A screening assay based on host-pathogen interaction models identifies a set of novel antifungal benzimidazole derivatives.

Fungal infections are a serious health problem in clinics, especially in the immune-compromised patient. Disease ranges from widespread superficial infections like vulvovaginal infections to life-threatening systemic candidiasis. Especially for systemic mycoses, only a limited arsenal of antifungals is available. The most commonly used classes of antifungal compounds used include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapy, significant side effects, and high costs for several antifungals, there is a medical need for new antifungals in the clinic and general practice. In order to expand the arsenal of compounds with antifungal activities, we screened a compound library including more than 35,000 individual compounds derived from organic synthesis as well as combinatorial compound collections representing mixtures of compounds for antimycotic activity. In total, more than 100,000 compounds were screened using a new type of activity-selectivity assay, analyzing both the antifungal activity and the compatibility with human cells at the same time. One promising hit, an (S)-2-aminoalkyl benzimidazole derivative, was developed among a series of lead compounds showing potent antifungal activity. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl) benzimidazole showed the highest antifungal activity and the best compatibility with human cells in several cell culture models and against a number of clinical isolates of several species of pathogenic Candida yeasts. Transcriptional profiling indicates that the newly discovered compound is a potential inhibitor of the ergosterol pathway, in contrast to other benzimidazole derivatives, which target microtubules.

Specific protein regions influence substrate specificity and product length in polyunsaturated fatty acid condensing enzymes.

We describe a condensing enzyme from Pythium irregulare (PirELO) that shows highest activity on the 18-carbon, Delta-6 desaturated fatty acids, stearidonic acid and gamma-linolenic acid. However, this enzyme is also capable of elongating a number of other fatty acids including the 20-carbon, Delta-5 desaturated fatty acid eicosapentaenoic acid. Surprisingly, a Phytophthora infestans condensing enzyme (PinELO) with very high homology to PirELO did not show activity with 20-carbon fatty acids. A series of chimeric proteins for these two enzymes were constructed to investigate the influence of different regions on substrate and product length. The substitution of a region from near the center of PirELO into PinELO resulted in an enzyme having EPA-elongating activity similar to that of PirELO. Only eight amino acids differed between the two proteins in this region; however, substitution of the same region from PinELO into PirELO produced a protein which was almost inactive. The addition of a small region from near the N-terminus of PinELO was sufficient to restore activity with GLA, indicating that amino acids from these two regions interact to determine protein structure or function. Predicted topology models for PirELO and PinELO placed the two regions described here near the luminal-proximal ends of the first and fourth/fifth transmembrane helixes, at the opposite end of the condensing enzyme from four conserved regions thought to form a catalytic ring. Thus, protein characteristics determined by specific luminal-proximal regions of fatty acid condensing enzymes have a major influence on substrate specificity and final product length.

Effects of biomaterials for Lab-on-a-chip production on cell growth and expression of differentiated functions of leukemic cell lines.

The rapid increase of the applications for Lab-on-a-chip devices has attracted the interest of researchers and engineers on standard process of the electronics industry for low production costs and large scale development, necessary for disposable applications. The printed circuit board technology could be used for this purpose, in particular for the wide range of materials available. In this paper, assays on biocompatibility of materials used for Lab-on-a-chip fabrication has been carried out using two tumor cell lines growing in suspension, the human chronic myelogenous leukemia K562 cell line, able to undergo erythroid differentiation when cultured with chemical inducers, and the lymphoblastoid cell line (LCL), extensively used for screening of cytotoxic T-lymphocytes (CTLs). We have demonstrated that some materials strongly inhibit cell proliferation of both the two cell lines to an extent higher that 70-75%, but only after a prolonged exposure of 3-6 days (Copper, Gold over Nickel, Aramid fiber filled epoxy uncured, b-stage epoxy die attach film, Tesa 4985 adhesive tape, Pyralux uncured, Copper + 1-octodecanethiol). However, when experiments were performed with short incubation time (1 h), only Aramid fiber filled epoxy uncured was cytotoxic. Variation of the results concerning the other materials was appreciable when the experiments performed on two cell lines were compared together. Furthermore, the effects of the materials on erythroid differentiation and CTL-mediated LCL lysis confirmed, in most of the cases, the data obtained in cytotoxic and antiproliferative tests.

Essential role of the G-domain in targeting of the protein import receptor atToc159 to the chloroplast outer membrane.

Two homologous GTP-binding proteins, atToc33 and atToc159, control access of cytosolic precursor proteins to the chloroplast. atToc33 is a constitutive outer chloroplast membrane protein, whereas the precursor receptor atToc159 also exists in a soluble, cytosolic form. This suggests that atToc159 may be able to switch between a soluble and an integral membrane form. By transient expression of GFP fusion proteins, mutant analysis, and biochemical experimentation, we demonstrate that the GTP-binding domain regulates the targeting of cytosolic atToc159 to the chloroplast and mediates the switch between cytosolic and integral membrane forms. Mutant atToc159, unable to bind GTP, does not reinstate a green phenotype in an albino mutant (ppi2) lacking endogenous atToc159, remaining trapped in the cytosol. Thus, the function of atToc159 in chloroplast biogenesis is dependent on an intrinsic GTP-regulated switch that controls localization of the receptor to the chloroplast envelope.

Rhamnolipids form drug-loaded nanoparticles for dermal drug delivery.

Bacterial biosurfactants are nature's strategy to solubilize and ingest hydrophobic molecules and nutrients using a fully biodegradable transport system. Eight structurally defined rhamnolipids were selected and investigated as potential drug carrier systems. Depending on the molecular structures defining their packing parameters, the rhamnolipids were found to form spherical nanoparticles with precisely defined average sizes between 5 and 100nm, low polydispersity, and stability over a broad concentration range as revealed from dynamic light scattering and electron microscopy. As rhamnolipids were tolerated well by the human skin, rhamnolipid nanoparticles were considered for dermal drug delivery and thus loaded with hydrophobic drug molecules. Using the drug model, Nile red, dexamethasone, and tacrolimus nanoparticles charged with up to 30% drug loading (w/w) were obtained. Nanoparticles loaded with Nile red were investigated for dermal drug delivery in a Franz cell using human skin. Fluoresence microscopy of skin slices indicated the efficient penetration of the model drug into human skin, both into the stratum corneum and although to a lesser extent into the lower epidermis. Rhamnolipid nanocarriers were found to be non-toxic to primary human fibroblasts in a proliferation assay and thus are considered candidates for the dermal delivery of drugs.

Biophysical characterization of synthetic rhamnolipids.

Synthetic rhamnolipids, derived from a natural diacylated glycolipid, RL-2,2(14), produced by Burkholderia (Pseudomonas) plantarii, were analyzed biophysically. Changes in the chemical structures comprised variations in the length, the stereochemistry and numbers of the lipid chains, numbers of rhamnoses, and the occurrence of charged or neutral groups. As relevant biophysical parameters, the gel (beta) to liquid crystalline (alpha) phase behavior of the acyl chains of the rhamnoses, their three-dimensional supramolecular aggregate structure, and the ability of the compounds to intercalate into phospholipid liposomes in the absence and presence of lipopolysaccharide-binding protein were monitored. Their biological activities were examined as the ability to induce cytokines in human mononuclear cells and to induce chemiluminescence in monocytes. Depending on the particular chemical structures, the physicochemical parameters as well as the biological test systems show large variations. This relates to the acyl chain fluidity, aggregate structure, and intercalation ability, as well as the bioactivity. Most importantly, the data extend our conformational concept of endotoxicity, based on the intercalation of naturally originating amphiphilic virulence factors into membranes from immune cells. This 'endotoxin conformation', produced by amphiphilic molecules with a hydrophilic charged backbone and apolar hydrophobic moiety, and adopting inverted cubic aggregate structures, causes high mechanical stress in target immune cells on integral proteins, eventually leading to cell activation. Furthermore, biologically inactive rhamnolipids with lamellar aggregate structures antagonize the endotoxin-induced activity in a way similar to lipid A-derived antagonists.

High-throughput-screening-based identification and structure-activity relationship characterization defined (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole as a highly antimycotic agent nontoxic to cell lines.

Novel nontoxic (S)-2-aminoalkylbenzimidazole derivatives were found to be effective against Candida spp. at low micromolar concentrations using high-throughput screening with infected HeLa cells. A collection of analogues defined the chemical groups relevant for activity. The most active compound was characterized by transcriptional analysis of the response of C. albicans Sc5314. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole had a strong impact on membrane biosynthesis. Testing different clinically relevant pathogenic fungi showed the selectivity of the antimycotic activity against Candida species.

Chemical synthesis of a glycolipid library by a solid-phase strategy allows elucidation of the structural specificity of immunostimulation by rhamnolipids.

The first synthesis of a glycolipid library by hydrophobically assisted switching phase (HASP) synthesis is described. HASP synthesis enables flexible switching between solution-phase steps and solid-supported reactions conducted with molecules attached to a hydrophobic silica support. A library of glycolipids derived from the lead compound 1-a strongly immunostimulatory rhamnolipid--with variations in the carbohydrate part, the lipid components, and the stereochemistry of the 3-hydroxy fatty acids was designed and synthesized. The enantioselective synthesis of the 3-hydroxy fatty acid building blocks was achieved by employing asymmetric hydrogenation of 3-oxo fatty acids. Glycolipids were prepared by this approach without any intermediary isolation steps, mostly in excellent yields. Final deprotection to the carboxylic acids was accomplished by enzymatic ester cleavage. All prepared rhamnolipids were tested for their immunostimulatory properties against human monocyte cells by assaying the secretion of the cytokine tumor necrosis factor alpha (TNFalpha) into the medium. The observed structure-activity relationships of rhamnolipids indicate a specific, recognition-based mode of action, with small structural variations in the rhamnolipids resulting in strong effects on the immunostimulatory activities of the rhamnolipids at low micromolar concentrations.

Hydrophobically assisted switching phase synthesis: the flexible combination of solid-phase and solution-phase reactions employed for oligosaccharide preparation.

Hydrophobically assisted switching phase (HASP) synthesis is a concept that allows the choice between the advantages of solid-supported chemistry and those of solution-phase synthesis. Starting from the examination of adsorption and desorption properties of hydrophobic molecules to and from reversed-phase silica, we designed a dilipid as a quantitative and fully reversible HASP anchor, permitting final product release. The utility of this new tool in synthetic organic chemistry was demonstrated on oligosaccharide preparation. The synthesis of a pentarhamnoside was accomplished by repetitive glycosylation reactions. Glycosylations were conducted preferably in solution, whereas all protecting group manipulations were performed on solid support. Without the need for chromatographic purification of intermediates, the HASP system furnished the final product after 12 linear steps with average yields of 94% per step at a scale of 0.1 mmol, thus overcoming several of the limitations encountered in the solid-phase synthesis of complex carbohydrates.

Frequently used plateletpheresis techniques result in variable target yields and platelet recruitment of donors.

BACKGROUND: Standard plateletpheresis techniques and effects on platelet (PLT) donors were investigated to provide an informative basis for advancement of apheresis software. STUDY DESIGN AND METHODS: Three paired groups with 33 male and 22 female blood donors were prospectively investigated by analyzing blood counts of donors and products. Four apheresis platforms, the COBE Spectra LRS and the Trima v4 (Gambro BCT) and the AS.TEC204 and the COM.TEC (Fresenius Hemocare), were compared. Deviations of the collected from programmed PLT targets and donor PLT recruitment were calculated for single-unit PLT concentrates (SU-PCs; 3 x 10(11) PLTs) and double-unit PLT concentrates (DU-PCs; 6 x 10(11) PLTs). RESULTS: Regarding SU-PCs, the productivity of the COM.TEC machine was superior to the AS.TEC204 machine, because of shorter processing time (54 min vs. 67 min) and higher yields (2.90 x 10(11) PLTs vs. 2.75 x 10(11) PLTs). Compared to the Spectra machine, the Trima v4 machine showed higher collection efficiencies (CEs) and shorter processing time and complied better with the programmed target (SU-PCs, 3.24 x 10(11) PLTs vs. 3.70 x 10(11) PLTs; DU-PCs, 6.87 x 10(11) PLTs vs. 7.56 x 10(11) PLTs). Harvests of the Spectra machine (DU-PCs) exceeded the target by 40 percent, which resulted in high PLT loss for donors. A longer processing time resulted in some higher CEs (SU-PCs, 53%; DU-PCs, 58%), which could contribute to this result. PLT recruitment compensated PLT loss to some extent. CONCLUSION: The major finding was that the newer devices (COM.TEC and Trima) gave more predictable yields than the older devices (AS.TEC204 and Spectra) and resulted in lower PLT deficit. PLT software should be improved to minimize relevant variations of collected yields regarding the programmed target.

Arabidopsis tic110 is essential for the assembly and function of the protein import machinery of plastids.

The translocon at the inner envelope membrane of chloroplasts (Tic) plays a central role in plastid biogenesis by coordinating the sorting of nucleus-encoded preproteins across the inner membrane and coordinating the interactions of preproteins with the processing and folding machineries of the stroma. Despite these activities, the precise roles of known Tic proteins in translocation, sorting, and preprotein maturation have not been defined. In this report, we examine the in vivo function of a major Tic component, Tic110. We demonstrate that Arabidopsis thaliana Tic110 (atTic110) is essential for plastid biogenesis and plant viability. The downregulation of atTic110 expression results in the reduced accumulation of a wide variety of plastid proteins. The expression of dominant negative mutants of atTic110 disrupts assembly of Tic complexes and the translocation of preproteins across the inner envelope membrane. Together, these data suggest that Tic110 plays a general role in the import of nuclear-encoded preproteins as a common component of Tic complexes.

AtToc90, a new GTP-binding component of the Arabidopsis chloroplast protein import machinery.

AtToc159 is a GTP-binding chloroplast protein import receptor. In vivo, atToc159 is required for massive accumulation of photosynthetic proteins during chloroplast biogenesis. Yet, in mutants lacking atToc159 photosynthetic proteins still accumulate, but at strongly reduced levels whereas non-photosynthetic proteins are imported normally: This suggests a role for the homologues of atToc159 (atToc132, -120 and -90). Here, we show that atToc90 supports accumulation of photosynthetic proteins in plastids, but is not required for import of several constitutive proteins. Part of atToc90 associates with the chloroplast surface in vivo and with the Toc-complex core components (atToc75 and atToc33) in vitro suggesting a function in chloroplast protein import similar to that of atToc159. As both proteins specifically contribute to the accumulation of photosynthetic proteins in chloroplasts they may be components of the same import pathway.

Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation.

Omega6- and omega3-polyunsaturated C20 fatty acids represent important components of the human diet. A more regular consumption and an accordingly sustainable source of these compounds are highly desirable. In contrast with the very high levels to which industrial fatty acids have to be enriched in plant oils for competitive use as chemical feedstocks, much lower percentages of very-long-chain polyunsaturated fatty acids (VLCPUFA) in edible plant oils would satisfy nutritional requirements. Seed-specific expression in transgenic tobacco (Nicotiana tabacum) and linseed (Linum usitatissimum) of cDNAs encoding fatty acyl-desaturases and elongases, absent from all agronomically important plants, resulted in the very high accumulation of Delta6-desaturated C18 fatty acids and up to 5% of C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acid. Detailed lipid analyses of developing seeds from transgenic plants were interpretated as indicating that, after desaturation on phosphatidylcholine, Delta6-desaturated products are immediately channeled to the triacylglycerols and effectively bypass the acyl-CoA pool. Thus, the lack of available Delta6-desaturated acyl-CoA substrates in the acyl-CoA pool limits the synthesis of elongated C20 fatty acids and disrupts the alternating sequence of lipid-linked desaturations and acyl-CoA dependent elongations. As well as the successful production of VLCPUFA in transgenic oilseeds and the identification of constraints on their accumulation, our results indicate alternative strategies to circumvent this bottleneck.

Novel fatty acid elongases and their use for the reconstitution of docosahexaenoic acid biosynthesis.

In algae, the biosynthesis of docosahexaenoic acid (22:6omega3; DHA) proceeds via the elongation of eicosapentaenoic acid (20:5omega3; EPA) to 22:5omega3, which is required as a substrate for the final Delta4 desaturation. To isolate the elongase specific for this step, we searched expressed sequence tag and genomic databases from the algae Ostreococcus tauri and Thalassiosira pseudonana, from the fish Oncorhynchus mykiss, from the frog Xenopus laevis, and from the sea squirt Ciona intestinalis using as a query the elongase sequence PpPSE1 from the moss Physcomitrella patens. The open reading frames of the identified elongase candidates were expressed in yeast for functional characterization. By this, we identified two types of elongases from O. tauri and T. pseudonana: one specific for the elongation of (Delta6-)C18-PUFAs and one specific for (Delta5-)C20-PUFAs, showing highest activity with EPA. The clones isolated from O. mykiss, X. laevis, and C. intestinalis accepted both C18- and C20-PUFAs. By coexpression of the Delta6- and Delta5-elongases from T. pseudonana and O. tauri, respectively, with the Delta5- and Delta4-desaturases from two other algae we successfully implemented DHA synthesis in stearidonic acid-fed yeast. This may be considered an encouraging first step in future efforts to implement this biosynthetic sequence into transgenic oilseed crops.