respectively. Furthermore, muscle fiber types and diameter, muscular concentration of phosphofructokinase 1 (PFK), succinate dehydrogenase (SDHa), and glucose transporter type 4 (GLUT4) were determined. In a further approach, the effect of ST on glucose intolerance was tested in diabetic mice. In mice of the ST group we observed an increase of MHT in isometric strength tests, a type II fiber hypertrophy, and an increased GLUT4 protein content in the membrane fraction. In contrast, in mice of the ET group an increase of VO_2max, a shift to oxidative muscle fiber type and an increase of oxidative enzyme content was measured. Furthermore strength training was effective in reducing glucose intolerance in mice fed a high fat diet. An effective murine strength training model was developed and evaluated, which revealed marked differences in adaptations known from endurance training. This approach seems also suitable to test for therapeutical effects of strength training.     

Cigarette Smoke-Induced Emphysema and Pulmonary Hypertension Can Be Prevented by Phosphodiesterase 4 and 5 Inhibition in Mice

Rationale

Chronic obstructive pulmonary disease (COPD) is a widespread disease, with no curative therapies available. Recent findings suggest a key role of NO and sGC-cGMP signaling for the pathogenesis of the disease. Previous data suggest a downregulation/inactivation of the cGMP producing soluble guanylate cyclase, and sGC stimulation prevented cigarette smoke-induced emphysema and pulmonary hypertension (PH) in mice. We thus aimed to investigate if the inhibition of the cGMP degrading phosphodiesterase (PDE)5 has similar effects. Results were compared to the effects of a PDE 4 inhibitor (cAMP elevating) and a combination of both.

Methods

C57BL6/J mice were chronically exposed to cigarette smoke and in parallel either treated with Tadalafil (PDE5 inhibitor), Piclamilast (PDE4 inhibitor) or both. Functional measurements (lung compliance, hemodynamics) and structural investigations (alveolar and vascular morphometry) as well as the heart ratio were determined after 6 months of tobacco smoke exposure. In addition, the number of alveolar macrophages in the respective lungs was counted.

Results

Preventive treatment with Tadalafil, Piclamilast or a combination of both almost completely prevented the development of emphysema, the increase in lung compliance, tidal volume, structural remodeling of the lung vasculature, right ventricular systolic pressure, and right ventricular hypertrophy induced by cigarette smoke exposure. Single, but not combination treatment prevented or reduced smoke-induced increase in alveolar macrophages.

Conclusion

Cigarette smoke-induced emphysema and PH could be prevented by inhibition of the phosphodiesterases 4 and 5 in mice.     

Early fibrosis inhibits hepatocellular carcinoma mediated by free radical effects

We studied the in-vitro/in-vivo interactions between HCC/HSCs in early and advanced fibrosis-models. Hep3B-mono-cultures secreted high levels of αFetoProtein (αFP). Human-HSCs co-cultured with Hep3B-cells significantly decreased αFP and increased their apoptosis. Confocal-microscopy demonstrated Hep3B-phagocytosis inside the HSCs suggesting a direct cellular-contact mediating anti-tumor effect. Leptin-activated HSCs further suppressed Hep3B-cells with increased ROS and decreased GSH. Following intrahepatic-Hep3B-cell injections, mice with established “advanced liver-fibrosis”; had higher tumor-size and αFP serum-levels as compared to non-fibrotic livers. Mice with “early liver-fibrosis”, which initiated post tumor induction had a significant decrease in tumor and high Malondialdehyde (MDA) serum levels compared to advanced-fibrosis animals.At early-fibrosis stages, activated-HSCs express direct anti-tumor effects by phagocytosis and apoptosis of tumor-cells mediated by oxidative stress.     

ARTS, the unusual septin: structural and functional aspects

The human Septin 4 gene (Sept4) encodes two major protein isoforms; Sept4_i1 (H5/PNUTL2) and Sept4_i2/ARTS. Septins have been traditionally studied for their role in cytokinesis and their filament-forming abilities, but subsequently have been implicated in diverse functions, including membrane dynamics, cytoskeletal reorganization, vesicle trafficking, and tumorigenesis. ARTS is localized at mitochondria and promotes programmed cell death (apoptosis). These features distinguish ARTS from any other known human septin family member. This review compares the structural and functional properties of ARTS with other septins. In addition, it describes how a combination of two distinct promoters, differential splicing, and intron retention leads to the generation of two different Sept4 variants with diverse biological activity.