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Sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells

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Chinese Medical Journal 2003年第12期116卷 1923-1929页

作者：赵进顺 Alexandra Schmid-Kotsas Hans-Juergen Gross Adolf Gruenert Max G. Bachem department of Occupational and Environmental Medicine department of Clinical Chemistry and pathobiochemistry

Objective To study the sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent *** Sensitivity and specificity of several common methods for apoptosis determination were evaluated (Apo2.7-expression, Annexin V-binding, TUNEL-reaction, poly-(ADP-ribose)-polymerase-(PARP) cleavage and single-stranded-DNA (ssDNA) staining). Apoptosis was induced by oxidative stress generated by hydrogen peroxide in 3 cultured cells types growing as adherent monolayer (MiaPaCa-2, Hep-G2 and human skin fibroblasts), necrosis was induced by depletion of cellular ATP using sodium azide. Cells positively stained by the respective apoptosis assay were quantified and alterations of cell morphology were monitored by fluorescence microscopy. The date was analyzed by one-way analysis of variance and significance test of correlation *** One hour after apoptosis induction significant cell fractions were positively stained for ssDNA (33% with MiaPaCa-2 cells, 35% with Hep-G2 cells, 56% with human skin fibroblasts). PARP-cleavage was less sensitive compared to the ssDNA-staining. Apo2.7-expression, Annexin V-binding and TUNEL-reaction were not applicable to detect early apoptosis induced by oxidative stress (below 2 hours), but were efficiently monitoring late apoptosis. Specificity of ssDNA-staining was complete with each cell type even 4 hs after induction of necrosis by the highest sodium azide concentration. In contrast, the same experimental conditions resulted in 50%-90% positively stained necrotic cells by using Apo2.7-expression, TUNEL-reaction or AnnexinV-binding. Surprisingly, specificity of PARP-cleavage was highly depending on the respective cell *** Our study prove that among the five methods investigated only ssDNA-staining allowed to completely differentiate apoptosis from necrosis, and is thus suitable to reliably detect early as well as late apoptosis. Therefore, the ssDNA-stainin

关键词： apoptosis staining method sensitivity specificity oxidative stress adherent cell

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The expression and antigenicity identification of recombinant rat TGF-β1 in bacteria

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Cell Research 2001年第2期11卷 95-100页

作者： GAO CHUN FANG, XIAN TAO KONG, AXEL M GRESSNER, RALF WEISKIRCHEN department of Laboratory Medicine, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China Institute of Clinical Chemistry and pathobiochemistry, Central Laboratory, R 1department of Laboratory Medicine Changzheng Hospital Second Military Medical University Shanghai 200003 China 1department of Laboratory Medicine Changzheng Hospital Second Military Medical University Shanghai 200003 China 2Institute of Clinical Chemistry and pathobiochemistry Central Laboratory RWTH-University Hospital Pauwelsstr 30 D-52074 Aachen Germany 2Institute of Clinical Chemistry and pathobiochemistry Central Laboratory RWTH-University Hospital Pauwelsstr 30 D-52074 Aachen Germany

In order to study structure-function details of TGF-β1, the recombinant mature form of rat TGF-β1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-β1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-β1. The molecular weight of expressed TGF-β1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-β1 antibody. The mature recombinant rat TGF-β1 expressed in this study provides a useful tool for future detailed structural and functional studies.

关键词： TGF-β1 recombinant expression inclusion bodies purification antigenicity.

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G6PT-H6PDH-11βHSD1 triad in the liver and its implication in the pathomechanism of the metabolic syndrome

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World Journal of Hepatology 2012年第4期4卷 129-138页

作者： Ibolya Czegle Miklós Csala József Mandl Angelo Benedetti István Karádi Gábor Bánhegyi 3rd department of Internal Medicine Semmelweis University 1125 Budapest Hungary pathobiochemistry Research Group Hungarian Academy of Sciences 1094 Budapest Hungary department of Medical Chemistry Molecular Biology and Pathobiochemistry Semmelweis University 1094 Budapest Hungary department of Pathophysiology Experimental Medicine and Public Health University of Siena 53100 Siena Italy

The metabolic syndrome, one of the most common clinical conditions in recent times, represents a combination of cardiometabolic risk determinants, including central obesity, glucose intolerance, insulin resistance, dyslipidemia, non-alcoholic fatty liver disease and hypertension. Prevalence of the metabolic syndrome is rapidly increasing worldwide as a consequence of common overnutrition and consequent obesity. Although a unifying picture of the pathomechanism is still missing, the key role of the pre-receptor glucocorticoid activation has emerged recently. Local glucocorticoid activation is catalyzed by a triad composed of glucose-6-phosphate-transporter, hexose-6-phosphate dehydrogenase and 11β-hydroxysteroid dehydrogenase type 1 in the endoplasmic reticulum. The elements of this system can be found in various cell types, including adipocytes and hepatocytes. While the contribution of glucocorticoid activation in adipose tissue to the pathomechanism of the metabolic syndrome has been well established, the relative importance of the hepatic process is less understood. This review summarizes the available data on the role of the hepatic triad and its role in the metabolic syndrome, by confronting experimental findings with clinical observations.

关键词： Metabolic syndrome Liver Glucocorticoid Glucose-6-phosphate-transporter Hexose-6-phosphate dehydrogenase 11β-hydroxysteroid dehydrogenase type 1

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Lipotoxicity in the liver

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World Journal of Hepatology 2013年第10期5卷 550-557页

作者： Veronika Zámbó Laura Simon-Szabó Péter Szelényi va Kereszturi Gábor Bánhegyi Miklós Csala department of Medical Chemistry Molecular Biology and PathobiochemistrySemmelweis University

Obesity due to excessive food intake and the lack of physical activity is becoming one of the most serious public health problems of the 21stcentury. With the increasing prevalence of obesity, non-alcoholic fatty liver disease is also emerging as a pandemic. While previously this pathophysiological condition was mainly attributed to triglyceride accumulation in hepatocytes,recent data show that the development of oxidative stress, lipid peroxidation, cell death, inflammation and fibrosis are mostly due to accumulation of fatty acids,and the altered composition of membrane phospholipids. In fact, triglyceride accumulation might play a protective role, and the higher toxicity of saturated or trans fatty acids seems to be the consequence of a blockade in triglyceride synthesis. Increased membrane saturation can profoundly disturb cellular homeostasis by impairing the function of membrane receptors,channels and transporters. However, it also inducesendoplasmic reticulum stress via novel sensing mechanisms of the organelle's stress receptors. The triggered signaling pathways in turn largely contribute to the development of insulin resistance and apoptosis. These findings have substantiated the lipotoxic liver injury hypothesis for the pathomechanism of *** minireview focuses on the metabolic and redox aspects of lipotoxicity and lipoapoptosis, with special regards on the involvement of endoplasmic reticulum stress responses.

关键词： Saturated fatty acid Lipotoxicity Steatosis Lipoapoptosis Endoplasmic reticulum stress

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Porcine model characterizing various parameters assessing the outcome after acetaminophen intoxication induced acute liver failure

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World Journal of Gastroenterology 2017年第9期23卷 1576-1585页

作者： Karolin Thiel Wilfried Klingert Kathrin Klingert Matthias H Morgalla Martin U Schuhmann Pamela Leckie Yalda Sharifi Nathan A Davies Rajiv Jalan Andreas Peter Christian Grasshoff Alfred Konigsrainer Martin Schenk Christian Thiel department of General Visceral and Transplant SurgeryTuebingen University Hospital department of Anaesthesiology Tuebingen University Hospital department of Neurosurgery Tuebingen University Hospital Institute for Liver and Digestive Health University College London Division of Endocrinology DiabetologyAngiologyNephrologyPathobiochemistry and Clinical ChemistryDepartment of Internal MedicineTuebingen University Hospital

To investigate the changes of hemodynamic and laboratory parameters during the course of acute liver failure following acetaminophen *** pigs underwent a midline laparotomy following jejunal catheter placement for further acetaminophen intoxication and positioning of a portal vein Doppler flow-probe. Acute liver failure was realized by intrajejunal acetaminophen administration in six animals, two animals were sham operated. All animals were invasively monitored and received standardized intensive care support throughout the study. Portal blood flow, hemodynamic and ventilation parameters were continuously recorded. Laboratory parameters were analysed every eight hours. Liver biopsies were sampled every 24 h following intoxication and upon *** liver failure (ALF) occurred after 28 ± 5 h resulted in multiple organ failure and death despite maximal support after further 21 ± 1 h (study end). Portal blood flow (baseline 1100 ± 156 mL/min) increased to a maximum flow of 1873 ± 175 mL/min at manifestation of ALF, which was significantly elevated (P 3/µL ± 34 × 103/µL) of intoxicated animals declined slowly to values of 145 × 103/µL ± 46 × 103/µL when liver failure occurred. Subsequent appearance of severe thrombocytopenia in liver failure resulted in values of 11 × 103/µL ± 3 × 103/µL preceding fatality within few hours which was significant (P > 0.01).CONCLUSIONDeclining portal blood flow and subsequent severe thrombocytopenia after acetaminophen intoxication precede fatality in a porcine acute liver failure model.

关键词： Acetaminophen intoxication Acute liver failure Portal blood flow Thrombocytopenia Animal model Porcine model

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Going only half the way:cell cycle exit after the G1 restriction point

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Signal Transduction and Targeted Therapy 2024年第1期9卷 1-2页

作者： Lisa Muller Tony Gutschner Mechthild Hatzfeld department of pathobiochemistry Institute of Molecular MedicineMartin Luther University Halle-WittenbergCharles Tanford Protein Research CenterHalleGermany department of RNA Biology and Pathogenesis Institute of Molecular MedicineMartin Luther University Halle-WittenbergCharles Tanford Protein Research CenterHalleGermany

In a recent article published in Nature,James *** et *** that cyclin A2 levels determine whether cells will make an entrance into mitosis or whether cells will leave the cell cycle after having passed the G1/restriction(R)-point implicating that the decision is fully reversible(Fig.1).This ground-breaking study could impact future approaches for cancer treatment by targeting the mitotic clock.

关键词： point cycle

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A Systems Biological Perspective of Cellular Stress-Directed Programmed Cell Death

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Computational Molecular Bioscience 2014年第1期4卷 28-34页

作者： Orsolya Kapuy Beáta Lizák Ibolya Stiller Gábor Bánhegyi department of Medical Chemistry Molecular Biology and Pathobiochemistry Semmelweis UniversityBudapest Hungary

Each eukaryotic cell of multicellular organisms must be able to maintain its integrity by sensing both external and internal stimuli. The primary goal of the generated response mechanism is to drive back the system to the former or to a new homeostatic state. Moreover, the response has to provide an accurate survival-or-death decision to avoid any “misunderstanding” and its unwanted consequences. New data revealed that a systems-level crosstalk of molecular networks has an essential role in achieving the correct characteristic of the response. Although many molecular components of these processes already have been revealed, several elements and regulatory connections of crosstalk are still missing. These “gaps” of the complex control networks make hardly impossible to present comprehensive models. Therefore we approach the questions from a systems biology aspect by combining the experimental results with the special technique of mathematical modelling. In this short report we discuss some novel and preliminary data gained by this approach on the crosstalk between life and death decisions under cellular stress, to get a systems biological view of these networks.

关键词： Systems Biology Cellular Stress Autophagy Apoptosis Crosstalk Mathematical Modelling

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Expression of tissue factor in pancreatic adenocarcinoma is associated with activation of coagulation

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World Journal of Gastroenterology 2006年第30期12卷 4843-4849页

作者： Stephan L Haas Ralf Jesnowski Michael Steiner Frank Hummel Jrg Ringel Christine Burstein Horst Nizze Stefan Liebe J Matthias Lhr department of Medicine II University of Heidelberg at Mannheim Germany department of Clinical Chemistry and pathobiochemistry University of Rostock Rostock Germany department of Pathology University of Rostock Rostock Germany department of Medicine University of Rostock Rostock Germany

AIM： To study expression of tissue factor （TF） in pancreatic cancer and its role in the development of thromboembolism.METHODS： TF expression was studied in eight human pancreatic carcinoma cell lines by Northern blot and indirect immunofluorescence. Expression of alternatively spliced TF （asTF） was assessed by RT-PCR. In addition, TF expression was determined by immunofluorescence in pancreatic tissues of 19 patients with pancreatic adenocarcinoma （PCa）, 9 patients with chronic pancreatitis （CP） and 20 normal controls. Plasma samples （30 PCa-patients, 13 CP-patients and 20 controls） were investigated for soluble TF levels and coagulation activation markers [thrombin-antithrombin Ⅲ complex （TAT）, prothrombin fragment 1 ＋ 2 （F1 ＋ 2）]. RESULTS： All pancreatic carcinoma cell lines expressed TF （8/8） and most of them expressed asTF （6/8）. TF expression at the protein level did not correlate with the differentiation of the carcinoma cell line. All but two pancreatic cancer tissue samples stained positive for TF （17/19）. In all samples of CP weak staining was restricted to pancreatic duct cells, whereas only a few subendothelial cells were positive in 9/20 of normal controls. TF and TAT levels in PCa patients were significantly elevated compared to controls whereas elevated F1 ＋ 2 levels did not reach statistical significance compared to controls. In CP patients TAT and F1 ＋ 2 levels proved to be significantly elevated compared to controls, although TAT elevation was less pronounced than in PCa patients. CONCLUSION： We conclude that in addition to the upregulated expression of TF on the cell membrane, soluble TF might contribute to activation of the coagulation system in pancreatic cancer.

关键词： Coagulation activation Pancreatic carcinoma Thromboembolism Thrombosis Tissue factor

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N-Propionylmannosamine stimulates axonal elongation in a murine model of sciatic nerve injury

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Neural Regeneration Research 2015年第6期10卷 976-981页

作者： Christian Witzel Werner Reutter G.Bj?rn Stark Georgios Koulaxouzidis Plastic and Reconstructive Surgery Interdisciplinary Breast CenterCharité–Universit?tsmedizin Berlin Institute of Laboratory Medicine Clinical Chemistry and PathobiochemistryCharité–Universit?tsmedizin Berlin department of Plastic and Hand Surgery University of Freiburg Medical Centre

Increasing evidence indicates that sialic acid plays an important role during nerve regeneration. Sialic acids can be modified in vitro as well as in vivo using metabolic oligosaccharide engineering of the N-awl side chain. N-Propionylmannosamine （ManNProp） increases neurite outgrowth and accelerates the reestablishment of functional synapses in vitro. We investigated the influence of systemic ManNProp application using a specific in vivo mouse model. Using mice expressing axonal fluorescent proteins, we quantified the extension of regenerating axons, the number of regenerating axons, the number of arborising axons and the number of branches per axon 5 days after injury. Sciatic nerves from non-expressing mice were grafted into those expressing yellow fluorescent protein. We began a twice-daily intraperitoneal application of either peracetylated ManNProp （200 mg/kg） or saline solution 5 days before injury, and continued it until nerve harvest （5 days after transection）. ManNProp significantly increased the mean distance of axonal regeneration （2.49 mm vs. 1.53 mm; P 〈 0.005） and the number of arborizing axons （21% vs. 16%; P = 0.008） 5 days after sciatic nerve grafting. ManNProp did not affect the number of regenerating axons or the number of branches per arborizing axon. The biochemical glycoengineering of the N-acyl side chain of sialic acid might be a promising approach for improving peripheral nerve regeneration.

关键词： N-propionylmannosamine sialic acid glycoengineering sciatic nerve peripheral nerve branching arborisation Thyl-YFP mouse nerve regeneration

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Occult celiac disease prevents penetrance of hemochromatosis

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World Journal of Gastroenterology 2005年第21期11卷 3323-3326页

作者： Andreas Geier Carsten Gartung Igor Theurl Guenter Weiss Frank Lammert Christoph G.Dietrich Ralf Weiskirchen Heinz Zoller Benita Hermanns Siegfried Matern department of Internal Medicine Ⅲ Aachen University (RWTH) AachenGermany department of Internal Medicine University of Innsbruck InnsbruckAustria department of Clinical Chemistry and pathobiochemistry Aachen University(RWTH) AachenGermany department of Pathology Aachen University (RWTH) AachenGermany department of Internal Medicine Ⅲ Aachen University(RWTH) Aachen Germany

AIM:To report a patient with C282Y homozygocity,depleted body iron and intestinal atrophy caused by celiac disease (CD) who experienced resolution of the enteropathy with subsequent normalization of iron metabolism upon gluten free diet. METHODS:To obtain information on the tissue distribution and quantitative expression of proteins involved in duodenal iron trafficking,we determined the expression of divalent-metal transporter 1 (DMT1),ferroportin 1 (FP1) and transferrin receptor (TfR1) by means of immunohist-ochemistry and real-time PCR in duodenal biopsies of this patient. RESULTS:Whereas in hereditary hemochromatosis patients without CD, DMT1 expression was up-regulated leading to excessive uptake of iron, we identified a significant reduction in protein ana mRNA expression of DMT1 as a compensatory mechanism in this patient with HH and CD. CONCLUSION:Occult CD may compensate for increased DMT1 expression in a specific subset of individuals with homozygous C282Y mutations in the hemochromatosis (HFE) gene,thus contributing to the low penetrance of HH.

关键词： Hemochromatosis Celiac disease Divalentmetal transporter 1 Transferrin receptor Iron metabolism

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