Osseous differentiation of human fat tissue grafts: From tissue engineering to tissue differentiation

Conventional bone tissue engineering approaches require isolation and in vitro propagation of autologous cells, followed by seeding on a variety of scaffolds. Those protracted procedures impede the clinical applications. Here we report the transdifferentiation of human fat tissue fragments retrieved from subcutaneous fat into tissue with bone characteristics in vitro without prior cell isolation and propagation. 3D collagen-I cultures of human fat tissue were cultivated either in growth medium or in osteogenic medium (OM) with or without addition of Bone Morphogenetic Proteins (BMPs) BMP-2, BMP-7 or BMP-9. Ca2+ depositions were observed after two weeks of osteogenic induction which visibly increased when either type of BMP was added. mRNA levels of alkaline phosphatase (ALP) and osteocalcin (OCN) increased when cultured in OM alone but addition of BMP-2, BMP-7 or BMP-9 caused significantly higher expression levels of ALP and OCN. Immunofluorescent staining for OCN, osteopontin and sclerostin supported the observed real-time-PCR data. BMP-9 was the most effective osteogenic inducer in this system. Our findings reveal that tissue regeneration can be remarkably simplified by omitting prior cell isolation and propagation, therefore removing significant obstacles on the way to clinical applications of much needed regeneration treatments

Photosynthetic Adaptation to Length of Day Is Dependent on S-Sulfocysteine Synthase Activity in the Thylakoid Lumen

Abstract Arabidopsis (Arabidopsis thaliana) chloroplasts contain two O-acetyl-serine(thiol)lyase (OASTL) homologs, OAS-B, which is an authentic OASTL, and CS26, which has S-sulfocysteine synthase activity. In contrast with OAS-B, the loss of CS26 function resulted in dramatic phenotypic changes, which were dependent on the light treatment. We have performed a detailed characterization of the photosynthetic and chlorophyll fluorescence parameters in cs26 plants compared with those of wild-type plants under short-day growth conditions (SD) and long-day growth conditions (LD). Under LD, the photosynthetic characterization, which was based on substomatal CO2 concentrations and CO2 concentration in the chloroplast curves, revealed significant reductions in most of the photosynthetic parameters for cs26, which were unchanged under SD. These parameters included net CO2 assimilation rate, mesophyll conductance, and mitochondrial respiration at darkness. The analysis also showed that cs26 under LD required more absorbed quanta per driven electron flux and fixed CO2. The nonphotochemical quenching values suggested that in cs26 plants, the excess electrons that are not used in photochemical reactions may form reactive oxygen species. A photoinhibitory effect was confirmed by the background fluorescence signal values under LD and SD, which were higher in young leaves compared with mature ones under SD. To hypothesize the role of CS26 in relation to the photosynthetic machinery, we addressed its location inside of the chloroplast. The activity determination and localization analyses that were performed using immunoblotting indicated the presence of an active CS26 enzyme exclusively in the thylakoid lumen. This finding was reinforced by the observation of marked alterations in many lumenal proteins in the cs26 mutant compared with the wild type.</jats:p

Hypertonicity-induced cation channels in HepG2 cells

Die hyperton aktivierte Kationen Leitfähigkeit ist der Hauptmechanismus des RVI. In der vorliegenden Arbeit konnte gezeigt werden, dass die HICC Ströme durch Amiloride, Flufenamat und Gd$^{3+}$ in dosisabhängiger Weise gehemmt werden. Die beobachtete Proliferationsrate zeigte ein pharmakologisches Profil, welches dem der Kanalinhibierung sehr ähnlich war. Dies wies bereits darauf hin, dass die Kationen Kanäle des RVI als Mediatoren der Proliferation fungieren. Mit der siRNA Technik und "$\textit {whole-cell patch-clamp}$" Ableitungen wurde die $\alpha$ Untereinheit des epithelialen Natrium Kanals (ENaC) als funktionelle Komponente des HICC in HepG2 Zellen identifiziert. Durch der "$\textit {Scanning Acoustic Microscopy}$" konnte die überragende Bedeutung der $\alpha$-ENaC Untereinheit für den RVI Prozess in HepG2 Zellen gezeigt werden. Zellzyklus-Analysen legt nahe, dass HepG2 Zellen die $\alpha$-ENaC Untereinheit benötigen, um von der G2 Phase in die M Phase des Zellzyklus fortschreiten zu können

Dynamics of GFP-SGLT1 in COS-7 Cells

We recently showed that in Caco-2 cells the high affinity Na+-/Dglucose cotransporter (SGLTl) resides in intracellular endosomes which are attached to microtubules and therefore proposed that the activity of SGLTl is regulated by intracellular trafficking (Kipp et al., 2003). To visualize the dynamics of the transporter, we expressed a GFP-SGLT1 fusion in COS-7 cells. Using fluorescence microscopy, we discovered the GFP-SGLT1 protein in vesicular stmctures resembling those of endogenous SGLTl in Caco-2 cells. We were able to show the SGLT1-containing vesicles moving rapidly within COS-7 cells transiently expressing GFP-SGLT1 by means of time-lapse fluorescence microscopy. This movement was completely abolished by the disruption of the microtubule network with nocodazole. Next, we compared the features of the SGLTl vesicles and vesicles containing the transferrin receptor (TR). Therefore we incubated COS-7 cells expressing GFP-SGLT1 with Alexa Fluor 546-transferrin to compare the localization and the trafficking of SGLTl and the TR. Fluorescence microscopy of these specimens revealed a localization of the TR in vesicular structures that do not colocalize with SGLT1-vesicles and move with a higher speed. Our presented results confirm a vesicular localization of SGLTl and display the microtubule-dependent transport of these vesicles in hing cells. The mechanism of SGLTl endo-/exocytosis is clearly distinguishable from the transport of the transferrin recepto