ATP-binding cassette transporters of Paracoccidiodes brasiliensis

ATP-binding cassette (ABC) transporters represent one of the largest recognised superfamilies of proteins. They are ubiquitous in nature and have been implicated in a wide range of physiological functions, including multi-drug resistance (MDR). All of these transporters are structurally related, with active transport complexes exhibiting the minimal complement of twelve transmembrane helices and two nucleotide-binding domains. Despite the structural similarity, the range of functions performed by these proteins is variable. In many cases the substrate range is equally diverse with the proteins displaying the capacity to translocate substrates which appear structurally unrelated. This intensifies the problem of MDR where a number of classes of drug are susceptible to efflux. A number of ABC transporters have been identified in fungi including Saccharomyces cerevisiae and Candida albicans. An increasing wealth of information is emerging for a few of these transporters. The current knowledge on the various characteristics of the S. cerevisiae transporter PDR5 approaches that relating to P-glycoprotein (Pgp) of mammalian cells. The ABC transporter CDRl from Candida albicans has been shown to confer clinical drug resistance in a number of disease cases. Paracoccidiodes brasiliensis is a thermally dimorphic human pathogen that is exclusive to South America. Inhalation is the route of infection with disease progression originating in the lungs and progressing to other parts of the body. Paracoccidiodiomycosis is the most prevalent systemic mycosis in Latin America. Exposure to the organism is most common in rural workers, particularly those involved in deforestation. Disease progression occurs when the host is immunocompromised and tends to follow and extended period of latency which can last over ten years. Outgrowth of the organism is dependent on a morphological transition from the mycelial to the yeast form of the organism. This transition is inhibited by the female hormone oestrogen resulting in the clinical syndrome displaying a bias towards males. The drug of choice is ketoconazole, a member of the azole family of anti-fungal agents. Azoles act on an intermediate enzyme in the ergosterol biosynthesis pathway, lanosterol demethylase, resulting in the perturbation of cell membrane integrity. Resistance to azole drugs can occur in two ways. A mutation in lanosterol demethylase can alter the drug-binding site so that the target is not recognised. Alternatively, the intracellular concentration of azole can be reduced by the ABC transporter mediated active efflux of the drug. The remit of this thesis was to screen the genome of P.brasiliensis for loci encoding ABC transporters. Particular interest would be paid to proteins that may be involved with drug resistance. Initially a strategy employed by other investigators was employed. Two degenerate oligonucleotides (MDRl and MDR2), designed on the basis of conserved motifs from the nucleotide binding domains of a number of ABC transporters were used in PCR. A single amplicon was obtained which showed homology to other sequences coding for nucleotide-binding domains. This amplicon was used to screen a lambda genomic library and a single phage was isolated. This phage clone contains part of a gene with displayed considerable identity to an ABC half-transporter from Aspergillus fumigatus. The complete sequence of the gene was then obtained using genome walking SSP-PCR. The gene, pftl, spans 2627bp and is interrupted by 2 introns of 68bp and 77bp. A similar approach was employed in a search for additional genes. The amino acid sequences from a number of PDR5-like fungal ABC transporters were aligned. This allowed the identification of conserved motifs that were specific to these fungal proteins. (Abstract shortened by ProQuest.)

A fully automated procedure for the parallel, multidimensional purification and nucleotide loading of the human GTPases KRas, Rac1 and RalB

Small GTPases regulate many key cellular processes and their role in human disease validates many proteins in this class as desirable targets for therapeutic intervention. Reliable recombinant production of GTPases, often in the active GTP loaded state, is a prerequisite for the prosecution of drug discovery efforts. The preparation of these active forms can be complex and often constricts the supply to the reagent intensive techniques used in structure base drug discovery. We have established a fully automated, multidimensional protein purification strategy for the parallel production of the catalytic G-domains of KRas, Rac1 and RalB GTPases in the active form. This method incorporates a four step chromatography purification with TEV protease-mediated affinity tag cleavage and a conditioning step that achieves the activation of the GTPase by exchanging GDP for the non-hydrolyzable GTP analogue GMPPnP. We also demonstrate that an automated method is efficient at loading of KRas with mantGDP for application in a SOS1 catalysed fluorescent nucleotide exchange assay. In comparison to more conventional manual workflows the automated method offers marked advantages in method run time and operator workload. This reduces the bottleneck in protein production while generating products that are highly purified and effectively loaded with nucleotide analogues

Assembly of multicellular constructs and microarrays of cells using magnetic nanowires

An approach is described for controlling the spatial organization of mammalian cells using ferromagnetic nanowires in conjunction with patterned micromagnet arrays. The nanowires are fabricated by electrodeposition in nanoporous templates, which allows for precise control of their size and magnetic properties. The high aspect ratio and large remanent magnetization of the nanowires enable suspensions of cells bound to Ni nanowires to be controlled with low magnetic fields. This was used to produce one- and two-dimensional field-tuned patterning of suspended 3T3 mouse fibroblasts. Self-assembled one-dimensional chains of cells were obtained through manipulation of the wires\u27 dipolar interactions. Ordered patterns of individual cells in two dimensions were formed through trapping onto magnetic microarrays of ellipsoidal permalloy micromagnets. Cell chains were formed on the arrays by varying the spacing between the micromagnets or the strength of fluid flow over the arrays. The positioning of cells on the array was further controlled by varying the direction of an external magnetic field. These results demonstrate the possibility of using magnetic nanowires to organize cells

Absolute physical calibration in the infrared

We determine an absolute calibration for the Multiband Imaging Photometer for Spitzer 24 μm band and recommend adjustments to the published calibrations for Two Micron All Sky Survey (2MASS), Infrared Array Camera (IRAC), and IRAS photometry to put them on the same scale. We show that consistent results are obtained by basing the calibration on either an average A0V star spectral energy distribution (SED), or by using the absolutely calibrated SED of the Sun in comparison with solar-type stellar photometry (the solar analog method). After the rejection of a small number of stars with anomalous SEDs (or bad measurements), upper limits of ~1.5% root mean square (rms) are placed on the intrinsic infrared (IR) SED variations in both A-dwarf and solar-type stars. These types of stars are therefore suitable as general-purpose standard stars in the IR. We provide absolutely calibrated SEDs for a standard zero magnitude A star and for the Sun to allow extending this work to any other IR photometric system. They allow the recommended calibration to be applied from 1 to 25 μm with an accuracy of ~2%, and with even higher accuracy at specific wavelengths such as 2.2, 10.6, and 24 μm, near which there are direct measurements. However, we confirm earlier indications that Vega does not behave as a typical A0V star between the visible and the IR, making it problematic as the defining star for photometric systems. The integration of measurements of the Sun with those of solar-type stars also provides an accurate estimate of the solar SED from 1 through 30 μm, which we show agrees with theoretical models

Force Output Comparison between Six U.S. Collegiate Athletic Teams.

The aim of the study was to compare allometrically scaled peak force and the force at 250 ms between six U.S. collegiate sport teams using isometric mid-thigh pull. Ninety subjects performed maximum effort of isometric mid-thigh pull to measure force output. The data were averaged within the teams, and statistically compared between teams using one-way ANOVA (p=.01). Significant difference was found that men’s soccer and baseball produced higher allometrically scaled peak force, and men’s soccer, tennis, and baseball produced higher allometrically scaled force at 250 ms. The data indicates that not all sports possess similar strength characteristics because of the nature of the sports, and observed separation between gender. Teams such as volleyball and baseball showed higher coefficient of variation due to the various positions within their sports

Identification of systemic immune response markers through metabolomic profiling of plasma from calves given an intra-nasally delivered respiratory vaccine

International audienceVaccination procedures within the cattle industry are important disease control tools to minimize economic and welfare burdens associated with respiratory pathogens. However, new vaccine, antigen and carrier technologies are required to combat emerging viral strains and enhance the efficacy of respiratory vaccines, particularly at the point of pathogen entry. New technologies, specifically metabolomic profiling, could be applied to identify metabolite immune-correlates representative of immune protection following vaccination aiding in the design and screening of vaccine candidates. This study for the first time demonstrates the ability of untargeted UPLC-MS metabolomic profiling to identify metabolite immune correlates characteristic of immune responses following mucosal vaccination in calves. Male Holstein Friesian calves were vaccinated with Pfizer Rispoval® PI3 + RSV intranasal vaccine and metabolomic profiling of post-vaccination plasma revealed 12 metabolites whose peak intensities differed significantly from controls. Plasma levels of glycocholic acid, N-[(3α,5β,12α)-3,12-Dihydroxy-7,24-dioxocholan-24-yl]glycine, uric acid and biliverdin were found to be significantly elevated in vaccinated animals following secondary vaccine administration, whereas hippuric acid significantly decreased. In contrast, significant upregulation of taurodeoxycholic acid and propionylcarnitine levels were confined to primary vaccine administration. Assessment of such metabolite markers may provide greater information on the immune pathways stimulated from vaccine formulations and benchmarking early metabolomic responses to highly immunogenic vaccine formulations could provide a means for rapidly assessing new vaccine formulations. Furthermore, the identification of metabolic systemic immune response markers which relate to specific cell signaling pathways of the immune system could allow for targeted vaccine design to stimulate key pathways which can be assessed at the metabolic level

Calsyntenin-1 mediates axonal transport of the amyloid precursor protein and regulates Aβ production

Understanding the mechanisms that control processing of the amyloid precursor protein (APP) to produce amyloid-β (Aβ) peptide represents a key area of Alzheimer's disease research. Here, we show that siRNA-mediated loss of calsyntenin-1 in cultured neurons alters APP processing to increase production of Aβ. We also show that calsyntenin-1 is reduced in Alzheimer's disease brains and that the extent of this reduction correlates with increased Aβ levels. Calsyntenin-1 is a ligand for kinesin-1 light chains and APP is transported through axons on kinesin-1 molecular motors. Defects in axonal transport are an early pathological feature in Alzheimer's disease and defective APP transport is known to increase Aβ production. We show that calsyntenin-1 and APP are co-transported through axons and that siRNA-induced loss of calsyntenin-1 markedly disrupts axonal transport of APP. Thus, perturbation to axonal transport of APP on calsyntenin-1 containing carriers induces alterations to APP processing that increase production of Aβ. Together, our findings suggest that disruption of calsyntenin-1-associated axonal transport of APP is a pathogenic mechanism in Alzheimer's disease