Microtubules and cyclic amp in human leukocytes: on the order of things

We have shown previously that the β-adrenergic agonist isoproterenol (2μM) and the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) produce a much greater increase in cyclic AMP in human leukocytes that have been pretreated with colchicine (or with other agents that affect microtubule assembly) than in control leukocytes. The effects of colchicines were both time- and dose-dependant. These and other data suggested that the generation of cyclic AMP is normally restricted by an intact system of cytoplasmic microtubules. If so, then the same time and dose dependencies might apply to other colchicines-induced changes in leukocyte function. We have now assayed the distribution of concanavalin A (Con A)-receptor complexes on the leukocyte membrane, taking into account that leukocytes competent to assemble microtubules show a uniform distribution of surface- bound Con A whereas microtubule-deficient cells accumulate Con A in surface caps. We have found that the effect of colchicine on capping is also both time- and dose dependent, and that the dose-response relationships conform to those required to increase cyclic AMP levels. These findings provide further evidence that both colchicine-induced Con-A capping and colchicine- induced cyclic AMP generation depend upon the relaxation of constraints normally imposed by cytoplasmic microtubules upon the plasma membrane, which limit, respectively, lateral mobility of the lectin-receptor complexes, and expression of hormone-sensitive adenylate cyclase. Moreover, colchicine-induced Con-A cap formation is not affected even by very large changes in leukocyte cyclic AMP levels. Thus, elevated cyclic AMP levels do not appear to promote the dissolution of microtubules; rather, the dissolution of microtubules permits the generation of increased amounts of cyclic AMP.     

Strong influence of long-distance edge effect on herb-layer vegetation in forest fragments in an agricultural landscape

In agricultural landscapes in central Europe, species richness of the herbaceous plant community may be compromised by processes associated with forest fragmentation, habitat loss, and management practices. We examined variability in species richness and composition of the herbaceous layer in 229 plots located in 23 forest fragments (0.1 to 255 ha), in a representative upland agricultural landscape in central Bohemia, in relation to the most important site environmental factors, edge effects, and site history. The influence of environmental factors on the composition of vegetation in the herb layer was evaluated using generalized additive models, which enabled us to analyze highly non-linear and non-monotonic relationships. Total species richness and number of red-listed and ancient forest species were significantly influenced by type of forest vegetation, light quality, soil pH, slope aspect, and distance from the forest edge. Implications of the significant explanatory variables corresponded well to previous findings, with the exception of distance from the forest edge, for which we found a positive relationship with species richness for distances up to 200 m toward the forest interior. Plant species with low colonization ability occupied plots with increasing frequency from edge to forest interior, while fast-colonizing species showed the opposite trend. Apart from the edge effect, forest continuity should be considered for its important contribution to the richness of ancient forest and red-listed species, whereas the effect of forest fragment size appeared to be generally weak. These results do not negate the importance of large forest fragments for the maintenance of herb layer species richness, but specifically emphasize the essential contribution of the core habitats of these forests. In summary, we showed that the negative effects of habitat fragmentation on the richness of ancient forest and red-listed species and on herb layer species in total can be largely attributed to either the edge effect itself or to aggregate effects of forest edge and forest continuity.     

Physiologically relevant oxidative degradation of oligo(proline) cross-linked polymeric scaffolds

Chronic inflammation-mediated oxidative stress is a common mechanism of implant rejection and failure. Therefore, polymer scaffolds that can degrade slowly in response to this environment may provide a viable platform for implant site-specific, sustained release of immunomodulatory agents over a long time period. In this work, proline oligomers of varying lengths (P(n)) were synthesized and exposed to oxidative environments, and their accelerated degradation under oxidative conditions was verified via high performance liquid chromatography and gel permeation chromatography. Next, diblock copolymers of poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) were carboxylated to form 100 kDa terpolymers of 4%PEG-86%PCL-10%cPCL (cPCL = poly(carboxyl-ε-caprolactone); i% indicates molar ratio). The polymers were then cross-linked with biaminated PEG-P(n)-PEG chains, where P(n) indicates the length of the proline oligomer flanked by PEG chains. Salt-leaching of the polymeric matrices created scaffolds of macroporous and microporous architecture, as observed by scanning electron microscopy. The degradation of scaffolds was accelerated under oxidative conditions, as evidenced by mass loss and differential scanning calorimetry measurements. Immortalized murine bone-marrow-derived macrophages were then seeded on the scaffolds and activated through the addition of γ-interferon and lipopolysaccharide throughout the 9-day study period. This treatment promoted the release of H(2)O(2) by the macrophages and the degradation of proline-containing scaffolds compared to the control scaffolds. The accelerated degradation was evidenced by increased scaffold porosity, as visualized through scanning electron microscopy and X-ray microtomography imaging. The current study provides insight into the development of scaffolds that respond to oxidative environments through gradual degradation for the controlled release of therapeutics targeted to diseases that feature chronic inflammation and oxidative stress.     

Richness of ancient forest plant species indicates suitable habitats for macrofungi

Macrofungal species richness generally increases with forest continuity as does the richness of so-called ancient forest plant species (AFS). Based on this assumption, we examined the ability of AFS to indicate macrofungal diversity in six study areas covering a range of elevations and environments in the Czech Republic. In total, we used data from 106 sampling plots (2,500 m² each) distributed over six types of forest stands reflecting different intensities and temporal stages of forest management. Species composition of vascular plants and macrofungi was recorded by a single inventory and regular 2-year monitoring, respectively. In total, we found 71 AFS and 1,413 macrofungal species, of which 150 were red-listed macrofungal species. We documented that AFS show potential for being used in the prediction of macrofungi species richness, including endangered species, at the local scale (α-diversity). Additionally, we found significant differences in macrofungal species richness depending on study area and type of forest management, which did not, however, derogate the effect of AFS. Spatial congruence between species composition of AFS and macrofungi communities (β-diversity) increased with forest age and decreased with intensity of forest management. If we consider the simplicity of monitoring AFS in comparison to regular monitoring of macrofungi, we found a widely usable tool for estimating macrofungal diversity in all dominant types of managed forest in central Europe. However, we should be aware of the limited ability of AFS to capture macrofungal diversity across a broader spatial context (γ-diversity), especially in areas with a low diversity of AFS.     

Assemblages of terrestrial isopods (Isopoda, Oniscidea) in a fragmented forest landscape in Central Europe

Terrestrial isopods were collected in 13 forest fragments differing in area (within the range of 0.1 and 254.5 ha), shape and composition of forest vegetation (thermophilous oak, mesophilous oak-hornbeam, thermophilous oak-hornbeam, acidophilous oak, basiphilous oak, beech oak-hornbeam, moist mixed deciduous forest, plantations of deciduous and coniferous trees), all situated in the Český kras Protected Landscape Area, Czech Republic, Central Europe. Number of sites sampled in each fragment of forest depended on its size and ranged from 1 to 7. Altogether 30 sites were sampled. Soil samples (5 per site collected twice a year) and pitfall trapping (5 traps per site in continuous operation throughout a year) during 2008–2009 yielded a total of 14 species of terrestrial isopods. The highest densities and highest epigeic activities of terrestrial isopods were recorded in the smallest fragments of woodland. Although a wider range of habitats were sampled in the larger fragments of woodland there was not a greater diversity of species there and the population densities and epigeic activities recorded there were lower. Porcellium collicola was most abundant in small fragments of woodland regardless the vegetation there. Armadillidium vulgare and Protracheoniscus politus were statistically more abundant in the larger fragments of woodland. The results indicate that forest fragmentation does not necessarily result in a decrease in the species richness of the isopod assemblages in such habitats.     

17β-Estradiol induces nongenomic effects in renal intercalated cells through G protein-coupled estrogen receptor 1

Steroid hormones such as 17β-estradiol (E2) are known to modulate ion transporter expression in the kidney through classic intracellular receptors. Steroid hormones are also known to cause rapid nongenomic responses in a variety of nonrenal tissues. However, little is known about renal short-term effects of steroid hormones. Here, we studied the acute actions of E2 on intracellular Ca(2+) signaling in isolated distal convoluted tubules (DCT2), connecting tubules (CNT), and initial cortical collecting ducts (iCCD) by fluo 4 fluorometry. Physiological concentrations of E2 induced transient increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in a subpopulation of cells. The [Ca(2+)](i) increases required extracellular Ca(2+) and were inhibited by Gd(3+). Strikingly, the classic E2 receptor antagonist ICI 182,780 also increased [Ca(2+)](i), which is inconsistent with the activation of classic E2 receptors. G protein-coupled estrogen receptor 1 (GPER1 or GPR30) was detected in microdissected DCT2/CNT/iCCD by RT-PCR. Stimulation with the specific GPER1 agonist G-1 induced similar [Ca(2+)](i) increases as E2, and in tubules from GPER1 knockout mice, E2, G-1, and ICI 182,780 failed to induce [Ca(2+)](i) elevations. The intercalated cells showed both E2-induced concanamycin-sensitive H(+)-ATPase activity by BCECF fluorometry and the E2-mediated [Ca(2+)](i) increment. We propose that E2 via GPER1 evokes [Ca(2+)](i) transients and increases H(+)-ATPase activity in intercalated cells in mouse DCT2/CNT/iCCD.