RESUMO
The complete nucleotide sequence encoding the high-molecular-mass amylase (HMMA) of Geobacillus stearothermophilus ATCC 12980 was established by PCR techniques. Based on the hmma gene sequence, the full-length rHMMA, four N- or C-terminal rHMMA truncations as well as three C-terminal rHMMA fragments were cloned and heterologously expressed in Escherichia coli. Purified rHMMA forms were used either for affinity studies with the recombinant (r) S-layer protein SbsC (rSbsC), peptidoglycan-containing sacculi (PGS) and pure peptidoglycan (PG) devoid of the secondary cell wall polymer (SCWP), or for surface plasmon resonance (SPR) studies using rSbsC and isolated SCWP. In the C-terminal part of the HMMA, three specific binding regions, one for each cell wall component (rSbsC, SCWP and PG), could be identified. The functionality of the PG-binding domain could be confirmed by replacing the main part of the SCWP-binding domain of an S-layer protein by the PG-binding domain of the HMMA. The present work describes a completely new and highly economic strategy for cell adhesion of an exoenzyme.
Assuntos
Amilases/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Geobacillus stearothermophilus/enzimologia , Amilases/genética , Proteínas de Bactérias/genética , Clonagem Molecular , Genes Bacterianos , Geobacillus stearothermophilus/genética , Peptidoglicano/metabolismo , Ligação Proteica , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
Osteoarthritis (OA) is one of the most common causes of disability and represents a major socio-economic burden. Despite intensive research, the molecular mechanisms responsible for the initiation and progression of OA remain inconclusive. In recent years experimental findings revealed elevated levels of reactive oxygen species (ROS) as a major factor contributing to the onset and progression of OA. Hence, we designed a hydrostatic pressure bioreactor system that is capable of stimulating cartilage cell cultures with elevated ROS levels. Increased ROS levels in the media did not only lead to an inhibition of glycosaminoglycans and collagen II formation but also to a reduction of already formed glycosaminoglycans and collagen II in chondrogenic mesenchymal stem cell pellet cultures. These effects were associated with the elevated activity of matrix metalloproteinases as well as the increased expression of several inflammatory cytokines. ROS activated different signaling pathways including PI3K/Akt and MAPK/ERK which are known to be involved in OA initiation and progression. Utilizing the presented bioreactor system, an OA in vitro model based on the generation of ROS was developed that enables the further investigation of ROS effects on cartilage degradation but can also be used as a versatile tool for anti-oxidative drug testing.
Assuntos
Cartilagem Articular/patologia , Condrogênese , Pressão Hidrostática/efeitos adversos , Células-Tronco Mesenquimais/patologia , Osteoartrite/etiologia , Espécies Reativas de Oxigênio/metabolismo , Cartilagem Articular/metabolismo , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Transdução de SinaisRESUMO
The control of bleeding is one of the most important interventions after a traumatic injury. Hemostatic devices delivering blood clotting accelerating agents such as fibrinogen are increasingly used due to their efficacy and their ease of application. In the present study, we describe a method to incorporate the coagulant supplements fibrinogen and thrombin in silk protein sponges by mixing the coagulants with an aqueous silk solution, followed by molding, freeze-drying, and water annealing. In this combination system, we demonstrate the delivery of fibrinogen while maintaining its hemostatic potential. Concentration ratios of silk to fibrinogen of 1.0%/2.8%, 2.3%/1.5%, and 3.0%/0.8% were used. The thrombin-induced fibrin polymeric network filled the space in and next to the silk spongy structure but also remained interconnected to the silk, providing an intact network. The mechanical characterization of the fibrinogen-releasing silk sponges before and after the induction of the fibrinogen polymerization demonstrated that the fibrin network resulted in reduced permanent deformation from 21.1% to 6.5%, 19.6% to 5.7%, and 12.7% to 9.4% for the 2.8%, 1.5%, and 0.8% fibrinogen-containing silk sponges, respectively. Moreover, the fibrin formation lead to a more linear elastic behavior over longer strain ranges. In combination, the Calcein-AM/PI staining and MTT assay results indicate uniform cell adhesion on the surface and cytocompatibility of the silk/fibrin sponges, respectively. Moreover, the co-delivery of thrombin with fibrinogen via silk as carrier material is described, offering a more mechanically robust and durable system while preserving hemostatic features of the coagulant substances for the generation of hemostatic devices. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 687-696, 2017.