Ostreolysin enhances fruiting initiation in the oyster mushroom (Pleurotus ostreatus)

Fruiting initiation in mushrooms can be triggered by a variety of environmental and biochemical stimuli, including substances of natural or synthetic origin. In this work ostreolysin, a cytolytic protein specifically expressed during the formation of primordia and fruit bodies of Pleurotus ostreatus, was applied to nutrient media inoculated with mycelium of P. ostreatus, and its effects on mycelial growth and fructification of the mushroom studied. The addition of ostreolysin slightly inhibited the growth of mycelium, but strongly induced the formation of primordia, which appeared 10 d earlier than in control plates supplemented with bovine serum albumin or with the dissolving buffer alone. Moreover, ostreolysin stimulated the subsequent development of primordia into fruit bodies. However, direct involvement of this protein in the sporulation of the mushroom is unlikely, as it was also detected in large amounts in the non-sporulating strain of P. ostreatus.     

Erythropoietin unfolding: thermodynamics and its correlation with structural features

Human erythropoietin (EPO) is a glycoprotein hormone considered to be the principal regulator of red blood cell formation. Although its recombinant version (rEPO) has been widely used for treatment of various anemias and its biological effects are relatively well-known, we know little about its biophysical properties and their relation to its structure. To gain a fuller understanding of the structural and functional properties of rEPO on the molecular level we followed its thermal and urea-induced unfolding at different pH (3.1-9.4) and urea concentrations (0-8 M) using spectropolarimetry, UV absorption, intrinsic emission fluorescence, and differential scanning calorimetry. Our results show that under a variety of conditions rEPO undergoes thermal or urea-induced denaturation that may be considered as a reversible two-state process characterized by unusually high (thermal) or moderate (urea-induced) extent of the residual structure. The highest thermal stability of the protein observed in aqueous solutions at physiological pH appears to be due to the largest difference in the extent of structure in the denatured and native state at this pH. The comparison between experimentally determined energetics of rEPO denaturation and its structure-based calculations indicates that the parametrization of thermodynamic quantities in terms of changes in solvent accessible nonpolar and polar surface areas resulting from protein unfolding can be successfully used provided that these changes are estimated from combination of experimentally determined deltaC(o)p and deltaH(o) values and not calculated from the structure of the protein's folded and assumingly fully unfolded state.     

Solution, solid state and biological characterization of ruthenium(III)-DMSO complexes with purine base derivatives

Two new complexes of Ru(III) with purine base derivatives, [mer-RuCl(3)(acv)(DMSO-S)(C(2)H(5)OH)].C(2)H(5)OH (1) (acv=acyclovir, DMSO=dimethyl sulfoxide) and [trans-RuCl(4)(guaH)(DMSO-S)].2H(2)O (2) (guaH=protonated molecule of guanine), were prepared from the same Ru(III) precursor, [trans-RuCl(4)(DMSO-S)(2)](-), by substitution of one DMSO-S. Coordination of acv induced also replacement of one chloride by an ethanol molecule. This reactivity difference was explained by striking contrasts in the hydrogen bonding schemes of the two complexes, evidenced in their X-ray crystal structures. In 1 the guanine derivative acyclovir is coordinated to ruthenium through the N(7) atom, while in 2 the protonated guanine molecule is bound through the N(9) atom. Both complexes were also characterized by various physico-chemical methods in the solid state and in the solution. In vitro, the biological activity of 2 and of the previously described complexes [mer-RuCl(3)(acv)(DMSO-S)(CH(3)OH)].0.5CH(3)OH (3) and [mer-RuCl(3)(acv)(DMSO-S)(H(2)O)].H(2)O (4) on tumour cells appear to be very similar to that of NAMI-A (NAMI-A=[ImH][trans-RuCl(4)(DMSO-S)Im]). All compounds are only weakly active on tumour cell proliferation but show an interesting proadhesive effect that suggest possible activity on tumour malignancy.     

Torsional regulation of hRPA-induced unwinding of double-stranded DNA

All cellular single-stranded (ss) DNA is rapidly bound and stabilized by single stranded DNA-binding proteins (SSBs). Replication protein A, the main eukaryotic SSB, is able to unwind double-stranded (ds) DNA by binding and stabilizing transiently forming bubbles of ssDNA. Here, we study the dynamics of human RPA (hRPA) activity on topologically constrained dsDNA with single-molecule magnetic tweezers. We find that the hRPA unwinding rate is exponentially dependent on torsion present in the DNA. The unwinding reaction is self-limiting, ultimately removing the driving torsional stress. The process can easily be reverted: release of tension or the application of a rewinding torque leads to protein dissociation and helix rewinding. Based on the force and salt dependence of the in vitro kinetics we anticipate that the unwinding reaction occurs frequently in vivo. We propose that the hRPA unwinding reaction serves to protect and stabilize the dsDNA when it is structurally destabilized by mechanical stresses.     

SDSL-ESR-based protein structure characterization

As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be seen in the number of protein structures published in the Protein Data Bank. This is especially the case for less-ordered, more hydrophobic and more flexible protein systems. The lack of efficient methods for structure determination calls for urgent development of a new class of biophysical techniques. This work attempts to address this problem with a novel combination of site-directed spin labelling electron spin resonance spectroscopy (SDSL-ESR) and protein structure modelling, which is coupled by restriction of the conformational spaces of the amino acid side chains. Comparison of the application to four different protein systems enables us to generalize the new method and to establish a general procedure for determination of protein structure.     

Carabelli's trait in contemporary Slovenes and inhabitants of a medieval settlement (Sredisce by the Drava River)

The objectives of this study were to determine the total frequency, expression and asymmetry of Carabelli's trait in permanent dentitions of contemporary Slovenes and a medieval skeletal population from northeastern Slovenia. A total of 254 dental casts from contemporary Slovene children were examined. The population of a medieval settlement (10th-15th centuries), was represented by 94 skeletons. A modification of the method of Alvesalo and associates was used to classify Carabelli's trait on a five-grade scale. The trait was expressed on the upper first molars of 79.7% of the contemporary subjects and 75.8% of the medieval sample. Positive expressions of the trait were found in 10.1% of the contemporary subjects and 15.2% of the medieval sample. While the observed total frequency of the trait in both samples is characteristic of Europeans, the rates of positive expressions are surprisingly low but consistent with data from a recently published worldwide literature survey. Both populations showed a low rate of left-right fluctuating asymmetry of the trait. This finding might reflect a pronounced ability of individuals in the medieval population to buffer unfavourable influences from the environment and a relatively low level of environmental stress in the contemporary population.     

Exopolymer Diversity and the Role of Levan in Bacillus subtilis Biofilms

Exopolymeric substances (EPS) are important for biofilm formation and their chemical composition may influence biofilm properties. To explore these relationships the chemical composition of EPS from Bacillus subtilis NCIB 3610 biofilms grown in sucrose-rich (SYM) and sucrose-poor (MSgg and Czapek) media was studied. We observed marked differences in composition of EPS polymers isolated from all three biofilms or from spent media below the biofilms. The polysaccharide levan dominated the EPS of SYM grown biofilms, while EPS from biofilms grown in sucrose-poor media contained significant amounts of proteins and DNA in addition to polysaccharides. The EPS polymers differed also in size with very large polymers (Mw>2000 kDa) found only in biofilms, while small polymers (Mw<200 kD) dominated in the EPS isolated from spent media. Biofilms of the eps knockout were significantly thinner than those of the tasA knockout in all media. The biofilm defective phenotypes of tasA and eps mutants were, however, partially compensated in the sucrose-rich SYM medium. Sucrose supplementation of Czapek and MSgg media increased the thickness and stability of biofilms compared to non-supplemented controls. Since sucrose is essential for synthesis of levan and the presence of levan was confirmed in all biofilms grown in media containing sucrose, this study for the first time shows that levan, although not essential for biofilm formation, can be a structural and possibly stabilizing component of B. subtilis floating biofilms. In addition, we propose that this polysaccharide, when incorporated into the biofilm EPS, may also serve as a nutritional reserve.     

Influence of CdCl2 and CdSO4 supplementation on Cd distribution and ligand environment in leaves of the Cd hyperaccumulator Noccaea (Thlaspi) praecox

Background and aims

The aim was to investigate whether different Cd salts in the nutrient solution of the Cd/Zn hyperaccumulator Noccaea (Thlaspi) praecox alter leaf Cd distribution and Cd ligand environment, and plant fitness.

Methods

Plants were grown for 8 weeks with 100/300 μM CdCl₂ or CdSO₄. Leaf biomass, and total chlorophyll, anthocyanin, Cd, Cl, S and P concentrations were monitored. Cd localisation and ligand environment in leaves were analysed using quantitative synchrotron-based micro-X-ray fluorescence imaging, and Cd K-edge X-ray absorption fine structure and Cd L₃-edge micro-X-ray absorption near-edge structure measurements.

Results

Cd uptake and plant fitness were comparable for CdCl₂ and CdSO₄ treatments, and depended on applied Cd concentration. In all treatments, Cd preferentially accumulated with high concentrations of Cl in vacuoles of large vacuolarised epidermal cells, bound mainly to oxygen-based (O)-ligands. In the mesophyll of CdCl₂? treated plants, Cd was preferentially sequestered in vacuoles, while for CdSO₄, Cd accumulated preferentially in the apoplast. In the symplast, O-ligands increased with increasing Cd concentrations; in the apoplast, sulphur-based (S)-ligands prevailed.

Conclusions

Cd partitioning between leaf mesophyll apoplast and symplast and the Cd ligand environment in N. praecox depend on the Cd salt type and concentration added to the nutrient solution.