Publications by authors named "Rudolf Tauber"

62 Publications

A Matter of Caution: Coagulation Parameters in COVID-19 Do Not Differ from Patients with Ruled-Out SARS-CoV-2 Infection in the Emergency Department.

TH Open 2021 Jan 6;5(1):e43-e55. Epub 2021 Feb 6.

Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

COVID-19 (coronavirus disease 2019) patients often show excessive activation of coagulation, associated with increased risk of thrombosis. However, the diagnostic value of coagulation at initial clinical evaluation is not clear. We present an in-depth analysis of coagulation in patients presenting to the emergency department (ED) with suspected COVID-19.  = 58 patients with clinically suspected COVID-19 in the ED were enrolled.  = 17 subsequently tested positive using SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) polymerase chain reaction (PCR) swabs, while in  = 41 COVID-19 was ruled-out. We analyzed both standard and extended coagulation parameters, including thromboplastin time (INR), activated partial thromboplastin time (aPTT), antithrombin, plasminogen, plasminogen activator inhibitor-1 (PAI-1), D-dimers, and fibrinogen at admission, as well as α2-antiplasmin, activated protein C -resistance, factor V, lupus anticoagulant, protein C, protein S, and von Willebrand diagnostics. These data, as well as mortality and further laboratory parameters, were compared across groups based on COVID-19 diagnosis and severity of disease. In patients with COVID-19, we detected frequent clotting abnormalities, including D-dimers. The comparison cohort in the ED, however, showed similarly altered coagulation. Furthermore, parameters previously shown to distinguish between severe and moderate COVID-19 courses, such as platelets, plasminogen, fibrinogen, aPTT, INR, and antithrombin, as well as multiple nonroutine coagulation analytes showed no significant differences between patients with and without COVID-19 when presenting to the ED. At admission to the ED the prevalence of coagulopathy in patients with COVID-19 is high, yet comparable to the non-COVID-19 cohort presenting with respiratory symptoms. Nevertheless, coagulopathy might worsen during disease progression with the need of subsequent risk stratification.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1055/s-0040-1722612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867413PMC
January 2021

Role of cell adhesion molecules for disease development of patients with and without COVID-19 in the emergency department.

J Infect Dis 2021 Jan 27. Epub 2021 Jan 27.

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health; Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Berlin, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/infdis/jiab042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928779PMC
January 2021

Novichok nerve agent poisoning.

Lancet 2021 01 23;397(10270):249-252. Epub 2020 Dec 23.

Department of Nephrology and Medical Intensive Care, Charité-Universitätsmedizin Berlin, Berlin, Germany. Electronic address:

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S0140-6736(20)32644-1DOI Listing
January 2021

Patients' subjective assessment as a decisive predictor of malignancy in pelvic masses: results of a multicentric, prospective pelvic mass study.

J Psychosom Obstet Gynaecol 2020 Nov 30:1-6. Epub 2020 Nov 30.

Department of Gynecology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

Objective: The prognosis for ovarian cancer patients remains poor. A key to maximizing survival rates is early detection and treatment. This requires an accurate prediction of malignancy. Our study seeks to improve the accuracy of prediction by focusing on early subjective assessment of malignancy. We therefore investigated the assessment of patients themselves in comparison to the assessment of physicians.

Methods: One thousand three hundred and thirty patients participated in a prospective and multicenter study in six hospitals in Berlin. Using univariate analysis and multivariate logistic regression models, we measured the accuracy of the early subjective assessment in comparison to the final histological outcome. Moreover, we investigated factors related to the assessment of patients and physicians.

Results: The patients' assessment of malignancy is remarkably accurate. With a positive predictive value of 58%, the majority of patients correctly assessed a pelvic mass as malignant. With more information available, physicians achieved only a slightly more accurate prediction of 63%.

Conclusions: For the first time, our study considered subjective factors in the diagnostic process of pelvic masses. This paper demonstrates that the patients' personal assessment should be taken seriously as it can provide a significant contribution to earlier diagnosis and thus improved therapy and overall prognosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/0167482X.2020.1850684DOI Listing
November 2020

Outcome prediction by serum calprotectin in patients with COVID-19 in the emergency department.

J Infect 2021 04 17;82(4):84-123. Epub 2020 Nov 17.

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Berlin, Germany; Labor Berlin - Charité Vivantes GmbH, Berlin, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jinf.2020.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670934PMC
April 2021

Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

Cell 2020 09 5;182(6):1419-1440.e23. Epub 2020 Aug 5.

Department of Infectious Diseases and Respiratory Medicine, Charité, Universitätsmedizin Berlin, Berlin, Germany; German Center for Lung Research (DZL).

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRCD11c inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DR monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405822PMC
September 2020

Assessment of serum ferritin as a biomarker in COVID-19: bystander or participant? Insights by comparison with other infectious and non-infectious diseases.

Biomarkers 2020 Dec 24;25(8):616-625. Epub 2020 Nov 24.

Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité - Universitätsmedizin Berlin, Berlin, Germany.

Background: The 2019 coronavirus disease (COVID-19) caused by the SARS-CoV-2 virus has an impact on all aspects of patient care. Serum ferritin generally represents a biomarker of choice when iron deficiency is suspected. However, ferritin is also an acute-phase-protein exhibiting elevated serum concentration in various inflammatory diseases. Here we focus on the role of serum ferritin for diagnostic and clinical management of patients with COVID-19 in comparison with other infectious and non-infectious diseases.

Methods: We examined scientific articles listed in PubMed reporting on ferritin in various infectious and non-infectious diseases. We then compared these results with nine current COVID-19 ferritin reports published in 2020.

Results: Several non-infectious, as well as non-COVID-19 infectious diseases, are characterised by a partly dramatic elevation of serum ferritin levels. All COVID-19 studies published between February and May 2020, which documented laboratory serum ferritin, indicate ferritin as a biomarker of COVID-19 severity in hospitalised patients.

Conclusions: Serum ferritin may be considered both a prognostic and stratifying biomarker that can also contribute to therapeutic decision-making concerning patients with COVID-19. It should be emphasised, however, that most scientific reports refer to cohorts in the Asian region. Further validation in other cohorts is urgently required.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/1354750X.2020.1797880DOI Listing
December 2020

Pooling, room temperature, and extended storage time increase the release of adult-specific biologic response modifiers in platelet concentrates: a hidden transfusion risk for neonates?

Transfusion 2020 Aug 27;60(8):1828-1836. Epub 2020 Apr 27.

Klinik für Neonatologie, Charité-Universitätsmedizin Berlin, Berlin, Germany.

Background: Adult donor platelets (PLTs) are frequently transfused to prevent or stop bleeding in neonates with thrombocytopenia. There is evidence for PLT transfusion-related morbidity and mortality, leading to the hypothesis on immunomodulatory effects of transfusing adult PLTs into neonates. Candidate factors are biologic response modifiers (BRMs) that are expressed at higher rates in adult than in neonatal PLTs. This study investigated whether storage conditions or preparation methods impact on the release of those differentially expressed BRMs.

Study Design And Methods: Pooled PLT concentrates (PCs) and apheresis PCs (APCs) were stored under agitation for up to 7 days at room temperature (RT) or at 2 to 8°C. The BRMs CCL5/RANTES, TGFβ1, TSP1, and DKK1 were measured in PCs' supernatant, lysate, and corresponding plasma. PLT function was assessed by light transmission aggregometry.

Results: Concerning the preparation method, higher concentrations of DKK1 were found in pooled PCs compared to APCs. In supernatants, the concentrations of CCL5, TGFβ1, TSP1, and DKK1 significantly increased, both over standard (≤4 days) and over extended storage times (7 days). Each of the four BRMs showed an up to twofold increase in concentration after storage at RT compared to cold storage (CS). There was no difference in the aggregation capacity.

Conclusion: This analysis shows that the release of adult-specific BRMs during storage is lowest in short- and CS APCs. Our study points to strategies for reducing the exposure of sick neonates to BRMs that can be specifically associated to PLT transfusion-related morbidity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/trf.15827DOI Listing
August 2020

Enhanced specificity due to method specific limits for relative ion intensities in a high-performance liquid chromatography - tandem mass spectrometry method for iohexol in human serum.

Clin Chem Lab Med 2020 04;58(5):709-718

Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute for Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Berlin, Germany.

Background Accurate assessment of kidney function is needed for a variety of clinical indications and for research. The measurement of the serum clearance of iohexol has emerged as a feasible method to reach this objective. We report the analytical validation and clinical application of a new high-performance liquid chromatography (HPLC) - tandem mass spectrometry (MS/MS) assay to quantify iohexol in human serum. Specificity was enhanced due to the use of method specific acceptance limits for relative ion (RI) intensities. Methods The internal standard ioversol was added to 50 μL serum prior to protein precipitation with methanol. Linear gradient elution was performed on a Waters Oasis® HLB column. Three transitions for both iohexol and ioversol were monitored allowing calculation of RIs. Measurements acquired during method validation were used as a training set to establish stricter acceptance criteria for RIs which were then tested retrospectively on clinical routine measurements (86 measurements) and on mathematically simulated interferences. Results The method was linear between 5.0 μg/mL (lower limit of quantification [LLOQ]) and 100.3 μg/mL iohexol. Intraday and interday imprecision were ≤2.6% and ≤3.2%, respectively. Bias was -1.6% to 1.5%. All validation criteria were met, including selectivity, recovery, extraction efficiency and matrix effects. Retrospectively acceptance limits for RIs could be narrowed to ±4 relative standard deviations of the corresponding RIs in the training set. The new limits resulted in an enhanced sensitivity for the simulated interferences. Conclusions Criteria for validation were met and the assay is now used in our clinical routine diagnostics and in research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1515/cclm-2019-1001DOI Listing
April 2020

Platelet-derived growth factor receptor β activation and regulation in murine myelofibrosis.

Haematologica 2020 08 31;105(8):2083-2094. Epub 2019 Oct 31.

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Berlin, Germany

There is prevailing evidence to suggest a decisive role for platelet-derived growth factors (PDGF) and their receptors in primary myelofibrosis. While PDGF receptor β (PDGFRβ) expression is increased in bone marrow stromal cells of patients correlating with the grade of myelofibrosis, knowledge on the precise role of PDGFRβ signaling in myelofibrosis is sparse. Using the Gata-1 mouse model for myelofibrosis, we applied RNA sequencing, protein expression analyses, multispectral imaging and, as a novel approach in bone marrow tissue, an proximity ligation assay to provide a detailed characterization of PDGFRβ signaling and regulation during development of myelofibrosis. We observed an increase in PDGFRβ and PDGF-B protein expression in overt fibrotic bone marrow, along with an increase in PDGFRβ-PDGF-B interaction, analyzed by proximity ligation assay. However, PDGFRβ tyrosine phosphorylation levels were not increased. We therefore focused on regulation of PDGFRβ by protein tyrosine phosphatases as endogenous PDGFRβ antagonists. Gene expression analyses showed distinct expression dynamics among PDGFRβ-targeting phosphatases. In particular, we observed enhanced T-cell protein tyrosine phosphatase protein expression and PDGFRβ-T-cell protein tyrosine phosphatase interaction in early and overt fibrotic bone marrow of Gata-1 mice. , T-cell protein tyrosine phosphatase () knockdown increased PDGFRβ phosphorylation at Y and Y, leading to enhanced downstream signaling in fibroblasts. Furthermore, knockdown cells showed increased growth rates when exposed to low-serum growth medium. Taken together, PDGF signaling is differentially regulated during myelofibrosis. Protein tyrosine phosphatases, which have so far not been examined during disease progression, are novel and hitherto unrecognized components in myelofibrosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2019.226332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395273PMC
August 2020

Underfilling of vacuum blood collection tubes leads to increased lactate dehydrogenase activity in serum and heparin plasma samples.

Clin Chem Lab Med 2020 01;58(2):213-221

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Berlin, Germany.

Background Lactate dehydrogenase (LD) activity is routinely monitored for therapeutic risk stratification of malignant diseases, but is also prone to preanalytical influences. Methods We systematically analyzed the impact of defined preanalytical conditions on the hemolysis-susceptible parameters LD, potassium (K) and hemolysis index in vacuum blood collection tubes (serum [SE], heparin plasma [HP]). Blood was collected by venipuncture from healthy volunteers. Tubes were either filled or underfilled to approximately 50%, then processed directly or stored at room temperature for 4 h. Potassium (K), sodium (Na), chloride (Cl), LD, creatine kinase (CK), total cholesterol, and indices for hemolysis, icterus, and lipemia were analyzed. Filling velocity was determined in a subset of tubes. Findings in healthy volunteers were reconfirmed in an in-patient cohort (n = 74,751) that was analyzed for plasma yield and LD data distribution. Results LD activity was higher in HP compared to SE. Underfilling led to higher LD values (SE: +21.6%; HP: +28.3%), K (SE: +4.2%; HP: +5.3%), and hemolysis index (SE: +260.8%; HP: +210.0%), while other analytes remained largely unchanged. Filling velocity of tubes was approximately 3-fold higher in the first half compared to the second half in both HP and SE collection tubes. Importantly, plasma yield also inversely correlated with LD in routine patients. By calculating reference limits, the lowest plasma yield quartile of the patient cohort displayed LD values clearly exceeding current reference recommendations. Conclusions Underfilling of tubes leads to a higher proportion of blood aspirated with high velocity and relevant elevations in LD. This finding should be considered in cases of clinically implausible elevated LD activities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1515/cclm-2019-0616DOI Listing
January 2020

Kidney Injury after Intravenous versus Intra-arterial Contrast Agent in Patients Suspected of Having Coronary Artery Disease: A Randomized Trial.

Radiology 2019 09 2;292(3):664-672. Epub 2019 Jul 2.

From the Department of Radiology, Charité-Universitätsmedizin Berlin, Humboldt-Universität and Freie Universität zu Berlin, Schumannstr 20/21, Berlin 10117, Germany (E.S., M.B., E.Z., R.T, M.L., M.D.); and Institute for Clinical Epidemiology and Applied Biometry, University of Tübingen, Tübingen, Germany (P.M.).

Background In the absence of randomized studies, it has been controversial whether the likelihood of acute kidney injury (AKI) differs between intravenous and intra-arterial contrast agent administration. Purpose To compare intravenous versus intra-arterial contrast agent administration in relationship to AKI and analyze the association between AKI and chronic kidney disease (defined as at least mildly decreased estimated glomerular filtration rates [eGFRs]). Materials and Methods This was a prospective study (: NCT00844220) that involved randomizing participants with atypical chest pain and suspected coronary artery disease (CAD) between February 2009 and August 2015 to undergo coronary CT angiography with intravenous contrast agent administration or cardiac catheterization angiography with intra-arterial contrast agent administration. This prespecified secondary analysis compared AKI (serum creatinine increase of ≥ 25% or 0.5 mg/dL after 18-24 or 46-50 hours) determined by blinded investigators using absolute differences and relative risks, including two-sided 95% confidence intervals (CIs). Results A total of 320 participants (163 [50.9%] women; mean age, 60 years ± 11) were included. Baseline eGFR did not differ between the CT angiography group (84.3 mL/min/1.73 m ± 17.2) and the catheterization group (87.1 mL/min/1.73 m ± 16.7) ( = .14). AKI occurred in nine of 161 participants in the CT angiography group (5.6%; 95% CI: 3%, 10%) and in 21 of 159 participants in the catheterization group (13.2%; 95% CI: 9%,19%) (relative risk, 2.4; 95% CI: 1.1, 5.0; = .02). Also in the subgroup of participants without obstructive CAD, in those not requiring coronary interventions, AKI was more common in the catheterization group (11.9%; 95% CI: 8%, 19%) than in the CT angiography group (4.3% [95% CI: 2%, 9%]; difference, 7.7% [95% CI: 1.3%, 14.1%]; relative risk, 2.8 [95% CI: 1.1, 7.0]; = .02). Obstructive CAD (odds ratio [OR]: 2.7 [95% CI: 1.1, 6.6]; = .02), femoral catheter access (OR: 2.5 [95% CI: 1.1, 5.6]; = .04), and cine ventriculography were associated with AKI (OR: 2.3 [95% CI: 1.0, 4.9]; = .03). In multivariable analysis, the presence of postcontrast AKI was associated with chronic kidney disease (hazard ratio: 12.4 [95% CI: 4.5, 34.6]; < .01). Conclusion Acute kidney injury was more common after cardiac catheterization than after CT angiography in this prospective randomized study of patients suspected of having coronary artery disease. © RSNA, 2019 See also the editorial by Einstein and Newhouse in this issue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1148/radiol.2019182220DOI Listing
September 2019

The effect of blood sampling and preanalytical processing on human N-glycome.

PLoS One 2018 11;13(7):e0200507. Epub 2018 Jul 11.

Charité -Universitätsmedizin Berlin, Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Berlin, Germany.

Glycome modulations have been described in the onset and progression of many diseases. Thus, many studies have proposed glycans from blood glycoproteins as disease markers. Astonishingly, little effort has been given unraveling preanalytical conditions potentially influencing glycan analysis prior to blood biomarker studies. In this work, we evaluate for the first time the effect of hemolysis, storage and blood collection, but also influence of various times and temperatures between individual processing steps on the total N-glycome and on a glycan-biomarker score. Venous blood was collected from 10 healthy donors in 11 blood collection tubes with different additives, processed variously to obtain 16 preanalytical variables and N-glycans released from serum or plasma were analyzed by MALDI-TOF-MS and capillary electrophoresis coupled with fluorescence detection (CE-LIF) for the first time. Long time storage of deep frozen samples at -20°C or -80°C exerted only a minor influence on the glycome as demonstrated by CE-LIF. The N-glycome was very stable evidenced by MALDI-TOF when stored at 4°C for at least 48 hours and blood collected in tubes devoid of additives. The glycome was stable upon storage after centrifugation and aliquoting, which is an important information considering future diagnostic applications. Hemolysis, however, negatively correlated with an established glycan score for ovarian cancer, when evaluated by MALDI-TOF-MS measurement by affecting relative intensities of certain glycans, which could lead to false negative / positive results in glycan biomarker studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200507PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040761PMC
January 2019

Werner Reutter: A Visionary Pioneer in Molecular Glycobiology.

Chembiochem 2017 07 16;18(13):1141-1145. Epub 2017 Jun 16.

Leibniz-Forschungsinstitut für Molekulare Pharmakologie FMP, Department of Chemical Biology, Robert-Roessle-Strasse 10, 13125, Berlin, Germany.

A creative pioneer: Werner Reutter (1937-2016) was a scientist who both made fundamental discoveries in glycobiology and reached out to disciplines beyond his core field. Many of his former colleagues and students will remember his desire to exchange research ideas, which ultimately contributed to the birth of new research fields.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201700277DOI Listing
July 2017

Reducing Macro- and Microheterogeneity of N-Glycans Enables the Crystal Structure of the Lectin and EGF-Like Domains of Human L-Selectin To Be Solved at 1.9 Å Resolution.

Chembiochem 2017 07 6;18(13):1338-1345. Epub 2017 Jun 6.

Institut für Chemie und Biochemie/Kristallographie, Freie Universität Berlin, Takustrasse 6, 14195, Berlin, Germany.

L-Selectin, a cell-adhesion receptor on the surface of most leukocytes, contains seven N-glycosylation sites. In order to obtain the crystal structure of human L-selectin, we expressed a shortened version of L-selectin comprising the C-type lectin and EGF-like domains (termed LE) and systematically analysed mutations of the three glycosylation sites (Asn22, Asn66 and Asn139) in order to reduce macroheterogeneity. After we further removed microheterogeneity, we obtained crystals that diffracted X-rays up to 1.9 Å from a variant (LE010) with exchanges N22Q and N139Q and one GlcNAc Man N-glycan chain attached to Asn66. Crystal-structure analysis showed that the terminal mannose of GlcNAc Man of one LE010 molecule was coordinated to Ca in the binding site of a symmetry-related LE010. The orientation of the lectin and EGF-like domain was similar to the described "bent" conformation of E- and P-selectins. The Ca -binding site reflects the binding mode seen in E- and P-selectin structures co-crystallised with ligands.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201700220DOI Listing
July 2017

The Cell-Surface N-Glycome of Human Embryonic Stem Cells and Differentiated Hepatic Cells thereof.

Chembiochem 2017 07 15;18(13):1234-1241. Epub 2017 May 15.

Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany.

Human embryonic stem cells (hESCs) are pluripotent stem cells that offer a wide range of applications in regenerative medicine. In addition, they have been proposed as an appropriate alternative source of hepatocytes. In this work, hESCs were differentiated into definitive endodermal cells (DECs), followed by maturation into hepatocyte-like cells (HLCs). Their cell-surface N-glycome was profiled and also compared with that of primary human hepatocytes (PHHs). Undifferentiated hESCs contained large amounts of high-mannose N-glycans. In contrast, complex-type N-glycans such as asialylated or monosialylated biantennary and triantennary N-glycans were dominant in HLCs, and fully galactosylated structures were significantly more abundant than in undifferentiated hESCs. The cell-surface N-glycosylation of PHHs was more biologically processed than that of HLCs, with bisialylated biantennary and trisialylated triantennary structures predominant. This is the first report of the cell surface N-glycome of PHHs and of HLCs being directly generated from hESCs without embryoid body formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201700001DOI Listing
July 2017

High incidence of cardiac dysfunction and response to antiviral treatment in patients with chronic hepatitis C virus infection.

Clin Res Cardiol 2017 Jul 24;106(7):551-556. Epub 2017 Feb 24.

Department of Cardiology, Campus Benjamin Franklin, CC11, Charité-Universitätsmedizin Berlin, Hindenburgdamm 30, 12200, Berlin, Germany.

Aims: Hepatitis C virus (HCV) has been associated with cardiomyopathies. Former anti-HCV therapies employing interferon could have serious side effects in patients with advanced heart failure since interferon may adversely impact upon cardiac function. We, therefore, examined whether the novel, interferon-free and highly virus-selective anti-HCV combination therapy might be applicable even in advanced or end-stage heart failure.

Methods And Results: In a retrospective series of HCV-positive patients admitted to our institution with suspected cardiac disease, coronary, valvular or hypertensive heart disease was diagnosed in 70/146 (47.9%). Among the others, 36/76 (47.4%) had myocardial disease: LV (32.9%)/RV (13.2%) hypertrophy, RV dysfunction (13.2%)/dilation (6.6%), severe diastolic dysfunction (7.9%), pulmonary hypertension (22.4%). One critically ill patient listed for heart transplantation (HTX) had previously not tolerated an interferon-based protocol. To still improve her chance of enduring transplant survival, we attempted an interferon-free virus-selective antiviral combination drug protocol under careful monitoring of possible side effects. Regarding clinical status she tolerated this treatment well, with the exception of transient severe hyponatremia requiring substitution. Her NYHA functional class improved from II-IV before to class II immediately after successful complete HCV elimination.

Conclusions: Whereas prevalence of cardiac dysfunction and potential benefit from antiviral treatment was reported previously, there is lack of data regarding the response of patients with advanced heart failure. Since the highly HCV-selective drugs used above do not eliminate other cardiotropic viruses and have no direct effect on inflammation, massive improvement in such critically ill patients indicates a causal role of HCV in their cardiac failure, and of HCV elimination in their functional recovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00392-017-1086-1DOI Listing
July 2017

The ascites N-glycome of epithelial ovarian cancer patients.

J Proteomics 2017 03 8;157:33-39. Epub 2017 Feb 8.

Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité Medical University, Augustenburger Platz 1, 13353 Berlin, Germany. Electronic address:

Epithelial ovarian cancer (EOC) is worldwide the sixth most lethal form of cancer occurring in women. More than one third of ovarian patients have ascites at the time of diagnosis and almost all of them have it when recurrence occurs. Although its effect on tumor cell microenvironment remains poorly understood, its presence is correlated with bad diagnosis. In previous studies, we proposed a novel glycan-based biomarker for the diagnosis of EOC, which showed an improved sensitivity and specificity at any stage of the disease and an improved discrimination between malignant and benign ovarian tumors. In this work, we report for the first time the N-glycome profiles of ascitic fluid from primary serous EOC patients and compare them with the serum N-glycomes of the same patients as well as of healthy controls. N-Glycans were digested from equivalent amount of ascites and serum from 18 EOC patients and from serum of 20 age-matched controls and measured by MALDI-TOF-MS. Ascites N-glycome showed increased antennarity, branching, sialylation and Lewis motives compared to healthy serum. In addition, a correlation was established between ascites volume and degree of sialylation.

Significance: Malignant ascitic fluid is the build-up of large volumes of fluid in the peritoneal cavity secondary to cancer. At least one-third of ovarian cancer patients develop ascites, a generally voluminous fluid containing cells of tumor origin, in the course of cancer and almost all when recurrence occurs. The proteome of ascites is known to be as complex as that of serum and contains high amount of proteins shed from inflammatory cells as well as from tumor cells. Although many attempts have been made to provide molecular insight into the proteomic and peptidomic content of malignant ascites, no data about the N-glycome of the ascitic fluid fraction from cancer patients has been reported to date. In this study, the N-glycosylation profile of ascites from 20 patients suffering from epithelial ovarian cancer (EOC) was analyzed by MALDI-TOF-mass spectrometry and compared to the pathologically modified N-glycan pattern obtained from serum of the same patients as well as to the pattern of serum from healthy individuals. Significant quantitative differences were observed in the ascites of EOC patients when compared to the serum of healthy subjects. The glycome of ascites shows typical features of inflammatory conditions, what was also found in the serum of patients suffering from EOC when compared to healthy serum. In addition, a correlation was established between ascites volume and degree of sialylation, showing that the high-volume ascites contains a higher amount of sialylated structures than the low-volume ascites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jprot.2017.02.001DOI Listing
March 2017

Structural requirements of mono- and multivalent L-selectin blocking aptamers for enhanced receptor inhibition in vitro and in vivo.

Nanomedicine 2016 May 7;12(4):901-908. Epub 2016 Jan 7.

Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Berlin, Germany. Electronic address:

Unlabelled: L-selectin mediates extravasation of leukocytes from blood into the surrounding tissue during inflammation and is therefore a therapeutical target in certain overwhelming immune reactions. In this study, we characterized an L-selectin specific blocking DNA aptamer with respect to nucleotide composition and target binding. Introduction of deletions and nucleotide exchanges resulted in an optimized DNA sequence but preservation of the IC50 in the low nanomolar range. The inhibitory potential was significantly increased when the aptamer was displayed as a di- and trimer connected via appropriate linker length. Similar to monoclonal antibodies, trimer yielded picomolar IC50 values in a competitive binding assay. In comparison to the monovalent aptamer, the trivalent assembly reduced PBMC interactions to L-selectin ligands 90-fold under shear and exerted superior inhibition of PBMC rolling in vivo. In conclusion, our work demonstrates the feasibility of optimizing aptamer sequences and shows that multivalent ligand presentation enables superior adhesion receptor targeting.

From The Clinical Editor: During inflammation, leukocytes extravasate from blood vessels under chemotaxic signals. The presence of L-selectin on endothelium acts as a mediator for the extravasation process. In this study, the authors investigated an L-selectin specific blocking DNA aptamer in various forms, as inhibitors to leukocyte binding and extravasation. This new approach confirmed the potential use of aptamers in clinical setting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.nano.2015.12.379DOI Listing
May 2016

Acute-phase glycoprotein N-glycome of ovarian cancer patients analyzed by CE-LIF.

Electrophoresis 2016 06 17;37(11):1461-7. Epub 2016 Feb 17.

Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité Medical University, Berlin, Germany.

Epithelial ovarian cancer (EOC) is the most frequent cause of death from all gynecological malignancies because of its late diagnosis. As N-glycosylation is modified in the course of ovarian cancer, it is a promising source of tumor biomarkers. In this work, serum glycoproteins, depleted from albumin and IgG, were separated by 2DE. Protein spots of acute-phase proteins were identified by peptide mapping and their corresponding glycan moieties were released enzymatically, fluorescently labeled and analyzed by CE-LIF. In the positive acute-phase proteins, haptoglobin, α1-antitrypsin, and α1-antichymotrypsin, an increase of antennarity and Lewis(X) motif could be measured in EOC patients on tri- and/or tetraantennary N-glycans. Tetraantennary N-glycans containing three Lewis(X) epitopes and triantennary N-glycans containing a β(1-6) branch and a Lewis(X) epitope were only present in EOC patients. We also showed for the first time that the core-fucosylated biantennary digalactosylated N-glycan of α1-acid glycoprotein is a potential biomarker for EOC. To conclude, core-fucosylated biantennary N-glycans on α1-acid glycoprotein as well as higher antennarity and increased amounts of Lewis(X) motif on haptoglobin, α1-antitrypsin, and α1-antichymotrypsin are promising biomarkers for EOC. Nevertheless, their specificity and selectivity for the early detection of EOC should be evaluated in a larger study.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/elps.201500518DOI Listing
June 2016

Cupulin is a zona pellucida-like domain protein and major component of the cupula from the inner ear.

PLoS One 2014 4;9(11):e111917. Epub 2014 Nov 4.

Klinik für Oto-Rhino-Laryngologie, Charité - Universitätsmedizin Berlin, Berlin, Germany.

The extracellular membranes of the inner ear are essential constituents to maintain sensory functions, the cupula for sensing torsional movements of the head, the otoconial membrane for sensing linear movements and accelerations like gravity, and the tectorial membrane in the cochlea for hearing. So far a number of structural proteins have been described, but for the gelatinous cupula precise data are missing. Here, we describe for the first time a major proteinogenic component of the cupula structure with an apparent molecular mass of 45 kDa from salmon. Analyses of respective peptides revealed highly conserved amino-acid sequences with identity to zona pellucida-like domain proteins. Immunohistochemistry studies localized the protein in the ampulla of the inner ear from salmon and according to its anatomical appearance we identified this glycoprotein as Cupulin. Future research on structure and function of zona pellucida-like domain proteins will enhance our knowledge of inner ear diseases, like sudden loss of vestibular function and other disturbances.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111917PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4219815PMC
December 2015

Rapid analysis of cell surface N-glycosylation from living cells using mass spectrometry.

J Proteome Res 2014 Dec 13;13(12):6144-51. Epub 2014 Nov 13.

Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin , Augustenburger Platz 1, 13353 Berlin, Germany.

Cell surfaces are covered with a dense carbohydrate layer referred to as the glycocalyx. Because different cell types express different glycan signatures, it is of paramount importance to have robust methods to analyze the glycome of living cells. To achieve this, a common procedure involves cell lysis and extraction of membrane (glyco)proteins and yields a major proportion of high-mannose N-glycans that most likely stem from intracellular proteins derived from the ER. Using HEK 293 cells as a model system, we developed a reproducible, sensitive, and fast method to profile surface N-glycosylation from living cells. We directly released glycopeptides from cell surfaces through tryptic digestion of freshly harvested and vital cells, thereby improving the detection and quantification of complex-type N-glycans by increasing their relative amount from 14 to 85%. It was also possible to detect 25 additional structures in HEK 293, 48 in AGE1.HN, 42 in CHO-K1, and 51 in Hep G2 cells. The additional signals provided deeper insight into cell-type-specific N-glycan features such as antennarity, fucosylation, and sialylation. Thus, this protocol, which can potentially be applied to any cells, will be useful in the fields of glycobiotechnology and biomarker discovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr5003005DOI Listing
December 2014

Improving compliance to colorectal cancer screening using blood and stool based tests in patients refusing screening colonoscopy in Germany.

BMC Gastroenterol 2014 Oct 17;14:183. Epub 2014 Oct 17.

Central Interdisciplinary Endoscopy Unit, Department for Internal Medicine with focus on Hepatology, Gastroenterology and Metabolic Diseases, University Hospitals Berlin, Charité-Virchow-Klinikum, Berlin, Germany.

Background: Despite strong recommendations for colorectal cancer (CRC) screening, participation rates are low. Understanding factors that affect screening choices is essential to developing future screening strategies. Therefore, this study assessed patient willingness to use non-invasive stool or blood based screening tests after refusing colonoscopy.

Methods: Participants were recruited during regular consultations. Demographic, health, psychological and socioeconomic factors were recorded. All subjects were advised to undergo screening by colonoscopy. Subjects who refused colonoscopy were offered a choice of non-invasive tests. Subjects who selected stool testing received a collection kit and instructions; subjects who selected plasma testing had a blood draw during the office visit. Stool samples were tested with the Hb/Hp Complex Elisa test, and blood samples were tested with the Epi proColon® 2.0 test. Patients who were positive for either were advised to have a diagnostic colonoscopy.

Results: 63 of 172 subjects were compliant to screening colonoscopy (37%). 106 of the 109 subjects who refused colonoscopy accepted an alternative non-invasive method (97%). 90 selected the Septin9 blood test (83%), 16 selected a stool test (15%) and 3 refused any test (3%). Reasons for blood test preference included convenience of an office draw, overall convenience and less time consuming procedure.

Conclusions: 97% of subjects refusing colonoscopy accepted a non-invasive screening test of which 83% chose the Septin9 blood test. The observation that participation can be increased by offering non-invasive tests, and that a blood test is the preferred option should be validated in a prospective trial in the screening setting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-230X-14-183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287474PMC
October 2014

Identification of 34 N-glycan isomers in human serum by capillary electrophoresis coupled with laser-induced fluorescence allows improving glycan biomarker discovery.

Anal Bioanal Chem 2014 Nov 19;406(28):7185-93. Epub 2014 Sep 19.

Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité Medical University, Augustenburger Platz 1, 13353, Berlin, Germany.

Alterations in glycosylation have been observed in many human diseases and specific changes in glycosylation have been proposed as relevant diagnostic information. Capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) is a robust method to quantify desialylated N-glycans that are labeled with 8-aminopyrene-1,3,6-trisulfonic acid prior to analysis. To date, only a maximum of 12 glycan structures, the most abundant ones, have been identified by CE-LIF to characterize glycome modulations of total serum in the course of the diseases. In most forms of cancer, findings using CE-LIF were limited to the increase of triantennary structures carrying a Lewis(x) epitope. In this work, we identified 32 linkage and positional glycan isomers in healthy human serum using exoglycosidase digestions as well as standard glycoproteins, for which we report the assignment of novel structures. It was possible to identify and quantify 34 glycan isomers in the serum of primary epithelial ovarian cancer patients (EOC). Reduced levels of diantennary structures and of high-mannose 5 were statistically significant in the EOC samples, and also, elevated branching as well as increased antennary fucosylation were observed. For the first time, we could demonstrate that not only antennary fucosylation was of relevance in tetraantennary structures but also core-fucosylated tetraantennary N-glycans were statistically increased in EOC patients. The results of the current study provide an improved dataset to be used in glycan biomarker discovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00216-014-8168-yDOI Listing
November 2014

The serum glycome to discriminate between early-stage epithelial ovarian cancer and benign ovarian diseases.

Dis Markers 2014 12;2014:238197. Epub 2014 Aug 12.

Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité Medical University, Augustenburger Platz 1, 13353 Berlin, Germany.

Epithelial ovarian cancer (EOC) is the sixth most common cause of cancer deaths in women because the diagnosis occurs mostly when the disease is in its late-stage. Current diagnostic methods of EOC show only a moderate sensitivity, especially at an early-stage of the disease; hence, novel biomarkers are needed to improve the diagnosis. We recently reported that serum glycome modifications observed in late-stage EOC patients by MALDI-TOF-MS could be combined as a glycan score named GLYCOV that was calculated from the relative areas of the 11 N-glycan structures that were significantly modulated. Here, we evaluated the ability of GLYCOV to recognize early-stage EOC in a cohort of 73 individuals comprised of 20 early-stage primary serous EOC, 20 benign ovarian diseases (BOD), and 33 age-matched healthy controls. GLYCOV was able to recognize stage I EOC whereas CA125 values were statistically significant only for stage II EOC patients. In addition, GLYCOV was more sensitive and specific compared to CA125 in distinguishing early-stage EOC from BOD patients, which is of high relevance to clinicians as it is difficult for them to diagnose malignancy prior to operation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2014/238197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4145549PMC
April 2015

Sialic acid methylation refines capillary electrophoresis laser-induced fluorescence analyses of immunoglobulin G N-glycans of ovarian cancer patients.

Electrophoresis 2014 Apr;35(7):1025-31

Alterations in IgG N-glycosylation coincide with the development of a number of diseases including cancer and could potentially be used as diagnostic markers. CE-LIF of 8-aminopyrene-1,3,6-trisulfonic acid labeled N-glycans is a well-established rapid method to characterize IgG N-glycans that needs only low amounts of starting material. However,sialylated N-glycans have short migration times due to their negative charge. As a result,some of them are not well resolved and co-migrate with neutral glycans. In this work, we neutralized the negative charge of sialic acids by methylation and optimized the protocol using the commercially available disialylated biantennary oligosaccharide (A2G2S2). IgGN-glycans isolated from healthy human serum were then analyzed using this method. We could demonstrate that co-migration of A2, FA2G2S1, and FA2B[3]G1S1 was prevented,which allowed an accurate quantification of these N-glycans. Finally, we investigated the IgG N-glycan profiles of patients suffering from ovarian cancer using the conventional and methylation methods.With both methods, we observed an increase of agalactosylated structures that was accompanied by a decrease in digalactosylated structures. Finally, using the methylation protocol, we could further demonstrate an increase of A2, which was technically impossible with the conventional method.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/elps.201300414DOI Listing
April 2014

Serum glycome profiling: a biomarker for diagnosis of ovarian cancer.

J Proteome Res 2013 Sep 29;12(9):4056-63. Epub 2013 Jul 29.

Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité Medical University, Augustenburger Platz 1, 13353 Berlin, Germany.

During the development of cancer, changes in cellular glycosylation are observed, indicating that alterations of the glycome occur in extracellular fluids as well as in serum and could therefore serve as tumor biomarkers. In the case of epithelial ovarian cancer (EOC), common tumor markers such as CA125 are known to have poor specificity; therefore, better biomarkers are needed. The aim of this work was to identify new potential glycan biomarkers in EOC-patients. N-Glycans were cleaved from serum glycoproteins from 63 preoperative primary EOC-patients along with 33 age-matched healthy women, permethylated, and analyzed using MALDI-TOF mass spectrometry. A value named GLYCOV was calculated from the relative areas of the 11 N-glycan biomarkers revealed by SPSS statistical analyses, namely four high-mannose and seven complex-type fucosylated N-glycans. GLYCOV diagnosed primary EOC with a sensitivity of 97% and a specificity of 98.4% whereas CA-125 showed a sensitivity of 97% and a specificity of 88.9%. Our study is the first one to compare glycan values with the established tumor marker CA125 and to give better results. Therefore, the N-glycome could potentially be used as a biomarker.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr400405xDOI Listing
September 2013

N-glycosylation profile of undifferentiated and adipogenically differentiated human bone marrow mesenchymal stem cells: towards a next generation of stem cell markers.

Stem Cells Dev 2013 Dec 20;22(23):3100-13. Epub 2013 Aug 20.

1 Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin , Berlin, Germany .

Mesenchymal stem cells (MSCs) are multipotent cells that are easy to isolate and expand, develop into several tissues, including fat, migrate to diseased organs, have immunosuppressive properties and secrete regenerative factors. This makes MSCs ideal for regenerative medicine. For application and regulatory purposes, knowledge of (bio)markers characterizing MSCs and their development stages is of paramount importance. The cell surface is coated with glycans that possess lineage-specific nature, which makes glycans to be promising candidate markers. In the context of soft tissue generation, we aimed to identify glycans that could be markers for MSCs and their adipogenically differentiated progeny. MSCs were isolated from human bone marrow, adipogenically stimulated for 15 days and adipogenesis was verified by staining the lipid droplets and quantitative real time polymerase chain reaction of the marker genes peroxisome proliferator-activated receptor gamma (PPARG) and fatty acid binding protein-4 (FABP4). Using matrix-assisted laser desorption-ionization-time of flight mass spectrometry combined with exoglycosidase digestions, we report for the first time the N-glycome of MSCs during adipogenic differentiation. We were able to detect more than 100 different N-glycans, including high-mannose, hybrid, and complex N-glycans, as well as poly-N-acetyllactosamine chains. Adipogenesis was accompanied by an increased amount of biantennary fucosylated structures, decreased amount of fucosylated, afucosylated tri- and tetraantennary structures and increased sialylation. N-glycans H6N5F1 and H7N6F1 were significantly overexpressed in undifferentiated MSCs while H3N4F1 and H5N4F3 were upregulated in adipogenically differentiated MSCs. These glycan structures are promising candidate markers to detect and distinguish MSCs and their adipogenic progeny.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/scd.2013.0108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3856714PMC
December 2013

Genotoxic effects of exposure to radiofrequency electromagnetic fields (RF-EMF) in HL-60 cells are not reproducible.

Mutat Res 2013 Aug 28;755(2):163-6. Epub 2013 Jun 28.

Universität Ulm, Institut für Humangenetik, D-89069 Ulm, Germany.

Conflicting results have been published regarding the induction of genotoxic effects by exposure to radiofrequency electromagnetic fields (RF-EMF). Various results indicating a genotoxic potential of RF-EMF were reported by the collaborative EU-funded REFLEX (Risk Evaluation of Potential Environmental Hazards From Low Energy Electromagnetic Field Exposure Using Sensitive in vitro Methods) project. There has been a long-lasting scientific debate about the reliability of the reported results and an attempt to reproduce parts of the results obtained with human fibroblasts failed. Another part of the REFLEX study was performed in Berlin with the human lymphoblastoid cell line HL-60; genotoxic effects of RF-EMF were measured by means of the comet assay and the micronucleus test. The plausibility and reliability of these results were also questioned. In order to contribute to a clarification of the biological significance of the reported findings, a repeat study was performed, involving scientists of the original study. Comet-assay experiments and micronucleus tests were performed under the same experimental conditions that had led to genotoxic effects in the REFLEX study. Here we report that the attempts to reproduce the induction of genotoxic effects by RF-EMF in HL-60 cells failed. No genotoxic effects of RF-EMF were measured in the repeat experiments. We could not find an explanation for the conflicting results. However, the negative repeat experiments suggest that the biological significance of genotoxic effects of RF-EMF reported by the REFLEX study should be re-assessed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mrgentox.2013.06.014DOI Listing
August 2013

CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity.

Cell Commun Signal 2013 Jun 10;11(1):40. Epub 2013 Jun 10.

Institute of Biochemistry II, Jena University Hospital, Friedrich-Schiller-University Jena, Nonnenplan 2, 07743 Jena, Germany.

Background: Casein kinase 2 (CK2) is a ubiquitously expressed Ser/Thr kinase with multiple functions in the regulation of cell proliferation and transformation. In targeting adherens and tight junctions (TJs), CK2 modulates the strength and dynamics of epithelial cell-cell contacts. Occludin previously was identified as a substrate of CK2, however the functional consequences of CK2-dependent occludin phosphorylation on TJ function were unknown.

Results: Here, we present evidence that phosphorylation of a Thr400-XXX-Thr404-XXX-Ser408 motif in the C-terminal cytoplasmic tail of human occludin regulates assembly/disassembly and barrier properties of TJs. In contrast to wildtype and T400A/T404A/S408A-mutated occludin, a phospho-mimetic Occ-T400E/T404E/S408E construct was impaired in binding to ZO-2. Interestingly, pre-phosphorylation of a GST-Occ C-terminal domain fusion protein attenuated binding to ZO-2, whereas, binding to ZO-1 was not affected. Moreover, Occ-T400E/T404E/S408E showed delayed reassembly into TJs in Ca2+-switch experiments. Stable expression of Occ-T400E/T404E/S408E in MDCK C11 cells augments barrier properties in enhancing paracellular resistance in two-path impedance spectroscopy, whereas expression of wildtype and Occ-T400A/T404A/S408A did not affect transepithelial resistance.

Conclusions: These results suggest an important role of CK2 in epithelial tight junction regulation. The occludin sequence motif at amino acids 400-408 apparently represents a hotspot for Ser/Thr-kinase phosphorylation and depending on the residue(s) which are phosphorylated it differentially modulates the functional properties of the TJ.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1478-811X-11-40DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3695765PMC
June 2013