The cellular and molecular basis of store-operated calcium entry.

The impact of calcium signalling on so many areas of cell biology reflects the crucial role of calcium signals in the control of diverse cellular functions. Despite the precision with which spatial and temporal details of calcium signals have been resolved, a fundamental aspect of the generation of calcium signals -- the activation of 'store-operated channels' (SOCs) -- remains a molecular and mechanistic mystery. Here we review new insights into the exchange of signals between the endoplasmic reticulum (ER) and plasma membrane that result in activation of calcium entry channels mediating crucial long-term calcium signals.

Ca2+ waves initiate antigen-stimulated Ca2+ responses in mast cells.

Ca(2+) mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca(2+) sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca(2+) waves that originate most frequently from the tips of extended cell protrusions, as well as Ca(2+) oscillations throughout the cell that usually follow the initiating Ca(2+) wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca(2+) puffs at the region of cell contact. Initiating Ca(2+) waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca(2+) release from endoplasmic reticulum stores and to extracellular Ca(2+), but they do not depend on store-operated Ca(2+) entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca(2+) signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca(2+) response.

Regulators of Ca(2+) signaling in mast cells: potential targets for treatment of mast cell-related diseases?

A calcium signal is essential for degranulation, generation of eicosanoids and optimal production of cytokines in mast cells in response to antigen and other stimulants. The signal is initiated by phospholipase C-mediated production of inositol1,4,5-trisphosphate resulting in release of stored Ca(2+) from the endoplasmic reticulum (ER) and Golgi. Depletion of these stores activates influx of extracellular Ca(2+), usually referred to as store-operated calcium entry (SOCE), through the interaction of the Ca(2+)-sensor, stromal interacting molecule-1 (STIM1 ), in ER with Orai1(CRACM1) and transient receptor potential canonical (TRPC) channel proteins in the plasma membrane (PM). This interaction is enabled by microtubular-directed reorganization of ER to form ER/PM contact points or "punctae" in which STIM1 and channel proteins colocalize. The ensuing influx of Ca(2+) replenishes Ca(2+) stores and sustains elevated levels of cytosolic Ca(2+) ions-the obligatory signal for mast-cell activation. In addition, the signal can acquire spatial and dynamic characteristics (e.g., calcium puffs, waves, oscillations) that encode signals for specific functional outputs. This is achieved by coordinated regulation of Ca(2+) fluxes through ATP-dependent Ca(2+)-pumps and ion exchangers in mitochondria, ER and PM. As discussed in this chapter, studies in mast cells revealed much about the mechanisms described above but little about allergic and autoimmune diseases although studies in other types of cells have exposed genetic defects that lead to aberrant calcium signaling in immune diseases. Pharmacologic agents that inhibit or activate the regulatory components of calcium signaling in mast cells are also discussed along with the prospects for development of novel SOCE inhibitors that may prove beneficial in the treatment inflammatory mast-cell related diseases.

Regulation of Ca2+ signaling with particular focus on mast cells.

Calcium signals mediate diverse cellular functions in immunological cells. Early studies with mast cells, then a preeminent model for studying Ca2+-dependent exocytosis, revealed several basic features of calcium signaling in non-electrically excitable cells. Subsequent studies in these and other cells further defined the basic processes such as inositol 1,4,5-trisphosphate-mediated release of Ca2+ from Ca2+ stores in the endoplasmic reticulum (ER); coupling of ER store depletion to influx of external Ca2+ through a calcium-release activated calcium (CRAC) channel now attributed to the interaction of the ER Ca2+ sensor, stromal interacting molecule-1 (STIM1), with a unique Ca2+-channel protein, Orai1/CRACM1, and subsequent uptake of excess Ca2+ into ER and mitochondria through ATP-dependent Ca2+ pumps. In addition, transient receptor potential channels and ion exchangers also contribute to the generation of calcium signals that may be global or have dynamic (e.g., waves and oscillations) and spatial resolution for specific functional readouts. This review discusses past and recent developments in this field of research, the pharmacologic agents that have assisted in these endeavors, and the mast cell as an exemplar for sorting out how calcium signals may regulate multiple outputs in a single cell.

Synergistic activation of phospholipases Cgamma and Cbeta: a novel mechanism for PI3K-independent enhancement of FcepsilonRI-induced mast cell mediator release.

Antigen/IgE-mediated mast cell activation via FcvarepsilonRI can be markedly enhanced by the activation of other receptors expressed on mast cells and these receptors may thus contribute to the allergic response in vivo. One such receptor family is the G protein-coupled receptors (GPCRs). Although the signaling cascade linking FcvarepsilonRI aggregation to mast cell activation has been extensively investigated, the mechanisms by which GPCRs amplify this response are relatively unknown. To investigate this, we utilized prostaglandin (PG)E2 based on initial studies demonstrating its greater ability to augment antigen-mediated degranulation in mouse mast cells than other GPCR agonists examined. This enhancement, and the ability of PGE2 to amplify antigen-induced calcium mobilization, was independent of phosphoinositide 3-kinase but was linked to a pertussis toxin-sensitive synergistic translocation to the membrane of phospholipase (PL)Cgamma and PLCbeta and to an enhancement of PLCgamma phosphorylation. This "trans-synergistic" activation of PLCbeta and gamma, in turn, enhanced production of inositol 1,4,5-trisphosphate, store-operated calcium entry, and activation of protein kinase C (PKC) (alpha and beta). These responses were critical for the promotion of degranulation. This is the first report of synergistic activation between PLCgamma and PLCbeta that permits reinforcement of signals for degranulation in mast cells.

The effects of physical activity on executive function in children with attention-deficit/hyperactivity disorder: A systematic review and meta-analysis protocol.

BACKGROUND: The effects of physical activity on executive function are well documented, but whether physical activity contributes to the executive function of attention deficit hyperactivity disorder (ADHD) children are still inconclusive. METHODS: The study is guided by the Preferred Reporting Items for Systematic Review and Meta-analysis Protocols (PRISMA-P). We will search the following databases PubMed, EMBASES, the Cochrane Library, CNKI, and Wanfang-Data to identify the Randomized Controlled Trials evaluating the effects of physical activity on executive function among ADHD children. The language of literature restricted in Chinese and English, which published from inception to January 2019. Two reviewers will screen the studies independently, while risk of bias assessment, data extraction, and inconsistent results will be discussed by the third reviewer. Revman 5.3 and Stata 12 software will be used to complete data analysis and synthesis. CONCLUSION: This study will be based on findings of previous studies, thus the ethics approval is not required. The final results will be presented at an international conference and submitted to a peer-reviewed journal of relative field for consideration of publication. PROSPERO REGISTRATION NUMBER: CRD42019118622.

[Investigation on inhibition of HIV III B virus with extractions of Juglans regia].

OBJECTIVE: To study the effect of anti-HIV III B virus with extractions from Juglans regia, so as to searching for the new and efficacious leading compound of AIDS therapy. METHOD: Phytochemical and chromatographical techniques were used to isolate compounds from J. regia; MT4 cells and HIV III B virus were used to study the effect of anti-HIV activity in vitro. BIACORE 3000 molecule coupled equipment were used for the target research also. RESULT: Two extractions (B&E) were isolated from J. regia which possess the effect of anti-HIV activity. Targets study found that extraction B could affected on HIV-1 gp-41 fusing protein and extraction E could affected on HIV-1 integrase respectively. CONCLUSION: J. regia is a traditional Chinese medicine with active anti-HIV activity and worth to be studied further.

[Screening of a sub-clone of human breast cancer cells with high metastasis potential].

OBJECTIVE: To screen a sub-clone of human breast cancer cell of the MCF-7 line with high metastasis potential. METHODS: Human breast cancer cells of the MCF-7 line were injected subcutaneously into 10 severe combined immunodeficiency (SCID) mice. Sixty-eight days after the mice were killed and their lungs were taken out. Primary cell culture was conducted. When the cells were passed on to the third generation a sub-clone was screened from the lung tissue and termed LM-MCF-7. Microscopy was performed on the lung tissues. The growth curve was drawn. Flow cytometry was used to examine the cell cycle. Chromosome analysis was done. Immunohistochemistry was used to detect the expression of breast cancer specific antigen CAI5-3. Western blotting was used to detect the protein expression of the protein associated with tumor metastasis: nm23 (a metastasis-suppressing gene), myosin light chain kinase (MLCK, a kinase related to cell movement), survivin, bcl-2 and p27 (a gene related to cell cycle). LM-MCF-7 cells were injected into other SCID mice and these mice were killed 30 days later to observe the metastasis of cancer so as to detect the tumorigenic ability of the LM-MCF-7 cells. RESULTS: When the cells from the mouse lung tissues were passed on to the third generation a sub-clone with high metastasis potential was screened and termed LM-MCF-7. The morphology of the new cell line was typically epithelioid. Flow cytometry showed that the DNA relatively contents were 53.40% of the LM-MCF-7 cells were in the G(0)/G(1) phase, a lower percentage than that of the MCF-7 cells, and 17.10% in the S phase and 23.20% in the G(2+)M phase, both percentages higher than those of the MCF-7 cells. The proliferating time of the LM-MCF-7 cell population was about 20 +/- 14 hours, much shorter than that of the parent strain cells. The chromosomes of the LM-MCF-7 cells, numbering 16-123, showed the morphology characteristic c of human chromosomes. The marker of human breast cancer CA15-3 was detected in both MCF-7 and LM-MCF-7 cells. The protein expression of nm23 and p27 was down-regulated, but the protein expression of MLCK, bcl-2 and survivin was up-regulated in LM-MCF-7 cells in comparison with those in MCF-7 cells. The tumorigenesis rate of LM-MCF-7 cells was 100% (5/5), with a latent period of 5.0 +/- 0.0 d, and the tumor metastasized to lung, kidney, spleen, bone marrow, lymph node and heart. CONCLUSION: A human breast cancer line, LM-MCF-7 cell line, with high metastasis potential has been derived from the human breast cancer cells of MCF-7 line.

Modification of phospholipase C-gamma-induced Ca2+ signal generation by 2-aminoethoxydiphenyl borate.

The mechanisms by which Ca(2+)-store-release channels and Ca(2+)-entry channels are coupled to receptor activation are poorly understood. Modification of Ca(2+) signals by 2-aminoethoxydiphenyl borate (2-APB), suggests the agent may target entry channels or the machinery controlling their activation. In DT40 B-cells and Jurkat T-cells, complete Ca(2+) store release was induced by 2-APB (EC(50) 10-20 microM). At 75 microM, 2-APB emptied stores completely in both lymphocyte lines, but had no such effect on other cells. In DT40 cells, 2-APB mimicked B-cell receptor (BCR) cross-linking, but no effect was observed in mutant DT40 lines devoid of inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) or phospholipase C-gamma2 (PLC-gamma2). Like the BCR, 2-APB activated transfected TRPC3 (canonical transient receptor potential) channels, which acted as sensors for PLC-gamma2-generated diacylglycerol in DT40 cells. The action of 2-APB on InsP(3)Rs and TRPC3 channels was prevented by PLC-inhibition, and required PLC-gamma2 catalytic activity. However, unlike BCR activation, no increased InsP(3) level could be measured in response to 2-APB. Also, calyculin A-induced cytoskeletal reorganization prevented 2-APB-induced InsP(3)R and TRPC3-channel activation, but not that induced by the BCR. 2-APB still activated TRPC3 channels in DT40 cells with fully depleted Ca(2+) stores, indicating its action was not via Ca(2+) release. Significantly, 2-APB-induced InsP(3)R and TRPC3 activation was prevented in DT40 knockout cells devoid of the BCR- and PLC-gamma2-coupled adaptor/kinases, Syk, Lyn, Btk or BLNK. The results suggest that 2-APB activates Ca(2+) signals in lymphocytes by initiating and enhancing coupling between components of the BCR-PLC-gamma2 complex and both Ca(2+)-entry and Ca(2+)-release channels.

Canonical transient receptor potential 5 channel in conjunction with Orai1 and STIM1 allows Sr2+ entry, optimal influx of Ca2+, and degranulation in a rat mast cell line.

Degranulation of mast cells in response to Ag or the calcium mobilizing agent, thapsigargin, is dependent on emptying of intracellular stores of Ca(2+) and the ensuing influx of external Ca(2+), also referred to as store-operated calcium entry. However, it is unlikely that the calcium release-activated calcium channel is the sole mechanism for the entry of Ca(2+) because Sr(2+) and other divalent cations also permeate and support degranulation in stimulated mast cells. In this study we show that influx of Ca(2+) and Sr(2+) as well as degranulation are dependent on the presence of the canonical transient receptor potential (TRPC) channel protein TRPC5, in addition to STIM1 and Orai1, as demonstrated by knock down of each of these proteins by inhibitory RNAs in a rat mast cell (RBL-2H3) line. Overexpression of STIM1 and Orai1, which are known to be essential components of calcium release-activated calcium channel, allows entry of Ca(2+) but not Sr(2+), whereas overexpression of STIM1 and TRPC5 allows entry of both Ca(2+) and Sr(2+). These and other observations suggest that the Sr(2+)-permeable TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca(2+) to generate a signal for degranulation.

[Immune response induced by vaccination with pseudotyped rAAV1 expressing HPV16 L1 protein].

To investigate the feasibility of using recombinant adeno-associated virus type 1 vector as prophylactic vaccine against HPV16 infection, rAAV1-mod. HPV16L1, the recombinant AAV1 vector containing codon-modified HPV16 L1 gene, was constructed. C57BL/6 mice were immunized with purified rAAV1 vector through intramuscular and intranasal inoculation routes, and the titer of neutralizing antibody was determined by neutralization assay based on HPV16 pseudovirus. The result shows that the single dose of rAAV1-mod. HPV16L1 can induce specific neutralizing antibody in serum through both inoculation routes. Compared with intranasal group, intramuscular group can induce higher titer of neutralizing antibody. Eliciting strong and prolonged neutralizing antibody in serum, the rAAV1-mod. HPV16L1 is one of promising HPV16 prophylactic vaccine candidates.

[Immune response induced by recombinant adenovirus combined with recombinant adeno-associated virus type 1 containing HPV16 L1 gene].

OBJECTIVE: To evaluate the immune potency of recombinant adenovirus combined with rAAV1 vector expressing HPV16L1 protein in mice. METHODS: The rAdV and rAAV1 vector containing codon-modified HPV16L1 gene was constructed using Admax and AAVmax packaging system respectively. C57 BL/6 mice were immunized with purified rAdV and rAAV1 vector through intramuscular and intranasal inoculation routes, and the titer of neutralizing antibody was determined by neutralization assay based HPV16 pseudovirus. RESULTS: Intramuscular immunization by rAAV1-mod. HPV16L1 or combined with rAd-mod. HPV16L1 can induce higher titer of neutralizing antibody in serum than that of other groups. The titer of neutralizing antibody of intranasal groups is significantly lower than that of intramuscular group, although the prime-boost strategy using in intranasal group was effective to enhance the specific humoral immunity. CONCLUSION: The rAAV1-mod. HPV16L1 combined with rAd-mod. HPV16L1 can induce higher titer of neutralizing antibody in serum through intramuscular route than that of other groups at the 16th week after the first immunization.

Protein kinase C beta and delta isoenzymes mediate cholesterol accumulation in PMA-activated macrophages.

Previously, we showed that PMA activation of human monocyte-derived macrophages stimulates macropinocytosis (i.e., fluid-phase endocytosis) of LDL and transforms these macrophages into foam cells. The current study aimed to learn which PKC isoenzymes mediate cholesterol accumulation in PMA-activated human macrophages incubated with LDL. Cholesterol accumulation by PMA-activated macrophages incubated with LDL was nearly completely inhibited (>85%) by the pan PKC inhibitors Go6850, Go6983, and RO 32-0432, but only was inhibited about 50% by the classical group PKC inhibitor, Go6976. This indicated that cholesterol accumulation was mediated by both a classical group and some other PKC isoenzyme. PKC beta was determined to be the classical group isoenzyme that mediated PMA-stimulated cholesterol accumulation. A pseudosubstrate myristoylated peptide inhibitor of PKC alpha and beta showed partial inhibition (congruent with 50%) of cholesterol accumulation. However, a small molecule inhibitor of PKC alpha, HBDDE, show minimal inhibition of cholesterol accumulation while a small molecule inhibitor of PKC beta, LY333513, could completely account for the inhibition of cholesterol accumulation by the classical group PKC isoenzyme. Thus, our findings show that beta and some other PKC isoenzyme, most likely delta, mediate cholesterol accumulation when macropinocytosis of LDL is stimulated in PMA-activated human monocyte-derived macrophages.

[Evaluation of mastication function after three-dimensional maxillary reconstruction].

OBJECTIVE: To evaluate the mastication function of the patient whose maxilla was reconstructed with individual titanium mesh and Chinese flap or combined with fibular flap. The superiority of this method on maxillary functional reconstruction was testified. METHODS: Since March of 2001, 10 cases of maxillary defect were reconstructed with individual titanium mesh and Chinese flap or combined with fibular flap, and routine removal partial dentures were fixed after 3 - 6 months postoperatively. Assessment of occlusal force was proceeded by T-Scan II system (Tekscan company, USA). RESULTS: The occlusal force analysis results indicated the asymmetry index of bite force and asymmetry index of occlusal contact area differed significantly between preoperation and postopertion (P < 0.05). The recovery rate of total occlusal force was between 27.05%-74.06%, and the average was 50.15%. CONCLUSION: This new three-dimensional maxillary functional reconstruction method had a satisfactory recovery in both of contour and function recently, especially restored the mastication function effectively.

Initiation of spontaneous epileptiform events in the rat neocortex in vivo.

We used voltage-sensitive dye imaging to visualize the distribution of initiation sites of the spontaneous interictal-like spikes (sISs) in rat neocortex, in vivo, induced by bicuculline or picrotoxin over the exposed cortex. The initiation site was small (approximately 200 microm diam). On average each initiation site initiated 2.0 +/- 0.8 sISs (9 animals, 499 sISs, 251 sites). This is significantly different from that in neocortical slices, where each initiation site initiated 30-100 sISs. The initiation sites were not randomly distributed. The distance between two consecutive sites tended to be either <800 or >1200 microm, suggesting a temporal "suppression annulus" surrounding each initiation site. Within the annulus, the likelihood for initiating the next sIS was reduced. Suppression annulus did not have a noticeable change in the presence of GABA(b) antagonist, suggesting it did not depend on the GABA(b) inhibition. We also applied bicuculline locally to a spot of 800 x 800 microm(2) for approximately 45 min. During this period approximately 1000 sISs occurred within the spot. Bicuculline or picrotoxin was then applied to the entire craniotomy window. The pretreatment created an obvious cluster of initiation sites. Around this cluster, the suppression annulus became obvious in individual animals. Our results suggest that, in disinhibited cortex, epileptiform events were initiated from small sites. The initiation sites may cluster in an area with increased local activity. Surrounding each initiation site there may be a temporal suppression annulus.

Modification of store-operated channel coupling and inositol trisphosphate receptor function by 2-aminoethoxydiphenyl borate in DT40 lymphocytes.

Store-operated channels (SOCs) provide an important means for mediating longer-term Ca(2+) signals and replenishment of Ca(2+) stores in a multitude of cell types. However, the coupling mechanism between endoplasmic reticulum stores to activate plasma membrane SOCs remains unknown. In DT40 chicken B lymphocytes, the permeant inositol trisphosphate receptor (InsP(3)R) modifier, 2-aminoethoxydiphenyl borate (2-APB), was a powerful activator of store-operated Ca(2+) entry between 1-10 microm. 2-APB activated authentic SOCs because the entry was totally selective for Ca(2+) (no detectable entry of Ba(2+) or Sr(2+) ions), and highly sensitive to La(3+) ions (IC(50) 30-100 nm). To assess the role of InsP(3)Rs in this response, we used the DT40 triple InsP(3)R-knockout (ko) cell line, DT40InsP(3)R-ko, in which the absence of full-length InsP(3)Rs or InsP(3)R fragments was verified by Western analysis using antibodies cross-reacting with N-terminal epitopes of all three chicken InsP(3)R subtypes. The 2-APB-induced activation of SOCs was identical in the DT40InsP(3)R-ko, cells indicating InsP(3)Rs were not involved. With both wild type (wt) and ko DT40 cells, 2-APB had no effect on Ca(2+) entry in store-replete cells, indicating that its action was restricted to SOCs in a store-coupled state. 2-APB induced a robust activation of Ca(2+) release from stores in intact DT40wt cells but not in DT40InsP(3)R-ko cells, indicating an InsP(3)R-mediated effect. In contrast, 2-APB blocked InsP(3)Rs in permeabilized DT40wt cells, suggesting that the stimulatory action of 2-APB was restricted to functionally coupled InsP(3)Rs in intact cells. Uncoupling of ER/PM interactions in intact cells by calyculin A-induced cytoskeletal rearrangement prevented SOC activation by store-emptying and 2-APB; this treatment completely prevented 2-APB-induced InsP(3)R activation but did not alter InsP(3)R activation mediated by phospholipase C-coupled receptor stimulation. The results indicate that the robust bifunctional actions of 2-APB on both SOCs and InsP(3)Rs are dependent on the coupled state of these channels and suggest that 2-APB may target the coupling machinery involved in mediating store-operated Ca(2+) entry.