142 research outputs found
Klonierung, Expression und Charakterisierung der Ektodomäne der MT1-MMP
No full textRoderfeld M. Klonierung, Expression und Charakterisierung der Ektodomäne der MT1-MMP. Bielefeld (Germany): Bielefeld University; 2001.Die gesamte, aus humanem Knorpel durch reverse Transkription gewonnene (Büttner et al., 1997) cDNA der humanen MT1-MMP wurde von Frau Dr. A. Lichte zur Verfügung gestellt (Lichte, 1997). In einer PCR konnte die für die Ektodomäne der MT1-MMP (Ala21-Glu523) codierende cDNA erfolgreich amplifiziert werden. Nach Subklonierungen in E. coli wurde das Genfragment in den Hefe-Expressionsvektor pPICZ[alpha]A einkloniert.
Als Wirtssystem zur rekombinanten Produktion der MT1-MMP wurde der Pichia pastoris Stamm KM71 gewählt und mit dem Konstrukt pPICZ[alpha]A/[Delta]MT1-MMP transformiert. Die Expression zur Herstellung der Ektodomäne zwecks nachfolgender Charakterisierung wurde im 1 L-Maßstab im Schüttelkolben durchgeführt. Nach Modifikation der Reinigungsmethode nach Moore & Spilberg konnten pro Aufarbeitung 0.2 - 0.5 mg des aktiven Enzyms [Delta]MT1-MMP (Tyr112-Glu523) hochrein hergestellt werden.
Die Identifizierung des Expressionsproduktes als MT1-MMP erfolgte über Western Blot unter Verwendung eines in Kaninchen produzierten Antikörpers gegen die katalytische Domäne. N-terminale Sequenzierung und Identifizierung des C-Terminus mittels MALDI-TOF erbrachten den Beweis für die Herstellung der vollständigen aktiven Ektodomäne der MT1-MMP (Tyr112-Glu523).
Die Untersuchung des autoproteolytischen Verhaltens führte zur Bestimmung von zwei Spaltstellen. Die erste Schnittstelle Gln281-Leu282 liegt am Ende der katalytischen Domäne in der letzten Windung einer [alpha]-Helix, während sich die zweite Stelle Pro294-Pro295 sehr exponiert in der Hinge-Region, dem Verbindungsloop zwischen katalytischer- und Hämopexin-ähnlicher Domäne, befindet.
Zum Vergleich der Substratspezifität der katalytischen und der Ektodomäne der MT1-MMP wurden Fibrinogen und das myelin basic protein im in vitro-Experiment proteolytisch abgebaut. Bei der Proteolyse des Fibrinogens durch die Ektodomäne konnten vier Spaltstellen ermittelt werden. Dabei konnte nur eine Spaltstelle beiden Enzymvarianten zugeordnet werden. Die aus dem Fibrinogenabbau resultierenden Fragmentierungsmuster wiesen keinerlei Übereinstimmung auf. Dagegen waren die Fragmentierungsmuster aus dem proteolytischen Abbau des myelin basic protein mit den beiden MT1-MMP-Varianten völlig identisch. Für die Spaltung durch die Ektodomäne konnten bisher allerdings nur zwei Schnittstellen bestimmt werden.
Für den proteolytischen Abbau eines synthetischen fluorogenen Peptids durch die katalytische Domäne, die Ektodomäne und das Gesamtenzym (auf Zellen) konnten kinetische Parameter bestimmt und verglichen werden. Weiterhin wurden für die Zerlegung von denaturiertem Kollagen durch die beiden löslichen Enzyme relative kinetische Konstanten ermittelt. Die katalytische Domäne besitzt demnach, verglichen mit der Ektodomäne, einen um den Faktor zwei größeren kcat/KM-Wert gegenüber kleinen Peptiden, wohingegen die relativen Aktivitäten gegenüber dem hochmolekularen Substrat nicht voneinander abwichen.
Durch die Untersuchung der Hemmung von beiden löslichen Enzymen mit den natürlichen Inhibitoren TIMP-2 und TIMP-4 konnten Hemmkonstanten bestimmt und verglichen werden. Die Inhibierung durch den TIMP-2 bestätigte die in der Literatur für andere MMP/TIMP-Paare beschriebene effektivere Hemmung der Ektodomäne, während die Hemmung durch die inhibitorische Domäne des TIMP-4 für die katalytische Domäne stärker war.
Weiterhin wurden ca. 40 Vertreter einer neuen Klasse von synthetischen Inhibitoren der Thiadiazin-Familie hinsichtlich ihrer Hemmwirkung gegenüber den beiden MT1-MMP-Varianten getestet. Viele der getesteten Stoffe wiesen Hemmkonstanten im nanomolaren Bereich auf.
Versuche zur Calcium-Abhängigkeit der proteolytischen Aktivität ergaben bei zunehmender Größe der Varianten eine sinkende Abhängigkeit. Für die löslichen Varianten konnte die reversible Aktivierung und Deaktivierung durch abwechselnde Gabe und Entzug von Calcium gezeigt werden
Effect of an innovative matrix metalloproteinase-9-antagonists (MMP-9-PEX) on migration and adhesion of colorectal cancer cells
No full textEffect of an innovative matrix metalloproteinase-9-antagonists (MMP-9-PEX) on migration and adhesion of colorectal cancer cells
No full textSerum proteome profiling identifies novel and powerful markers of cystic fibrosis liver disease.
No full textCystic Fibrosis associated liver disease (CFLD) develops in approximately 30% of CF patients. However, routine sensitive diagnostic tools for CFLD are lacking. Within this study, we aimed to identify new experimental biomarkers for the detection of CFLD.
45 CF patients were included in the study and received transient elastography. Differential regulation of 220 different serum proteins was assessed in a subgroup of patients with and without CFLD. Most interesting candidate proteins were further quantified and validated by ELISA in the whole patient cohort. To assess a potential relation of biomarker expression to the degree of hepatic fibrosis, serum biomarkers were further determined in 18 HCV patients where liver histology was available.
43 serum proteins differed at least 2-fold in patients with CFLD compared to those without liver disease as identified in proteome profiling. In ELISA quantifications, TIMP-4 and Endoglin were significantly up-regulated in patients with CFLD as diagnosed by clinical guidelines or increased liver stiffness. Pentraxin-3 was significantly decreased in patients with CFLD. Serum TIMP-4 and Endoglin showed highest values in HCV patients with liver cirrhosis compared to those with fibrosis but without cirrhosis. At a cut-off value of 6.3 kPa, transient elastography compassed a very high diagnostic accuracy and specificity for the detection of CFLD. Among the biomarkers, TIMP-4 and Endoglin exhibited a high diagnostic accuracy for CFLD. Diagnostic sensitivities and negative predictive values were increased when elastography and TIMP-4 and Endoglin were combined for the detection of CFLD.
Serum TIMP-4 and Endoglin are increased in CFLD and their expression correlates with hepatic staging. Determination of TIMP-4 and Endoglin together with transient elastography can increase the sensitivity for the non-invasive diagnosis of CFLD
Serum Chemerin Is Decreased by Roux-en-Y Gastric Bypass and Low Calorie-Formula Diet in Obese Individuals
No full textThe pleiotropic chemokine chemerin is involved in multiple processes in metabolism and inflammation. The present study aimed to elucidate its regulation in morbid obesity and during therapy-induced rapid weight loss. A total of 128 severely obese patients were enrolled, and their basal anthropometric and clinical parameters were assessed. In total, 64 individuals attended a conservative 12-month weight loss program that included a low calorie-formula diet (LCD), and 64 patients underwent bariatric surgery (Roux-en-Y gastric bypass, RYGB). Blood serum was obtained at study baseline and at follow-up visits after 3, 6, and 12 months. Systemic chemerin concentrations, as well as metabolic and immunological parameters, were quantified. During the 12-month period studied, serum chemerin levels decreased significantly with weight loss after bariatric surgery, as well as with conservative low calorie therapy; however, the effects of RYGB were generally stronger. No substantial associations of systemic chemerin concentrations with therapy-induced improvement of type 2 diabetes and with indicators of liver function and fibrosis were observed. We conclude that systemic chemerin levels decrease in obese individuals during weight loss, regardless of the therapeutic strategy. A potential involvement in weight loss-associated improvement of metabolic disorders and liver fibrosis remains to be further investigated.Deutsche Forschungsgemeinschaft (DFG); ROR-ID:018mejw6
Circulating Adipokines and Hepatokines Serve as Diagnostic Markers during Obesity Therapy
No full textAllocation of morbidly obese patients to either conservative therapy options—such as lifestyle intervention and/or low-calorie diet (LCD)—or to bariatric surgery—preferably sleeve gastrectomy or Roux-en-Y gastric bypass (RYGB)—represents a crucial decision in order to obtain sustainable metabolic improvement and weight loss. The present study encompasses 160 severely obese patients, 81 of whom participated in an LCD program, whereas 79 underwent RYGB surgery. The post-interventional dynamics of physiologically relevant adipokines and hepatokines (ANGPTL4, CCL5, GDF15, GPNMB, IGFBP6), as well as their correlation with fat mass reduction and improvement of liver fibrosis, were analyzed. Systemic GDF15 was characterized as an excellent predictive marker for hepatic fibrosis as well as type 2 diabetes mellitus. Of note, baseline GDF15 serum concentrations were positively correlated with NFS and HbA1c levels after correction for BMI, suggesting GDF15 as a BMI-independent marker of hepatic fibrosis and T2D in obese individuals. Specific GDF15 cut-off values for both diseases were calculated. Overall, the present data demonstrate that circulating levels of specific adipokines and hepatokines are regulated with therapy-induced fat loss and metabolic improvement and might, therefore, serve as biomarkers for the success of obesity therapy strategies
Serum Chemerin Is Decreased by Roux-en-Y Gastric Bypass and Low Calorie-Formula Diet in Obese Individuals
No full textThe pleiotropic chemokine chemerin is involved in multiple processes in metabolism and inflammation. The present study aimed to elucidate its regulation in morbid obesity and during therapy-induced rapid weight loss. A total of 128 severely obese patients were enrolled, and their basal anthropometric and clinical parameters were assessed. In total, 64 individuals attended a conservative 12-month weight loss program that included a low calorie-formula diet (LCD), and 64 patients underwent bariatric surgery (Roux-en-Y gastric bypass, RYGB). Blood serum was obtained at study baseline and at follow-up visits after 3, 6, and 12 months. Systemic chemerin concentrations, as well as metabolic and immunological parameters, were quantified. During the 12-month period studied, serum chemerin levels decreased significantly with weight loss after bariatric surgery, as well as with conservative low calorie therapy; however, the effects of RYGB were generally stronger. No substantial associations of systemic chemerin concentrations with therapy-induced improvement of type 2 diabetes and with indicators of liver function and fibrosis were observed. We conclude that systemic chemerin levels decrease in obese individuals during weight loss, regardless of the therapeutic strategy. A potential involvement in weight loss-associated improvement of metabolic disorders and liver fibrosis remains to be further investigated
Expression of MMPs and TIMPs in liver fibrosis – a systematic review with special emphasis on anti-fibrotic strategies
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