To study the effect of adenosine 3',5'-cyclic monophosphate (cAMP) on respiratory ciliary activity, we measured ciliary beat frequency (CBF) of rabbit tracheal epithelium by a photoelectric method in response to cAMP analogues and agents that can increase endogenous cAMP production. Addition of 8-bromo-cAMP dose dependently enhanced CBF, with the maximal increase and the concentration necessary to produce a half-maximal response (KD) being 31.0 +/- 3.4% (SE) (P less than 0.001) and 3.2 +/- 1.5 x 10(-7) M, respectively. Other structurally dissimilar cAMP analogues dibutyryl cAMP and chlorophenylthio-cAMP likewise caused increases in CBF. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the adenylate cyclase stimulator forskolin also augmented CBF in a dose-dependent fashion and were accompanied by the increases in intracellular concentrations of cAMP. Ciliary discoordination was not observed in any of the experiments. These results suggest that cAMP may accelerate mucociliary clearance through the activation of ciliary motility and that intracellular cAMP levels appear to be an important determinant for the lung mucociliary transport functions.
Differentiation of osteoclasts (OCs) from hematopoietic cells requires cellular interaction with osteoblasts (OBs). Due to the difficulty of live-imaging in the bone, however, the cellular and molecular mechanisms underlying intercellular communication involved in OC differentiation are still elusive. Here, we develop a fracture healing model using the scale of trap: GFP; osterix:mCherry transgenic zebrafish to visualize the interaction between OCs and OBs. Transplantation assays followed by flow cytometric analysis reveal that most trap:GFP high OCs in the fractured scale are detected in the osterix:mCherry + fraction because of uptake of OB-derived extracellular vesicles (EVs). In vivo live-imaging shows that immature OCs actively interact with osterix:mCherry + OBs and engulf EVs prior to convergence at the fracture site. In vitro cell culture assays show that OB-derived EVs promote OC differentiation via Rankl signaling. Collectively, these data suggest that EV-mediated intercellular communication with OBs plays an important role in the differentiation of OCs in bone tissue.
Hematopoietic stem cells (HScs) maintain the entire blood system throughout life and are utilized in therapeutic approaches for blood diseases. Prospective isolation of highly purified HSCs is crucial to understand the molecular mechanisms underlying regulation of HSCs. The zebrafish is an elegant genetic model for the study of hematopoiesis due to its many unique advantages. it has not yet been possible, however, to purify HSCs in adult zebrafish due to a lack of specific HSC markers. Here we show the enrichment of zebrafish HSCs by a combination of two HSC-related transgenes, gata2a:GFP and runx1:mCherry. the double-positive fraction of gata2a:GFP and runx1:mCherry (gata2a + runx1 + ) was detected at approximately 0.16% in the kidney, the main hematopoietic organ in teleosts. transcriptome analysis revealed that gata2a + runx1 + cells showed typical molecular signatures of HScs, including upregulation of gata2b, gfi1aa, runx1t1, pbx1b, and meis1b. transplantation assays demonstrated that long-term repopulating HScs were highly enriched within the gata2a + runx1 + fraction. in contrast, colony-forming assays showed that gata2a − runx1 + cells abundantly contain erythroid-and/or myeloid-primed progenitors. Thus, our purification method of HSCs in the zebrafish kidney is useful to identify molecular cues needed to regulate self-renewal and differentiation of HSCs.Hematopoietic stem cells (HSCs) are self-renewing multipotent cells that can generate all types of blood cells over the lifetime of an individual and can be used therapeutically to treat hematopoietic diseases 1 . In the adult, most HSCs present in bone marrow are quiescent and divide rarely under homeostatic conditions. HSCs produce a heterogeneous pool of hematopoietic progenitor cells (HPCs), which have limited or no self-renewal ability, but rapidly proliferate and differentiate to satisfy the requirements for new mature blood cells 2,3 . Although the frequency of HSCs is extremely rare in bone marrow, HSC potential can be evaluated by transplantation assays, whereby the relative hematopoietic reconstitution activity of co-transplanted donor and competitor cells are compared in a recipient 4 . Purification of HSCs from murine and human bone marrow has been facilitated via transplantation assays using combinations of multiple cell-surface markers [5][6][7][8][9] . Studies in mice revealed that a single CD150 + CD34 − c-kit + Sca-1 + Lineage-marker − cell in the bone marrow showed long-term and multilineage hematopoietic reconstitution following transplantation 10,11 . Prospective isolation of highly purified HSCs thus elucidated many aspects of HSC biology, including self-renewal, differentiation, and HSC niches.The zebrafish is an excellent model for the study of HSCs due to its many unique advantages. Many valuable tools and experimental methods have been established for the study of hematopoietic cells in zebrafish (e.g. transgenic/mutant animals, transplantation assays, cell culture assays, etc.) 12,13 . Moreover, genome-editing technology based on...
Copper (Cu) is an essential trace element and acts as a redox cofactor for many enzymes; however, excess Cu is toxic to cells.
Copper (Cu) is an essential trace element that plays an important role in maintaining neuronal functions such as the biosynthesis of neurotransmitters. In contrast, exposure to excess Cu results in cell injury. Therefore, intracellular Cu levels are strictly regulated by proteins related to Cu-trafficking, including ATP7A. Parkinson's disease (PD) is a neurodegenerative disorder and is characterized by the loss of dopaminergic neurons in the substantia nigra. Recently, the abnormality of Cu homeostasis was demonstrated to be related to the pathogenesis of PD. However, the association between Cu dyshomeostasis and PD remains unclear. In this study, we examined the effects of 6-hydroxydopamine (6-OHDA), a neurotoxin used for the production of PD model animals, on cellular Cu trafficking in human neuroblastoma SH-SY5Y cells. 6-OHDA reduced the protein levels of the Cu exporter ATP7A and the Cu chaperone Atox1, but not CTR1, a Cu importer; however, it did not affect the expression of ATP7A and Atox1 mRNAs. The decreased levels of ATP7A and Atox1 proteins were restored by the antioxidant N-acetylcysteine and the lysosomal inhibitor bafilomycin A1. This suggests that 6-OHDA-induced oxidative stress facilitates the degradation of these proteins. In addition, the amount of intracellular Cu after exposure to CuCl2 was significantly higher in cells pretreated with 6-OHDA than in untreated cells. Moreover, 6-OHDA reduced the protein levels of the cuproenzyme dopamine β-hydroxylase, which converts dopamine to noradrenaline. Thus, this study suggests that 6-OHDA disrupts Cu homeostasis through the dysregulation of cellular Cu trafficking, resulting in the dysfunction of neuronal cells.
The creep deformation mechanism of SnAgCu alloy doped with Bi and Sb (SACBiSb) alloys is theoretically and experimentally analyzed in order to clarify the effect of solid solution additives in SnAgCu alloys. The theoretical prediction results and test results are found to mostly agree with each other. The breakaway stress which is the stress at the transition from Class II to Class I in particular is finely reproduced in the theoretical prediction, with a stress of 25 MPa found both by the prediction and test results. In the stress range where the stress is higher than the breakaway stress, the creep strength of SACBiSb is higher than that of SnAgCu, while it was predicted that the creep strength of SnAgCu would be superior to that of SACBiSb in the range of stress lower than the breakaway stress. The thermal fatigue life of SACBiSb was predicted to be longer than that of SnAgCu in the temperature profile mainly used above the breakaway stress. However, in the temperature profile mainly used in the low-stress range, a reversal of the creep strength between SACBiSb and SnAgCu was predicted to occur and the loss of the superiority of SACBiSb in the thermal fatigue life was also predicted.
The maintenance and proliferation of hematopoietic stem cells (HSCs) are tightly regulated by their niches in the bone marrow. The analysis of niche cells or stromal cell lines that can support HSCs has facilitated the finding of novel supporting factors for HSCs. Despite large efforts in the murine bone marrow, however, HSC expansion is still difficult ex vivo, highlighting the need for new approaches to elucidate the molecular elements that regulate HSCs. The zebrafish provides a unique model to study hematopoietic niches as HSCs are maintained in the kidney, allowing for a parallel view of hematopoietic niches over evolution. Here, using a stromal cell line from the zebrafish kidney, zebrafish kidney stromal (ZKS), we uncover that an inhibitor of canonical Wnt signaling, IWR-1-endo, is a potent regulator of HSCs. Co-culture assays revealed that ZKS cells were in part supportive of maintenance, but not expansion, of gata2a:GFP+runx1:mCherry+ (gata2a+runx1+) HSCs. Transcriptome analysis revealed that, compared to candidate niche cells in the kidney, ZKS cells weakly expressed HSC maintenance factor genes, thpo and cxcl12, but highly expressed canonical Wnt ligand genes, wnt1, 7bb, and 9a. Thpo supplementation in ZKS culture slightly increased, but inhibition of canonical Wnt signaling by IWR-1-endo treatment largely increased the number of gata2a+runx1+ cells (> 2-fold). Moreover, we found that gata2a+runx1+ cells can be maintained by supplementing both IWR-1-endo and Thpo without stromal cells. Collectively, our data provide evidence that IWR-1-endo can be used as a novel supporting factor for HSCs.
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