Biochemical characterization of recombinant dihydroorotate dehydrogenase from the opportunistic pathogenic yeast Candida albicans

Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine synthesis, as a new target for controlling infection. We propose that the enzyme is a member of the DHODH family 2, which comprises mitochondrially bound enzymes, with quinone as the direct electron acceptor and oxygen as the final electron acceptor. Full-length DHODH and N-terminally truncated DHODH, which lacks the targeting sequence and the transmembrane domain, were subcloned from C. albicans, recombinantly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity. An inhibitor screening with 28 selected compounds was performed. Only the dianisidine derivative, redoxal, and the biphenyl quinoline-carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced C. albicans DHODH activity. This study provides a background for the development of antipyrimidines with high efficacy for decreasing in situ pyrimidine nucleotide pools in C. albicans.     

Helices in peptoids of alpha- and beta-peptides

Peptoids of alpha- and beta-peptides (alpha- and beta-peptoids) can be obtained by shifting the amino acid side chains from the backbone carbon atoms of the monomer constituents to the peptide nitrogen atoms. They are, therefore, N-substituted poly-glycines and poly-beta-alanines, respectively. Due to the substituted nitrogen atoms, the ability for hydrogen bond formation between peptide bonds gets lost. It may be very interesting to see whether such non-natural oligomers could be regarded as foldamers, which fold into definite backbone conformers. In this paper, we provide a complete overview on helix formation in alpha- and beta-peptoids on the basis of systematic theoretical conformational analyses employing the methods of ab initio molecular orbital (MO) theory. It can be shown that the alpha- and beta-peptoid structures form helical structures with both trans and cis peptide bonds despite the missing hydrogen bonds. Obviously, the conformational properties of the backbone are more important for folding than the possibility of hydrogen bonding. There are close relationships between the helices of alpha-peptoids and poly-glycine and poly-proline helices of alpha-peptides, whereas the helices of beta-peptoids correspond to the well-known helical structures of beta-peptides as, for instance, the 3(1)-helix of beta-peptides with 14-membered hydrogen-bonded rings. Thus, alpha- and beta-peptoids enrich the field of foldamers and may be used as useful tools in peptide and protein design.     

Effects of reproductive status and management on cortisol secretion and fertility of oestrous horse mares

Stressful events may contribute to low reproductive efficiency due to glucocorticoid-mediated inhibition of hormone secretion in a variety of species. We therefore investigated effects of stress related to management of mares around artificial insemination on secretion of cortisol and fertility parameters. To avoid further disturbance of mares by frequent blood sampling, faecal cortisol metabolites (fCM) were determined instead (sample collection at 8-h intervals). A total of 50 mares (16 maiden, 17 barren, 12 foaling, 5 teaching mares) were included in the study. Mares were brought to the AI centre in vans or trailers (driving time between 30 min and 5 h). Teaching mares were housed in the clinic and had therefore not to be transported. Mares were inseminated either with fresh/cooled-shipped or frozen semen. Rectal palpations and ultrasound examinations were performed at 24- to 48-h intervals, in animals inseminated with frozen semen at 6-h intervals during the last 48 h before ovulation. In maiden, barren and foaling mares, fCM concentrations in faeces tended to be higher than in teaching mares at all times after arrival at the AI centre. At 24 and 48 h after arrival, fCM concentrations in maiden mares were significantly higher than in teaching mares (24h: maiden mares 12.3+/-3.1 ng/g, barren mares 8.5+/-1.2 ng/g, foaling mares 11.0+/-2.4 ng/g, teaching mares 3.8+/-0.6 ng/g, p<0.05). The time from arrival at the AI centre to detection of ovulation did not differ among the different groups of mares and was 4.5+/-0.4, 5.0+/-0.5, 3.8+/-0.5 and 5.6+/-0.9 days in maiden, barren, foaling and teaching mares, respectively (n.s.). Pregnancy rates were 53, 53, 55 and 60%, respectively (n.s.). The time from arrival at the AI centre to detection of ovulation was 4.4+/-0.3 days and 4.9+/-0.3 days in mares inseminated with fresh/shipped (n=39) or frozen semen (n=11; n.s.), respectively. The frequency of follicular checks influenced fCM secretion and was statistically significant at 16 h before ovulation (fresh/shipped semen: fCM 6.9+/-0.7 ng/g faeces, frozen semen: fCM 16.9+/-5.2 ng/g faeces, p<0.01). In the mare, gynaecological examinations seem to act as stressors and may increase cortisol secretion. However, this does not negatively influence fertility and in animals familiar with that procedure fCM concentrations are not elevated.     

Binding-site identification and genotypic profiling of hepatitis C virus polymerase inhibitors

The search for hepatitis C virus polymerase inhibitors has resulted in the identification of several nonnucleoside binding pockets. The shape and nature of these binding sites differ across and even within diverse hepatitis C virus genotypes. These differences confront antiviral drug discovery with the challenge of finding compounds that are capable of inhibition in variable binding pockets. To address this, we have established a hepatitis C virus mutant and genotypic recombinant polymerase panel as a means of guiding medicinal chemistry through the elucidation of the site of action of novel inhibitors and profiling against genotypes. Using a genotype 1b backbone, we demonstrate that the recombinant P495L, M423T, M414T, and S282T mutant enzymes can be used to identify the binding site of an acyl pyrrolidine analog. We assess the inhibitory activity of this analog and other nonnucleoside inhibitors with our panel of enzyme isolates generated from clinical sera representing genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a.     

Lambda interferon (IFN-lambda) in serum is decreased in hantavirus-infected patients, and in vitro-established infection is insensitive to treatment with all IFNs and inhibits IFN-gamma-induced nitric oxide production

Hantaviruses, causing hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), are known to be sensitive to nitric oxide (NO) and to pretreatment with type I and II interferons (alpha interferon [IFN-alpha]/IFN-beta and IFN-gamma, respectively). Elevated serum levels of NO and IFN-gamma have been observed in HFRS patients, but little is known regarding the systemic levels of other IFNs and the possible effects of hantaviruses on innate antiviral immune responses. In Puumala virus-infected HFRS patients (n = 18), we report that the levels of IFN-alpha and IFN-beta are similar, whereas the level of IFN-lambda (type III IFN) is significantly decreased, during acute (day of hospitalization) compared to the convalescent phase. The possible antiviral effects of IFN-lambda on the prototypic hantavirus Hantaan virus (HTNV) replication was then investigated. Pretreatment of A549 cells with IFN-lambda alone inhibited HTNV replication, and IFN-lambda combined with IFN-gamma induced additive antiviral effects. We then studied the effect of postinfection treatment with IFNs. Interestingly, an already-established HTNV infection was insensitive to subsequent IFN-alpha, -beta, -gamma, and -lambda stimulation, and HTNV-infected cells produced less NO compared to noninfected cells when stimulated with IFN-gamma and IL-1beta. Furthermore, less phosphorylated STAT1 after IFN treatment was observed in the nuclei of infected cells than in those of noninfected cells. The results suggest that hantavirus can interfere with the activation of antiviral innate immune responses in patients and inhibit the antiviral effects of all IFNs. We believe that future studies addressing the mechanisms by which hantaviruses interfere with the activation and shaping of immune responses may bring more knowledge regarding HFRS and HCPS pathogenesis.     

Personalized medicine and proteomics: lessons from non-small cell lung cancer

Personalized medicine allows the selection of treatments best suited to an individual patient and disease phenotype. To implement personalized medicine, effective tests predictive of response to treatment or susceptibility to adverse events are needed, and to develop a personalized medicine test, both high quality samples and reliable data are required. We review key features of state-of-the-art proteomic profiling and introduce further analytic developments to build a proteomic toolkit for use in personalized medicine approaches. The combination of novel analytical approaches in proteomic data generation, alignment and comparison permit translation of identified biomarkers into practical assays. We further propose an expanded statistical analysis to understand the sources of variability between individuals in terms of both protein expression and clinical variables and utilize this understanding in a predictive test.     

IncP-1-beta plasmid pGNB1 isolated from a bacterial community from a wastewater treatment plant mediates decolorization of triphenylmethane dyes

Plasmid pGNB1 was isolated from bacteria residing in the activated sludge compartment of a wastewater treatment plant by using a transformation-based approach. This 60-kb plasmid confers resistance to the triphenylmethane dye crystal violet and enables its host bacterium to decolorize crystal violet. Partial sequencing of pGNB1 revealed that its backbone is very similar to that of previously sequenced IncP-1beta plasmids. The two accessory regions of the plasmid, one located downstream of the replication initiation gene trfA and the other located between the conjugative transfer modules Tra and Trb, were completely sequenced. Accessory region L1 contains a transposon related to Tn5501 and a gene encoding a Cupin 2 conserved barrel protein with an unknown function. The triphenylmethane reductase gene tmr and a truncated dihydrolipoamide dehydrogenase gene that is flanked by IS1071 and another putative insertion element were identified in accessory region L2. Subcloning of the pGNB1 tmr gene demonstrated that this gene is responsible for the observed crystal violet resistance phenotype and mediates decolorization of the triphenylmethane dyes crystal violet, malachite green, and basic fuchsin. Plasmid pGNB1 and the associated phenotype are transferable to the alpha-proteobacterium Sinorhizobium meliloti and the gamma-proteobacterium Escherichia coli. This is the first report of a promiscuous IncP-1beta plasmid isolated from the bacterial community from a wastewater treatment plant that harbors a triphenylmethane reductase gene. The pGNB1-encoded enzyme activity is discussed with respect to bioremediation of sewage polluted with triphenylmethane dyes.     

Label-free detection of single nucleotide polymorphisms utilizing the differential transfer function of field-effect transistors

We present a label-free method for the detection of DNA hybridization, which is monitored by non-metallized silicon field-effect transistors (FET) in a microarray approach. The described method enables a fast and fully electronic readout of ex situ binding assays. The label-free detection utilizing the field-effect is based on the intrinsic charge of the DNA molecules and/or on changes of the solid–liquid interface impedance, when biomolecules bind to the sensor surface. With our sensor system, usually a time-resolved, dc readout is used. In general, this FET signal suffers from sensor drift, temperature drift, changes in electrolyte composition or pH value, influence of the reference electrode, etc. In this article, we present a differential ac readout concept for FET microarrays, which enables a stable operation of the sensor against many of these side-parameters, reliable readout and a possibility for a quick screening of large sensor arrays. We present the detection of point mutations in short DNA samples with this method in an ex situ binding assay.     

Seasonal Viral Loop Dynamics in Two Large Ultraoligotrophic Antarctic Freshwater Lakes

The effect of viruses on the microbial loop, with particular emphasis on bacteria, was investigated over an annual cycle in 2003–2004 in Lake Druzhby and Crooked Lake, two large ultraoligotrophic freshwater lakes in the Vestfold Hills, Eastern Antarctica. Viral abundance ranged from 0.16 to 1.56 × 10⁹ particles L^-1;1 and bacterial abundances ranged from 0.10 to 0.24 × 10⁹ cells L^-1;1, with the lowest bacterial abundances noted in the winter months. Virus-to-bacteria ratios (VBR) were consistently low in both lakes throughout the season, ranging from 1.2 to 8.4. lysogenic bacteria, determined by induction with mitomycin C, were detected on three sampling occasions out of 10 in both lakes. In Lake Druzhby and Crooked Lake, lysogenic bacteria made up between 18% and 73% of the total bacteria population during the lysogenic events. Bacterial production ranged from 8.2 to 304.9 × 10⁶ cells L^-1;1 day^-1;1 and lytic viral production ranged from 47.5 to 718.4 × 10⁶ viruslike particles L^-1;1 day^-1;1. When only considering primary production, heterotrophic nanoflagellate (HNF) grazing and viral lysis as the major contributors to the DOC pool (i.e., autochthonous sources), we estimated a high contribution from viruses during the winter months when >60% of the carbon supplied to the DOC pool originated from viral lysis. In contrast, during the summer <20% originated from viral lysis. Our study shows that viral process in ultraoligotrophic Antarctic lakes may be of quantitative significance with respect to carbon flow especially during the dark winter period.