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Hannover, Germany

Tarasov V.,Max Planck Institute of Biochemistry | Schwaiger R.,Institute For Klinische Chemie | Furtwangler K.,Max Planck Institute of Molecular Plant Physiology | Dyall-Smith M.,Charles Sturt University | Oesterhelt D.,Max Planck Institute of Biochemistry
BMC Molecular Biology

Background: The halophilic archaeon Halobacterium salinarum expresses bacteriorhodopsin, a retinal-protein that allows photosynthetic growth. Transcription of the bop (bacterioopsin) gene is controlled by two transcription factors, Bat and Brz that induce bop when cells are grown anaerobically and under light.Results: A new gene was identified that is transcribed together with the brz gene that encodes a small basic protein designated as Brb (bacteriorhodopsin-regulating basic protein). The translation activity of the start codon of the brb gene was confirmed by BgaH reporter assays. In vivo site-directed mutagenesis of the brb gene showed that the Brb protein cooperates with Brz in the regulation of bop expression. Using a GFP reporter assay, it was demonstrated that Brb cooperates with both Brz and Bat proteins to activate bop transcription under phototrophic growth conditions.Conclusions: The activation of the bop promoter was shown to be dependent not only on two major factors, Bat and Brz, but is also tuned by the small basic protein, Brb. © 2011 Tarasov et al; licensee BioMed Central Ltd. Source

Muller B.,Ruhr University Bochum | Muller B.,Institute For Klinische Chemie | Prante C.,Ruhr University Bochum | Knabbe C.,Ruhr University Bochum | And 2 more authors.
Glycoconjugate Journal

Recently, we demonstrated that the human xylosyltransferase II (XT-II) has enzymatic activity and is able to catalyze the initial and rate-limiting step in the biosynthesis of glycosaminoglycans (GAGs) like chondroitin and dermatan sulfate, as well as heparan sulfate and heparin. Therefore, this enzyme also very likely assumes a crucial regulatory role in the biosynthesis of proteoglycans (PGs). In this study, we identified and characterized for the first time the XYLT2 gene promoter region and transcription factors involved in its regulation. Several binding sites for members of the Sp1 family of transcription factors were identified as being necessary for transcriptional regulation of the XYLT2 gene. This was determined by mithramycin A treatment, electrophoretic mobility shift and supershift assays, as well as numerous site-directed mutagenesis experiments. Different 5′ and 3′ deletion constructs of the predicted GC rich promoter region, which lacks a canonical TATA and CAAT box, revealed that a 177 nts proximal promoter element is sufficient and indispensable to drive the constitutive transcription in full strength in HepG2 hepatoma cells. In addition, we also detected the transcriptional start site using 5′-RACE (rapid amplification of cDNA ends). Our results provide an insight into transcriptional regulation of the XYLT2 gene and may contribute to understanding the manifold GAG-involving processes in health and disease. © 2012 Springer Science+Business Media, LLC. Source

Haeckel R.,Katrepeler Landstrasse 45e | Wosniok W.,University of Bremen | Klauke R.,Institute For Klinische Chemie

A well-accepted tool for method validation is a method comparison study. Results are usually assessed on a scatter plot of which the fitting line is calculated by several approaches, for example, ordinary (vertical) linear regression (OLR), orthogonal regression (OR), Deming regression (DR), Passing-Bablok method (PBR) or standardized principal component regression (SPCR). DR was applied in its general form (gDR), requiring information of the imprecision of at least two different quantities and as simple DR (sDR) with imprecision information of only one quantity. The equation of the regression line calculated by these concepts varies depending on range of measurement, analytical variation and on imprecision ratio ( s AY / s AX ). There is still a global debate about which statistical concept is the most adequate for validating purposes. Various paired random samples with a size of 100 were simulated in 5000 replicates and evaluated with different regression models. The behavior of the slope and intercept of the regression lines were compared under various conditions. Two extreme ranges of measurement and several variance ratios in the absence and presence of bias were studied. The results clearly demonstrated that DR is the only model which can be applied without any precautions under conditions which usually occur in method comparison studies, and therefore should be preferred in laboratory medicine. Other models require restrictions with regard to range of measurement and/ or imprecision profile. Differences of the concentrations at different positions of the measurement interval calculated with regression coefficients of both DRs did not deviate more than the permissible bias. Therefore, the advantage of using gDR does not justify its greater disadvantages in comparison with sDR. © 2013 Walter de Gruyter GmbH. Source

Luppa P.B.,Institute For Klinische Chemie
Methods in molecular biology (Clifton, N.J.)

Surface plasmon resonance (SPR) is a novel biophysical detection method. In combination with sophisticated surface chemistries and sensing instrumentations, SPR biosensors are approved as tools for molecular interaction studies. SPR plays also a role in interaction proteomics. Once being detected in urine, SPR helps to unravel the functions of new proteins. Due to its outstanding analytical characteristics, SPR also moves more and more into the realm of quantitative analyses in the clinical laboratory. Complex urine determinations of proteins and/or metabolites will bring the SPR biosensor both to the core lab and to point-of-care-testing.This review delineates first the optical phenomena of SPR near to the gold surface, and also the main features of bioconjugation chemistry on a solid-state surface. Then the kinetic calculation of molecular interaction analysis using SPR is introduced. In order to portray the capability of the method, new applications in urine proteomics and proteinuria diagnostics are finally described in detail. Source

Schumann G.,Institute For Klinische Chemie | Canalias F.,Autonomous University of Barcelona | Joergensen P.J.,Kolding Hospital | Kang D.,Kyushu University | And 2 more authors.
Clinical Chemistry and Laboratory Medicine

The primary reference measurement procedures (PRMPs) for the international standardization of catalytic concentration measurements of α-amylase, alanine aminotransferase, aspartate aminotransferase (AST), creatine kinase (CK), γ-glutamyltransferase and lactate dehydrogenase have been performed in reference laboratories for several years. The IFCC Committee on Reference Systems for Enzymes and two reference laboratories, with official accreditation for the PRMPs, have collected useful information on some of the steps of the reference procedures that require special attention. This document comprises errata corrige for minor mistakes in published PRMPs for AST and CK. Several notes on the PRMPs are emphasized. This includes details that are very important for improved standardization, and general suggestions for reducing measurement uncertainty. © 2010 by Walter de Gruyter Berlin New York. Source

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