Peptitergent PD1 affects the GTPase activity of rat brain cortical membranes.

Peptitergent PD1 shows complex effects on GTPase activity of rat brain cortical membranes: inhibition in the presence of lower concentrations of GTP and activation at a higher concentration, above 0.5 microM, of GTP. Its effect is dose dependent and is characterized by an EC50 of 1.8 +/- 0.2 microM and a Hill coefficient of 1.6 +/- 0.3, and it increases both Km and Vmax of the GTP hydrolysis. PD1 that was unable to solubilize G-proteins from the membranes probably acts on them by direct binding near the C-terminal alpha-helical region of the Galpha subunit, similarly to mastoparan.     

Aging does not alter the mechanosensitivity of the p38, p70S6k, and JNK2 signaling pathways in skeletal muscle.

The capacity for skeletal muscle to recover its mass following periods of unloading (regrowth) has been reported to decline with age. Although the mechanisms responsible for the impaired regrowth are not known, it has been suggested that aged muscles have a diminished capacity to sense and subsequently respond to a given amount of mechanical stimuli (mechanosensitivity). To test this hypothesis, extensor digitorum longus muscles from young (2-3 mo) and old (26-27 mo) mice were subjected to intermittent 15% passive stretch (ex vivo) as a source of mechanical stimulation and analyzed for alterations in the phosphorylation of stress-activated protein kinase (p38), ribosomal S6 kinase (p70S6k), and the p54 jun N-terminal kinase (JNK2). The results indicated that the average magnitude of specific tension (mechanical stimuli) induced by 15% stretch was similar in muscles from young and old mice. Young and old muscles also revealed similar increases in the magnitude of mechanically induced p38, p70S6k (threonine/serine 421/424 and threonine 389), and JNK2 phosphorylation. In addition, coincubation experiments demonstrated that the release of locally acting growth factors was not sufficient for the induction of JNK2 phosphorylation, suggesting that JNK2 was activated by a mechanical rather than a mechanical/growth factor-dependent mechanism. Taken together, the results of this study demonstrate that aging does not alter the mechanosensitivity of the p38, p70S6k, and JNK2 signaling pathways in skeletal muscle.     

The quality of the aquatic environment and macrophytes of karstic watercourses

The quality of the aquatic environment and macrophytes were surveyed in the case of the remarkable karstic river Ljubljanica s. lat. (Slovenia). It consists of seven intermittent and permanent surface watercourses, connected by groundwater flows. We assessed 11 parameters describing the land use beyond the riparian zone, the structure of the riparian zone and stream channel morphology, as well as the distribution and abundance of macrophytes. The intermittent watercourses exhibited a relatively high level of naturalness, because the variable water regime prevents intensive human activity. Greater influence of the human activity on the permanent watercourses resulted in a modified riverine environment. The share of different ecological groups of macrophytes, i.e. submerged, emerged and amphibious species, reflected the water regimes of the different watercourses. A total of 62 taxa of macrophytes were identified in about 100 km of length examined. Of these, amphibious macrophytes dominated as a consequence of intermittent water regime. The relationship between environmental parameters and macrophytes, and the effects of the intermittent hydrology were investigated. All environmental parameters explained 28% of the variance in the distribution of macrophytes in watercourses, as determined by canonical correspondence analysis. Completeness of the riparian zone, bank undercutting and sediment accumulation were found to be the most influential.     

Biocompatibility of different nanostructured TiO<Subscript>2</Subscript> scaffolds and their potential for urologic applications

Despite great efforts in tissue engineering of the ureter, urinary bladder, and urethra, further research is needed in order to improve the patient’s quality of life and minimize the economic burden of different lower urinary tract disorders. The nanostructured titanium dioxide (TiO₂) scaffolds have a wide range of clinical applications and are already widely used in orthopedic or dental medicine. The current study was conducted to synthesize TiO₂ nanotubes by the anodization method and TiO₂ nanowires and nanospheres by the chemical vapor deposition method. These scaffolds were characterized with scanning electron microscopy (SEM) and X-ray diffraction (XRD) methods. In order to test the urologic applicability of generated TiO₂ scaffolds, we seeded the normal porcine urothelial (NPU) cells on TiO₂ nanotubes, TiO₂ nanowires, TiO₂ nanospheres, and on the standard porous membrane. The viability and growth of the cells were monitored everyday, and after 3 weeks of culturing, the analysis with scanning electron microscope (SEM) was performed. Our results showed that the NPU cells were attached on all scaffolds; they were viable and formed a multilayered epithelium, i.e., urothelium. The apical plasma membrane of the majority of superficial NPU cells, grown on all three different TiO₂ scaffolds and on the porous membrane, exhibited microvilli; thus, indicating that they were at a similar differentiation stage. The maximal caliper diameter measurements of superficial NPU cells revealed significant alterations, with the largest cells being observed on nanowires and the smallest ones on the porous membrane. Our findings indicate that different nanostructured TiO₂ scaffolds, especially nanowires, have a great potential for tissue engineering and should be further investigated for various urologic applications.     

Cell-Penetrating Mimics of Agonist-Activated G-Protein Coupled Receptors

Cell-penetrating peptides have proven themselves as valuable vectors for intracellular delivery. Relatively little is known about the frequency of cell-penetrating sequences in native proteins and their functional role. By computational comparison of peptide sequences, we recently predicted that intracellular loops of G-protein coupled receptors (GPCR) have high probability for occurrence of cell-penetrating motifs. Since the loops are also receptor and G-protein interaction sites, we postulated that the short cell-penetrating peptides, derived from GPCR, when applied extracellularly can pass the membrane and modulate G-protein activity similarly to parent receptor proteins. Two model systems were analyzed as proofs of the principle. A peptide based on the C-terminal intracellular sequence of the rat angiotensin receptor (AT1AR) is shown to internalize into live cells and elicit blood vessel contraction even in the presence of AT1AR antagonist Sar¹-Thr⁸-angiotensin II. The peptide interacts with the same selectivity towards G-protein subtypes as agonist-activated AT1AR and blockade of phospholipase C abolishes its effect. Another cell-penetrating peptide, G53-2 derived from human glucagon-like peptide receptor (GLP-1R) is shown to induce insulin release from isolated pancreatic islets. The mechanism was again found to be shared with the original GLP-1R, namely G₁₁-mediated inositol 1,4,5-triphosphate release pathway. These data reveal a novel possibility to mimic the effects of signalling transmembrane proteins by application of shorter peptide fragments.     

Spatial pattern of native species Myriophyllum spicatum and invasive alien species Elodea nuttallii after introduction of the latter one into the Drava River (Slovenia)

Invasive alien species Elodea nuttallii was introduced into the Drava River (Slovenia) in 2007. The spatial distribution of native macrophyte species Myriophyllum spicatum and the invasive alien species E. nuttallii was studied in two impoundments, HPP Vuhred and HPP Mariborski otok in the years 2009, 2010 and 2011. The very heterogeneous environment of the Drava River had resulted in dynamic and non-uniform distributions of E. nuttalli and M. spicatum in both impoundments. The distribution of E. nuttalli was shown to be suppressed in river stretches exposed to great turbulence. It had, however, successfully invaded areas with gentle slope of littoral, low exposition to main water flow and muddy sediment and prevailed against the native species M. spicatum. The latter was more abundant than E. nuttallii in stretches exposed to higher flow velocity. Thus, the competitive success of M. spicatum and E. nuttallii depended on environmental conditions. Our results lead to the prediction that E. nuttallii will increase further in the river Drava, causing serious economic problems.     

Selective binding of lectins to normal and neoplastic urothelium in rat and mouse bladder carcinogenesis models

Bladder cancer adjuvant intravesical therapy could be optimized by more selective targeting of neoplastic tissue via specific binding of lectins to plasma membrane carbohydrates. Our aim was to establish rat and mouse models of bladder carcinogenesis to investigate in vivo and ex vivo binding of selected lectins to the luminal surface of normal and neoplastic urothelium. Male rats and mice were treated with 0.05 % N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water and used for ex vivo and in vivo lectin binding experiments. Urinary bladder samples were also used for paraffin embedding, scanning electron microscopy and immunofluorescence labelling of uroplakins. During carcinogenesis, the structure of the urinary bladder luminal surface changed from microridges to microvilli and ropy ridges and the expression of urothelial-specific glycoproteins uroplakins was decreased. Ex vivo and in vivo lectin binding experiments gave comparable results. Jacalin (lectin from Artocarpus integrifolia) exhibited the highest selectivity for neoplastic compared to normal urothelium of rats and mice. The binding of lectin from Amaranthus caudatus decreased in rat model and increased in mouse carcinogenesis model, indicating interspecies variations of plasma membrane glycosylation. Lectin from Datura stramonium showed higher affinity for neoplastic urothelium compared to the normal in rat and mouse model. The BBN-induced animal models of bladder carcinogenesis offer a promising approach for lectin binding experiments and further lectin-mediated targeted drug delivery research. Moreover, in vivo lectin binding experiments are comparable to ex vivo experiments, which should be considered when planning and optimizing future research.