Influence of the extraction mode on the yield of some furanocoumarins from Pastinaca sativa fruits

Analysis of plant material is an important task in chemotaxonomical investigations, in search of plants with pharmacological activity or in standardisation of plant drugs. The choice of optimal conditions for the analysis of plant material and effect of extraction method on the yield of furanocoumarins from Pastinaca sativa fruits were examined. The following extraction methods were used in experiments: exhaustive extraction in Soxhlet apparatus, ultrasonification (USAE) at 25 and 60 degrees C, microwave-assisted solvent extraction in open and closed system (MASE) and accelerated solvent extraction (ASE). In most cases, the yield of furanocoumarins was highest by use of ASE method as well as by ultrasonification at 60 degrees C.     

CFP10 discriminates between nonacetylated and acetylated ESAT-6 of Mycobacterium tuberculosis by differential interaction

ESAT-6 (the 6 kDa early secreted antigenic target) protein species in short-term culture filtrate of Mycobacterium tuberculosis were separated in a 4-5 narrow range pI gradient two-dimensional gel electrophoresis (2-DE). Eight ESAT-6 protein species were analyzed in detail by peptide mass fingerprinting matrix-assisted laser desorption/ionization-mass spectrometry as well as by electrospray ionization-tandem mass spectrometry. An N-terminal Thr acetylation was identified in four species and a C-terminal truncation was identified in two species. In 2-DE blot overlay assays, the recombinant 10 kDa culture filtrate protein (CFP10) discriminated N-terminal acetylated and nonacetylated ESAT-6 by differential interaction, whereas removal of the C-terminal 11 residues of ESAT-6 had no effects thereon. This example shows that the access to the protein species level can be a prerequisite to understand regulation of protein-protein interaction.     

Modulation of drug sensitivity in yeast cells by the ATP-binding domain of human DNA topoisomerase IIalpha

Epipodophyllotoxins are effective antitumour drugs that trap eukaryotic DNA topoisomerase II in a covalent complex with DNA. Based on DNA cleavage assays, the mode of interaction of these drugs was proposed to involve amino acid residues of the catalytic site. An in vitro binding study, however, revealed two potential binding sites for etoposide within human DNA topoisomerase IIα (htopoIIα), one in the catalytic core of the enzyme and one in the ATP-binding N-terminal domain. Here we have tested how N-terminal mutations that reduce the affinity of the site for etoposide or ATP affect the sensitivity of yeast cells to etoposide. Surprisingly, when introduced into full-length enzymes, mutations that lower the drug binding capacity of the N-terminal domain in vitro render yeast more sensitive to epipodophyllotoxins. Consistently, when the htopoIIα N-terminal domain alone is overexpressed in the presence of yeast topoII, cells become more resistant to etoposide. Point mutations that weaken etoposide binding eliminate this resistance phenotype. We argue that the N-terminal ATP-binding pocket competes with the active site of the holoenzyme for binding etoposide both in cis and in trans with different outcomes, suggesting that each topoisomerase II monomer has two non-equivalent drug-binding sites.     

Reconstruction of the muscle system in Antygomonas sp. (Kinorhyncha,Cyclorhagida) by means of phalloidin labeling and cLSM

In the present investigation the entire muscle system of the cyclorhagid kinorhynch Antygomonas sp. was three-dimensionally reconstructed from whole mounts by means of FITC-phalloidin labeling and confocal scanning microscopy. With this technique, which proved to be especially useful for microscopically small species, we wanted to reinvestigate and supplement the findings obtained by histological and electron microscopical methods. The organization of the major muscle systems can be summarized as follows: 1) All muscle fibers, apart from the intestinal ones, the spine, and the mouth cone muscles, show a cross-striated pattern; 2) Dorsal longitudinal muscle fibers as well as segmentally arranged dorsoventral fibers occur from segment III to XIII; 3) Diagonal muscle fibers are located laterally in segments III to X; 4) Two rings of circular fibers are present in segment II, forming the closing apparatus in Cyclorhagida. Further circular muscles are present in segment I, forming the mouth cone and the eversible introvert, and in the pharyngeal bulb.     

Genomic effects of IFN-beta in multiple sclerosis patients

The purpose of this report was to characterize the dynamics of the gene expression cascades induced by an IFN-beta-1a treatment regimen in multiple sclerosis patients and to examine the molecular mechanisms potentially capable of causing heterogeneity in response to therapy. In this open-label pharmacodynamic study design, peripheral blood was obtained from eight relapsing-remitting multiple sclerosis patients just before and at 1, 2, 4, 8, 24, 48, 120, and 168 h after i.m. injection of 30 micro g of IFN-beta-1a. The total RNA was isolated from monocyte-depleted PBL and analyzed using cDNA microarrays containing probes for >4000 known genes. IFN-beta-1a treatment resulted in selective, time-dependent effects on multiple genes. The mRNAs for genes implicated in the anti-viral response, e.g., double-stranded RNA-dependent protein kinase, myxovirus resistance proteins 1 and 2, and guanylate binding proteins 1 and 2 were rapidly induced within 1-4 h of IFN-beta treatment. The mRNAs for several genes involved in IFN-beta signaling, such as IFN-alpha/beta receptor-2 and Stat1, were also increased. The mRNAs for lymphocyte activation markers, such as IFN-induced transmembrane protein 1 (9-27), IFN-induced transmembrane protein 2 (1-8D), beta(2)-microglobulin, and CD69, were also increased in a time-dependent manner. The findings demonstrate that IFN-beta treatment induces specific and time-dependent changes in multiple mRNAs in lymphocytes of multiple sclerosis patients that could provide a framework for rapid monitoring of the response to therapy.     

Analysis of the interaction of small heat shock proteins with unfolding proteins

The ubiquitous small heat shock proteins (sHsps) are efficient molecular chaperones that interact with nonnative proteins, prevent their aggregation, and support subsequent refolding. No obvious substrate specificity has been detected so far. A striking feature of sHsps is that they form large complexes with nonnative proteins. Here, we used several well established model chaperone substrates, including citrate synthase, alpha-glucosidase, rhodanese, and insulin, and analyzed their interaction with murine Hsp25 and yeast Hsp26 upon thermal unfolding. The two sHsps differ in their modes of activation. In contrast to Hsp25, Hsp26 undergoes a temperature-dependent dissociation that is required for efficient substrate binding. Our analysis shows that Hsp25 and Hsp26 reacted in a similar manner with the nonnative proteins. For all substrates investigated, complexes of defined size and shape were formed. Interestingly, several different nonnative proteins could be incorporated into defined sHsp-substrate complexes. The first substrate protein bound seems to determine the complex morphology. Thus, despite the differences in quaternary structure and mode of activation, the formation of large uniform sHsp-substrate complexes seems to be a general feature of sHsps, and this unique chaperone mechanism is conserved from yeast to mammals.