Analytical challenges of glycosaminoglycans at biological interfaces

The analysis of glycosaminoglycans (GAGs) is a challenging task due to their high structural heterogeneity, which results in diverse GAG chains with similar chemical properties. Simultaneously, it is of high importance to understand their role and behavior in biological systems. It has been known for decades now that GAGs can interact with lipid molecules and thus contribute to the onset of atherosclerosis, but their interactions at and with biological interfaces, such as the cell membrane, are yet to be revealed. Here, analytical approaches that could yield important knowledge on the GAG-cell membrane interactions as well as the synthetic and analytical advances that make their study possible are discussed. Due to recent developments in laser technology, we particularly focus on nonlinear spectroscopic methods, especially vibrational sum-frequency generation spectroscopy, which has the potential to unravel the structural complexity of heterogeneous biological interfaces in contact with GAGs, in situ and in real time

Puhatestű közösségek térbeli mintázata és diverzitása eltérő vízterek mentén

In this study we examined the structure and diversity of malacological communities at human disturbed and undisturbed sites of oxbow lakes and medium-sized rivers in the water system of the River Danube and Tisza. Sampling was carried out at total 33 sites in 2011 and 2012. According to the malacological species composition and the water chemistry the sampling sites of the water system of the River Danube and Tisza, and certain water bodies can be distinguished. An unambiguous pattern cannot be observed according to the presence-absence of human disturbance. However the Shannon-Wiener diversity and the number of endangered and rare species was significantly higher in the undisturbed Mosoni-Danube, and the number of invasive species was higher at the antropogenically disturbed sampling sites. A total of 49 species were collected, among them 29 species are common, 6 species are endangered and protected in Hungary (Unio crassus, Pseudanodonta complanata, Anisus vorticulus, Borysthenia naticina, Esperiana daudebartii, Esperiana esperi, Theodoxus danubialis), 7 species are rare in Hungary (Pisidium amnicum, Sphaerium rivivola, Sphaerium solidum, Acroloxus lacustris, Anisus spirorbis, Bathyomphalus contortus, Physa fontinalis), and 6 species are invasive. The two endangered bivalve species cannot be found in the oxbow lakes, and the occurrence frequency and density of P. complanata was very low in the studied rivers. The invasive C. fluminea was recorded in the tributary Ipoly, Rába, Bodrog only at sampling sites located close to the mouth of river, and it was not found in the reaches of Tisza above Tokaj. The study was supported by the Hungarian Scientific Fund (KTIA-OTKA) under the contract No. CNK80140

Production of extracellular proteases by human pathogenic Trichoderma longibrachiatum strains

Species belonging to the filamentous fungal genus Trichoderma are well known as potential candidates for the biological control of plant pathogenic fungi and as cellulase producers of biotechnological importance. Several data were published in the last decade also about the clinical importance of this genus, indicating that Trichoderma strains may be potential opportunistic pathogens in immunocompromised patients. However, there is a lack of information about the potential virulence factors of clinical Trichoderma strains. This study was designed to examine the extracellular proteolytic enzymes of six clinical T. longibrachiatum isolates. Supernatants from induced liquid cultures of the examined strains were screened for proteolytic enzyme activities with 11 different chromogenic p-nitroaniline substrates. The production of trypsin-like, chymotrypsin-like and chymoelastase-like protease activities cleaving N-Benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide, N-Succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide,and N-Succinyl-L- Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, respectively, was common among the strains examined. Separation of trypsin- and chymotrypsin-like activities by column chromatography revealed, that both systems are complex consisting of several isoenzymes. The pH-dependence of these two protease systems was also studied. Based on the results, the different isoenzymes seem to have different optimal pH values. Extracellular proteolytic enzymes may be involved in the pathogenecity of Trichoderma strains as facultative human pathogens

A kalcium tranziens kialakulásának mechanizmusa működő szívben = Development of the calcium transient in the beating heart

Kutatásaink a ciklikus intracelluláris kalciumion koncentráció (Ca2+i) változásait kialakító transzport folyamatok felderítésére irányultak normál és kardiomiopátiás szívben. A mért primer jel a Ca2+i tranziens volt, melynek kvantitív analízisére matematikai modellt fejlesztettünk ki. Ennek segítségével lehetővé vált a rianodin csatorna és a SERCA2a kinetikai paramétereinak kiszámítása. Megfigyeléseink szerint a posztiszkémiás szívben a tartósan megemelkedett Ca2+i szint kontraktúrát, valamint a PLA2 aktiválásával membrán degradációt okoz. Ezek a károsodások hő-sokk előkezeléssel részlegesen kivédhetők. További megfigyelésünk szerint a diabéteszes és toxikus kardiomiopátia Ca2+i felszabadulási és szekvesztrációs zavarokat okoz, valamint a nukleáris poli(ADP-ribóz)polimeráz enzim gátlásával a toxikus kardiomiopátia ezen hatásai a Ca2+i homeosztázisra mérsékelhetők. Összefoglalva; a T038121 sz. OTKA támogatás segítségével jelentős előrelépést tettünk a myocardium Ca2+i tranziensét kialakító mechanizmusok feltérképezésében mind normál, mind patofiziológiás körülmények között. | During the grant period of 2002-2005 our research focused on the delineation of the processes responsible for the cyclic changes of the intracellular calcium concentration (Ca2+i) in the normal and diseased heart. A mathematical model was developed to allow the quantitative analysis of the observed Ca2+i signal. Using this novel mathematical approach we have calculated the kinetic parameters of the sarcoplasmic reticulum calcium release channel (RyR2) and SERCA2a. We have observed that the persistent increase in end-diastolic Ca2+i is associated with contracture of the myocardium and activation of PLA2 resulting in membrane degradation. These pathological events can be ameliorated with heat shock pretreatment. We have found that cardiomyopathy of either diabetes or toxic origin results in malfunction of Ca2+ release and sequestration. We have also shown that the disruptive effects of toxic cardiomyopathy on Ca2+i handling can be suppressed with the inhibition of the nuclear enzyme ply(ADP-ribose)polymerase. In summary; funds provided by OTKA T038121 spawned major progress in our research to delineate the processes involved in Ca2+i handling both under normal and pathological circumstances

Coadministration of antigen-conjugated and free CpG : Effects of in vitro and in vivo interactions in a murine model

CpG oligodeoxynucleotides (CpG) are widely studied as promising adjuvants in vaccines against a range of diseases including infection, cancer or allergy. Conjugating antigen to CpG has been shown to potentiate the adjuvant effect via enhancing antigen uptake and danger signaling by the very same cell. In the present study, using biotinylated CpG and streptavidin as a model system, we demonstrate that CpG motif containing free and antigen-conjugated oligonucleotides do not compete in terms of cell activation via TLR9, but do compete for cellular uptake. Antigen-conjugated CpG enhances cellular association and uptake of the antigen by antigen-presenting cells (APC) and T cells. Free CpG efficiently competes with antigen-CpG conjugates in BMDC and T cells, but shows weak or no competition in B cells that have higher TLR9 expression. Vaccination with antigen-conjugated CpG or with a mixture of antigen and CpG elevates the level of antigen-specific antibodies but co-administration of CpG-antigen conjugates and free CpG adversely effects immunogenicity. These observations may help optimize CpG-based vaccine formulation. © 2014 Elsevier B.V

The interaction of chondroitin sulfate with a lipid monolayer observed by using nonlinear vibrational spectroscopy

The first vibrational sum-frequency generation (VSFG) spectra of chondroitin sulfate (CS) interacting with dipalmitoyl phosphatidylcholine (DPPC) at air-liquid interface are reported here, collected at a laser repetition rate of 100 kHz. By studying the VSFG spectra in the regions of 1050-1450 cm(-1), 2750-3180 cm(-1), and 3200-3825 cm(-1), it was concluded that in the presence of Ca2+ ions, the head groups together with the head-group-bound water molecules in the DPPC monolayer are strongly influenced by the interaction with CS, while the organization of the phospholipid tails remains mostly unchanged. The interactions were observed at a CS concentration below 200 nM, which exemplifies the potential of VSFG in studying biomolecular interactions at low physiological concentrations. The VSFG spectra recorded in the O-H stretching region at chiral polarization combination imply that CS molecules are organized into ordered macromolecular superstructures with a chiral secondary structure

A complete nicotinate degradation pathway in the microbial eukaryote Aspergillus nidulans

Several strikingly different aerobic and anaerobic pathways of nicotinate breakdown are extant in bacteria. Here, through reverse genetics and analytical techniques we elucidated in Aspergillus nidulans, a complete eukaryotic nicotinate utilization pathway. The pathway extant in this fungus and other ascomycetes, is quite different from bacterial ones. All intermediate metabolites were identified. The cognate proteins, encoded by eleven genes (hxn) mapping in three clusters are co-regulated by a specific transcription factor. Several enzymatic steps have no prokaryotic equivalent and two metabolites, 3-hydroxypiperidine-2,6-dione and 5,6-dihydroxypiperidine-2-one, have not been identified previously in any organism, the latter being a novel chemical compound. Hydrolytic ring opening results in α-hydroxyglutaramate, a compound not detected in analogous prokaryotic pathways. Our earlier phylogenetic analysis of Hxn proteins together with this complete biochemical pathway illustrates convergent evolution of catabolic pathways between fungi and bacteria