Local atrial natriuretic peptide signaling prevents hypertensive cardiac hypertrophy in endothelial nitric-oxide synthase-deficient mice

The crucial functions of atrial natriuretic peptide (ANP) and endothelial nitric oxide/NO in the regulation of arterial blood pressure have been emphasized by the hypertensive phenotype of mice with systemic inactivation of either the guanylyl cyclase-A receptor for ANP (GC-A-/-) or endothelial nitric-oxide synthase (eNOS-/-). Intriguingly, similar levels of arterial hypertension are accompanied by marked cardiac hypertrophy in GC-A-/-, but not in eNOS-/-, mice, suggesting that changes in local pathways regulating cardiac growth accelerate cardiac hypertrophy in the former and protect the heart of the latter. Our recent observations in mice with conditional, cardiomyocyte-restricted GC-A deletion demonstrated that ANP locally inhibits cardiomyocyte growth. Abolition of these local, protective effects may enhance the cardiac hypertrophic response of GC-A-/- mice to persistent increases in hemodynamic load. Notably, eNOS-/- mice exhibit markedly increased cardiac ANP levels, suggesting that increased activation of cardiac GC-A can prevent hypertensive heart disease. To test this hypothesis, we generated mice with systemic inactivation of eNOS and cardiomyocyte-restricted deletion of GC-A by crossing eNOS-/- and cardiomyocyte-restricted GC-A-deficient mice. Cardiac deletion of GC-A did not affect arterial hypertension but significantly exacerbated cardiac hypertrophy and fibrosis in eNOS-/- mice. This was accompanied by marked cardiac activation of both the mitogen-activated protein kinase (MAPK) ERK 1/2 and the phosphatase calcineurin. Our observations suggest that local ANP/GC-A/cyclic GMP signaling counter-regulates MAPK/ERK- and calcineurin/nuclear factor of activated T cells-dependent pathways of cardiac myocyte growth in hypertensive eNOS-/- mice.     

Differential Impacts of Copepods and Cladocerans on Lake Seston,and Resulting Effects on Zooplankton Growth

In an enclosure study in Schöhsee, a small mesotrophic lake in Northern Germany, the impact of copepods and daphniids on the seston community was studied. In general, these two guilds differ in their feeding behaviour. Copepods actively select their food, with a preference for larger particles, whereas most cladocerans are unselective filter-feeders. In this study we investigate how the impact of the two different grazers affects zooplankton growth. We combine results obtained in the laboratory with results measured in situ in the enclosures. Copepods and cladocerans were cultured on seston from enclosures that were inhabited by density gradients of copepods or daphniids. We observed that Daphnia grew faster on seston that was pre-handled by copepods than on seston that was pre-handled by daphniids, and that somatic growth decreased with increasing densities of daphniids in the enclosures. In contrast, we observed no differences in development rates for copepods grown on the different media. The population growth rates of Daphnia in the Daphnia treatments were determined in the enclosures. Growth differences in both somatic- and population growth of Daphnia were correlated to food quality aspects of the seston. In the laboratory we found that Daphnia growth was correlated with several fatty acids. The strongest regression was with the concentration of 20:43 (r ²= 0.37). This particular fatty acid also showed the highest correlation with growth after normalisation of the fatty acids to the carbon content of the enclosures (r ²= 0.33). On the other hand, in the enclosure the population growth correlated most to the particulate nitrogen content (r ²= 0.78) and only to the N:C ratio, when normalised to carbon (r ²= 0.51).     

One Enzyme,Two Functions: PON2 PREVENTS MITOCHONDRIAL SUPEROXIDE FORMATION AND APOPTOSIS INDEPENDENT FROM ITS LACTONASE ACTIVITY*

The human enzyme paraoxonase-2 (PON2) has two functions, an enzymatic lactonase activity and the reduction of intracellular oxidative stress. As a lactonase, it dominantly hydrolyzes bacterial signaling molecule 3OC12 and may contribute to the defense against pathogenic Pseudomonas aeruginosa. By its anti-oxidative effect, PON2 reduces cellular oxidative damage and influences redox signaling, which promotes cell survival. This may be appreciated but also deleterious given that high PON2 levels reduce atherosclerosis but may stabilize tumor cells. Here we addressed the unknown mechanisms and linkage of PON2 enzymatic and anti-oxidative function. We demonstrate that PON2 indirectly but specifically reduced superoxide release from the inner mitochondrial membrane, irrespective whether resulting from complex I or complex III of the electron transport chain. PON2 left O₂^˙̄ dismutase activities and cytochrome c expression unaltered, and it did not oxidize O₂^˙̄ but rather prevented its formation, which implies that PON2 acts by modulating quinones. To analyze linkage to hydrolytic activity, we introduced several point mutations and show that residues His¹¹⁴ and His¹³³ are essential for PON2 activity. Further, we mapped its glycosylation sites and provide evidence that glycosylation, but not a native polymorphism Ser/Cys³¹¹, was critical to its activity. Importantly, none of these mutations altered the anti-oxidative/anti-apoptotic function of PON2, demonstrating unrelated activities of the same protein. Collectively, our study provides detailed mechanistic insight into the functions of PON2, which is important for its role in innate immunity, atherosclerosis, and cancer.     

Antiamoebins, myrocin B and the basis of antifungal antibiosis in the coprophilous fungus Stilbella erythrocephala (syn. S. fimetaria)

Antiamoebins I, III and XVI as well as several others in minor amounts were produced by four strains of the coprophilous fungus Stilbella erythrocephala (syn. S. fimetaria) in its natural substrate and in liquid culture. The total antiamoebin concentration in dung was 126-624 microg g(-1) fresh weight, with minimum inhibitory concentrations against most other coprophilous fungi being at or below 100 microg mL(-1). Myrocin B, not previously described from S. erythrocephala, was also produced, but only at low, nonfungicidal levels (< 5.3 microg g(-1)). No other antifungal substances were detected. It is concluded that antiamoebins are responsible for antibiosis in dung colonized by S. erythrocephala.     

White-rots, chlorine and the environment - a tale of many twists

White-rot fungi possess a unique oxidative mechanism by which the recalcitrant lignin component of wood is mineralised. The activity of lignin-degrading enzymes, chiefly lignin and manganese peroxidases, depends on several small organic molecules. Some of these (e.g. chloroanisyl alcohols) are chloroaromatics and may act as environmental pollutants in the forest soil, whereas the synthesis of others (e.g. veratryl alcohol) requires chloromethane. Certain white-rot genera, notably Phellinus and Inonotus, release excess quantities of chloromethane into the atmosphere where it acts as a greenhouse gas. On the other hand, their powerful ligninolytic system enables white-rot fungi to degrade a wide range of man-made environmental pollutants, including recalcitrant chloroaromatics such as DDT, PCP, 2,4-D and 2,4,5-T. This review describes the multifarious interactions of white-rot fungi with their environment via the chlorine cycle.     

An NADPH and FAD dependent enzyme catalyzes hydroxylation of flavonoids in position 8

Yellow flavonols contribute to flower pigmentation in Asteraceae. In contrast to common flavonols, they show additional hydroxyl groups in position 6 and/or 8 of the aromatic A-ring in addition to the basic 5,7-hydroxylation pattern. An enzyme introducing a hydroxyl group in position 8 of flavonols and flavones was demonstrated for the first time with enzyme preparations from petals of Chrysanthemum segetum. Flavanones, dihydroflavonols and glucosylated flavonols and flavones were not accepted as substrates. The enzyme was localized in the microsomal fraction and uses NADPH and FAD as cofactors. Experiments with carbon monoxide/blue light and with antibodies specific for cytochrome P450 reductase did not indicate the involvement of a classical cytochrome P450 dependent monooxygenase in the reaction. Thus, the flavonoid 8-hydroxylase represents a novel type of hydroxylating enzyme in the flavonoid pathway. Apart from flavonoid 8-hydroxylase activity, the presence of all enzymes involved in the formation of flavonoid 7-O-glucosides in C. segetum was demonstrated. The pathway leading to 8-hydroxyflavonoids in C. segetum has been derived from enzyme activities and substrate specificities observed.     

A versatile library of activity-based probes for fluorescence detection and/or affinity isolation of lipolytic enzymes

This work describes the synthesis of a library of fluorescent and/or biotinylated alkylphosphonate inhibitors being reactive towards serine hydrolases, especially lipases and esterases. Fluorescent inhibitors can be used for sensitive and rapid detection of active proteins by gel electrophoresis. Biotinylated inhibitors are applicable for the enrichment and isolation of active enzymes. Functionality as well as the different detection methods of the synthesized inhibitors were successfully tested with an enzyme preparation, namely cholesterol esterase from porcine pancreas (ppCE). Moreover, a biotinylated inhibitor was employed to enrich ppCE on avidin beads. Hence, our set of phosphonate inhibitors can be used for the detection and/or isolation of active serine hydrolases.