Quantification of rosuvastatin in human plasma by automated solid-phase extraction using tandem mass spectrometric detection

An assay employing automated solid-phase extraction (SPE) followed by high-performance liquid chromatography with positive ion TurboIonspray tandem mass spectrometry (LC-MS-MS) was developed and validated for the quantification of rosuvastatin (Crestor) in human plasma. Rosuvastatin is a hydroxy-methyl glutaryl coenzyme A reductase inhibitor currently under development by AstraZeneca. The standard curve range in human plasma was 0.1-30 ng/ml with a lower limit of quantification (LLOQ) verified at 0.1 ng/ml. Inaccuracy was less than 8% and imprecision less than +/-15% at all concentration levels. There was no interference from endogenous substances. The analyte was stable in human plasma following three freeze/thaw cycles and for up to 6 months following storage at both -20 and -70 degrees C. The assay was successfully applied to the analysis of rosuvastatin in human plasma samples derived from clinical trials, allowing the pharmacokinetics of the compound to be determined.     

Isolation,identification and localization of a second beetle antidiuretic peptide

We isolated from head extracts of Tenebrio molitor a peptide that inhibits fluid secretion by the Malpighian tubules of this insect. This second antidiuretic factor, ADFb, like the previously published ADFa, works through cyclic GMP as a second messenger. It has primary structure Tyr-Asp-Asp-Gly-Ser-Tyr-Lys-Pro-His-Ile-Tyr-Gly-Phe-OH with an EC(50) of approximately 240 pM in a fluid secretion assay. This peptide is now the second sequenced endogenous insect ADF which inhibits Malpighian tubule fluid secretion. Immunohistochemical techniques show that the peptide is localized in the brain; it appears to be produced mainly in two pairs of bilaterally symmetrical cells in the protocerebrum.     

Bacterial expression and purification of the Arabidopsis NADPH-cytochrome P450 reductase ATR2

An N-terminally modified form of the Arabidopsis NADPH-cytochrome P450 ATR2 (ATR2mod) was expressed from the tactac promoter in Escherichia coli to obtain high yields of the enzyme. The N-terminal modification eliminates the predicted chloroplast transit peptide of ATR2 allowing for more efficient expression. ATR2mod was purified from membrane extracts using a 2',5'-ADP-agarose affinity column. The specific activity of the purified ATR2mod for cytochrome c reduction was 9.4 micromol min(-1) mg(-1) and the K(m) for cytochrome c reduction was 15 +/- 2 microM. The purified NADPH-cytochrome P450 reductase was able to support function of CYP79B2.     

Strain inhomogeneity in the anterior cruciate ligament under application of external and muscular loads.

To determine whether mathematical relations between strains in different bundles and loads would be needed to predict injury of the anterior cruciate ligament (ACL), this work tested the hypothesis that strains developed in two bundles of the ACL were significantly different under the application of a number of loads important to injury etiology of the ACL. To provide the data for testing this hypothesis, liquid mercury strain gages were installed on both the anteromedial (AMB) and posterolateral (PLB) bundles of the ACL of ten specimens, which were then subjected to passive flexion/extension, hyperextension moment, anterior force, internal and external axial moments, quadriceps, and hamstrings forces. Various combinations of these loads were also applied. Flexion angles ranged from 8 deg of hyperextension through 120 deg of flexion. The data were analyzed using a repeated measures analysis of variance. The analyses indicated that significant strain differences existed between the two bundles only for passive flexion/extension. However, the analyses did not support the hypothesis that AMB and PLB strains are significantly different from each other under the application of external and muscular loads. Because noticeable differences (> 3 percent) between bundle strains did exist in some load cases for limited ranges of flexion and the PLB strain was consistently higher than the AMB strain, it may be sufficient to consider strain in only the PLB when predicting ligament damage based on strain-load relations.     

Rapid recovery of benthic invertebrates downstream of hyperalkaline steel slag discharges

This study assesses the physical and chemical characteristics of hyperalkaline steel slag leachate from a former steelworks on two streams in England and their impacts on benthic invertebrate communities. Using multivariate methods (CCA), we related invertebrate richness and diversity with chemical parameters along the environmental gradient from point sources to less impacted sites downstream. Point discharges are characterised by high pH (10.6–11.5), high ionic strength (dominated by Ca–CO₃–OH waters), elevated trace elements (notably Li, Sr and V) and high rates of calcium carbonate precipitation. This combination of stressors gives rise to an impoverished benthic invertebrate community in source areas. The total abundance, taxonomic richness and densities of most observed organisms were strongly negatively correlated with water pH. Analysis using biological pollution monitoring indices (e.g. BMWP and Functional Feeding Groups) shows the system to be highly impacted at source, but when pH approaches values close to aquatic life standards, some 500 m downstream, complex biological communities become established. In addition to showing the rapid recovery of invertebrate communities downstream of the discharges, this study also provides a baseline characterisation of invertebrate communities at the extreme alkaline range of the pH spectrum.     

Market Perceptions and Opportunities for Native Plant Production on the Southern Colorado Plateau

Increases in revegetation activities have created a large demand for locally adapted native plant materials (NPM) in the southwestern United States. Currently, there is a minimal supply of local genotypes to meet this demand. We investigated the potential for the initiation of a native plant market in the southern Colorado Plateau. Through a literature search, interviews, and site visits, we identified existing native plant markets outside of the region as useful models to help initiate a regional market. We used web‐based surveys to identify and analyze current and future NPM needs and concerns. Survey results indicate that management policy strongly drives decisions regarding the use and purchase of NPM. From a demand perspective , lack of availability and cost of NPM has kept purchasing minimal, despite policy changes favoring the use of natives. For suppliers, further development of NPM is limited by inconsistent and unreliable demand and lack of production knowledge. The knowledge and tools necessary to initiate an NPM market are available, but inadequate funding sources and insufficient information sharing hinder its development. Communication among producers, land managers, buyers, and researchers, as well as partnerships with local growers, appear to be vital to initiating a functional market.     

Characterizing the role of the microtubule binding protein Bim1 in Cryptococcus neoformans

During sexual development the human fungal pathogen Cryptococcus neoformans undergoes a developmental transition from yeast-form growth to filamentous growth. This transition requires cellular restructuring to form a filamentous dikaryon. Dikaryotic growth also requires tightly controlled nuclear migration to ensure faithful replication and dissemination of genetic material to spore progeny. Although the gross morphological changes that take place during dikaryotic growth are largely known, the molecular underpinnings that control this process are uncharacterized. Here we identify and characterize a C. neoformans homolog of the Saccharomyces cerevisiae BIM1 gene, and establish the importance of BIM1 for proper filamentous growth of C. neoformans. Deletion of BIM1 leads to truncated sexual development filaments, a severe defect in diploid formation, and a block in monokaryotic fruiting. Our findings lead to a model consistent with a critical role for BIM1 in both filament integrity and nuclear congression that is mediated through the microtubule cytoskeleton.     

beta-Hydroxy acyl-CoA inhibition of mitochondrial ATP production

beta-Hydroxypalmitoyl-CoA and beta-hydroxystearoyl-CoA were synthesized, purified and quantitated. beta-Hydroxypalmitoyl-CoA and beta-hydroxystearoyl-CoA instantly and reversibly inhibited oxidative phosphorylation by rabbit heart mitochondria oxidizing pyruvate. [8-14C]ADP uptake studies showed that the beta-hydroxy acyl-CoA species linearly inhibited the adenine nucleotide translocase system. Free beta-hydroxy fatty acids at comparable concentrations (0.005 mM) did not affect ADP uptake or state III respiration.     

An MRI-based method to align the compressive loading axis for human cadaveric knees

There is a need to align the mechanical axis of the tibia with the axis of loading for studies involving tibiofemoral compression to interpret results and to ensure repeatability of loading within and among specimens. Therefore, the objectives of this study were (1) to develop a magnetic resonance imaging (MRI)-based alignment method for use with apparatuses applying tibiofemoral joint compression, (2) to demonstrate the usefulness of the method by aligning cadaveric knees in an apparatus that could apply tibiofemoral joint compression, and (3) to quantify the error associated with the alignment method. A four degree-of-freedom adjustable device was constructed to allow determination and alignment of the mechanical axis of the tibia of cadaveric knee joints with the axis of loading of an apparatus applying tibiofemoral joint compression. MRI was used to determine the locations of bony landmarks in three dimensions defining the mechanical axis of the tibia relative to an initial orientation of the four degree-of-freedom device. Adjustment values of the device were then computed and applied to the device to align the mechanical axis of the tibia with the axis of a compressive loading apparatus. To demonstrate the usefulness of the method, four cadaveric knees were aligned in the compressive loading apparatus. The vectors describing the mechanical axis of the tibia and the loading axis of the apparatus before and after adjustment of the four degree-of-freedom device were computed for each cadaveric knee. After adjustment of the four degree-of-freedom device, the mechanical axis of the tibia was collinear with the loading axis of the apparatus for each cadaveric knee. The errors in the adjustment values introduced by inaccuracies in the MR images were quantified using the Monte Carlo technique. The precisions in the translational and rotational adjustments were 1.20 mm and 0.90 deg respectively. The MR-based alignment method will allow consistent interpretation of results obtained during tibiofemoral compressive studies conducted using the apparatus described in this paper by providing a well-defined loading axis. The alignment method can also be adapted for use with other apparatuses applying tibiofemoral compression.     

Immunological responses and viral modulatory effects of vaccination with recombinant modified vaccinia virus Ankara (rMVA) expressing structural and regulatory transgenes of simian immunodeficiency virus (SIVmac32H/J5M)

Background The immunogenicity and protective efficacy of recombinant modified vaccinia virus Ankara (rMVA) vectors expressing structural (gag/pol, env) and regulatory (tat, rev, nef) genes of SIVmac251/32H‐J5 (rMVA‐J5) were assessed. Methods Immunization with rMVA constructs (2.5 × 10⁷ IU) 32, 20 and 8 weeks pre‐challenge was compared with 32 and 20 weeks but with a final boost 8 weeks pre‐challenge with 2 × 10⁶ fixed‐inactivated HSC‐F4 cells infected with SIVmac32H. Controls received rMVA vectors expressing an irrelevant transgene or were naïve challenge controls. All received 10 MID₅₀ SIVmac32H/J5 intravenously. Results Vaccinates immunized with rMVA‐J5 exhibited significant, albeit transient, control of peak primary viraemia despite inconsistent and variable immune responses elicted by vaccination. Humoral and cellular responses to Env were most consistent, with lower responses to Nef, Rev and Tat. Increasing titres of anti‐vaccinia neutralizing antibodies reflected the number and dose of rMVA inoculations. Conclusions Improved combinations of viral vectors are required to elicit appropriate immune responses to control viral replication.