Phosphopeptides with improved cellular uptake properties as ligands for the polo-box domain of polo-like kinase 1

Human polo-like kinase 1 (Plk1) is involved in cell proliferation and overexpressed in a broad variety of different cancer types. Due to its crucial role in cancerogenesis Plk1 is a potential target for diagnostic and therapeutic applications. Peptidic ligands can specifically interact with the polo-box domain (PBD) of Plk1, a C-terminal located phosphoepitope binding motif. Recently, phosphopeptide MQSpTPL has been identified as ligand with high binding affinity. However, a radiolabeled version of this peptide showed only insufficient cellular uptake. The present study investigated peptide dimers consisting of PBD-targeting phosphopeptide MQSpTPL and a cell-penetrating peptide (CPP) moiety. The new constructs demonstrate superior uptake in different cancer cell-lines compared to the phosphopeptide alone. Furthermore, we could demonstrate binding of phosphopeptide-CPP dimers to PBD of Plk1 making the compounds interesting leads for the development of molecular probes for imaging Plk1 in cancer.     

Purification and characterization of a thermodynamic stable serine protease from Aspergillus fumigatus

A thermostable extracellular serine protease from Aspergillus fumigatus was purified 8.8-fold using a 4-step protocol. The enzyme was produced using a 36 h solid-state culture, had a molecular weight of 88 kDa and exhibited maximal enzyme activity at pH 7 and 60 °C. Structural analysis revealed that the protease is monomeric and non-glycosylated. Thermal inactivation of the pure enzyme followed first-order kinetics. The half-life (t_1/2) of the pure enzyme at 50, 60 and 70 °C was 65, 34 and 14 min, respectively. The denaturation and activation energies were 69 and 62 kJ mol⁻¹, respectively. Thermodynamic parameters (entropy and enthalpy) suggested that the protease was highly thermostable. This is the first report on the thermodynamic parameters of proteases produced by A. fumigatus.     

The effect of nutrient level on plant structure and production in a wet grassland: a field study

Since the 1950s, agricultural intensification has affected the structure and functioning of ecological systems including wet grasslands. Our study site, a wet grassland near Třeboň, Czech Republic, was historically a sedge meadow, but increased nutrient additions, a long-lasting flood in 2002 and changed mowing patterns resulted in domination by Phalaris arundinacea. The aim of the study was to determine how different nutrient conditions may affect plant structure and production in a wet grassland used for hay production. Species composition and percent cover were determined from line intercepts. Aboveground biomass was harvested six times each in 2007 and 2008 and primary production then calculated. Ingrowth core bags were used to determine belowground production. Aboveground production was two times greater in the high nutrient versus the low nutrient area in both years, while belowground production was very similar. The high nutrient area was still dominated by P. arundinacea, but Carex gracilis was now a co-dominant in the low nutrient area. However, other factors, such as water level and mowing regime, may interact with nutrient level to govern wet grassland structure and function.     

Latent microsporidial infection in immunocompetent individuals - a longitudinal study

Background

Microsporidia (Fungi) have been repeatedly identified as the cause of opportunistic infections predominantly in immunodeficient individuals such as AIDS patients. However, the global epidemiology of human microsporidiosis is poorly understood and the ability of microsporidia to survive and multiply in immunocompetent hosts remains unsolved.

Aims

To determine the presence of latent microsporidia infections in apparently healthy humans in the Czech Republic, the authors tested sera, urine and stool originating from fifteen persons within a three month period examined on a weekly basis.

Methods

Sera, stool and urine samples originating from fifteen HIV-negative people at risk with occupational exposure to animals, aged 22–56 years, living in the Czech Republic were tested by indirect immunofluorescence assay (IFA) for the presence of specific anti-microsporidial antibodies, standard Calcofluor M2R staining for the detection of microsporidian spores in all urine sediments and stool smears and molecular methods for the microsporidial species determination.

Results

Specific anti-microsporidial antibodies were detected in fourteen individuals, asymptomatic Encephalitozoon spp. infection was found in thirteen and E. bieneusi infection was detected in seven of those examined. While E. hellem 1A and E. cuniculi II were the major causative agents identified, seven different genotypes of E. bieneusi were recorded.

Conclusions

These findings clearly show that exposure to microsporidia is common and chronic microsporidiosis is not linked to any clinical manifestation in healthy population. Moreover, our results indicate much higher incidence of microsporidial infections among an apparently healthy population than previously reported. These results open the question about the potential risk of reactivation of latent microsporidiosis in cases of immunosupression causing life-threatening disease.     

Comparing and combining distance-based and character-based approaches for barcoding turtles

Molecular barcoding can serve as a powerful tool in wildlife forensics and may prove to be a vital aid in conserving organisms that are threatened by illegal wildlife trade, such as turtles (Order Testudines). We produced cytochrome oxidase subunit one (COI) sequences (650 bp) for 174 turtle species and combined these with publicly available sequences for 50 species to produce a data set representative of the breadth of the order. Variability within the barcode region was assessed, and the utility of both distance-based and character-based methods for species identification was evaluated. For species in which genetic material from more than one individual was available (n = 69), intraspecific divergences were 1.3% on average, although divergences greater than the customary 2% barcode threshold occurred within 15 species. High intraspecific divergences could indicate species with a high degree of internal genetic structure or possibly even cryptic species, although introgression is also probable in some of these taxa. Divergences between species of the same genus were 6.4% on average; however, 49 species were <2% divergent from congeners. Low levels of interspecific divergence could be caused by recent evolutionary radiations coupled with the low rates of mtDNA evolution previously observed in turtles. Complementing distance-based barcoding with character-based methods for identifying diagnostic sets of nucleotides provided better resolution in several cases where distance-based methods failed to distinguish species. An online identification engine was created to provide character-based identifications. This study constitutes the first comprehensive barcoding effort for this seriously threatened order.     

Galantamine antiacetylcholinesterase activity in rat brain influenced by L-carnitine

Galantamine (GAL) is a selective, competitive and reversible acetylcholinesterase (AChE) inhibitor, which increases the activity of the cholinergic system and hence gives rise to an improvement of cognitive functions in patients suffering from dementia of Alzheimer type. L-Carnitine (CAR) is a natural component of the mammalian tissue and is known to increase penetration of some chemical compounds/groups across biological membranes. The aim of this study was to evaluate the influence of pretreatment with CAR on AChE inhibition caused by GAL in selected brain parts in rat (basal ganglia, septum, frontal cortex, hippocampus) and in hypophysis, which does not lay beyond the blood-brain-barrier. During the first stage of the study, GAL was administered i.m. in different doses ranging from 2.5 to 10 mg/kg. The highest degree of AChE dose dependent inhibition was observed in hypophysis, while that in CNS was lower and became apparent in frontal cortex and hippocampus only after the administration of the dose of 10 mg/kg i.m. In the second stage, CAR was administered daily during 3 consecutive days at a dose of 250 mg/kg p.o. prior to the administration of GAL (10 mg/kg i.m.). Pretreatment with CAR enhanced trend of AChE inhibition in all selected brain parts comparing with single GAL administration, however, significant difference was not observed. Comparing these results with control group, statistical significance was found in frontal cortex, hippocampus and hypophysis.