Effect of Cannabis sativa L. root,leaf and inflorescence ethanol extracts on the chemotrophic response of entomopathogenic nematodes

Plant and Soil - Soils represent the natural habitat of entomopathogenic nematodes (EPNs). When moving in soil, EPNs are oriented to follow a chemical signal (chemotaxis). Cannabis sativa L. is...     

Genotyping of Chlamydia trachomatis directly from urogenital and conjunctiva samples using an ompA gene pyrosequencing-based assay

The aim of the present study was to develop and validate a multitarget pyrosequencing-based protocol for basic Chlamydia trachomatis genotyping directly from clinical samples and to characterize the distribution of genotypes among Slovenian sexually active population. The newly developed combination of assays that targets the variable domains VD-I and VD-IV of the C. trachomatis ompA gene, was optimized and validated with 11 reference C. trachomatis strains and by comparison to complete ompA conventional sequencing. In addition, 183 clinical specimens which were previously diagnosed as C. trachomatis positive were evaluated by pyrosequencing. The pyrosequencing products showed a 100% match to corresponding sections of the respective conventional ompA sequences. Based on our results the most frequent genotype in urogenital samples was E (51.1%) followed by F (21.4%), G and K (6.9%), D (6.1%), H (3.8%), J (2.3%) and Ia and Ja (0.8%). In conjunctiva samples the genotype distribution was E (63.3%), D and F (13.3%), K (6.7%) and G (3.3%). Pyrosequencing thus proved itself to be a rapid method for C. trachomatis typing, which is important for better understanding the pathogenesis and epidemiology of this pathogen.     

Direct analysis of carbohydrates in animal plasma by ion chromatography coupled with mass spectrometry and pulsed amperometric detection for use as a non-invasive diagnostic tool

The present paper demonstrates that electrochemical detection (ECD) coupled to ion chromatography and electrospray ionization tandem mass spectrometry (IC-ECD-ESI/MS/MS) can be used to rapidly estimate some indications of the health status of organisms. The lactulose to mannitol ratio (L/M) is used as a non-invasive assay to investigate small intestinal absorption pathways and mucosal integrity. In the present study, an evaluation of the negative effects of nonsteroidal anti-inflammatory drug meloxicam perorally administrated to a group of dogs was carried out by determining the lactulose/mannitol index using the IC-ECD-ESI/MS/MS hyphenated technique. According to the results of the study, meloxicam altered gastrointestinal permeability. Coenzyme Q(10) (CoQ(10)) was tested to determine if it could prevent meloxicam induced gastrointestinal damage and it was found that CoQ(10) could be an effective preventive treatment. Furthermore, plasma glucose concentration level was determined to be an indirect indicator of the oxidative state in the blood. To find out the beneficial effects of a double antioxidant combination (α-lipoic acid (ALA) and CoQ(10)) on the total glucose level in chickens, ALA and CoQ(10) were provided as food additives in factory farm raised chicken. The results of the pilot study indicate that the glucose level in the plasma of chickens group fed with CoQ(10) and ALA was significantly decreased compared to the control group. Ion chromatography (IC) utilizing pulsed amperometric detection (PAD) was compared to ion chromatography coupled with tandem mass spectrometry (MS/MS) as an analytical tool for monitoring the carbohydrate level in biological fluids. In electrochemical detection, the newly developed two-pulse waveform successfully withstands matrix effects in biological samples. Continuous on-line desalting of the high salt concentrations used as the eluent for carbohydrate separation from the anion-exchange column allows coupling of IC and MS techniques. A make-up solution (0.5mM LiCl) was delivered prior to MS detection for efficient ionization of eluted carbohydrates. Method validation showed that both used techniques are practically comparable and some advantages of each are presented.     

Bioreactor engineering of stem cell environments

Stem cells hold promise to revolutionize modern medicine by the development of new therapies, disease models and drug screening systems. Standard cell culture systems have limited biological relevance because they do not recapitulate the complex 3-dimensional interactions and biophysical cues that characterize the in vivo environment. In this review, we discuss the current advances in engineering stem cell environments using novel biomaterials and bioreactor technologies. We also reflect on the challenges the field is currently facing with regard to the translation of stem cell based therapies into the clinic.     

Surface-plasmon-resonance-based biosensor with immobilized bisubstrate analog inhibitor for the determination of affinities of ATP- and protein-competitive ligands of cAMP-dependent protein kinase

Interactions between adenosine-oligoarginine conjugates (ARC), bisubstrate analog inhibitors of protein kinases, and catalytic subunits of cAMP-dependent protein kinase (cAPK Calpha) were characterized with surface-plasmon-resonance-based biosensors. ARC-704 bound to the immobilized kinase with subnanomolar affinity. The immobilization of ARC-704 to the chip surface via streptavidin-biotin complex yielded a high-affinity surface (K(D)=16nM). The bisubstrate character of ARC-704 was demonstrated with various ligands targeted to ATP-binding pocket (ATP and inhibitors H89 and H1152P) and protein-substrate-binding domain of Calpha (RIIalpha and GST-PKIalpha) in competition assays. The experiments performed on surfaces with different immobilization levels of ARC-704 produced similar results. The closeness of the obtained affinities of the tested compounds to the inhibitory potencies and affinities of the compounds measured with other methods demonstrates the applicability of the chip with the immobilized biligand inhibitor for the characterization of both ATP- and substrate protein-competitive ligands of basophilic protein kinases.     

Carbocyclic 3'-deoxyadenosine-based highly potent bisubstrate-analog inhibitor of basophilic protein kinases

Carbocyclic analogs of 3'-deoxyadenosine were synthesized as racemates and the resulting stereoisomers were separated by chromatography on a chiral column. The conjugation of obtained compounds with hexa-(D-arginine) via 6-aminohexanoic acid linker led to a highly potent inhibitor of several basophilic protein kinases with some selectivity towards cAMP-dependent protein kinase.     

Tracking of human Y receptors in living cells--a fluorescence approach

Non-invasive methods for studying biological processes in living cells have become very important, also in the field of GPCR biochemistry. Great advancements in the application of fluorescence techniques as well as in the development and improvement of novel fluorophores allow the visualization of dynamic processes. Using these technologies, problems concerning receptor biosynthesis, internalization, recycling and degradation can be investigated. Here we compare the application of the different fluorescent tags EYFP, Lumiotrade mark and SNAPtrade mark to track hY(1) and hY(5) receptors in living cells.     

Bacillus licheniformis bacitracin-resistance ABC transporter: relationship to mammalian multidrug resistance

The nucleotide sequence of the Bacillus licheniformis bacitracin-resistance locus was determined. The presence of three open reading frames, bcrA, bcrB and bcrC, was revealed. The BcrA protein shares a high degree of homology with the hydrophilic ATP-binding components of the ABC family of transport proteins. The bcrB and bcrC genes were found to encode hydro-phobic proteins, which may function as membrane components of the permease. Apart from Bacillus subtilis, these genes also confer resistance upon the Gram-negative Escherichia coli. The presumed function of the Bcr transporter is to remove the bacitracin molecule from its membrane target. In addition to the homology of the nucleotide-binding sites, BcrA protein and mammalian multidrug transporter or P-glycoprotein share collateral detergent sensitivity of resistant cells and possibly the mode of Bcr transport activity within the membrane. The advantage of the resistance phenotype of the Bcr transporter was used to construct deletions within the nucleotide-binding protein to determine the Importance of various regions in transport.