Direct coupled column separation and determination of the diastereomeric glucuronides of almokalant, a new class III antiarrhythmic drug, in human urine.

A reversed-phase coupled column separation (CCS) system for the analysis of two diastereomeric glucuronides of almokalant, a new class III antiarrhythmic drug, in human urine is described. After direct injection of urine samples (50 microliters) the glucuronides were isolated by complex formation on a terbium(III) loaded strong cation exchanger at alkaline pH. The solutes were eluted from the precolumn by an acidic mobile phase, enriched and separated on Hypercarb (porous graphitic carbon) as analytical column with 0.1 M acetic acid pH 2.8 and 30% acetonitrile as mobile phase. The calibration graph was linear (r2 = 0.9999) and the detection limits were in the low picomole (UV) or femtomole (fluorescence) range. Optimization of the analytical column revealed that elution order and selectivity for the glucuronides were dependent on the buffer agent and temperature used. By appropriate choice of mobile phase conditions all four diastereomers could be separated.     

The influence of three unchanging photoperiods on growth and parr-smolt transformation in Atlantic salmon, Salmo salar L.

Groups of pre-smolt Atlantic salmon were reared under three experimental photoperiods. Growth rate was significantly enhanced under 24 h of light: 0 h of darkness per diem (24 L: 0 D) compared with 16 L: 8 D and 8 L: 16 D from early January to early May. From the beginning of May until the termination of the experiment on 27 May, growth rate was highest under 8 L: 16 D.
All groups developed bimodal length-frequency distributions during the experiment. The proportion of the population in each of the two growth modes was significantly affected by photoperiod treatment.
The level of plasma cortisol increased significantly from February to May. There were no differences in levels of plasma cortisol among photoperiod treatments.
Judged by development in plasma cortisol, changes in condition factor and external appearance, the parr-smolt transformation was not completed under any of the experimental photoperiods.     

A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo.

14-3-3 proteins play an important role in a multitude of signalling pathways. The interactions between 14-3-3 and other signalling proteins, such as Raf and KSR (kinase suppressor of Ras), occur in a phospho-specific manner. Recently, a phosphorylation-independent interaction has been reported to occur between 14-3-3 and several proteins, for example 5-phosphatase, p75NTR-associated cell death executor (NADE) and the bacterial toxin Exoenzyme S (ExoS), an ADP-ribosyltransferase from Pseudomonas aeruginosa. In this study we have identified the amino acid residues on ExoS, which are responsible for its specific interaction with 14-3-3. Furthermore, we show that a peptide derived from ExoS, containing the 14-3-3 interaction site, effectively competes out the interaction between ExoS and 14-3-3. In addition, competition with this peptide blocks ExoS modification of Ras in our Ras modification assay. We show that the ExoS protein interacts with all isoforms of the 14-3-3 family tested. Moreover, in vivo an ExoS protein lacking the 14-3-3 binding site has a reduced capacity to ADP ribosylate cytoplasmic proteins, e.g. Ras, and shows a reduced capacity to change the morphology of infected cells.     

A novel MALDI-TOF based methodology for genotyping single nucleotide polymorphisms

A new MALDI-TOF based detection assay was developed for analysis of single nucleotide polymorphisms (SNPs). It is a significant modification on the classic three-step minisequencing method, which includes a polymerase chain reaction (PCR), removal of excess nucleotides and primers, followed by primer extension in the presence of dideoxynucleotides using modified thermostable DNA polymerase. The key feature of this novel assay is reliance upon deoxynucleotide mixes, lacking one of the nucleotides at the polymorphic position. During primer extension in the presence of depleted nucleotide mixes, standard thermostable DNA polymerases dissociate from the template at positions requiring a depleted nucleotide; this principal was harnessed to create a genotyping assay. The assay design requires a primer- extension primer having its 3'-end one nucleotide upstream from the interrogated site. The assay further utilizes the same DNA polymerase in both PCR and the primer extension step. This not only simplifies the assay but also greatly reduces the cost per genotype compared to minisequencing methodology. We demonstrate accurate genotyping using this methodology for two SNPs run in both singleplex and duplex reactions. We term this assay nucleotide depletion genotyping (NUDGE). Nucleotide depletion genotyping could be extended to other genotyping assays based on primer extension such as detection by gel or capillary electrophoresis.     

Gene therapy vector-mediated expression of insulin-like growth factors protects cardiomyocytes from apoptosis and enhances neovascularization

IGF-I and IGF-II are single-chain polypeptide growth factors that regulate pleiotropic cellular responses. We have characterized the effect of recombinant IGF proteins, as well as third-generation adenoviral vectors encoding either IGF-I or IGF-II genes, on cardiomyocyte apoptosis and on angiogenesis. We found that endothelial cells cultured in the presence of the extracellular protein laminin exhibit a robust response to IGF-I and -II proteins via enhanced cell migration and angiogenic outgrowth. Furthermore, IGF vectors greatly enhanced neovascularization in an in vivo Matrigel model. Transduction of cardiomyocytes with the IGF adenoviral vectors resulted in a dose- and time-dependent increase in the expression of IGF-I or IGF-II protein. This correlated with abrogation of apoptosis induced by ischemia-reoxygenation, ceramide, or heat shock with optimal inhibition of approximately 80%. We conclude that gene transfer of IGF-I and IGF-II is a plausible strategy for the local delivery of IGFs to treat ischemic heart disease and heart failure by stimulating angiogenesis and protecting cardiomyocytes from cell death.     

Localization of a susceptibility gene for type 2 diabetes to chromosome 5q34-q35.2

We report a genomewide linkage study of type 2 diabetes (T2D [MIM 125853]) in the Icelandic population. A list of type 2 diabetics was cross-matched with a computerized genealogical database clustering 763 type 2 diabetics into 227 families. The diabetic patients and their relatives were genotyped with 906 microsatellite markers. A nonparametric multipoint linkage analysis yielded linkage to 5q34-q35.2 (LOD = 2.90, P=1.29 x 10(-4)) in all diabetics. Since obesity, here defined as body mass index (BMI) > or =30 kg/m(2), is a key risk factor for the development of T2D, we studied the data either independently of BMI or by stratifying the patient group as obese (BMI > or =30) or nonobese (BMI <30). A nonparametric multipoint linkage analysis yielded linkage to 5q34-q35.2 (LOD = 3.64, P=2.12 x (10)-5) in the nonobese diabetics. No linkage was observed in this region for the obese diabetics. Linkage analysis conditioning on maternal transmission to the nonobese diabetics resulted in a LOD score of 3.48 (P=3.12 x 10(-5)) in the same region, whereas conditioning on paternal transmission led to a substantial drop in the LOD score. Finally, we observed potential interactions between the 5q locus and two T2D susceptibility loci, previously mapped in other populations.     

The crystal structure of guinea pig 11beta-hydroxysteroid dehydrogenase type 1 provides a model for enzyme-lipid bilayer interactions

The metabolic reduction of 11-keto groups in glucocorticoid steroids such as cortisone leads to the nuclear receptor ligand cortisol. This conversion is an example of pre-receptor regulation and constitutes a novel pharmacological target for the treatment of metabolic disorders such as insulin resistance and possibly other derangements observed in the metabolic syndrome, such as hyperlipidemia, hypertension, and lowered insulin secretion. This reaction is carried out by the NADPH-dependent type 1 11beta-hydroxysteroid dehydrogenase (11beta-HSD1), an enzyme attached through an integral N-terminal transmembrane helix to the lipid bilayer and located with its active site within the lumen of the endoplasmic reticulum. Here we report the crystal structure of recombinant guinea pig 11beta-HSD1. This variant was determined in complex with NADP at 2.5 A resolution and crystallized in the presence of detergent and guanidinium hydrochloride. The overall structure of guinea pig 11beta-HSD1 shows a clear relationship to other members of the superfamily of short-chain dehydrogenases/reductases but harbors a unique C-terminal helical segment that fulfills three essential functions and accordingly is involved in subunit interactions, contributes to active site architecture, and is necessary for lipid-membrane interactions. The structure provides a model for enzyme-lipid bilayer interactions and suggests a funneling of lipophilic substrates such as steroid hormones from the hydrophobic membrane environment to the enzyme active site.     

A Small-Scale Comparison of Iceland Scallop Size Distributions Obtained from a Camera Based Autonomous Underwater Vehicle and Dredge Survey

An approach is developed to estimate size of Iceland scallop shells from AUV photos. A small-scale camera based AUV survey of Iceland scallops was conducted at a defined site off West Iceland. Prior to height estimation of the identified shells, the distortions introduced by the vehicle orientation and the camera lens were corrected. The average AUV pitch and roll was and deg that resulted in error in ground distance rendering these effects negligible. A quadratic polynomial model was identified for lens distortion correction. This model successfully predicted a theoretical grid from a frame photographed underwater, representing the inherent lens distortion. The predicted shell heights were scaled for the distance from the bottom at which the photos were taken. This approach was validated by height estimation of scallops of known sizes. An underestimation of approximately cm was seen, which could be attributed to pixel error, where each pixel represented cm. After correcting for this difference the estimated heights ranged from cm. A comparison of the height-distribution from a small-scale dredge survey carried out in the vicinity showed non-overlapping peaks in size distribution, with scallops of a broader size range visible in the AUV survey. Further investigations are necessary to evaluate any underlying bias and to validate how representative these surveys are of the true population. The low resolution images made identification of smaller scallops difficult. Overall, the observations of very few small scallops in both surveys could be attributed to low recruitment levels in the recent years due to the known scallop parasite outbreak in the region.     

Points of view: A framework for multispecies modelling of Arcto-boreal systems

Reviews in Fish Biology and Fisheries -