CiteULike: jyuh Metzger

Inflammation and outcome in end-stage renal failure: does female gender constitute a survival advantage?

P Stenvinkel — 2008-06-30T14:59:44-00:00

Kidney international, Vol. 62, No. 5. (November 2002), pp. 1791-1798.

BACKGROUND: Elevated C-reactive protein (CRP) is a strong predictor of cardiovascular events and all-cause mortality in end-stage renal disease (ESRD) patients. However, although sex hormones may influence serum levels of inflammatory proteins, gender has not been taken into consideration in previous studies of inflammation and outcome in ESRD patients. METHODS: We included 663 (374 males) ESRD patients (59 +/- 1 year) from three European renal centers (Sweden, Germany and Italy) in which CRP levels and outcome data (follow-up 33 +/- 1 months) were available. The relation between outcome and serum levels of the soluble intercellular adhesion molecule (sICAM-1) was evaluated in 312 of the patients. RESULTS: The present study shows that elevated CRP is a strong predictor of outcome, but whereas no difference in all-cause mortality was observed between non-inflamed (CRP <or=3.4 mg/L) males and females, inflamed males had a significantly (log rank 6.1; P = 0.01) higher mortality rate than inflamed females. A strong positive correlation between CRP and sICAM-1 was found in the combined patient material (rho = 0.37; P < 0.0001) as well as in the male (rho = 0.25; P < 0.01) and female (rho = 0.52; P < 0.0001) subgroups. The Cox proportional hazard model showed that whereas both elevated sICAM-1 and log CRP predicted outcome in males, neither predicted outcome significantly in females. CONCLUSIONS: As inflamed female patients have a better outcome that inflamed males the present observation suggests that sex hormones may have important cardioprotective effects that limit the effect of inflammation on vascular injury in female ESRD patients.

Characterization of the Amino-terminal Transcriptional Activation Function of the Human Estrogen Receptor in Animal and Yeast Cells

Daniel Metzger — 2008-02-04T02:09:32-00:00

J. Biol. Chem., Vol. 270, No. 16. (21 April 1995), pp. 9535-9542.

We have previously reported that the transcriptional activation function AF-1, located in the A/B region of the human estrogen receptor, exhibits cell-type and promoter context specificity in both animal cells and yeast. To further characterize AF-1, we have constructed a number of deletion mutants spanning the A/B region in the context of either the whole human estrogen receptor or the A/B region linked to the GAL4 DNA binding domain, and tested their transcriptional activity in chicken embryo fibroblasts and in yeast cells, two cell types in which AF-1 efficiently activates transcription on its own. Additionally, we utilized HeLa cells in which AF-1 is poorly active but can synergize with the transcriptional activation function AF-2 located in the hormone binding domain. We show that in animal cells the “independent” activity of AF-1 is embodied in a rather hydrophobic proline-rich 99-amino acid activating domain (amino acids 51-149), whereas amino acids 51-93 and 102-149 can independently synergize with AF-2. Interestingly, in yeast, three discrete activating domains (amino acids 1-62, 80-113, and 118-149) are almost as active on their own as the whole A/B region, indicating that multiple activating domains can operate independently in yeast. Our study also demonstrates that, within the context of the whole human estrogen receptor, the same AF-1 activating domains are “induced” by either estradiol or 4-hydroxytamoxifen. 10.1074/jbc.270.16.9535

Inducible Cre/loxP recombination in the mouse proximal tubule.

B Dworniczak — 2008-01-20T09:01:07-00:00

Nephron Exp Nephrol, Vol. 106, No. 1. (2007)

Transgenic technologies in mice became invaluable experimental tools to identify the in vivo function of proteins. However, conventional knockout technology often results in embryonic lethality and because genes are frequently expressed in multiple cell types, the resulting knockout phenotypes can be complex and difficult or impossible to dissect. These issues are particularly important for gene-targeting strategies used to examine renal function. The kidney contains quite a number of different cell types, the function of many of which impacts that of other renal cells. To avoid these limitations conditional knockout strategies have been designed. As one important part of this system we describe the development of a mouse line expressing the tamoxifen-activatable Cre recombinase Cre-ER(T2) specifically in renal proximal tubules. The expression of Cre-ER(T2) is driven by a promoter fragment of the mouse gamma-glutamyl transpeptidase type II gene resulting in the generation of the activatable recombinase in S3 segments of the proximal tubules from which over 80% were positive for Cre activity. In combination with loxP-based conditional mutant mice as a second tool this tamoxifen-inducible Cre-ER(T2) line allows functional analysis of a variety of genes important for renal development and function in a precisely controlled spatiotemporal manner.

Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis.

K-F Becker — 2007-01-12T15:59:53-00:00

J Pathol (28 November 2006)

Owing to its cross-linking effects, it is currently believed that formalin fixation of routinely processed tissues in the clinic prevents protein extraction and profiling. The aim of our study was to develop a robust, fast, standardized, and easy to use technique for the solubilization of non-degraded, full length, and immunoreactive proteins from formalin-fixed tissues for western blot and protein microarray analysis. Sections of routinely processed formalin-fixed and paraffin-embedded tissues of various origin were analysed. After deparaffination, tissues were manually dissected from the slides and transferred into an optimized protein extraction buffer system. Proteins were solubilized and subsequently analysed by western blot and reverse phase protein microarrays. We succeeded in isolating non-degraded, soluble, and immunoreactive proteins from routinely processed formalin-fixed tissues. We were able to detect membrane, cytoplasmic and nuclear proteins at the expected molecular weight. No differences were found in the protein yield and protein abundances between fresh frozen and formalin-fixed tissues. Using western blots and reverse phase protein microarrays, the receptor tyrosine kinase HER2, an important protein target for antibody based cancer treatment, was reliably measured in formalin-fixed breast cancer biopsy samples when compared with measurement by immunohistochemistry and fluorescence in situ hybridization; remarkably, immunohistochemically equivocal cases (score 2 + ) can be categorized according to HER2 protein abundance. Our new clinically orientated multiplexed protein measurement system may be generally applicable to determine the relative abundances of known disease-related proteins in small amounts of routinely processed formalin-fixed tissue samples for research and diagnosis. This technique may also be used to identify, characterize, and validate known and new protein markers in a variety of human diseases. Copyright (c) 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.