Copper-Ligand Interactions and Physiological free Radical Processes. Part 2. Influence of Cu2+ Ions on Cu+-Driven Oh Generation and Comparison with Their Effects on Fe2+-Driven Oh Production

In our search to establish a reference ·OH production system with respect to which the reactivity of copper(II) complexes could then be tested, the influence of free Cu²⁺ ions on the Cu⁺/H₂O₂ reaction has been investigated.

This influence depends on the C_Cu²⁺/C_Cu⁺ ratio. At low Cu²⁺ concentrations, ·OH damage to various detector molecules decreases with increasing Cu²⁺ concentrations until C_Cu²⁺/C_Cu⁺ reaches unity. Above this value, ·OH damage increases sharply until C_Cu²⁺/C_Cu⁺ becomes equal to 5 with salicylate and 2 with deoxyribose, ratios for which the protective effect of Cu²⁺ cancels. Finally, at higher concentrations, Cu²⁺ ions logically add their own ·OH production to that normally expected from Cu⁺ ions. The possible origin of this unprecedented alternate effect has been discussed. The possible influence of Cu⁺ ions on the generation of ·OH radicals by water gamma radiolysis has also been tested and, as already established for Cu²⁺ in a previous work, shown to be nonexistent. This definitely confirms that either form of ionised copper cannot scavenge ·OH radicals in the absence of a Iigand.     

Slo1 tail domains,but not the Ca2+ bowl,are required for the beta 1 subunit to increase the apparent Ca2+ sensitivity of BK channels

Functional large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels can be assembled from four alpha subunits (Slo1) alone, or together with four auxiliary beta1 subunits to greatly increase the apparent Ca(2+) sensitivity of the channel. We examined the structural features involved in this modulation with two types of experiments. In the first, the tail domain of the alpha subunit, which includes the RCK2 (regulator of K(+) conductance) domain and Ca(2+) bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a Ca(2+) bowl and high affinity Ca(2+) sensitivity. In the second, the Ca(2+) bowl was disrupted by mutations that greatly reduce the apparent Ca(2+) sensitivity. We found that the beta1 subunit increased the apparent Ca(2+) sensitivity of Slo1 channels, independently of whether the alpha subunits were expressed as separate cores (S0-S8) and tails (S9-S10) or full length, and this increase was still observed after the Ca(2+) bowl was mutated. In contrast, beta1 subunits no longer increased Ca(2+) sensitivity when Slo1 tails were replaced by Slo3 tails. The beta1 subunits were still functionally coupled to channels with Slo3 tails, as DHS-I and 17 beta-estradiol activated these channels in the presence of beta1 subunits, but not in their absence. These findings indicate that the increase in apparent Ca(2+) sensitivity induced by the beta1 subunit does not require either the Ca(2+) bowl or the linker between the RCK1 and RCK2 domains, and that Slo3 tails cannot substitute for Slo1 tails. The beta1 subunit also induced a decrease in voltage sensitivity that occurred with either Slo1 or Slo3 tails. In contrast, the beta1 subunit-induced increase in apparent Ca(2+) sensitivity required Slo1 tails. This suggests that the allosteric activation pathways for these two types of actions of the beta1 subunit may be different.     

microRNA-143 is associated with the survival of ALDH1+CD133+ osteosarcoma cells and the chemoresistance of osteosarcoma

This study investigated the role of miR-143 in the chemoresistance of osteosarcoma tumor cells and the associated mechanisms. Real-time PCR was used to measure miR-143 levels. Western blot was used to detect protein expression. Cell proliferation was analyzed by MTT assay and Matrigel colony formation assay. Forced miR-143 expression was established by adenoviral vector infection. Cell death was detected by Hoechst33342 staining. Loss of miR-143 expression was observed in osteosarcomas, which correlated with shorter survival of patients with osteosarcomas underlying chemotherapy. In chemoresistant SAOS-2 and U2OS osteosarcomas cells, miR-143 levels were significantly downregulated and accompanied by increases in ATG2B, Bcl-2, and/or LC3-II protein levels, high rate of ALDH1⁺CD133⁺ cells, and an increase in Matrigel colony formation ability. H₂O₂ upregulated p53 and miR-143, but downregulated ATG2B, Bcl-2, and LC3-I expression in U2OS cells (wild-type p53) but not in SAOS-2 (p53-null) cells. Forced miR-143 expression significantly reversed chemoresistance as well as downregulation of ATG2B, LC3-I, and Bcl-2 expression in SAOS-2- and U2OS-resistant cells. Forced miR-143 expression significantly inhibited tumor growth in xenograft SAOS-2-Dox and U2OS-Dox animal models. Loss of miR-143 expression is associated with poor prognosis of patients with osteosarcoma underlying chemotherapy. The chemoresistance of osteosarcoma tumor cells to doxorubicin is associated with the downregulation of miR-143 expression, activation of ALDH1⁺CD133⁺ cells, activation of autophagy, and inhibition of cell death. miR-143 may play a crucial role in the chemoresistance of osterosarcoma tumors.     

Interdomain interactions within ryanodine receptors regulate Ca2+ spark frequency in skeletal muscle.

DP4 is a 36-residue synthetic peptide that corresponds to the Leu(2442)-Pro(2477) region of RyR1 that contains the reported malignant hyperthermia (MH) mutation site. It has been proposed that DP4 disrupts the normal interdomain interactions that stabilize the closed state of the Ca(2)+ release channel (Yamamoto, T., R. El-Hayek, and N. Ikemoto. 2000. J. Biol. Chem. 275:11618-11625). We have investigated the effects of DP4 on local SR Ca(2)+ release events (Ca(2)+ sparks) in saponin-permeabilized frog skeletal muscle fibers using laser scanning confocal microscopy (line-scan mode, 2 ms/line), as well as the effects of DP4 on frog SR vesicles and frog single RyR Ca(2)+ release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in Ca(2)+ spark frequency in muscle fibers. However, the mean values of the amplitude, rise time, spatial half width, and temporal half duration of the Ca(2)+ sparks, as well as the distribution of these parameters, remained essentially unchanged in the presence of DP4. Thus, DP4 increased the opening rate, but not the open time of the RyR Ca(2)+ release channel(s) generating the sparks. DP4 also increased [(3)H]ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR Ca(2)+ release channels reconstituted in bilayers, without changing the amplitude of the current through those channels. However, unlike in Ca(2)+ spark experiments, DP4 produced a pronounced increase in the open time of channels in bilayers. The same peptide with an Arg(17) to Cys(17) replacement (DP4mut), which corresponds to the Arg(2458)-to-Cys(2458) mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. Mg(2)+ dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially Mg(2)+-free RyR(s), thus promoting channel opening and production of Ca(2)+ sparks.     

Substantial increase in the frequency of circulating CD4+NKG2D+ T cells in patients with cervical intraepithelial neoplasia grade 1

Background

The NKG2D receptor confers important activating signals to NK cells via ligands expressed during cellular stress and viral infection. This receptor has generated great interest because not only is it expressed on NK cells, but it is also seen in virtually all CD8⁺ cytotoxic T cells and is classically considered absent in CD4⁺ T cells. However, recent studies have identified a distinctive population of CD4⁺ T cells that do express NKG2D, which could represent a particular cytotoxic effector population involved in viral infections and chronic diseases. On the other hand, increased incidence of human papillomavirus-associated lesions in CD4⁺ T cell-immunocompromised individuals suggests that CD4⁺ T cells play a key role in controlling the viral infection. Therefore, this study was focused on identifying the frequency of NKG2D-expressing CD4⁺ T cells in patients with cervical intraepithelial neoplasia (CIN) 1. Additionally, factors influencing CD4⁺NKG2D⁺ T cell expansion were also measured.

Results

Close to 50% of patients with CIN 1 contained at least one of the 37 HPV types detected by our genotyping system. A tendency for increased CD4⁺ T cells and CD8⁺ T cells and decreased NK cells was found in CIN 1 patients. The percentage of circulating CD4⁺ T cells co-expressing the NKG2D receptor significantly increased in women with CIN 1 versus control group. Interestingly, the increase of CD4⁺NKG2D⁺ T cells was seen in patients with CIN 1, despite the overall levels of CD4⁺ T cells did not significantly increase. We also found a significant increase of soluble MICB in CIN 1 patients; however, no correlation with the presence of CD4⁺NKG2D⁺ T cells was seen. While TGF-beta was significantly decreased in the group of CIN 1 patients, both TNF-alpha and IL-15 showed a tendency to increase in this group.

Conclusions

Taken together, our results suggest that the significant increase within the CD4⁺NKG2D⁺ T cell population in CIN 1 patients might be the result of a chronic exposure to viral and/or pro-inflammatory factors, and concomitantly might also influence the clearance of CIN 1-type lesion.     

亚硒酸钠对巨噬细胞内游离Ca2+和Mg2+的影响

用单细胞阳离子测定系统研究了SeO^2-₃对巨噬细胞内游离Ca²⁺和Mg²⁺的影响.实验结果表明:SeO^2-₃高于10^-4mol/L时,有显著的细胞毒性.SeO^2-₃对细胞的毒性作用使细胞内游离Ca²⁺和Mg²⁺的浓度升高但Ca²⁺浓度的升高速率比Mg²⁺快.还有,高于10^-4mol/L的SeO^2-₃对红细胞膜上的Ca²⁺-ATP酶活性有明显抑制作用.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

敲低EDAG加强NPM1蛋白的降解并增加AML病人对药物的敏感性

Nucleophosmin (NPM1或B23.1)是在细胞核内广泛表达的蛋白磷酸酶,在多方面发挥重要作用,如核糖体合成、中心体复制、细胞周期控制、细胞增殖及转化．NPM1是急性粒细胞白血病(acute myeloid leukemia, AML)中最常见的突变基因之一．红系分化相关基因(erythroid differentiation associated gene, EDAG)是在造血组织特异表达的基因,在造血细胞的增殖与谱系分化调节方面发挥重要作用．在AML病人中,高表达的EDAG与较差的预后相关联．我们前期研究结果显示,EDAG与NPM1相结合并调节NPM1稳定性,但在AML病人体内EDAG与NPM1的关系,及EDAG与NPM突变体(NPMc⁺)的关系尚未明确．在本文中发现：在AML病人骨髓CD34⁺细胞中,敲低EDAG表达导致NPM1蛋白稳定性降低并提高了对柔红霉素的敏感性;EDAG虽不与突变体NPMc⁺相互作用,但在蛋白出核抑制剂(leptomycin B, LMB)作用下,过表达EDAG提高NPMc⁺蛋白稳定性;表达突变NPMc⁺的AML病人与表达NPM1蛋白的病人相比,其骨髓CD34⁺细胞对柔红霉素具有更高的敏感性,且敲低EDAG能微弱提高其敏感性．上述结果表明,EDAG在AML病人药物治疗中发挥的可能作用以及NPMc⁺ “逃脱”,使EDAG无法保护其稳定性,这提示了在AML病人药物治疗过程中EDAG的潜在作用,同时也提示,携带NPMc⁺蛋白的AML患者具有较好预后,可能与NPMc⁺蛋白“逃脱”出EDAG对其稳定性的保护有关．     

Roles of transmembrane segment M1 of Na+,K+-ATPase and Ca2+-ATPase, the gatekeeper and the pivot

In this review we summarize mutagenesis work on the structure–function relationship of transmembrane segment M1 in the Na⁺,K⁺-ATPase and the sarco(endo)plasmic reticulum Ca²⁺-ATPase. The original hypothesis that charged residues in the N-terminal part of M1 interact with the transported cations can be rejected. On the other hand hydrophobic residues in the middle part of M1 turned out to play crucial roles in Ca²⁺ interaction/occlusion in Ca²⁺-ATPase and K⁺ interaction/occlusion in Na⁺,K⁺-ATPase. Leu⁶⁵ of the Ca²⁺-ATPase and Leu⁹⁹ of the Na⁺,K⁺-ATPase, located at homologous positions in M1, function as gate-locking residues that restrict the mobility of the side chain of the cation binding/gating residue of transmembrane segment M4, Glu³⁰⁹/Glu³²⁹. A pivot formed between a pair of a glycine and a bulky residue in M1 and M3 seems critical to the opening of the extracytoplasmic gate in both the Ca²⁺-ATPase and the Na⁺,K⁺-ATPase. All numbering of Na⁺,K⁺-ATPase amino acid residues in this article refers to the sequence of the rat α₁-isoform.     

Ca2+ regulation of ion transport in the na+/ca2+ exchanger

The binding of Ca(2+) to two adjacent Ca(2+)-binding domains, CBD1 and CBD2, regulates ion transport in the Na(+)/Ca(2+) exchanger. As sensors for intracellular Ca(2+), the CBDs form electrostatic switches that induce the conformational changes required to initiate and sustain Na(+)/Ca(2+) exchange. Depending on the presence of a few key residues in the Ca(2+)-binding sites, zero to four Ca(2+) ions can bind with affinities between 0.1 to 20 μm. Importantly, variability in CBD2 as a consequence of alternative splicing modulates not only the number and affinities of the Ca(2+)-binding sites in CBD2 but also the Ca(2+) affinities in CBD1.     

Early effects of decapitation on the Mg2+-K+ ATPase and cation contents in lateral buds of the aquatic fern,Marsilea drummondii A. Br.

Summary A detailed comparison of Mg²⁺-K⁺ ATPase activity and cation fluxes in terminal buds, inhibited buds or buds released from apical dominance was carried out. Light microscope observations indicate intense reaction at the plasmalemma of stelar cells, pericyclic, phloem and xylem transfer cells at nodes along the main rhizome. In intact plants, with the exception of pericyclic cells, bud branches show no ATPase activity. Excision of the terminal bud results in rapid (within 5–15 minutes) stimulation of ATPase activity at nodes and all bud axes. As the subapical bud gains precedence, ATPase stimulation ceases and returns to its initial level in the older median and basal buds. Enzyme activation is kinetically correlated with K⁺ flux. X-ray microanalysis confirms that K⁺ accumulates in the stele at the node and the bud branch with the same lag period. This data increases the evidence for close association between ATP (Sossountzov et al. 1982), ATPase activity and K⁺ flux. The kinetics strengthen the impression that these factors may be involved very early in bud outgrowth regulation.     

Differential response of BDCA-1+ and BDCA-3+ myeloid dendritic cells to respiratory syncytial virus infection

Background

Respiratory syncytial virus (RSV) is the leading cause of respiratory infections in children, elderly, and immunocompromised individuals. Severe infection is associated with short- and long-term morbidity including pneumonia, recurrent wheezing, and abnormal pulmonary function, and several lines of evidence indicate that impaired adaptive immune responses during infection are critical in the pathophysiology of RSV-mediated disease. Myeloid Dendritic cells (mDCs) play a pivotal role in shaping antiviral immune responses in the respiratory tract; however, few studies have examined the interactions between RSV and individual mDC subsets. In this study, we examined the effect of RSV on the functional response of primary mDC subsets (BDCA-1⁺ and BDCA-3⁺) isolated from peripheral blood.

Methods

BDCA-1⁺ and BDCA-3⁺ mDCs were isolated from the peripheral blood of healthy adults using FACS sorting. Donor-matched BDCA-1⁺ and BDCA-3⁺ mDCs were infected with RSV at a multiplicity of infection (MOI) of 5 for 40 hours. After infection, cells were analyzed for the expression of costimulatory molecules (CD86, CD80, and PD-L1), cytokine production, and the ability to stimulate allogenic CD4⁺ T cell proliferation.

Results

Both BDCA-1⁺ and BDCA-3⁺ mDCs were susceptible to infection with RSV and demonstrated enhanced expression of CD86, and the inhibitory costimulatory molecules CD80 and PD-L1. Compared to BDCA-3⁺ mDCs, RSV-infected BDCA-1⁺ mDC produced a profile of cytokines and chemokines predominantly associated with pro-inflammatory responses (IL-1β, IL-6, IL-12, MIP-1α, and TNF-α), and both BDCA-1⁺ and BDCA-3⁺ mDCs were found to produce IL-10. Compared to uninfected mDCs, RSV-infected BDCA-1⁺ and BDCA-3⁺ mDCs demonstrated a reduced capacity to stimulate T cell proliferation.

Conclusions

RSV infection induces a distinct pattern of costimulatory molecule expression and cytokine production by BDCA-1⁺ and BDCA-3⁺ mDCs, and impairs their ability to stimulate T cell proliferation.The differential expression of CD86 and pro-inflammatory cytokines by highly purified mDC subsets in response to RSV provides further evidence that BDCA-1⁺ and BDCA-3⁺ mDCs have distinct roles in coordinating the host immune response during RSV infection. Findings of differential expression of PD-L1 and IL-10 by infected mDCs, suggests possible mechanisms by which RSV is able to impair adaptive immune responses.     

Dikkopf-1 promotes matrix mineralization of osteoblasts by regulating Ca+-CAMK2A-CREB1 pathway

Dickkopf-1 (DKK1) is a secreted protein that acts as an antagonist of the canonical WNT/β-catenin pathway, which regulates osteoblast differentiation. However, the role of DKK1 on osteoblast differentiation has not yet been fully clarified. Here, we investigate the functional role of DKK1 on osteoblast differentiation. Primary osteoprogenitor cells were isolated from human spinal bone tissues. To examine the role of DKK1 in osteoblast differentiation, we manipulated the expression of DKK1, and the cells were differentiated into mature osteoblasts. DKK1 overexpression in osteoprogenitor cells promoted matrix mineralization of osteoblast differentiation but did not promote matrix maturation. DKK1 increased Ca⁺ influx and activation of the Ca⁺/calmodulin-dependent protein kinase II Alpha (CAMK2A)-cAMP response element-binding protein 1 (CREB1) and increased translocation of p-CREB1 into the nucleus. In contrast, stable DKK1 knockdown in human osteosarcoma cell line SaOS2 exhibited reduced nuclear translocation of p-CREB1 and matrix mineralization. Overall, we suggest that manipulating DKK1 regulates the matrix mineralization of osteoblasts by Ca⁺-CAMK2A-CREB1, and DKK1 is a crucial gene for bone mineralization of osteoblasts.     

Analysis of the Ca2+ domain in the Arabidopsis H+/Ca2+ antiporters CAX1 and CAX3

Ca²⁺ levels in plants are controlled in part by H⁺/Ca²⁺ exchangers. Structure/function analysis of the Arabidopsis H⁺/cation exchanger, CAX1, revealed that a nine amino acid region (87–95) is involved in CAX1-mediated Ca²⁺ specificity. CAX3 is 77% identical (93% similar) to CAX1, and when expressed in yeast, localizes to the vacuole but does not suppress yeast mutants defective in vacuolar Ca²⁺ transport. Transgenic tobacco plants expressing CAX3 containing the 9 amino acid Ca²⁺ domain (Cad) from CAX1 (CAX3-9) displayed altered stress sensitivities similar to CAX1-expressing plants, whereas CAX3-9-expressing plants did not have any altered stress sensitivities. A single leucine-to-isoleucine change at position 87 (CAX3-I) within the Cad of CAX3 allows this protein to weakly transport Ca²⁺ in yeast (less than 10% of CAX1). Site-directed mutagenesis of the leucine in the CAX3 Cad demonstrated that no amino acid change tested could confer more activity than CAX3-I. Transport studies in yeast demonstrated that the first three amino acids of the CAX1 Cad could confer twice the Ca²⁺ transport capability compared to CAX3-I. The entire Cad of CAX3 (87–95) inserted into CAX1 abolishes CAX1-mediated Ca²⁺ transport. However, single, double, or triple amino acid replacements within the native CAX1 Cad did not block CAX1 mediated Ca²⁺ transport. Together these findings suggest that other domains within CAX1 and CAX3 influence Ca²⁺ transport. This study has implications for the ability to engineer CAX-mediated transport in plants by manipulating Cad residues.