Determination of hydroxyproline.

Age-associated increases in collagen cross-linking and accumulation of advanced glycosylation products are both accelerated by diabetes, suggesting that glucose-derived cross-link formation may contribute to the development of chronic diabetic complications as well as certain physical changes of aging. Aminoguanidine, a nucleophilic hydrazine compound, prevented both the formation of fluorescent advanced nonenzymatic glycosylation products and the formation of glucose-derived collagen cross-links in vitro. Aminoguanidine administration to rats was equally effective in preventing diabetes-induced formation of fluorescent advanced nonenzymatic glycosylation products and cross-linking of arterial wall connective tissue protein in vivo. The identification of aminoguanidine as an inhibitor of advanced nonenzymatic glycosylation product formation now makes possible precise experimental definition of the pathogenetic significance of this process and suggests a potential clinical role for aminoguanidine in the future treatment of chronic diabetic complications.

http://science.sciencemag.org/content/sci/232/4758/1629.full.pdf

Aminoguanidine prevents diabetes-induced arterial wall protein cross-linking

A simplified method for the analysis of hydroxyproline in biological tissues.

Transforming growth factor (TGF)-beta1 has been implicated in the pathogenesis of fibrosis based upon its matrix-inducing effects on stromal cells in vitro, and studies demonstrating increased expression of total TGF-beta1 in fibrotic tissues from a variety of organs. The precise role in vivo of this cytokine in both its latent and active forms, however, remains unclear. Using replication-deficient adenovirus vectors to transfer the cDNA of porcine TGF-beta1 to rat lung, we have been able to study the effect of TGF-beta1 protein in the respiratory tract directly. We have demonstrated that transient overexpression of active, but not latent, TGF-beta1 resulted in prolonged and severe interstitial and pleural fibrosis characterized by extensive deposition of the extracellular matrix (ECM) proteins collagen, fibronectin, and elastin, and by emergence of cells with the myofibroblast phenotype. These results illustrate the role of TGF-beta1 and the importance of its activation in the pulmonary fibrotic process, and suggest that targeting active TGF-beta1 and steps involved in TGF-beta1 activation are likely to be valuable antifibrogenic therapeutic strategies. This new and versatile model of pulmonary fibrosis can be used to study such therapies.

/pdf/adenovector-mediated-gene-transfer-of-active-transforming-4ssshu2gs8.pdf

Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung.

Due to its avascular nature, articular cartilage exhibits a very limited capacity to regenerate and to repair. Although much of the tissue-engineered cartilage in existence has been successful in mimicking the morphological and biochemical appearance of hyaline cartilage, it is generally mechanically inferior to the natural tissue. In this study, we tested the hypothesis that the application of dynamic deformational loading at physiological strain levels enhances chondrocyte matrix elaboration in cell-seeded agarose scaffolds to produce a more functional engineered tissue construct than in free swelling controls. A custom-designed bioreactor was used to load cell-seeded agarose disks dynamically in unconfined compression with a peak-to-peak compressive strain amplitude of 10 percent, at a frequency of 1 Hz, 3 x (1 hour on, 1 hour off)/day, 5 days/week for 4 weeks. Results demonstrated that dynamically loaded disks yielded a sixfold increase in the equilibrium aggregate modulus over free swelling controls after 28 days of loading (100 +/- 16 kPa versus 15 +/- 8 kPa, p < 0.0001). This represented a 21-fold increase over the equilibrium modulus of day 0 (4.8 +/- 2.3 kPa). Sulfated glycosaminoglycan content and hydroxyproline content was also found to be greater in dynamically loaded disks compared to free swelling controls at day 21 (p < 0.0001 and p = 0.002, respectively).

Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels.

Estimation of amino acids using ninhydrin reduced with dithionite.

[Microdetermination of nitrogen as indophenol blue by chloramine-T oxidation].

Amylases of bacteria, barley, sweet potatoes, and white potatoes were separated by electrophoresis in polyacrylamide gels containing soluble starch. After incubation in acetate buffer and staining in iodine solution, the zones of enzyme activity remained unstained. The zones of crystalline α-amylase (bacillus subtilis) and of crude α-amylase (bacteriae) were found to be identical. The main zone of the crystalline β-amylase (lpomoea batatas) was identical with the zone of crude β-amylase (barley) and with the amylase zone of the potato (solanum tuberosum) extracts. Using 5 mm slots, 1 ng of amylase is traceable. Crystalline β-amylase yielded, in addition to the main zone, a minor zone nearer to the anode. The α-amylases had three zones of different properties. Phosphorylases were demonstrated as starch synthesizing and as starch degrading enzymes by making use of the reaction: “glycogen +glucose-1-phosphat⇌starch +phosphate”. Glycogen was polymerized into the gel to demonstrate the synthesis of starch, and incubation was carried out with glucose-1-phosphate. The starch formed was stained with iodine. The extracts of freshly harvested tubers yielded four zones, whereas those of stored tubers yielded two zones, and leaves and sprouts each showed one zone. - To observe starch degradation by phosphorylases, starch was polymerized into the polyacrylamide, and the gels were incubated in phosphate buffer. After treatment with iodine, an unstained zone was observed which corresponded to the most intense phosphorlase zone (close to the anode) in the glycogen gel. This zone was more intense in the tuber extracts than in leaves and sprouts. Pig heart extract contained the phosphorylase in the b form. Three zones have been observed. - Extracts of rat muscle and heart yielded phosphorylases which corresponded in their migration rate and in their requirement for adenosine-5′-monophosphate to the b form of pig heart phosphorylase. The same was true for the migration rate of liver phosphorylase. Crystalline α-phosphorylase of rabbit muscle required the addition of cystein to the electrophoresis buffer during separation. Two zones of activity could be demonstrated.

Hermann Stegemann

Papers

Determination of hydroxyproline.

Estimation of amino acids using ninhydrin reduced with dithionite.

[Microdetermination of nitrogen as indophenol blue by chloramine-T oxidation].

[Ezyme electrophoresis in embedding polymerisates of acrylamide. A. Amylases, phosphorylases].

Proteine der kartoffelknollen in abhängigkeit von sorte und virosen (polyacrylamid-elektrophorese)