The local concentration of extracellular Ca(2+) ([Ca(2+)]o) in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca(2+)]o induced store-operated calcium entry (SOCE) in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca(2+)]o to osteoblastic proliferation.
Fluo-3 is widely used to study cell calcium. Two traditional approaches: (1) direct injection and (2) Fluo-3 acetoxymethyl ester (AM) loading, often bring conflicting results in cytoplasmic calcium ([Ca(2+)]c) and nuclear calcium ([Ca(2+)]n) imaging. AM loading usually yields a darker nucleus than in cytoplasm, while direct injection always induces a brighter nucleus which is more responsive to [Ca(2+)]n detection. In this work, we detailedly investigated the effects of loading and de-esterification temperatures on the fluorescence intensity of Fluo-3 in response to [Ca(2+)]n and [Ca(2+)]c in adherent cells, including osteoblast, HeLa and BV2 cells. Interestingly, it showed that fluorescence intensity of nucleus in osteoblast cells was about two times larger than that of cytoplasm when cells were loaded with Fluo-3 AM at 4°C and allowed a subsequent step for de-esterification at 20°C. Brighter nuclei were also acquired in HeLa and BV2 cells using the same experimental condition. Furthermore, loading time and adhesion quality of cells had effect on fluorescence intensity. Taken together, cold loading and room temperature de-esterification treatment of Fluo-3 AM selectively yielded brighter nucleus in adherent cells.
The high-level production of nitric oxide (NO) induced by inflammatory cytokines has been shown to play a key role in the pathogenesis of inflammation-mediated osteoporosis. In the present work, we observed that 1mM of the NO donor sodium nitroprusside (SNP) induced an increase of the cytosolic calcium concentration ([Ca(2+)]c) in osteoblasts, which was completely abolished by applying an extracellular Ca(2+)-free buffer. Further experiments showed that the SNP-induced [Ca(2+)]c increase was specifically blocked by potent antagonists of the transient receptor potential vanilloid subtype 1 (TRPV1) channel: capsazepine, ruthenium red, and La(3+) in Ca(2+)-containing buffer. However, nifedipine, an L-type voltage sensitive Ca(2+)-channel blocker, failed to suppress the [Ca(2+)]c elevation caused by SNP. Additionally, 1mM SNP induced osteoblast apoptosis, which was largely inhibited by the blockers of TRPV1, capsazepine and ruthenium red. Interestingly, our data showed that the SNP-induced [Ca(2+)]c increase was significantly inhibited by N-ethylmaleimide, the blocker of S-nitrosylation modification, instead of inhibitors of the NO-cGMP-PKG pathway. Taken together, our data clearly demonstrated that the NO donor SNP resulted in apoptosis associated with TRPV1 channel-mediated Ca(2+) entry via S-nitrosylation in osteoblasts.
As the first and main form of active immune defense in the central nervous system, microglial cells usually exhibit complicated intracellular calcium (Ca²?) activity that can regulate the downstream components of signaling cascades. In the present work, spontaneous oscillations of the cytosolic calcium concentration ([Ca²?]c) in multi-BV-2 microglial cells were observed by video microscopy. These cells exhibited random spikes of Ca²? oscillations. Cross-correlation analysis of the temporal dependence of the oscillations indicated the existence of cell-cell communication mediated by extracellular messengers. Numerical simulations based on a simple mathematical model suggested that these communications could induce random spikes of spontaneous Ca oscillations in the multi-cell system. Short-time imaging analysis of random spikes in different regions of a single cell showed that spontaneous Ca²? oscillations resulted from Ca²? wave generated by other cells as well as from calcium elevation inside the cell. Taken together, our data demonstrate that cell-cell communication existed between the BV-2 microglial cells in vitro and further resulted in the random spikes of spontaneous Ca²? oscillations.
A fungal immunomodulatory protein isolated from Flammulina velutipes (FIP-fve) has structural similarity to the variable region of the immunoglobulin heavy chain. In the present study, the recombinant bioactive FIP-fve protein with a His-tag in N-terminal of recombinant protein was expressed in transetta (DE3) at a high level under the optimized culturing conditions of 0.2 mM IPTG and 28 °C. The efficiency of the purification was improved with additional ultrasonication to the process of lysozyme lysis. The yield of the bioactive FIP-fve protein with 97.1% purity reached 29.1 mg/L with a large quantity for industrial applications. Enzyme-linked immunosorbent assay showed a maximum increase in interleukin-2 (IL-2) and gamma interferon (IFN-?) for the mice serum group of 5 mg/kg body mass (p < 0.01) with three doses of His-FIP-fve. However, the production of IL-4 had no apparent difference compared to the control.
Nitrogen is one of the most important limiting factors for plant growth. However, as ammonium is readily converted into ammonia (NH3) when soil pH rises above 8.0, this activity depletes the availability of ammonium (NH4 (+)) in alkaline soils, consequently preventing the growth of most plant species. The perennial wild grass Puccinellia tenuiflora is one of a few plants able to grow in soils with extremely high salt and alkaline pH (>9.0) levels. Here, we assessed how this species responds to ammonium under such conditions by isolating and analyzing the functions of a putative ammonium transporter (PutAMT1;1). PutAMT1;1 is the first member of the AMT1 (ammonium transporter) family that has been identified in P. tenuiflora. This gene (1) functionally complemented a yeast mutant deficient in ammonium uptake (2), is preferentially expressed in the anther of P. tenuiflora, and (3) is significantly upregulated by ammonium ions in both the shoot and roots. The PutAMT1;1 protein is localized in the plasma membrane and around the nuclear periphery in yeast cells and P. tenuiflora suspension cells. Immunoelectron microscopy analysis also indicated that PutAMT1;1 is localized in the endomembrane. The overexpression of PutAMT1;1 in A. thaliana enhanced plant growth, and increased plant susceptibility to toxic methylammonium (MeA). Here, we confirmed that PutAMT1;1 is an ammonium-inducible ammonium transporter in P. tenuiflora. On the basis of the results of PutAMT1;1 overexpression in A. thaliana, this gene might be useful for improving the root to shoot mobilization of MeA (or NH4 (+)).
As the first line of host defense, neutrophils are stimulated by pro-inflammatory cytokines from resting state, facilitating the execution of immunomodulatory functions in activation state. Sulfhydryl modification has a regulatory role in a wide variety of physiological functions through mediation of signaling transductions in various cell types. Recent research suggested that two kinds of sulfhydryl modification, S-nitrosylation by exogenous nitric oxide (NO) and alkylation by N-ethylmaleimide (NEM), could induce calcium entry through a non-store-operated pathway in resting rat neutrophils and DDT?MF-2 cells, while in active human neutrophils a different process has been observed by us. In the present work, data showed that NEM induced a sharp rising of cytosolic calcium concentration ([Ca²?](c)) without external calcium, followed by a second [Ca²?](c) increase with readdition of external calcium in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. Meanwhile, addition of external calcium did not cause [Ca²?](c) change of Ca²?-free PMA-activated neutrophils before application of NEM. These data indicated that NEM could induce believable store-operated calcium entry (SOCE) in PMA-activated neutrophils. Besides, we found that sodium nitroprusside (SNP), a donor of exogenous NO, resulted in believable SOCE in PMA-activated human neutrophils via S-nitrosylation modification. In contrast, NEM and SNP have no effect on [Ca²?](c) of resting neutrophils which were performed in suspension. Furthermore, 2-Aminoethoxydiphenyl borate, a reliable blocker of SOCE and an inhibitor of inositol 1,4,5-trisphosphate (IP?) receptor, evidently abolished SNP and NEM-induced calcium entry at 75 µM, while preventing calcium release in a concentration-dependent manner. Considered together, these results demonstrated that NEM and SNP induced calcium entry through an IP?-sensitive store-operated pathway of human neutrophils via sulfhydryl modification in a PMA-induced activation-dependent manner.
Microfluidics is a convenient platform to study the influences of fluid shear stress on calcium dynamics. Fluidic shear stress has been proven to affect bone cell functions and remodelling. We have developed a microfluidic system which can generate four shear flows in one device as a means to study cytosolic calcium concentration ([Ca(2+)](c)) dynamics of osteoblasts. Four shear forces were achieved by having four cell culture chambers with different widths while resistance correction channels compensated for the overall resistance to allow equal flow distribution towards the chambers. Computational simulation of the local shear stress distribution highlighted the preferred section in the cell chamber to measure the calcium dynamics. Osteoblasts showed an [Ca(2+)](c) increment proportional to the intensity of the shear stress from 0.03 to 0.30 Pa. A delay in response was observed with an activation threshold between 0.03 and 0.06 Pa. With computational modelling, our microfluidic device can offer controllable multishear stresses and perform quantitative comparisons of shear stress-induced intensity change of calcium in osteoblasts.
Several studies have been undertaken to elucidate the effects of electromagnetic field (EMF) on intracellular calcium ([Ca(2+)](i)) in the past 20 years. However, still there were controversies of electromagnetic pollution within the scientific community. In this work, we studied the effects of alternative magnetic fields on intracellular calcium. Osteoblastic cells were used as a model both to test the hypothesis that extremely low-frequency (ELF) magnetic fields can alter the concentrations of the intracellular calcium, and to examine the window effect predicted by our previous theoretical work. The outcome of this experiment demonstrated that 50 Hz, 0.8 mT magnetic field can induce the uptake of [Ca(2+)](i) in osteoblasts. The empirical evidences of the specified window effects of [Ca(2+)](i) in osteoblastic cells were reported for the first time in this work.
Fluid shear stress (FSS) is widely explored regarding its influence on osteoblasts. In vitro studies have shown that the cytoskeleton is very important in cellular responses to FSS. However, morphological changes, which would reflect the cytoskeleton changes as well as other cellular responses, were rarely quantitatively studied in the past years. Therefore, FSS-induced morphological changes in osteoblasts were quantified in this study. Real-time rapid morphological responses were observed by exposing osteoblasts to FSS with magnitude of 1.2, 1.6, and 1.9 Pa for 1 h. Afterward, osteoblast actin cytoskeleton was labeled with rhodamine phalloidin and observed using fluorescence microscopy. The results showed that 1.6 and 1.9 Pa FFS resulted in significant cellular elongation and reorientation along the direction of fluid flow. Besides, along with the enhancement of FSS magnitude, cytoskeleton aggregated more remarkably. Furthermore, extracellular Ca(2+)-depleted fluid flow was also used to stimulate osteoblasts for 1 h with magnitude of 1.6 and 1.9 Pa. No morphological change was observed after removing extracellular calcium. Our study suggested that the level of FSS from 1.2 to 1.9 Pa is capable of influencing cellular morphology, and extracellular calcium might play a role in osteoblasts response to FSS stimulation.
Ultraviolet (UV) light has a potent effect on biological organisms. Hemoglobin, an oxygen-transport protein, plays an irreplaceable role in sustaining life of all vertebrates. In this study we scrutinize the effects of ultraviolet irradiation (UVI) as well as visible irradiation on the fluorescence characteristics of bovine hemoglobin (BHb) in vitro. Data show that UVI results in fluorescence enhancement of BHb in a dose-dependent manner. Furthermore, UVI-induced fluorescence enhancement is significantly increased when BHb is pretreated with hydrogen peroxide (H(2)O(2)), a type of reactive oxygen species (ROS). Meanwhile, The water-soluble antioxidant vitamin C suppresses this UVI-induced fluorescence enhancement. In contrast, green light irradiation does not lead to fluorescence enhancement of BHb no matter whether H(2)O(2) is acting on the BHb solution or not. Taken together, these results indicate that catalysis of ROS and UVI-dependent irradiation play two key roles in the process of UVI-induced fluorescence enhancement of BHb.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.