Effect of AQP4 and its palmitoylation on the permeability of exogenous reactive oxygen species: Insights from computational study.

Aquaporin 4 (AQP4) facilitates the transport of reactive oxygen species (ROS). Both cancer cells and the ionizing radiation microenvironment can induce posttranslational modifications (PTMs) in AQP4, which may affect its permeability to ROS. Because this ROS diffusion process is rapid, microscopic, and instantaneous within and outside cells, conventional experimental methods are inadequate for elucidating the molecular mechanisms involved. In this study, computational methods were employed to investigate the permeability of exogenous ROS mediated by radiation in AQP4 at a molecular scale. We constructed a simulation system incorporating AQP4 and AQP4-Cysp13 in a complex lipid environment with ROS. Long-timescale molecular dynamics simulations were conducted to assess the structural stability of both AQP4 and AQP4-Cysp13. Free energy calculations were utilized to determine the ROS transport capability of the two AQP4 proteins. Computational electrophysiology and channel structural analysis quantitatively evaluated changes in ROS transport capacity under various radiation-induced transmembrane voltage microenvironments. Our findings demonstrate the distinct transport capabilities of AQP4 channels for water molecules and various types of ROS and reveal a decrease in transport efficiency when AQP4 undergoes palmitoylation modification. In addition, we have simulated the radiation-induced alteration of cell membrane voltage, which significantly affected the ROS transport capacity. We propose that this research will enhance the understanding of the molecular mechanisms governing the transport of exogenous ROS by AQP4 and elucidate the influence of palmitoylation on ROS transport. This study will also help clarify how different structural features of AQP4 affect the transport of exogenous ROS mediated by radiotherapy, thereby providing a theoretical molecular basis for the development of new treatment strategies that combine with radiotherapy.

Effects of membrane lipids on phospholamban pentameric channel structure and ion transportation mechanisms.

Protein-lipid interactions are an essential element of the function of many membrane ion-channel proteins. These potential interactions should be considered alongside the diversity and complexity of membrane lipid composition. Phospholamban (PLN) is an inhibitor of sarcoplasmic reticulum Ca2+ ATPase (SERCA). PLN is a 52-residue transmembrane protein encoded by lncRNA, and PLN monomers form stable pentamers of biological function in a lipid bilayer membrane. Some earlier studies suggest that it can form a cationic selective channel, while others suggest that it can only store ions. Here, we report the distribution of different lipids in the membrane and the structural dynamics and conductance properties of PLN pentamers after coarse-grained (CG) and all-atom (AA) molecular dynamics simulations of different systems. The results show that cholesterol is highly enriched around the protein and stabilizes the structure of the PLN pentamer. The absence of cholesterol increases the flexibility of the protein backbone. The conductance properties of monovalent ions and water suggest that they cannot spontaneously permeate through the PLN pentamer channel pore. However, the energy barrier to overcome is much lower in the absence of cholesterol, underlining the need to fully consider multiple lipid species when investigating small transmembrane protein oligomer ion-channel structure and conductance.

Calcium response of spatially arranged cell networks to shear stress by confined single cell patterned microfluidic chips.

Osteoblasts in multicellular organisms are sensitive to fluid shear stress (Fss) and respond smartly with versatile patterns of intracellular calcium signal ([Ca2+]i). In this study, a spatial-single cell patterning method was developed by combining micro-contact printing (µCP) and reversible microfluidic chip mounted with vacuum together. Based on this well-defined patterning platform, it's possible to investigate calcium response to Fss modulated by spatial factors, and to characterize multiple calcium patterns quantitatively in terms of cell spacing and cell orientation. The result showed that the Fss-induced [Ca2+]i profiles revealed oscillational signal patterns in non-connected cells such as those in physical-contacted cells. Close-arrayed osteoblasts showed remarkably more [Ca2+]i oscillations than sparse-arrayed cells. The circular shape of the cells was sensitive to oscillational [Ca2+]i as a potential major cause. The consistency of cell orientation and shear stress promoted temporal homogeneity of calcium oscillations.

Probing the formation, structure and free energy relationships of M protein dimers of SARS-CoV-2.

The M protein of the novel coronavirus 2019 (SARS-CoV-2) is the major structural component of the viral envelope and is also the minimum requirement for virus particle budding. M proteins generally exist as dimers. In virus assembly, they are the main driving force for envelope formation through lateral interactions and interactions with other viral structural proteins that play a central role. We built 100 candidate models and finally analyzed the six most convincing structural features of the SARS-CoV-2 M protein dimer based on long-timescale molecular dynamics (MD) simulations, multiple free energy analyses (potential mean force (PMF) and molecular mechanics Poisson-Boltzmann surface area (MMPBSA)) and principal component analysis (PCA) to obtain the most reasonable structure. The dimer stability was found to depend on the Leu-Ile zipper motif and aromatic amino acids in the transmembrane domain (TMD). Furthermore, the C-terminal domain (CTD) effects were relatively small. These results highlight a model in which there is sufficient binding affinity between the TMDs of M proteins to form dimers through the residues at the interface of the three transmembrane helices (TMHs). This study aims to help find more effective inhibitors of SARS-CoV-2 M dimers and to develop vaccines based on structural information.

Characterization of the SARS-CoV-2 E Protein: Sequence, Structure, Viroporin, and Inhibitors.

The COVID-19 epidemic is one of the most influential epidemics in history. Understanding the impact of coronaviruses (CoVs) on host cells is very important for disease treatment. The SARS-CoV-2 envelope (E) protein is a small structural protein involved in many aspects of the viral life cycle. The E protein promotes the packaging and reproduction of the virus, and deletion of this protein weakens or even abolishes the virulence. This review aims to establish new knowledge by combining recent advances in the study of the SARS-CoV-2 E protein and by comparing it with the SARS-CoV E protein. The E protein amino acid sequence, structure, self-assembly characteristics, viroporin mechanisms and inhibitors are summarized and analyzed herein. Although the mechanisms of the SARS-CoV-2 and SARS-CoV E proteins are similar in many respects, specific studies on the SARS-CoV-2 E protein, for both monomers and oligomers, are still lacking. A comprehensive understanding of this protein should prompt further studies on the design and characterization of effective targeted therapeutic measures.

Prediction of LncRNA-encoded small peptides in glioma and oligomer channel functional analysis using in silico approaches.

Glioma is a lethal malignant brain cancer, and many reports have shown that abnormalities in the behavior of water and ion channels play an important role in regulating tumor proliferation, migration, apoptosis, and differentiation. Recently, new studies have suggested that some long noncoding RNAs containing small open reading frames can encode small peptides and form oligomers for water or ion regulation. However, because the peptides are difficult to identify, their functional mechanisms are far from being clearly understood. In this study, we used bioinformatics methods to identify and evaluate lncRNAs, which may encode small transmembrane peptides in gliomas. Combining ab initio homology modeling, molecular dynamics simulations, and free energy calculations, we constructed a predictive model and predicted the oligomer channel activity of peptides by identifying the lncRNA ORFs. We found that one key hub lncRNA, namely, DLEU1, which contains two smORFs (ORF1 and ORF8), encodes small peptides that form pentameric channels. The mechanics of water and ion (Na+ and Cl-) transport through this pentameric channel were simulated. The potential mean force of the H2O molecules along the two ORF-encoded peptide channels indicated that the energy barrier was different between ORF1 and ORF8. The ORF1-encoded peptide pentamer acted as a self-assembled water channel but not as an ion channel, and the ORF8 permeated neither ions nor water. This work provides new methods and theoretical support for further elucidation of the function of lncRNA-encoded small peptides and their role in cancer. Additionally, this study provides a theoretical basis for drug development.

Effects of amino acid modifications on the permeability of the pentameric sarcolipin channel.

Sarcolipin (SLN) is an important transmembrane (TM) protein encoded by long noncoding RNA. SLN is expressed in the sarcoplasmic reticulum and regulates cardiac and skeletal muscle contractions. SLN forms a pentameric hydrophobic ligand-gated ion channel. The protonation of Glu7 (protonated SLN, pSLN) and mutation of Thr18 to Ala18 (T18A) have been reported to exert a significant influence on the permeability of the channel. In this study, the altered permeability of both the pSLN and T18A pentameric channels was simulated. Combined with molecular dynamics simulation, the free-energy landscape for single ions, computational electrophysiology, diffusion coefficient, and pore geometrical characteristic analyses were performed to further understand the properties of amino acid modifications in the SLN pentameric channel. The results suggest that both the pSLN and T18A pentameric channels form stable hydrophobic ligand-gated channels. The TM voltage has a positive effect on the permeability of water molecules and ions. By using pSLN and T18A, our study provides helpful information on the pore-forming mechanism of SLN and furthers our understanding of the regulatory mechanisms underlying the permeation of ions and water molecules in the pentameric SLN channel.

Computational Study of the Ion and Water Permeation and Transport Mechanisms of the SARS-CoV-2 Pentameric E Protein Channel.

Coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus (SARS-CoV-2) and represents the causative agent of a potentially fatal disease that is a public health emergency of international concern. Coronaviruses, including SARS-CoV-2, encode an envelope (E) protein, which is a small, hydrophobic membrane protein; the E protein of SARS-CoV-2 shares a high level of homology with severe acute respiratory syndrome coronavirus (SARS-CoV). In this study, we provide insights into the function of the SARS-CoV-2 E protein channel and the ion and water permeation mechanisms using a combination of in silico methods. Based on our results, the pentameric E protein promotes the penetration of cation ions through the channel. An analysis of the potential mean force (PMF), pore radius and diffusion coefficient reveals that Leu10 and Phe19 are the hydrophobic gates of the channel. In addition, the pore exhibits a clear wetting/dewetting transition with cation selectivity under transmembrane voltage, indicating that it is a hydrophobic voltage-dependent channel. Overall, these results provide structure-based insights and molecular dynamic information that are needed to understand the regulatory mechanisms of ion permeability in the pentameric SARS-CoV-2 E protein channel.

Design and Characterization of a Cancer-Targeted Drug Co-Delivery System Composed of Liposomes and Selenium Nanoparticles.

A drug co-delivery system composed of selenium nanoparticles (SeNPs) has attracted increasing interest due to its ability to increase the anticancer efficacy against multidrug-resistant cancer cells. In this study, a cancer-targeted drug co-delivery system combining fluorescein-loaded liposomes and SeNPs was designed and evaluated. The system was developed by coating SeNPs and fluorescein-loaded liposomes with folic acid-chitosan conjugates (FA-CS-SeNPs-Lips). Folic acid-chitosan conjugates (FA-CS) were synthesized by coupling folic acid (FA) with chitosan (CS), and the structure was confirmed by performing Fourier transform spectroscopy (FT-IR) and nuclear magnetic resonance (1H-NMR) spectroscopy. Dynamic light scattering (DLS) measurements and transmission electron microscopy (TEM) were used to evaluate the particle size, Zeta potential, and morphology. The cytotoxicity of SeNPs coated with FA-CS conjugates (FA-CS-SeNPs) toward A549 cells and HeLa cells was examined using the MTT assay. The cancer-targeting ability and drug release behaviors were evaluated in vitro by measuring the cellular uptake of fluorescein and dialysis, respectively. The FA-CS-SeNPs were uniform, spherical particles with a ~50 nm diameter and high positive Zeta potential (+57.7 mV). Based on the results of the MTT assay, FA-CS-SeNPs displayed a more significant increase in the anticancer efficacy in HeLa cells than CS-SeNPs. FA-CS-SeNPs-Lips not only slowly released fluorescein but also specifically targeted HeLa cells through selective binding between folate and folate receptors to increase the cellular uptake of fluorescein.

Rhein inhibits ATP-triggered inflammatory responses in rheumatoid rat fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disorder, which may lead to joint disabilities. So far the pathogenesis of RA remains largely undetermined, and there are still no potent drugs for clinical treatment. Rhein, a natural bioactive anthraquinone derivative, exhibited significant anti-inflammatory activities demonstrated by previous studies. Here we aimed to investigate the effects of rhein on ATP-induced inflammation responses in fibroblast-like synoviocytes isolated from a rat model of collagen induced arthritis (CIA). Our results showed that ATP triggered rapid cytosolic calcium concentration ([Ca2+]c) increase depending on extracellular Ca2+ entry. Given the major P2 subtypes expressed in rat synoviocytes were P2X4 and P2Y2 receptors, ATP-elicited calcium entry should be mainly resulted from activating P2X4. Interestingly, rhein could effectively block the ATP-induced [Ca2+]c increases in a dose-dependent manner. Besides, rhein also suppressed the production of intracellular reactive oxygen species (ROS) induced by ATP in synoviocytes that was resulted from P2X4-mediated Ca2+ entry. Brilliant blue G (BBG), which can block P2X4 receptor at high concentration, showed similar suppressive effects on above responses. Furthermore, in lipopolysaccharide-primed cells, application of ATP synergistically promoted the gene expression of cyclooxygenase-2, interleukin-6 and matrix metalloproteinase-9. Both rhein and BBG attenuated these inflammatory gene expressions enhanced by ATP. Above data together suggested a potential anti-arthritic role of rhein by inhibiting ATP-induced [Ca2+]c increase, ROS production and inflammatory gene expression targeting P2X4 in CIA rat synoviocytes, which will provide a novel insight in the therapy of RA.

Changes of cationic transport in AtCAX5 transformant yeast by electromagnetic field environments.

The electromagnetic field (EMF) is newly considered as an exogenous environmental stimulus that is closely related to ion transportation on the cellular membrane, maintaining the internal ionic homeostasis. Cation transports of Ca2+ and other metal ions, Cd2+, Zn2+, and Mn2+were studied in terms of the external Ca2+ stress, [Ca2+]ext, and exposure to the physical EMF. A specific yeast strain K667 was used for controlling CAX5 (cation/H+ exchanger) expression. Culture samples were exposed to 60 Hz, 0.1 mT sinusoidal or square magnetics waves, and intracellular cations of each sample were measured and analyzed. AtCAX5 transformant yeast grew normally under the metallic stress. However, the growth of the control group was significantly inhibited under the same cation concentration; 60 Hz and 0.1 mT magnetic field enhanced intracellular cation concentrations significantly as exposure time increased both in the AtCAX5 transformed yeast and in the control group. However, the AtCAX5-transformed yeast showed higher concentration of the intracellular cations than the control group under the same exposure EMF. AtCAX5-transformed yeasts displayed an increment in [Ca2+]int, [K+]int, [Na+]int, and [Zn2+]int concentration under the presence of both sinusoidal and square-waved EMF stresses compared to the control group, which shows that AtCAX5 expressed in the vacuole play an important role in maintaining the homeostasis of intracellular cations. These findings could be utilized in the cultivation of the crops which were resistant to excessive exogenous ions or in the production of biomass containing a large proportion of ions for nutritional food or in the bioremediation process in metal-polluted environments.

Genome-Wide Pathway Analysis of Microarray Data Identifies Risk Pathways Related to Salt Stress in Arabidopsis Thaliana.

Salt stress is a common abiotic stress in agricultural production, which is affected by multiple genes and environmental factors. Although transcriptome analyses have detected some salt-related genes in Arabidopsis thaliana, these genes are often major genes and can not adequately explain the molecular mechanism of salt tolerance. Some genes related to salt stress, but does not reach significant threshold in gene expression analysis (called modest effect genes), are often ignored. Therefore, we took full account of the role of modest effect genes and performed a pathway-based analysis of three gene microarray datasets to identify the pathways related to salt stress. We also compared these results with the pathways identified by major genes. Finally, three pathways were identified as salt-related pathways, some of which were previously reported to be related to salt stress in plants, while others are novel. These findings will help us to study the molecular mechanism of salt stress, but also provide a new perspective for the study of salt tolerance in Arabidopsis thaliana.

Multiple calcium patterns of rat osteoblasts under fluidic shear stress.

The intracellular calcium ([Ca2+ ]i ) response induced by external forces can be diverse and complex. Using primary osteoblasts from Wistar rats, we found multiple patterns of [Ca2+ ]i responses induced by fluidic shear stress (Fss), including homogeneous non-oscillation and heterogeneous oscillations. These multiple-patterned [Ca2+ ]i responses could be influenced by Fss intensity, cell density, and cell differentiation. Our real-time measurements with free calcium, ATP, ATP without calcium, suramin, apyrase, and thapsigargin confirmed homogeneous [Ca2+ ]i patterns and/or heterogeneous [Ca2+ ]i oscillations with respect to the combined degree of external Ca2+ influx, and intracellular Ca2+ release. Our theoretical model supported diverse Fss-induced calcium activities as well. We concluded that a singular factor of Ca2+ influx or release dominated to produce smooth homogeneous patterns, but combined factors produced oscillatory heterogeneous patterns. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2039-2051, 2018.

Nucleotide transmitters ATP and ADP mediate intercellular calcium wave communication via P2Y12/13 receptors among BV-2 microglia.

Nerve injury is accompanied by a liberation of diverse nucleotides, some of which act as 'find/eat-me' signals in mediating neuron-glial interplay. Intercellular Ca2+ wave (ICW) communication is the main approach by which glial cells interact and coordinate with each other to execute immune defense. However, the detailed mechanisms on how these nucleotides participate in ICW communication remain largely unclear. In the present work, we employed a mechanical stimulus to an individual BV-2 microglia to simulate localized injury. Remarkable ICW propagation was observed no matter whether calcium was in the environment or not. Apyrase (ATP/ADP-hydrolyzing enzyme), suramin (broad-spectrum P2 receptor antagonist), 2-APB (IP3 receptor blocker) and thapsigargin (endoplasmic reticulum calcium pump inhibitor) potently inhibited these ICWs, respectively, indicating the dependence of nucleotide signals and P2Y receptors. Then, we detected the involvement of five naturally occurring nucleotides (ATP, ADP, UTP, UDP and UDP-glucose) by desensitizing receptors. Results showed that desensitization with ATP and ADP could block ICW propagation in a dose-dependent manner, whereas other nucleotides had little effect. Meanwhile, the expression of P2Y receptors in BV-2 microglia was identified and their contributions were analyzed, from which we suggested P2Y12/13 receptors activation mostly contributed to ICWs. Besides, we estimated that extracellular ATP and ADP concentration sensed by BV-2 microglia was about 0.3 µM during ICWs by analyzing calcium dynamic characteristics. Taken together, these results demonstrated that the nucleotides ATP and ADP were predominant signal transmitters in mechanical stimulation-induced ICW communication through acting on P2Y12/13 receptors in BV-2 microglia.

Self-assembling study of sarcolipin and its mutants in multiple molecular dynamic simulations.

The Sarcolipin (SLN) is a single trans-membrane protein that can self-assembly to dimer and oligomer for playing importantphysiological function. In this work, we addressed the dimerization of wild type SLN (wSLN) and its mutants (mSLNs) - I17A and I20A, using both coarse-grained (CG) and atomistic (AT) molecular dynamics (MD) simulations. Our results demonstrated that wSLN homodimer assembled as a left-handed helical complex, while mSLNs heterodimers assembled as right-handed complexes. Analysis of residue-residue contacts map indicated that isoleucine (Ile)-leucione (Leu) zipper domain played an important role in dimerization. The potential of mean force (PMF) demonstrated that wSLN homodimer was more stable than mSLNs heterodimers. Meanwhile, the mSLNs heterodimers preferred right-handed rather than left-handed helix. AT-MD simulations for wSLN and mSLNs were also in line with CG-MD simulations. These results provided the insights for understanding the mechanisms of SLNs self-assembling. Proteins 2017; 85:1065-1077. © 2017 Wiley Periodicals, Inc.

Transmembrane dynamics of the Thr-5 phosphorylated sarcolipin pentameric channel.

Sarcolipin (SLN), an important membrane protein expressed in the sarcoplasmic reticulum (SR), regulates muscle contractions in cardiac and skeletal muscle. The phosphorylation at amino acid Thr5 of the SLN protein modulates the amount of Ca(2+) that passes through the SR. Using molecular dynamics simulation, we evaluated the phosphorylation at Thr5 of pentameric SLN (phospho-SLN) channel's energy barrier and pore characteristics by calculating the potential of mean force (PMF) along the channel pore and determining the diffusion coefficient. The results indicate that pentameric phospho-SLN promotes penetration of monovalent and divalent ions through the channel. The analysis of PMF, pore radius and diffusion coefficient indicates that Leu21 is the hydrophobic gate of the pentameric SLN channel. In the channel, water molecules near the Leu21 pore demonstrated a clear hydrated-dehydrated transition; however, the mutation of Leu21 to an Alanine (L21A) destroyed the hydrated-dehydrated transitions. These water-dynamic behaviors and PMF confirm that Leu21 is the key residue that regulates the ion permeability of the pentameric SLN channel. These results provide the structural-basis insights and molecular-dynamic information that are needed to understand the regulatory mechanisms of ion permeability in the pentameric SLN channel.

NaCl stress-induced transcriptomics analysis of Salix linearistipularis (syn. Salix mongolica).

BACKGROUND: Salix linearistipularis (syn. S. mongolica) is a woody halophyte, which is distributed naturally in saline-alkali soil of Songnen plain, Heilongjiang, China. It plays an important role in maintaining ecological balance and in improving saline soil. Furthermore, S. linearistipularis is also a genetic resource; however, there is no available information of genomic background for salt tolerance mechanism. We conducted the transcriptome analysis of S. linearistipularis to understand the mechanisms of salt tolerance by using RNA-seq technology. RESULTS: The transcription profiles of both the salt stress (SLH-treated) and the control (SLH-control) sample for S. linearistipularis were obtained by using RNA-seq in this study. By comparative analysis, only 3034 of 53,362 all-unigenes between two samples were expressed differently at more than 1.5-fold ([Formula: see text], FDR ≤ 0.05), including 1397 up-regulated genes and 1637 down-regulated genes. In total, 2199 genes were classified into 50 Gene Ontology (GO) terms and 1103 genes were involved in 116 biological pathways. To find salt stress related genes, all-unigenes of S. linearistipularis were classified into three categories according to their degree of the differentially expressed genes (DEGs) at 0-1.5-fold (non differently expressed genes, N-DEGs), at 1.5-4.0-fold and more than 4.0-fold. The pathways of three categorized genes were compared with the DEGs of Arabidopsis thaliana, showing that 22, 10 and 1 pathway of S. linearistipularis were overlapped with A. thaliana. Degree of the overlapping was categorized as 0-1.5-fold, 1.5-4.0-fold and more than 4.0-folds. CONCLUSION: Our study revealed that the N-DEGs of 22 pathways in S. linearistipularis were overlapped with the DEGs of A. thaliana. This result suggests that those overlapped genes that contrasted with the up- or down-regulated genes in A. thaliana were possibility evolved into housekeeping genes in S. linearistipularis under salt stress.

Molecular dynamics of water and monovalent-ions transportation mechanisms of pentameric sarcolipin.

The Sarcolipin (SLN) is a transmembrane protein that can form a self-assembled pentamer. In this work, the homology modeling and all-atom molecular dynamic (MD) simulation was performed to study the model of SLN pentamer in POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membrane. The potential of mean force (PMF) was calculated for transmembrane transportation of Na(+), Cl(-) and water molecule along the pore channel of penta-SLN complex. The root mean square deviation (RMSD) of the SLN pentamer in POPC membrane showed that the stabilized SLN protein complex could exist in the membrane and that the Na(+) and Cl(-) could not permeate through the channel when the pore was under the vacuum state, but the water could permeate through from cytoplasm to lumen. Under the aqueous state, our simulation demonstrated that hydrated state of Na(+) and Cl(-) could pass through the channel. The PMF and radii of the pore showed that the channel had a gate at Leu(21) that is a key hydrophobicity residue in the channel. Our simulations help to clarify and to understand better the SLN pentamer channel that had a hydrophobic gate and could switch Na(+) and Cl(-) ion permeability by hydrated and vacuum states.

Rhein antagonizes P2X7 receptor in rat peritoneal macrophages.

P2X7 receptor plays important roles in inflammation and immunity, and thereby it serves as a potential therapeutic target for inflammatory diseases. Rhein, an anthraquinone derivative, exhibits significant anti-inflammatory and immunosuppressive activities in therapy. However, the underlying mechanisms are largely unclear. Here, we aimed to investigate the effects of rhein on P2X7 receptor-mediated responses in vitro. In HEK293 cells expressing rat P2X7 receptor, we first found that rhein concentration-dependently blocked ATP-induced cytosolic calcium concentration ([Ca(2+)]c) elevation and pore formation of the plasma membrane, two hallmarks of the P2X7 receptor activation. These two inhibitory effects of rhein were also observed in rat peritoneal macrophages. Furthermore, rhein counteracted macrophage phagocytosis attenuation and suppressed reactive oxygen species (ROS) production triggered by ATP/BzATP. Meanwhile, rhein reduced ATP/BzATP-induced IL-1ß release in lipopolysaccharide-activated macrophages. Prolonged application of ATP caused macrophage apoptosis, while the presence of rhein suppressed this cell cytotoxicity. Such ATP/BzATP-induced cellular reactions were also inhibited by a well-known rat P2X7 receptor antagonist, brilliant blue G, in a similar way to rhein. Together, our results demonstrate that rhein inhibit ATP/BzATP-induced [Ca(2+)]c increase, pore formation, ROS production, phagocytosis attenuation, IL-1ß release and cell apoptosis by antagonizing the P2X7 receptor in rat peritoneal macrophages.

Saline-induced changes of epicuticular waxy layer on the Puccinellia tenuiflora and Oryza sativa leave surfaces.

BACKGROUND: The epicuticular waxy layer of plant leaves enhances the extreme environmental stress tolerance. However, the relationship between waxy layer and saline tolerance was not established well. The epicuticular waxy layer of rice (Oryza sativa L.) was studied under the NaHCO3 stresses. In addition, strong saline tolerance Puccinellia tenuiflora was chosen for comparative studies. RESULTS: Scanning electron microscope (SEM) images showed that there were significant changes in waxy morphologies of the rice epicuticular surfaces, while no remarkable changes in those of P. tenuiflora epicuticular surfaces. The NaHCO3-induced morphological changes of the rice epicuticular surfaces appeared as enlarged silica cells, swollen corns-shapes and leaked salt columns under high stress. Energy dispersive X-ray (EDX) spectroscopic profiles supported that the changes were caused by significant increment and localization of [Na(+)] and [Cl(-)] in the shoot. Atomic absorption spectra showed that [Na(+)]shoot/[Na(+)]root for P. tenuiflora maintained stable as the saline stress increased, but that for rice increased significantly. CONCLUSION: In rice, NaHCO3 stress induced localization and accumulation of [Na(+)] and [Cl(-)] appeared as the enlarged silica cells (MSC), the swollen corns (S-C), and the leaked columns (C), while no significant changes in P. tenuiflora.