Fluo-3检测细胞内钙离子的条件优化

目的:优化用于检测细胞内钙离子的Fluo-3浓度条件和羧苯磺胺浓度条件,以得到最适的检测用浓度组合。方法:应用析因实验设计方法,选择32种不同的Fluo-3和羧苯磺胺浓度组合孵育CHO-K1细胞,通过FlexStation检测平台检测细胞内的钙离子浓度。结果与结论:检测信号随着Fluo-3浓度的升高而增强,同时随着羧苯磺胺浓度的升高而减弱。4μmol/L的Fluo-3和1mmol/L的羧苯磺胺浓度组合是最适用的检测条件。     

Implication of the nucleus in excitation contraction coupling of heart cells

In the present study, Fluo-3 Ca²⁺ measurement and confocal microscopy techniques were used in order to localize cytosolic [ ]_c and nuclear [ ]_n free Ca²⁺ distribution in resting and spontaneously contracting single heart cells from 10-day-old chick embryos. In resting single cells, the concentration of Ca²⁺ in the cytoplasm was lower than that in the nucleus. Increasing cytosolic free Ca²⁺ from 100–1600 nM gradually increased [Ca²⁺]_n with a maximum capacity near 1200 nM. Results from Fura-2 microfluorometry and Fluo-3 confocal microscopy suggest a potential cross talk between the increase of cytosolic free Ca²⁺ and the uptake and release of Ca²⁺ by the nucleus during spontaneous contraction of single myocytes. Calcium waves in spontaneously contracting cells were found to spread from one cell to the next with the nucleus acting as a fluorescent beacon in which Ca²⁺ levels remained elevated for several milliseconds even after cytosolic Ca²⁺ had returned to near basal values. These results strongly suggest that the nucleus plays a negative and positive feedback role in controlling cytosolic free Ca²⁺ concentration during excitation-contraction coupling in heart cells.     

Sequential phases of Ca<Superscript>2+</Superscript> alterations in pre-apoptotic cells

The very early events of the intrinsic, damage-induced apoptotic pathway, i.e., upstream to Bax activation, probably consist of physico-chemical alterations (i.e., redox, pH or Ca²⁺ changes) rather then subtle molecular interactions, and in spite of many studies they remain unclear. One problem is that cells undergo apoptosis in an asynchronous way, leading to heterogeneity in the cell population that impairs the results of bulk analyses. In this study, we present a flow cytometric approach for studying Ca²⁺ alteration in apoptosis at the single cell level. By means of a multiparametric analysis, we could discriminate different sub-populations, i.e., viable and apoptotic cells and cells in secondary necrosis, and separately analyse static as well as dynamic Ca²⁺ parameters in each sub-population. With this approach, we have identified a set of sequential Ca²⁺ changes; two very early ones occur prior to any other apoptotic alterations, whereas a later change coincides with the appearance of apoptosis. Interestingly, the two pre-apoptotic changes occur simultaneously in all treated cells, i.e., at fixed times post-treatment, whereas the later one occurs at varying times, i.e., within a wide time range, concomitantly with the other apoptotic events.     

Nuclear localisation of wild type and mutant galectin-3 in transfected cells

Galectin-3, a member of a family of carbohydrate-binding proteins, is present generally in the cytoplasm of cells. However, galectin 3 can also be located in nuclei under certain conditions although it lacks any known nuclear localisation signal and the mechanism by which the protein is sequestered in nuclei is unknown. Here we describe that Cos-7 cells or rabbit smooth muscle Rb-1 cells transfected with cDNA encoding hamster galectin-3 sequester the protein in nuclei whereas untransfected BHK cells expressing the endogenous hamster lectin or transfected BHK cells over-expressing the protein, do not. Confocal immunofluorescence microscopy of Cos-7 cells or rabbit smooth muscle Rb-1 cells transfected with cDNAs encoding mutants of hamster galectin-3 containing N-terminal or internal deletions shows that nuclear localisation does not require the first 103 amino acid residues of the protein. Further deletion of residues 104-110 dramatically prevents sequestration in nuclei. However, the sequence A104PTGALT110 by itself is not obligatory for nuclear localisation and can be substituted by other unrelated sequences. A truncated galectin-3 protein, that is blocked in nuclear expression, retains carbohydrate-binding activity, making less likely the possibility that severe N-terminal truncations of galectin-3 induce mis-folding leading to aggregation and cytoplasmic sequestration and an incidental effect on nuclear trafficking. These studies indicate that nuclear import and retention of galectin-3 is a property of the CRD domain and is independent of N-terminal domains that others have shown to contain binding domains for various nuclear components.     

Real-time monitoring of adherent Vero cell density and apoptosis in bioreactor processes

This study proposes an easy to use in situ device, based on multi-frequency permittivity measurements, to monitor the growth and death of attached Vero cells cultivated on microporous microcarriers, without any cell sampling. Vero cell densities were on-line quantified up to 10⁶ cell mL⁻¹. Some parameters which could potentially impact Vero cell morphological and physiological states were assessed through different culture operating conditions, such as media formulation or medium feed-harvest during cell growth phase. A new method of in situ cell death detection with dielectric spectroscopy was also successfully implemented. Thus, through permittivity frequency scanning, major rises of the apoptotic cell population in bioreactor cultures were detected by monitoring the characteristic frequency of the cell population, f_c, which is one of the culture dielectric parameters. Both cell density quantification and cell apoptosis detection are strategic information in cell-based production processes as they are involved in major events of the process, such as scale-up or choice of the viral infection conditions. This new application of dielectric spectroscopy to adherent cell culture processes makes it a very promising tool for risk-mitigation strategy in industrial processes. Therefore, our results contribute to the development of Process Analytical Technology in cell-based industrial processes.     

Induction of calcium influx from extracellular fluid by beauvericin in human leukemia cells

Beauvericin, a cyclic hexadepsipeptide, is a mycotoxin that can induce cell death in human lymphoblastic leukemia CCRF-CEM cells. Our previous data have shown that beauvericin induces cell death in CCRF-CEM cells in a dose- and time-dependent manner, and that this beauvericin-induced cell death can be prevented by administration of intracellular calcium chelator-BAPTA. Therefore, the intracellular Ca2+ concentration ([Ca2+]i) may play an important role in beauvericin-induced cell death in CCRF-CEM cells. In this study, the effect of beauvericin on [Ca2+]i and the possible mechanism responsible for the changes of [Ca2+]i in CCRF-CEM cells were investigated. Beauvericin caused a rapid and sustained [Ca2+]i rise in a dose-dependent manner. Excess extracellular Ca2+ facilitated beauvericin-induced [Ca2+]i rise by adding 1 mM CaCl2 in the bathing medium. On the other hand, beauvericin-induced [Ca2+]i rise was prevented in Ca2+-free Tyrode's solution by 200 microM EGTA. In addition, beauvericin-induced [Ca2+]i rise was also attenuated by intracellular Ca2+ chelator-BAPTA/AM. It is worthy to note that neither the voltage-dependent Ca2+ channel blocker, nimodipine, nor depletion of intracellular Ca2+ with thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor, has any effect on beauvericin-induced [Ca2+]i rise. The data from present study indicate that beauvericin acts as a potent Ca2+ mobilizer by stimulating extracellular Ca2+ influx CCRF-CEM cells.     

Tracing of intracellular zinc(II) fluorescence flux to monitor cell apoptosis by using FluoZin‐3AM

Changes in the free zinc(II) concentration are closely related to cell proliferation and apoptosis, especially during the early apoptotic process. In the present paper, we demonstrated that zinc(II) probe FluoZin‐3AM owns sensitive properties to distinguish different stages of apoptotic cell (induced by an anticancer agent, etoposide) according to trace intracellular zinc(II) fluorescence flux. When apoptosis in HeLa or K562 cells was artificially induced, FluoZin‐3AM selectively and strongly stained apoptotic cells only at early and middle stages, which was attributed to significantly increased free zinc(II) flux during these stages. This conclusion was further verified by comparing it with the conventional apoptosis detector probe Annexin‐V‐FITC and PI. Furthermore, FluoZin‐3AM was found cell permeable to detect the intracellular zinc(II) fluorescence enhancement to threefolds within 120 s with low cytotoxicity when zinc(II) was incorporated into the cell by zinc(II) ionophore pyrithione. All the above implied that monitoring intracellular zinc fluorescence flux was an effective method to distinguish cell apoptosis from necrosis, and FluoZin‐3AM was found to be a suitable probe acting alone to fulfill the work. Copyright © 2009 John Wiley & Sons, Ltd.     

Carbohydrate sulfotransferase 3 (CHST3) overexpression promotes cartilage endplate-derived stem cells (CESCs) to regulate molecular mechanisms related to repair of intervertebral disc degeneration by rat nucleus pulposus

To investigate the regulatory effect of carbohydrate sulfotransferase 3 (CHST3) in cartilage endplate-derived stem cells (CESCs) on the molecular mechanism of intervertebral disc degeneration after nucleus pulposus repair in rats. We performed GO and KEGG analysis of GSE15227 database to select the differential genes CHST3 and CSPG4 in grade Ⅱ, Ⅲ and Ⅳ intervertebral disc degeneration, IHC and WB to detect the protein profile of CHST3 and CSPG4, Co-IP for the interaction between CHST3 and CSPG4. Then, immunofluorescence was applied to measure the level of CD90 and CD105, and flow cytometry indicated the level of CD73, CD90 and CD105 in CESCs. Next, Alizarin red staining, Alcian blue staining and TEM were performed to evaluate the effects of CESCs into osteoblasts and chondroblasts, respectively, CCK8 for the cell proliferation of osteoblasts and chondroblasts after induction for different times; cell cycle of osteoblasts or chondroblasts was measured by flow cytometry after induction, and WB for the measurement of specific biomarkers of OC and RUNX in osteoblasts and aggrecan, collagen II in chondroblasts. Finally, colony formation was applied to measure the cell proliferation of CESCs transfected with ov-CHST3 or sh-CHST3 when cocultured with bone marrow cells, WB for the protein expression of CHST3, CSPG4 and ELAVL1 in CSECs, transwell assay for the migration of CESCs to bone marrow cells, TEM image for the cellular characteristics of bone marrow cells, and WB for the protein profile of VCAN, VASP, NCAN and OFD1 in bone marrow cells. CHST3 and CSPG4 were differentially expressed and interacted in grade Ⅱ, Ⅲ and Ⅳ intervertebral disc degeneration; CD73, CD90 and CD105 were lowly expressed in CESCs, osteogenic or chondroblastic induction changed the characteristics, proliferation, cell cycle and specific biomarkers of osteoblasts and chondroblasts after 14 or 21 days,; CHST3 affected the cell proliferation, protein profile, migration and cellular features of cocultured CESCs or bone marrow cells. CHST3 overexpression promoted CESCs to regulate bone marrow cells through interaction with CSPG4 to repair the grade Ⅱ, Ⅲ and Ⅳ intervertebral disc degeneration.     

Effect of regulatory T cells and adherent cells on the expansion of HBcAg-specific CD8 T cells in patients with chronic hepatitis B virus infection

Patients with chronic HBV infection show poor immune response to HBV-specific CD8⁺ T cells. Several studies demonstrate that regulatory T cells (Treg) and dendritic cells (DC) are important to maintain peripheral immune tolerance. In this study, we investigated the effects of CD4⁺CD25⁺Treg and/or the adherent cells (AC) on the proliferation of HBc18-27-specific CD8⁺ T cells (c18-27-CD8Ts) in response to in vitro stimulation. The frequency of c18-27-CD8Ts in four different mixed leukocyte reactions (MLRs) were analyzed using an HLA-A2-HBc18-27 tetramer. The data indicated that the median percentage of c18-27-CD8Ts in four different MLRs were significant difference in patients with chronic HBV infection. Our results showed that Treg and/or AC might suppress the frequency of HBc18-27-specific CD8⁺ T cell proliferation in response to in vitro stimulation in chronic HBV patients, and AC might be more effective than Treg.     

Synaptology of neurosecretory cells in the nucleus paraventricularis of the domestic fowl

Summary Processes of magnocellular neurosecretory cells (MNCs) are easily identifiable on the basis of their content in neurosecretory granules in the neuropil of the rostral division of the paraventricular nucleus (PVN) of the domestic fowl. In specimens sacrificed during the winter the synaptic organization of the neuropil and the pattern of synapses ending on neurosecretory processes were studied at the ultrastructural level. Synapses in the rostral part of the PVN neuropil may be divided into three main categories on the basis of their morphology and their content of clear and dense-core synaptic vesicles. These different types of terminals can be attributed to aminergic, peptidergic or other types of synapses. The percent distribution of synapses within these categories differs when all synapses observed in the neuropil or only those ending on MNC processes are compared. Present ultrastructural data obtained in birds support two physiological hypotheses already suggested for mammals, i.e., the probable existence of a recurrent pathway to MNCs via an interneuron, and the importance of aminergic and peptidergic input in regulating the electrical activity of MNCs.This work was partly supported by a CNR grant (n. 81.00377.04)     

Role of calcium in differentiation of murine erythroleu-kemia cells

Calcium plays a crucial role in the normal and abnomal cell metabolism.The role of calcium in the differentiation process of murine erythroleukemia cells(MELC)remains controversial.Here,based upon quantitative measurement of fluorescence in single cells,a method was developed to investigate the intracellular free calcium[Ca^2 ]i concentration and DNA contents simultaneously,by employing the fluorescent probe,fluo-3 acetoxymethyl ester and DNA dye Hoechst 33342.During MELC differentiation.[Ca^2 ]i concentration incresed.We also demonstrated that calcium ionophore,A23187,enhanced the HMB-induced MELC differentiation,while verapamil,an inhibitor of calcuim uptake,slightly reduced differentiation.These results suggested that an increase in the [Ca^2 ]i level was an essential step in HMBA-induced MELC differentiation.     

丹参悬浮培养细胞原生质体的制备和活力检测

对丹参悬浮培养细胞原生质体制备条件进行了研究,并利用FDA染色和钙离子荧光探针Fluo-3/AM装载对制备得到的原生质体的活力和功能进行了检测。丹参悬浮培养细胞原生质体的制备条件为:悬浮培养细胞酶解的适宜酶液组合为纤维素酶1.5%、果胶酶0.3%和离析酶0.5%;适宜的甘露醇浓度为0.4 mol/L;酶解时间为12 h;在600 r/min转速下离心5 min收集,纯化得到原生质体,其产量为1.1×106/g FW,FDA检测显示其活力为95%以上,荧光探针Fluo-3/AM可成功装载到原生质体中。     

Carbamylcholine-induced morphological changes and spatial dynamics of [Ca2+]c in Harderian glands of guinea pigs: calcium-dependent lipid secretion and contraction of myoepithelial cells

To determine whether lipid-secreting cells have cytosolic Ca²⁺ concentration ([Ca²⁺]_c)-related secretory mechanisms, morphological changes and intracellular calcium dynamics of Harderian glands of guinea pigs stimulated by secretagogs were studied by electron microspy and Fura-2/AM digital image analysis. Control glandular cells contained large lipid vacuoles that were bordered by multi-layered membranes. Rough-surfaced endoplasmic reticulum, mitochondria, and smooth-surfaced endoplasmic reticulum may be involved in lipid vacuole formation. Myoepithelial cells surrounded alveoli. After carbamylcholine (CCh, 10^–6, 10^–5, and 10^–3 M) stimulation, lipid materials within the membranous structures were frequently discharged by an exocytotic mechanism. Conspicuous deformation of glandular cells caused by vigorous contraction of myoepithelial cells was observed in isolated alveoli after 10^–6M CCh stimulation, whereas the deformaties of glandular tissues perfused via vessels were small even after 10^–3M CCh stimulation. Connective tissue between glandular alveoli inhibited unbridled myoepithelial-cell contraction. Fura-2/AM digital imaging analysis revealed that CCh stimulation caused an increase in [Ca²⁺]_c in isolated alveoli. The morphological reactions and changes in [Ca²⁺]_c were prevented by atropine. When extracellular calcium ions were absent, enhanced extrusion of lipid vacuoles, myoepithelial-cell contraction, and a rise in [Ca²⁺]_c after CCh stimulation were not observed. Nicotine and catecholamines had no effect on the secretion or on the dynamics of [Ca²⁺]_c. It can be concluded that acetylcholine elicits exocytosis in glandular cells and contraction of the myoepithelial cells of Harderian glands, accompanied by an increase in [Ca²⁺]_c. The dynamics of [Ca²⁺]_c of the gland alveoli are mostly dependent on extracellular Ca²⁺.     

Estrogen-dependent changes in the functional interrelationships among neurons,ependymal cells and glial cells of the arcuate nucleus

Summary The synchronizing effect of ethinylestradiol (4 g/g b.w.) on neurons of the arcuate nucleus 700–950 m caudal to the posterior edge of the optic chiasma was studied by karyometry in 6-week-old albino mice during proestrus.The caudal portion of the arcuate nucleus was identified as the most estrogen-sensitive subdivision; all neurons showed an increase in their nuclear area (mean transect, profile area of the nucleus) 1 h following administration of ethinylestradiol. This hypothalamic region was selected for the subsequent electron-microscopic cytometric study to analyze functional interrelationships among neurons, ependymal cells and glial cells. Six and 12 days after ovariectomy no significant change in the nuclear area of neurons and ependymal cells was found 850–950 m behind the posterior slope of the optic chiasma, but the neurons exhibited a decrease in the number of polyribosomes, the volume fraction (V_Vmi) and the surface density of the inner membrane of mitochondria (S_Vmi). A similar decrease in V_Vmi and S_Vmi was measured in the apical part of ependymal cells and in the pericapillary profiles of ependymal and glial cells, which was accompanied by a reduction in the surface density of ependymal processes extending into the ventricular lumen. In addition, no change of V_Vmi and S_Vmi was seen in the basal subnuclear part of ependymal cells.This bipolar functional reaction of ependymal cells after ovariectomy is discussed as an indicator of ependymal control of neuronal activity by sequestering biologically active agents, e.g., transmitters of neurohormones, in their apical and basal extensions facing the ventricular surface or the pericapillary space.     

Advanced glycation end products regulate anabolic and catabolic activities via NLRP3‐inflammasome activation in human nucleus pulposus cells

Intervertebral disc degeneration is widely recognized as a cause of lower back pain, neurological dysfunction and other musculoskeletal disorders. The major inflammatory cytokine IL‐1β is associated with intervertebral disc degeneration; however, the molecular mechanisms that drive IL‐1β production in the intervertebral disc, especially in nucleus pulposus (NP) cells, are unknown. In some tissues, advanced glycation end products (AGEs), which accumulate in NP tissues and promote its degeneration, increase oxidative stress and IL‐1β secretion, resulting in disorders, such as obesity, diabetes mellitus and ageing. It remains unclear whether AGEs exhibit similar effects in NP cells. In this study, we observed significant activation of the NLRP3 inflammasome in NP tissues obtained from patients with degenerative disc disease compared to that with idiopathic scoliosis according to results detected by Western blot and immunofluorescence. Using NP cells established from healthy tissues, our in vitro study revealed that AGEs induced an inflammatory response in NP cells and a degenerative phenotype in a NLRP3‐inflammasome‐dependent manner related to the receptor for AGEs (RAGE)/NF‐κB pathway and mitochondrial damage induced by mitochondrial reactive oxygen species (mtROS) generation, mitochondrial permeability transition pore (mPTP) activation and calcium mobilization. Among these signals, both RAGE and mitochondrial damage primed NLRP3 and pro‐IL‐1β activation as upstream signals of NF‐κB activity, whereas mitochondrial damage was critical for the assembly of inflammasome components. These results revealed that accumulation of AGEs in NP tissue may initiate inflammation‐related degeneration of the intervertebral disc via activation of the NLRP3 inflammasome.     

Effect of O-superfamily conotoxin SO3 on synchronized spontaneous calcium spikes in cultured hippocampal networks

SO3 belongs to the O-superfamily of conotoxins and is known to have analgesic effects in experimental animals. In order to explore the mechanism of its potential pharmacological actions, the effect of SO3 on synchronized spontaneous calcium spikes was examined in cultured hippocampal networks by calcium imaging. Spontaneous oscillations of intracellular concentrations of calcium (Ca²⁺) in the form of waves and spikes are found in cultured hippocampal networks. Exposure to increasing concentrations of SO3 resulted in a progressive decrease in synchronized spontaneous calcium spikes. The higher concentrations (0.1 μmol/L and 1 μmol/L) of SO3 showed the strongest inhibition. The rank order of inhibition was 1 μmol/L > 0.1 μmol/L > 10 μmol/L > 0.01 μmol/L. This action of SO3 in reducing synchronized calcium spikes suggests a possible application for therapeutic treatment of epilepsy.     

Identification of ribosomal protein S3a as a candidate for a novel PI 3-kinase target in the nucleus

Phosphatidylinositol 3,4,5-trisphosphate (PIP₃) is an important lipid second messenger that mediates various cell responses. We have searched for the nuclear PIP₃ binding proteins using PIP₃ analogue beads. A 33 kD protein was detected in this method, which was identified as ribosomal protein S3a by the mass spectrometric analysis. The recombinant S3a protein bound specifically to PIP₃. S3a localized not only in the cytosol but also in the nucleus. Interestingly, not cytosolic but nuclear S3a bound to PIP₃, suggesting different roles of S3a in the cytosol and the nucleus. This revised version was published online in August 2006 with corrections to the Cover Date.