Effects of chronic food restriction and treatments with leptin or ghrelin on different reproductive parameters of male rats

The existence of a close relationship between energy status and reproductive function is well-documented, especially in females, but its underlying mechanisms remain to be fully unfolded. This study aimed to examine the effects of restriction of daily calorie intake, as well as chronic treatments with the metabolic hormones leptin and ghrelin, on the secretion of different reproductive hormones, namely pituitary gonadotropins and prolactin, as well as testosterone, in male rats. Restriction (50%) in daily food intake for 20 days significantly reduced body weight as well as plasma PRL and T levels, without affecting basal LH and FSH concentrations and testicular weight. Chronic administration of leptin to rats fed ad libitum increased plasma PRL levels and decreased circulating T, while it did not alter other hormonal parameters under analysis. In contrast, in rats subjected to 50% calorie restriction, leptin administration increased plasma T levels and reduced testis weight. Conversely, ghrelin failed to induce major hormonal changes but tended to increase testicular weight in fed animals, while repeated ghrelin injections in food-restricted males dramatically decreased plasma LH and T concentrations and reduced testis weight. In sum, we document herein the isolated and combined effects of metabolic stress (50% food restriction) and leptin or ghrelin treatments on several reproductive hormones in adult male rats. Overall, our results further stress the impact and complex way of action of different metabolic cues, such as energy status and key hormones, in reproductive function also in the male.     

TGF-β binding in human Wharton’s jelly

Our previous study reported that TGF-β may be isolated from human Wharton’s jelly (WJ) in a form of soluble, high molecular complex(es). We decided to study the effect of extracellular matrix degradation and reduction of disulphide bridges reduction on the release of TGF-β from WJ. The WJ prepared from the umbilical cords of newborns delivered at term by healthy mothers was homogenised and treated with hyaluronidase, collagenase, heparinase, chondroitinase and β-mercaptoethanol, the resulting extracts were then submitted to TGF-β immunoassay and SDS/PAGE followed by Western immunoblotting. The effect of metalloproteinase activation on TGF-β was also studied. Pre-treatment of WJ homogenates with hyaluronidase or collagenase markedly increased the extractability of TGF-β, but did not dissociate the complexes. In contrast, the action of β-mercaptoethanol resulted in the release of free TGF-β; but activation of metalloproteinases resulted in the disappearance of this factor. We conclude that TGF-β1 is bound through disulphide bonds to an extracellular matrix component of WJ. The large amount of collagen fibrils and hyaluronate molecules which surround the cells scattered in WJ may prevent the access of extracting solution to TGF-β causing a low extractability of this factor. Although hyaluronate and collagen do not bind TGF-β directly, they may present a barrier that prevents the diffusion of TGF-β in WJ and results in its concentration around the cells thereby facilitating its interaction with membrane receptors and subsequent stimulation of cell division and synthesis of extracellular matrix components.     

Effects of Proteus mirabilis lipopolysaccharides with different O-polysaccharide structures on the plasma membrane of human erythrocytes

The effects of O33 and O49 P. mirabilis lipopolysaccharides (LPSs) on human erythrocyte membrane properties were examined. Physical parameters of the plasma membrane, such as membrane lipid fluidity, physical state of membrane proteins, and osmotic fragility, were determined. The fluidity of the lipids was estimated using three spin-labeled stearic acids of doxyl derivatives: 5-doxylstearic acid, 12-doxylstearic acid, and 16-doxylstearic acid. All the applied labels locate to different depths of the lipid layer and provide information on the ordering of phospholipid fatty acyl chain mobility. LPSs O49 increased the membrane lipid fluidity in the polar region of the lipid bilayer as indicated by spin-labeled 5-doxylstearic acid. An increase in fluidity was also observed in the deeper region using 12-doxylstearic acid only for O33 LPSs. The highest concentration of O33 LPSs (1 mg/ml) increased the motion of membrane proteins detected by the spin-label residue of iodoacetamide. These results showed different actions of O33 and O49 LPSs on the plasma membrane due to the different chemical structures of O-polysaccharides. P. mirabilis O33 and O49 LPSs did not induce changes in the membrane cytoskeleton, osmotic fragility and lipid peroxidation of erythrocytes. On the other hand a rise in the content of carbonyl compounds was observed for the highest concentrations of O33 LPS. This result indicated protein oxidation in the erythrocyte membrane. Lipid A, the hydrophobic part of LPS, did not change the membrane lipid fluidity and osmotic fragility of erythrocytes. Smooth and rough forms of P. mirabilis LPSs were tested for their abilities for complement-mediated immunohemolysis of erythrocytes. Only one out of seven LPSs used was a potent agent of complement-mediated hemolysis. It was rough, Ra-type of P. mirabilis R110 LPS. The O-polysaccharide-dependent scheme of reaction is presented.     

Phospholipid-induced structural changes to an erythroid beta spectrin ankyrin-dependent lipid-binding site

The region of beta-spectrin that is responsible for interactions with ankyrin was shown to comprise an ankyrin-sensitive lipid-binding site. Structural studies indicate that it exhibits a mixed 3(10)/alpha helical conformation and is highly amphipathic. These features together with the distinctively conserved sequence of the lipid-binding site motivated us to explore the mechanism of its interactions with biological membranes. A series of singly and doubly spin-labeled erythroid beta-spectrin-derived peptides was constructed, and the spin-label mobility and spin-spin distances were analyzed via electron paramagnetic resonance spectroscopy and two different calculation methods. The results indicate that in beta-spectrin, the lipid-binding domain, which is part of the 14(th) segment, has the topology of typical triple-helical spectrin repeat. However, it undergoes significant changes when interacting with phospholipids or detergents. A mechanism for these interactions is proposed in this paper.     

Photodynamic effect of protoporphyrin diarginate (PPArg2) on methicillin-resistant Staphylococcus aureus and human dermal fibroblasts

The worldwide rise in the antibiotic resistance of bacteria forces the development of alternative antimicrobial treatments. A potential approach is photodynamic inactivation (PDI). The aim of the present study was to determine the phototoxicity of protoporphyrin diarginate (PPArg(2)) against methicillin-resistant Staphylococcus aureus and human dermal fibroblasts. Different concentrations (0 to 20 microM) of PPArg(2) and light dose of 6 J cm(-2) were tested. Cell viability was evaluated using the methylthiazoletetrazolium (MTT) assay. Incubation with 10 microM followed by illumination yielded a 3.6 log(10)-unit reduction in the viable count for Staphylococcus aureus. At the same experimental conditions, only 22.5% of the fibroblasts were photoinactivated. Protoporphyrin diarginate at concentrations up to 20 microM demonstrated no toxicity towards S. aureus or fibroblasts when not irradiated. These results suggest that the protoporphyrin diarginate exerts a high bactericidal effect against methicillin-resistant S. aureus strain without harming eukaryotic cells.     

Behavioural and spatial adaptation of the Eurasian lynx to a decline in prey availability

The distribution and abundance of food resources is a major factor influencing animal populations. I studied the effect of a roe and red deer population decline on diet composition, home range size and foraging pattern in the Eurasian lynxLynx lynx (Linnaeus, 1758) in the Bia?owie?a Primeval Forest (BPF), eastern Poland. The population of cervids in BPF experienced a nearly two-fold reduction in size from 1991 through 2006 due to severe hunting pressure between 1991 and 1996. Comparison of published data on lynx diet during the high abundance of ungulates with new data obtained for the low abundance period showed that despite a significant decline in their availability, cervids (roe and red deer) continued to form the majority of the diet of lynx, with roe deer being most preferred in both periods. Home range sizes of lynx showed a tendency to increase with declining prey densities, as indicated by relative percentage increases in average yearly home range sizes amongst different sex/age groups. In response to lower availability of their main prey, lynx increased their daily straight-line movement distances by 44% and doubled the ranges covered in 5-day periods. This illustrated that, with declining prey abundance, the lynx increased their hunting efforts by either spending more time actively searching for prey or continuing foraging even after a successful hunt. Spatial analysis of the distribution of ungulates and lynx indicated that deer were evenly distributed throughout lynx ranges in BPF and spatial proximity of the predator to prey sites did not play an important role in the efficiency of hunting. Lynx may adapt to changing prey availability by increasing search effort, but this was not sufficient to prevent the negative influences of the prey decline on the lynx population. Prey depletion has an immediate effect on lynx spatial organization and, in consequence, on their density. This information has to be considered in prioritizing lynx conservation measures and management of ungulates.     

Microhabitat selection by Eurasian lynx and its implications for species conservation

We studied microhabitat selection of the Eurasian lynxLynx lynx (Linnaeus, 1758) at 116 hunting and 88 resting sites in Białowieża Primeval Forest (Poland) to describe its characteristics and determine the importance of habitat structure for stalking prey and for security during resting. We identified lynx-used sites by radio-tracking 3 male and 3 female lynx. When hunting, the lynx did not select for any type or age class of forest. During both summer and winter, the lynx selected sites characterized by high complexity (number of structures useful for stalking: fallen logs and branches, root plates, patches of dense bushes) and low visibility. In summer, hunting sites were often located in the vicinity of small forest glades that provided good stalking opportunities for lynx and rich foraging resources for roe deer — the main prey of lynx. The habitat at kill sites was more open than at sites where the prey was cached, with higher visibility, lower density of trees and poorer undergrowth. The most important characteristic of resting sites was very low visibility that resulted mainly from using young pine or spruce thickets in the winter and dense undergrowth of oak-lime-hornbeam and ash-alder forests in the summer. The information provided by this study could have direct implications for Eurasian lynx conservation by guiding forest restructuring to better suit the species’ biological requirements.     

Design of selective substrates of proteinase 3 using combinatorial chemistry methods

In this study, chemical synthesis of the selective chromogenic/fluorogenic substrates for proteinase 3 is described. The substrates’ sequence was obtained using combinatorial chemistry methods. Deconvolution of the tripeptide library against proteinase 3 with general formula ABZ-X₃-X₂-X₁-ANB-NH₂ yielded the active sequence. Selected peptide was further modified on its C terminus to investigate the impact of chromophore moiety modification on enzyme-substrate interaction. To determine specificity, activity of selected substrates was characterized against proteinase 3 and neutrophil elastase. Finally, the peptide ABZ-Tyr-Tyr-Abu-ANB-NH₂ displayed the highest value of specificity constant (k_cat/K_M = 189 × 10³ M⁻¹ s⁻¹) for proteinase 3. To the best of our knowledge, this is the first short peptide that undergoes selective proteolysis by proteinase 3 and displays no significant hydrolysis in the presence of human neutrophil elastase and cathepsin G.