Revealing the three dimensional architecture of focal adhesion components to explain Ca2 +-mediated turnover of focal adhesions

BackgroundFocal adhesions (FAs) are large, dynamic protein complexes located close to the plasma membrane, which serve as the mechanical linkages and a biochemical signaling hub of cells. The coordinated and dynamic regulation of focal adhesion is required for cell migration. Degradation, or turnover, of FAs is a major event at the trailing edge of a migratory cell, and is mediated by Ca^2 +/calpain-dependent proteolysis and disassembly. Here, we investigated how Ca^2 + influx induces cascades of FA turnover in living cells.MethodsImages obtained with a total internal reflection fluorescence microscope (TIRFM) showed that Ca^2 + ions induce different processes in the FA molecules focal adhesion kinase (FAK), paxillin, vinculin, and talin. Three mutated calpain-resistant FA molecules, FAK-V744G, paxillin-S95G, and talin-L432G, were used to clarify the role of each FA molecule in FA turnover.ResultsVinculin was resistant to degradation and was not significantly affected by the presence of mutated calpain-resistant FA molecules. In contrast, talin was more sensitive to calpain-mediated turnover than the other molecules. Three-dimensional (3D) fluorescence imaging and immunoblotting demonstrated that outer FA molecules were more sensitive to calpain-mediated proteolysis than internal FA molecules. Furthermore, cell contraction is not involved in degradation of FA.ConclusionsThese results suggest that Ca^2 +-mediated degradation of FAs was mediated by both proteolysis and disassembly. The 3D architecture of FAs is related to the different dynamics of FA molecule degradation during Ca^2 +-mediated FA turnover.General significanceThis study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca^2 +.     

Structures and stability of N<Subscript>13</Subscript><Superscript>+</Superscript> and N<Subscript>13</Subscript><Superscript>−</Superscript> clusters

The structures and stabilities of eleven N₁₃ ⁺ and N₁₃ ^– isomers have been investigated with second-order Møller–Plesset (MP2) and density functional theory (DFT) methods. Five N₁₃ ⁺ isomers and six N₁₃ ^– isomers are all reasonable local minima on their potential energy hypersurfaces. The most stable N₁₃ ⁺ cation is structure C-2 with C_2v symmetry, which contains a pentazole ring and two N₄ open chains. It is different from those of the N₇ ⁺ and N₉ ⁺ clusters, but similar to the N₁₁ ⁺ cluster. Meanwhile, the most stable N₁₃ ^– structure A-2 is composed of a pentazole ring and a six-membered ring connected by two nitrogen atoms. It is not only different from those of the N₇ ^– and N₉ ^– clusters, but also from the N₁₁ ^– cluster. The decomposition pathways of structures C-2 and A-2 were investigated at the B3LYP/(aug)-cc-pVDZ level. From the barrier heights of the structures C-2 and A-2 decomposition processes, it is suggested that C-2 is difficult to observe experimentally and A-2 may be observed as a short-lived species. Figure Optimized geometrical parameters of N₁₃ ⁺ isomer C-2      

Energy Profile of Cs+ in Gramicidin A in the Presence of Water. Problem of the Ion Selectivity of the Channel

Abstract

The effect of water present at the mouth and inside the channel of Gramicidin A on the energy profile calculated for a caesium ion is determined. The total optimal interaction energy computed for the system GA-Cs⁺-(22 waters) leads to an energy profile characterized by a deep minimum at 11Å followed by an entrance energy barrier of 7 Kcal/mol expanding until 9 Å from the center. After this point, a second minimum less deep than the previous one is observed, itself followed by a central barrier. The shape of the profile at the entrance is governed by the balance between the progressive desolvation process of the ion and the increase of favorable hydrogen bond interactions implying both the water molecules and GA. The comparison of this energy profile with that obtained in vacuo shows that the presence of water molecules does not modify the pathway of the ion which, owing to its size, is constrained essentially to remain on the channel axis. The comparison Na⁺ versus Cs⁺ indicates that although the phenomena involved are globally the same, differences between the two profiles appear due firstly to the difference in the affinity of the two ions for water and secondly to their respective size. This last difference implies that the number of water molecules present in the interior of the channel during the cation progression is reduced roughly by one in the case of caesium.

The desolvation barrier computed for Cs⁺ is half the corresponding value for Na⁺, a result in agreement with the observed selectivity.     

Crystal Structure of 04-Methyl Uridine: Stacking Induced Changes in the Geometry of the Pyrimidine Ring and Its Mutagenic Role

Abstract

The geometric properties of the pyrimidine ring of O⁴-methyl uridine more closely resemble those of cytidine than diketo uridine. Differences between the independent molecules of O⁴-methyl uridine are observed in the C(7)-O(4)-C(4)-C(5)-C(6) bond orders and the planarity of the pyrimidine rings. These differences are attributed to the monopole-induced dipole interactions between the ribose ring oxygen atom and a neighboring base of molecule A. A survey of the literature reveals that similar stacking-induced effects occur in other structures, involving both pyrimidine and purines. Finally, two base pairing schemes between O⁴-methyl uridine and guanosine, in which two hydrogen bonds can form, have been presented. Of these two the mispair with Watson-Crick geometry is favored.     

Conserved Water Molecular Dynamics of the Different X-ray Structures of Rusticyanin: An Unique Aquation Potentiality of the Ligand Bonded Cu++ Center

Abstract

The invariant water molecular interaction involving in the Rusticyanin of Thiobacillus ferrooxidans is thought to be important for its molecular complexation with other proteins at differential acidophilic situation. The comparative analysis of the different x-ray, energy minimized, and auto solvated structures of Rusticyanin revealed the presence of five specific invariant bound water molecules (among the ~ 150 water molecules per monomer) in the crystals. The five W 205, W 206, W 112, W 214, and W 221 water molecules (in Rusticyanin PDB code: 1RCY) were seem to be invariant in all the seven structures (PDB codes: 1RCY, 1A3Z, 1A8Z, 1E3O, 1GY1, 1GY2, 2CAL). Among the five conserved water molecules the W 221 (of 1 RCY or the equivalent water molecules in the other oxidized form of Rusticyanin structures) had endowed an interesting coordination potentiality to Cu⁺² ion during the energy minimization. The W 221 was observed to approach toward the tetrahedrally bonded Cu⁺² ion through the opposite (or trans) route of metal-bonded Met 148. This direct water molecular coordination affected the tetrahedral geometry of Cu⁺² to trigonal bipyramidal. Presumably this structural dynamics at the Cu⁺² center could involve in the electron transport process during protein-protein complexation.     

A proportion of CD4+ T cells from patients with chronic Chagas disease undergo a dysfunctional process,which is partially reversed by benznidazole treatment

BackgroundSigns of senescence and the late stages of differentiation associated with the more severe forms of Chagas disease have been described in the Trypanosoma cruzi antigen-specific CD4⁺ T-cell population. However, the mechanisms involved in these functions are not fully known. To date, little is known about the possible impact of benznidazole treatment on the T. cruzi-specific functional response of CD4⁺ T cells.Methodology/Principal findingsThe functional capacity of CD4⁺ T cells was analyzed by cytometric assays in chronic Chagas disease patients, with indeterminate form (IND) and cardiac alterations (CCC) (25 and 15, respectively) before and after benznidazole treatment. An increase in the multifunctional capacity (expression of IFN-γ, IL-2, TNF-α, perforin and/or granzyme B) of the antigen-specific CD4⁺ T cells was observed in indeterminate versus cardiac patients, which was associated with the reduced coexpression of inhibitory receptors (2B4, CD160, CTLA-4, PD-1 and/or TIM-3). The functional profile of these cells shows statistically significant differences between IND and CCC (p<0.001), with a higher proportion of CD4⁺ T cells coexpressing 2 and 3 molecules in IND (54.4% versus 23.1% and 4.1% versus 2.4%, respectively). A significant decrease in the frequencies of CD4⁺ T cells that coexpress 2, 3 and 4 inhibitory receptors was observed in IND after 24–48 months of treatment (p<0.05, p<0.01 and p<0.05, respectively), which was associated with an increase in antigen-specific multifunctional activity. The IND group showed, at 9–12 months after treatment, an increase in the CD4⁺ T cell subset coproducing three molecules, which were mainly granzyme B⁺, perforin⁺ and IFN-γ⁺ (1.4% versus 4.5%).Conclusions/SignificanceA CD4⁺ T cell dysfunctional process was detected in chronic Chagas disease patients, being more exacerbated in those patients with cardiac symptoms. After short-term benznidazole treatment (9–12 months), indeterminate patients showed a significant increase in the frequency of multifunctional antigen-specific CD4⁺ T cells.     

人树突状细胞的大量培养及鉴定

摘要 目的：探讨人树突状细胞体外大量培养及鉴定方法。方法：采用免疫磁珠法分离纯化CD34⁺干细胞;采用含有TPO、SCF、Flt3L和IL-3的扩增培养基培养1周,以及含有SCF、Flt3L、GM-CSF和IL-4的分化培养基培养2-3周,获得CD34⁺细胞来源树突状细胞。采用普通光学显微镜观察细胞形态,牛鲍氏血细胞计数板进行细胞计数,荧光抗体标记、流式细胞仪检测细胞纯度和细胞表面共刺激分子的表达情况。结果：以含有TPO、SCF、Flt3L和IL-3的培养基扩展培养一周,及含有SCF、Flt3L、GM-CSF和IL-4的培养基诱导分化3周,可获得大量悬浮细胞;细胞数目扩增倍数约达50倍;普通光学显微镜下可见悬浮细胞有明显的树突状凸起;流式细胞术检测结果显示悬浮细胞中CD141和CD11c双阳性细胞（等同于单核细胞来源树突状细胞）比例达30%,此群细胞高表达HLA-DR和CD209,低表达共刺激分子CD80和CD86;细胞寿命较短,40天时培养体系中悬浮细胞和CD34⁺细胞来源树突状细胞数目急剧减少。结论：采用多细胞因子联合刺激可获得大量的树突状细胞,为树突状细胞的特性及功能学研究奠定了基础。     

Control of the pro-oxidant-dependent calcium release from intact liver mitochondria

Summary

The reduction of molecular oxygen to water provides most of the energy that enables higher organisms to exist. Oxygen reduction is a mixed blessing because incompletely reduced oxygen species are more reactive than molecular oxygen in the ground state and can, when out of control, damage biological molecules. However, incompletely reduced oxygen species may also serve useful functions, as exemplified by their control of mitochondrial Ca²⁺ homeostasis, the understanding of which has improved greatly during the last few years. Hydrogen peroxide can stimulate a specific Ca²⁺ release pathway from intact mitochondria by oxidizing mitochondrial pyridine nucleotides through the activities of glutathione peroxidase, glutathione reductase, and the energy-linked transhydrogenase. Other pro-oxidants such as menadione, alloxan, or divicine also stimulate the specific Ca²⁺ release, because they furnish NAD⁺. The specific Ca²⁺ release requires for its activation the hydrolysis of intramitochondrial NAD⁺ to ADPribose and nicotinamide, and is prevented by inhibitors of NAD⁺ hydrolysis and protein monoADPribosylation. Recent experiments reveal that NAD⁺ hydrolysis and therefore Ca²⁺ release is regulated by vicinal thiols in mitochondria. When reduced or alkylated, the thiols prevent hydrolysis, but when they are cross-linked hydrolysis takes place. Cyclosporine A, which also prevents NAD⁺ hydrolysis, acts distal of these vicinal thiols. Since mitochondrial Ca²⁺ handling is physiologically relevant, its control by pro-oxidants must be added to the growing list of their useful functions.     

The interaction of adenine with its binding site in rabbit muscle glyceraldehyde 3-phosphate dehydrogenase studied by fluorescence decay.

The fluorescence decay mechanism of 1, N⁶-ethenoadenosine diphosphoribose bound to rabbit muscle glyceraldehyde 3-phosphate dehydrogenase markedly differs from that of the intact coenzyme analog (εNAD⁺) bound to the same enzyme. In the latter case the fluorescence is partially quenched by interactions between the ethenoadenine ring and amino acid residues in its binding site. Binding of the nicotinamide moiety of the coenzyme thus affects the relative orientation of the adenine ring within its binding site leading to the quenching interactions. The interactions of the adenine group with its binding site induce conformational changes in the enzyme which affect the binding of additional coenzyme molecules. The nicotinamide base thus determines, indirectly, the negative cooperativity found in NAD⁺ binding.     

Alteration of the groove width of DNA induced by the multimodal hydrogen bonding of denaturants with DNA bases in its grooves affects their stability

BackgroundDenaturants, namely, urea and guanidinium chloride (GdmCl) affect the stability as well as structure of DNA. Critical assessment of the role of hydrogen bonding of these denaturants with the different regions of DNA is essential in terms of its stability and structural aspect. However, the understanding of the mechanistic aspects of structural change of DNA induced by the denaturants is not yet well understood.MethodsIn this study, various spectroscopic along with molecular dynamics (MD) simulation techniques were employed to understand the role of hydrogen bonding of these denaturants with DNA bases in their stability and structural change.Results and conclusionIt has been found that both, GdmCl and urea intrude into groove region of DNA by striping surrounding water. The hydrogen bonding pattern of Gdm⁺ and urea with DNA bases in its groove region is multimodal and distinctly different from each other. The interaction of GdmCl with DNA is stabilized by electrostatic interaction whereas electrostatic and Lennard-Jones interactions both contribute for urea. Gdm⁺ forms direct hydrogen bond with the bases in the minor groove of DNA whereas direct and water assisted hydrogen bond takes place with urea. The hydrogen bond formed between Gdm⁺ with bases in the groove region of DNA is stronger than urea due to strong electrostatic interaction along with less self-aggregation of Gdm⁺ than urea. The distinct hydrogen bonding capability of Gdm⁺ and urea with DNA bases in its groove region affects its width differently. The interaction of Gdm⁺ decreases the width of the minor and major groove which probably increases the strength of hydrogen bond between the Watson-Crick base pairs of DNA leading to its stability. In contrast, the interaction of urea does not affect much to the width of the grooves except the marginal increase in the minor groove width which probably decreases the strength of hydrogen bond between Watson Crick base pairs leading to the destabilization of DNA.General significanceOur study clearly depicts the role of hydrogen bonding between DNA bases and denaturants in their stability and structural change which can be used further for designing of the guanidinium based drug molecules.     

PI3K and Notch signal pathways coordinately regulate the activation and proliferation of T lymphocytes in asthma

AimsIn the present study, we determined whether Phosphoinositide 3-kinase (PI3K) and Notch signal pathways are involved in the expression of cyclinD1, cyclinA and p27kip1 which were key molecules in controlling cell cycling from CD4⁺ T lymphocyte in animal model of asthma.Main methodsOvalbumin (OVA) sensitized murine model of asthma was used to investigate the expression of cyclin D1, cyclin A, and p27kip1 by splenic CD4⁺ T lymphocytes. We further observed the effect of specific inhibitor of PI3K(LY294002) and specific inhibitor of Notch(DAPT) on the proliferation of such CD4⁺ T lymphocytes.Key findingsWe found that the expression of cyclinD1 and cyclinA was upregulated at both protein and mRNA levels in asthma group while p27kip1 was down-regulated. Both LY294002 and DAPT inhibit the proliferation of CD4⁺ T lymphocytes in a time- and dose-dependent manner. Furthermore, LY294002 and DAPT have additive effect in down-regulation of cyclinD1 and upregulation of p27kip1. An upregulation of cyclinA, although not statistically significant, was also observed.SignificanceThese data suggested that PI3K signal pathway and Notch signal pathway may coordinately regulate the cell proliferation and differentiation processes through up-regulating cyclinD1 and down-regulating p27kip1 of CD4⁺ T lymphocytes.     

Molecular Dynamics Simulation of Limiting Conductance for Na2+, Cl2−, Na°, and Cl° in Supercritical Water

Abstract

We report results of molecular dynamics simulations of the limiting conductance of Na²⁺, Cl^2?, Na°, and Cl° in supercritical water using the SPC/E model for water in conjuction with our previous study (Lee et al., Chem. Phys. Lett. 293, 289 (1998)). The behavior of the limiting conductances of Na²⁺ and Cl^2? in the whole range of water density shows almost the same trend as those of Na⁺ and Cl^?, but the deviation from the assumed linear dependence of limiting conductances of Na²⁺ and Cl^2? on the water density is smaller than that of Na⁺ and Cl^?. The ratio of the limiting conductance of the divalentions to that of the corresponding monovalentions over the whole range of water density is almost constant. In the cases of Na²⁺ and Cl^2?, the dominating factor of the number of hydration water molecules around ions in the higher-density region and the dominating factor of the interaction strength between the ions and the hydration water molecules in the lower-density region are also found as was the cases for Na⁺ and Cl^?. These factors, however, are not so strong as for the corresponding monovalent ions because the change in the energetics, structure, and dynamics are very small mainly due to the strong Coulomb interaction of the divalent ions with the hydration water molecules. The diffusion coefficient of Na° and Cl° monotonically increases with decreasing water density over the whole range of water density. The increase of the diffusion coefficient with decreasing water density is attributed only to the dramatic decrease of the hydration number of water in the first solvation shell around the uncharged species. Among the two important competing factors in the limiting conductance of Na⁺ and Cl^?, the effect of the number of hydration water molecules around the uncharged species is the only existing factor over the whole range of water density since the interaction strength between the uncharged species and the hydration water molecules very small through the LJ interaction. This result has confirmed the dominating factor of the number of hydration water molecules around ions in the higher-density region in the explanation of the limiting conductance of Na⁺ and Cl^? in supercritical water at 673 K.     

The use of transgenic mouse models to reveal the functions of Ca2 + buffer proteins in excitable cells

BackgroundCytosolic Ca^2 + buffers are members of the large family of Ca^2 +-binding proteins and are essential components of the Ca^2 + signaling toolkit implicated in the precise regulation of intracellular Ca^2 + signals. Their physiological role in excitable cells has been investigated in vivo by analyzing the phenotype of mice either lacking one of the Ca^2 + buffers or mice with ectopic expression.Scope of ReviewIn this review, results obtained with knockout mice for the three most prominent Ca^2 + buffers, parvalbumin, calbindin-D28k and calretinin are summarized.Major ConclusionsThe absence of Ca^2 + buffers in specific neuron subpopulations, and for parvalbumin additionally in fast-twitch muscles, leads to Ca^2 + buffer-specific changes in intracellular Ca^2 + signals. This affects the excitation–contraction cycle in parvalbumin-deficient muscles, and in Ca^2 + buffer-deficient neurons, properties associated with synaptic transmission (e.g. short-term modulation), excitability and network oscillations are altered. These findings have not only resulted in a better understanding of the physiological function of Ca^2 + buffers, but have revealed that the absence of Ca^2 + signaling toolkit components leads to protein-and neuron-specific adaptive/homeostatic changes that also include changes in neuron morphology (e.g. altered spine morphology, changes in mitochondria content) and network properties.General SignificanceThe complex phenotype of Ca^2 + buffer knockout mice arises from the direct effect of these proteins on Ca^2 + signaling and moreover from the homeostatic mechanisms induced in these mice. For a better mechanistic understanding of neurological diseases linked to disturbed/altered Ca^2 + signaling, a global view on Ca^2 + signaling is expected to lead to new avenues for specific therapies. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

Abatacept decreases disease activity in a absence of CD4+ T cells in a collagen-induced arthritis model

IntroductionAbatacept is a fusion protein of human cytotoxic T-lymphocyte–associated protein (CTLA)-4 and the Fc portion of human immunoglobulin G1 (IgG1). It is believed to be effective in the treatment of rheumatoid arthritis by inhibiting costimulation of T cells via blocking CD28–B7 interactions as CTLA-4 binds to both B7.1 (CD80) and B7.2 (CD86). However, the interaction of CD28 with B7 molecules is crucial for activation of naive cells, whereas it is unclear whether the action of already activated CD4⁺ T cells, which are readily present in established disease, also depends on this interaction. The aim of this study was to determine whether the mode of action of abatacept depends solely on its ability to halt T cell activation in established disease.MethodsArthritis was induced in thymectomized male DBA/1 mice by immunisation with bovine collagen type II. The mice were subsequently depleted for CD4⁺ T cells. Abatacept or control treatment was started when 80 % of the mice showed signs of arthritis. Arthritis severity was monitored by clinical scoring of the paws, and anti-collagen antibody levels over time were determined by enzyme-linked immunosorbent assay.ResultsTreatment with abatacept in the absence of CD4⁺ T cells resulted in lower disease activity. This was associated with decreasing levels of collagen-specific IgG1 and IgG2a antibodies, whereas the antibody levels in control or CD4⁺ T cell–depleted mice increased over time.ConclusionsThese results show that abatacept decreased disease activity in the absence of CD4⁺ T cells, indicating that the mode of action of abatacept in established arthritis does not depend entirely on its effects on CD4⁺ T cell activation.     

Four-Stranded Intercalated Cytosine-Rich Molecules: Novel Insights into DNA Structure and Stability

Abstract

DNA fragments with stretches of cytosine residues can form four-stranded intercalated i-DNA molecules stabilized by hemiprotonated cytosine·cytosine⁺ (C·C⁺) base pairs. Intriguing features of this motif are the accomodation of base stacking that is unfavorable due to electrostatic repulsion and the close approach of phosphates in narrow grooves of the molecule. Unusual sources of stability in this structure involve sugar-base stacking and CH-O interribose short contacts between the backbones of adjacent strands.     

老年支气管哮喘患者血清 MC-CP、S1P水平与 T细胞免疫分子及气道基底膜厚度的关系

摘要 目的：探讨老年支气管哮喘患者血清肥大细胞羧肽酶（MC-CP）、1-磷酸鞘氨醇（S1P）水平,并分析与 T细胞免疫分子及气道基底膜厚度的关系。方法：选择 2016年 8月至 2018年 12月我院收治的老年支气管哮喘患者 86例,根据哮喘控制情况分为急性发作期组 48例和缓解期组 38例,选择同期医院体检的健康老年人 40例作为对照组,检测并比较各组血清 MC-CP、S1P水平、肺功能、T细胞免疫分子、支气管黏膜网状基底膜厚度（BMT）,并分析血清 MC-CP、S1P水平与肺功能指标、T细胞免疫分子及BMT的相关性。结果：急性发作组、缓解组患者血清 MC-CP、S1P水平及 BMT、CD8⁺均高于对照组,且急性发作组患者血清MC-CP、S1P、CD8+水平高于缓解期组（P＜0.05）。急性发作期组和缓解期组 FEV1%pred、CD4⁺、CD4⁺/ CD8⁺,血清免疫球蛋白 A（IgA）、免疫球蛋白 M（IgM）、免疫球蛋白 G（IgG）水平低于对照组,且急性发作期组上述指标低于缓解期组（P＜0.05）。老年支气管哮喘患者血清 MC-CP、S1P与 CD8⁺、BMT呈正相关（P＜0.05）,与 FEV1%pred、CD4⁺、CD4⁺/CD8⁺、IgA、IgM、IgG呈负相关（P＜0.05）。结论：老年支气管哮喘患者的血清 MC-CP、S1P水平显著升高,其水平与与肺功能、免疫功能和气道重塑相关,且血清MC-CP与 S1P水平密切相关。     

A Nanosecond Molecular Dynamics Study of Antiparallel d(G)7 Quadruplex Structures: Effect of the Coordinated Cations

Abstract

Nanosecond scale molecular dynamics simulations have been performed on antiparallel Greek key type d(G₇) quadruplex structures with different coordinated ions, namely Na⁺ and K⁺ ion, water and Na⁺ counter ions, using the AMBER force field and Particle Mesh Ewald technique for electrostatic interactions. Antiparallel structures are stable during the simulation, with root mean square deviation values of ～ 1.5 Å from the initial structures. Hydrogen bonding patterns within the G-tetrads depend on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate different cations. However, alternating syn-anti arrangement of bases along a chain as well as in a quartet is maintained through out the MD simulation. Coordinated Na+ ions, within the quadruplex cavity are quite mobile within the central channel and can even enter or exit from the quadruplex core, whereas coordinated K⁺ ions are quite immobile. MD studies at 400K indicate that K⁺ ion cannot come out from the quadruplex core without breaking the terminal G-tetrads. Smaller grooves in antiparallel structures are better binding sites for hydrated counter ions, while a string of hydrogen bonded water molecules are observed within both the small and large grooves. The hydration free energy for the K⁺ ion coordinated structure is more favourable than that for the Na⁺ ion coordinated antiparallel quadruplex structure.