In skeletal muscle the activation of phosphorylase b is catalyzed by phosphorylase kinase. Both enzymes occur in vivo as part of a multienzyme complex. The two enzymes have been imaged by atomic force microscopy and the results compared to those previously found by scanning tunneling microscopy. Scanning tunneling microscopy and atomic force microscopy have been used to view complexes between the activating enzyme phosphorylase kinase and its substrate phosphorylase b. Changes in the size and shape of phosphorylase kinase were observed when it bound phosphorylase b.
These results demonstrated that the abated microRNA-195 expression protected mesangial cells from apoptosis, suggesting that the antiapoptosis in a microRNA-regulated manner may play an important role in the early stages of diabetic nephropathy.
Background
LL-37 peptide is a member of the human cathelicidin family, and has been shown to promote the healing of pressure ulcers. However, the low stability of this peptide within the wound environment limits its clinical use. Chitosan (CS) hydrogel is commonly used as a base material for wound dressing material.
Methods
CS hydrogel (2.5% w/v) was encapsulated with LL-37. Cytotoxicity of the product was examined in cultured NIH3T3 fibroblasts. Effects on immune response was examined by measuring tumor necrosis factor-α (TNF-α) release from RAW 264.7 macrophages upon exposure to lipopolysaccharides. Antibacterial activity was assessed using Staphylococcus aureus. Potential effect on pressure ulcers was examined using a mouse model. Briefly, adult male C57BL/6 mice were subjected to skin pressure using magnets under a 12/12 h schedule for 21 days. Mice were randomized to receive naked LL-37 (20 μg), chitosan gel containing 20-μg LL-37 (LL-37/CS hydrogel) or hydrogel alone under the ulcer bed (n = 6). A group of mice receiving no intervention was also included as a control.
Results
LL-37/CS hydrogel did not affect NIH3T3 cell viability. At a concentration of 1–5 μg/ml, LL-37/CS inhibited TNF-α release from macrophage. At 5 μg/ml, LL-37/CS inhibited the growth of Staphylococcus aureus. The area of the pressure ulcers was significantly lower in mice receiving LL-37/CS hydrogel in comparison to all other 3 groups on days 11 (84.24% ± 0.25%), 13 (56.22% ± 3.91%) and 15 (48.12% ± 0.28%). Histological examination on days 15 and 21 showed increased epithelial thickness and density of newly-formed capillary with naked LL-37 and more so with LL-37/CS. The expression of key macromolecules in the process of angiogenesis (i.e., hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor-A (VEGF-A)) in wound tissue was increased at both the mRNA and protein levels.
Conclusion
Chitosan hydrogel encapsulated with LL-37 is biocompatible and could promote the healing of pressure ulcers.
The molecular structures of phosphorylase b and phosphorylase kinase have been visualized by scanning tunneling microscopy (STM). STM is a near field technique that can resolve structures at the nanometer level and thus can image individual molecules. Phosphorylase b can be seen in dimeric and tetrameric forms as well as linear and globular aggregates. The linear arrays consist of side by side dimers with the long axis of the dimer perpendicular to the aggregated chain. Individual molecules of phosphorylase kinase appear to be planar, bilobate structures with a 2-fold axis of symmetry and a central depression.
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