Quantification of 8-iso-prostaglandin-F(2alpha) and 2,3-dinor-8-iso-prostaglandin-F(2alpha) in human urine using liquid chromatography-tandem mass spectrometry

Quantification of 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) has been suggested to be a reliable indicator of lipid peroxidation that may be related to in vivo free radical generation, oxidative damage, and antioxidant deficiency. We have developed a LC-MS/MS method to quantify 8-iso- PGF(2alpha) and its dinor metabolite, 2,3-dinor-8-iso-prostaglandin F(2alpha) (2,3-dinor-8-iso-PGF(2alpha)), in human urine samples. After an initial purification step using an automated C18 solid phase extraction procedure, the urine sample was injected directly into a liquid chromatography (LC) system and detected with tandem mass spectrometry. The detection limit of the assay was 9 pg for 8-iso-PGF(2alpha) and 3 pg for 2,3-dinor-8-iso-PGF(2alpha) with both inter- and intraday variations of less than 12%. The inaccuracies were less than 3% for both analytes at three different levels. The urinary excretion rate of 2,3-dinor-8-iso-PGF(2alpha) was higher than that of 8-iso-PGF(2alpha), and changed in proportion to the parent compound (R = 0.70, n = 60). Values obtained with this method showed good linear correlation to duplicate 8-iso-PGF(2alpha) measurements performed with GCMS (R = 0.97, n = 15). The mean excretion rates of 8-iso-PGF(2alpha) and 2,3-dinor-8-iso-PGF(2alpha) were significantly higher in smokers than in nonsmokers (0.53 +/- 0.37 vs. 0.25 +/- 0.15 microg/g creatinine, p = 0.002 for 8-iso-PGF(2alpha) and 8.9 +/- 3.8 vs. 4.6 +/- 2.6 microg/g creatinine, p = 0.003 for 2,3-dinor-8-iso-PGF(2alpha), respectively). The excellent accuracy, reproducibility, and high throughput of this method should permit it to be used in large clinical studies and standard clinical laboratories.     

Understanding the overdispersed molecular clock

Rates of molecular evolution at some protein-encoding loci are more irregular than expected under a simple neutral model of molecular evolution. This pattern of excessive irregularity in protein substitutions is often called the "overdispersed molecular clock" and is characterized by an index of dispersion, R(T) > 1. Assuming infinite sites, no recombination model of the gene R(T) is given for a general stationary model of molecular evolution. R(T) is shown to be affected by only three things: fluctuations that occur on a very slow time scale, advantageous or deleterious mutations, and interactions between mutations. In the absence of interactions, advantageous mutations are shown to lower R(T); deleterious mutations are shown to raise it. Previously described models for the overdispersed molecular clock are analyzed in terms of this work as are a few very simple new models. A model of deleterious mutations is shown to be sufficient to explain the observed values of R(T). Our current best estimates of R(T) suggest that either most mutations are deleterious or some key population parameter changes on a very slow time scale. No other interpretations seem plausible. Finally, a comment is made on how R(T) might be used to distinguish selective sweeps from background selection.     

Weibel-Palade body membrane proteins exhibit differential trafficking after exocytosis in endothelial cells

Weibel-Palade bodies, the secretory granules of endothelial cells, possess two different membrane proteins. However, P-selectin is seen only in Weibel-Palade bodies in HUVECs, whereas CD63 is also seen in late endosomes/lysosomes. Since P-selectin is targeted to lysosomes in heterologous expression studies, we have determined whether a lysosomal targeting signal also operates within HUVECs. We have also examined the trafficking of CD63 to its two different intracellular locations. By following antibodies bound at the plasma membrane during stimulation, we have discovered that while half of the P-selectin recycles to the WPBs, 50% is rapidly delivered to a lamp-1-positive compartment. Thus, the lysosomal targeting signal of this protein also operates in HUVECs. CD63 is found constitutively at the cell surface of HUVECs and most of it is delivered to the late endosomes/lysosomes after internalisation. However, stimulation causes both a rise in the CD63 plasma membrane level and in the amount that recycles to the WPBs. Our data strongly suggest that the CD63 that originates in the WPB preferentially recycles to the granule rather than being delivered to the late endosome/lysosome, and that there are, therefore, two separate pools of this protein within HUVECs. Our findings indicate that although P-selectin and CD63 are both targeted to the same compartments from the PM, the kinetics and the ratio of their targeting to Weibel-Palade bodies versus lysosomes are very different.     

Anthracenone ABA analogue as a potential photoaffinity reagent for ABA-binding proteins

An anthracenone analogue of abscisic acid (ABA) was synthesized as a potential photoaffinity reagent and tested for biological activity. Reaction between 10,10'-dimethoxy-9-anthrone with two equivalents of the lithiated dianion of cis-3-methylpent-2-en-4-yn-1-ol afforded an acetylenic alcohol key intermediate. Subsequent reduction of the triple bond, functional group manipulation of the side chain alcohol and deprotection of the dimethoxy protected anthrone provided anthracenone ABA analogue 7 as a potential photoaffinity reagent for ABA-binding proteins. The effect of natural ABA and the potential photoaffinity anthracenone ABA 7 on corn cell growth was determined at various concentrations. The results show that anthracenone ABA 7 is perceived as ABA-like, although producing less inhibition than ABA itself. For example, 7 at 33 microM produces approximately the same inhibition as ABA at 10 microM.     

The correlation of protein structure and evolution of a protein-coding gene: phylogenetic inference using cytochrome oxidase III

Discriminating phylogenetic signal from noise in DNA sequence data is a difficult problem in phylogenetic inference at higher systematic levels. For protein-coding genes, noise at synonymous (silent) positions can be filtered by deleting entire codon positions or types of change at a codon position. This method is not appropriate for replacement sites, because changes at each site within a codon may not be independent. This research presents a method using information from protein structure to evaluate variation in replacement sites. Analysis of the correlation of amino acid variation with protein structure identified rapidly evolving codons in the COIII gene. In a series of phylogenetic analyses attempting to recover a known set of vertebrate relationships, downweighting these labile codons produced the most accurate results. Structural correlates of variable and invariant residues identified in this study can be used to increase the accuracy of models used for phylogenetic inference. Viewing amino acid variation within a phylogenetic framework provided insight into residue changes important in the secondary and tertiary structures of the molecule, changes that were correlated between pairs of neighboring residues or between residues in neighboring helices.      

Rsu1 contributes to cell adhesion and spreading in MCF10A cells via effects on P38 map kinase signaling

The ILK, PINCH, Parvin (IPP) complex regulates adhesion and migration via binding of ILK to β1 integrin and α−parvin thus linking focal adhesions to actin cytoskeleton. ILK also binds the adaptor protein PINCH which connects signaling proteins including Rsu1 to the complex. A recent study of Rsu1 and PINCH1 in non-transformed MCF10A human mammary epithelial cells revealed that the siRNA-mediated depletion of either Rsu1 or PINCH1 decreased the number of focal adhesions (FAs) and altered the distribution and localization of FA proteins. This correlated with reduced adhesion, failure to spread or migrate in response to EGF and a loss of actin stress fibers and caveolae. The depletion of Rsu1 caused significant reduction in PINCH1 implying that Rsu1 may function in part by regulating levels of PINCH1. However, Rsu1, but not PINCH1, was required for EGF-induced activation of p38 Map kinase and ATF2 phosphorylation, suggesting a Rsu1 function independent from the IPP complex. Reconstitution of Rsu1-depleted cells with a Rsu1 mutant (N92D) that does not bind to PINCH1 failed to restore FAs or migration but did promote IPP-independent spreading and constitutive as well as EGF-induced p38 activation. In this commentary we discuss p38 activity in adhesion and how Rsu1 expression may be linked to Map kinase kinase (MKK) activation and detachment-induced stress kinase signaling.     

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Background

Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.

Results

This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.

Conclusions

Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.     

Impact of insurance and supply of health professionals on coverage of treatment for hypertension in Mexico: population based study

Objective To examine the independent and combined contributions of insurance status and supply of health professionals on coverage of antihypertensive treatment among adults in Mexico.Design Population based study.Setting Mexico.Participants 4032 hypertensive adults (2967 uninsured and 1065 insured): 1065 uninsured adults matched with 1065 adults insured through Seguro Popular, a programme to expand health insurance coverage to uninsured people in Mexico.Main outcome measures Coverage of antihypertensive treatment and coverage of antihypertensive treatment with control of blood pressure.Results Rates of treatment for hypertension varied by insurance status and supply of health professionals. Hypertensive adults insured through Seguro Popular had a significantly higher probability of receiving antihypertensive treatment (odds ratio 1.50, 95% confidence interval 1.27 to 1.78) and receiving antihypertensive treatment with control of blood pressure (1.35, 1.00 to 1.82). Greater supply of health professionals in areas with coverage through Seguro Popular was a significant predictor of antihypertensive treatment after adjusting for covariates (1.49, 1.00 to 2.20).Conclusions Expansion of healthcare coverage to uninsured people in Mexico was associated with greater use of antihypertensive treatment and blood pressure control, particularly in areas with a greater supply of health professionals.