Cyclic Peptide D7 Promotes Osteogenesis and Inhibits Osteoclastogenesis by Regulating Redox Balance of Bone Remodeling

BackgroundMilitary training usually causes excessive physical burden to the bones and joints of soldiers, which in turn disturbs the balance of bone homeostasis and eventually induces bone metabolic diseases. In our previous studies, a cyclic peptide D7 in was characterized through phage display technology from the peptide phage display library (Ph.D.‑C7C) and was proven to enhance the adhesion, expansion, and proliferation of bone marrow mesenchymal stem cells (BMSCs) on the biomaterial scaffold. However, we found that cyclic peptide D7 also has a certain affinity for mouse calvarial osteoblast precursor cells (OPCs) and bone marrow-derived monocytes/macrophages (BMMs). To investigate whether D7 can protect physiological bone remodeling and maintain bone homeostasis, we elucidated the mechanisms by which D7 promotes osteogenesis and inhibits osteoclastogenesis. MethodsThe affinity of D7 peptide towards calvarial OPCs and BMMs was investigated using fluorescence cytochemistry. The roles of D7 in osteogenesis and osteoclastogenesis by regulating redox balance was confirmed by cell counting kit-8, special stain assays (alkaline phosphatase stain, alizarin red stain and tartrate‐resistant alkaline phosphatase stain), fluorescence cytochemistry, western blotting, quantitative real-time polymerase chain reaction. ResultsThe results demonstrated that D7 promoted the osteogenesis of calvarial OPCs by upregulating the expression of osteogenic differentiation factors. In contrast, D7 inhibited osteoclast differentiation and resorption by downregulating the expression of osteoclast phenotype marker. We further identified that these phenomenons may be due to the suppressive effect of D7 on reactive oxygen species production and the enhancing effect on antioxidant expression in both OPCs and BMMs, thereby regulating the redox balance of bone remodeling.ConclusionsCyclic peptide D7 could maintain bone homeostasis by regulating redox balance and provide a potential approach to strengthen the skeletal fitness of soldiers and prevent the occurrence of bone metabolic diseases.

Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport

Inhibitor studies have implicated microtubules in at least three important developmental processes during Drosophila oogenesis: oocyte determination and growth during stages 1 through 6, positioning of the anterior determinant bicoid mRNA during stages 9 through 12, and ooplasmic streaming during stages 10b through 12. We have used fluorescence cytochemistry together with laser scanning confocal microscopy to identify distinct microtubule structures at each of the above three periods that are likely to be involved in these processes. During stages 1 through 7, maternal components synthesized in nurse cells are transported through cytoplasmic bridges to the oocyte. At this time, microtubules that appear to originate in the oocyte pass through these cytoplasmic bridges into the adjacent nurse cells; these microtubules are likely to serve as a polarized scaffold on which maternal RNAs and proteins are transported. During stages 7 and 8, microtubules in the oocyte cortex reorganize to form an anterior-to-posterior gradient, suggesting a role for microtubules in the localization of morphogenetic determinants. Finally, when ooplasmic streaming begins during stage 10 b, it is accompanied by the assembly of subsurface microtubule arrays that spiral around the oocyte; these arrays disassemble as the oocyte matures and streaming stops. During ooplasmic streaming, many vesicles are closely associated with the subsurface microtubules, suggesting that streaming is driven by vesicle translocation along microtubules. We believe that actin plays a secondary role in each of these morphogenetic events, based on our parallel studies of actin organization during each of the above stages of oogenesis.

Cytochemical localization of ornithine decarboxylase with rhodamine or biotin-labeled alpha-difluoromethylornithine. An example for the use of labeled irreversible enzyme inhibitors as cytochemical markers.

The present work describes a new method for cytochemical localization of enzymes using ornithine decarboxylase (ODC) as an example. The method is based on the preservation of the characteristic-specific and irreversible binding of the inhibitor alpha-difluoromethylornithine (alpha-DFMO) following its conjugation to "label" molecules. The inhibitor has been conjugated to the fluorescent molecule rhodamine-B-isothiocyanate, and its localization in tissue sections was detected directly by fluorescence cytochemistry. Alternatively, alpha-DFMO has been conjugated to biotin and its cytochemical localization determined indirectly following its binding with avidin conjugated to horseradish peroxidase (HRP) and visualization of the HRP reaction product. Both labeled inhibitor molecules were successfully localized cytochemically within specific cells of the developing rat cerebellum and rat liver following thioacetamide injection where ODC activity is greatly enhanced. This novel technique should be of general application 1) in other tissues, 2) for other enzymes, and 3) in electron microscopic studies for ultrastructural localization of the enzyme.