Surface property and in vitro toxicity effect of insoluble particles given by protein corona: Implication for PM cytotoxicity assessment.

In vitro toxicological assessment helps explore key fractions of particulate matter (PM) in association with the toxic mechanism. Previous studies mainly discussed the toxicity effects of the water-soluble and organic-soluble fractions of PM. However, the toxicity of insoluble fractions is relatively poorly understood, and the adsorption of proteins is rarely considered. In this work, the formation of protein corona on the surface of insoluble particles during incubation in a culture medium was investigated. It was found that highly abundant proteins in fetal bovine serum were the main components of the protein corona. The adsorbed proteins increased the dispersion stability of insoluble particles. Meanwhile, the leaching concentrations of some metal elements (e.g., Cu, Zn, and Pb) from PM increased in the presence of proteins. The toxicity effects and potential mechanisms of the PM insoluble particle-protein corona complex on macrophage cells RAW264.7 were discussed. The results revealed that the PM insoluble particle-protein corona complex could influence the phagosome pathway in RAW264.7 cells. Thus, it promoted the intracellular reactive oxygen species generation and induced a greater degree of cell differentiation, significantly altering cell morphology. Consequently, this work sheds new light on the combination of insoluble particles and protein corona in terms of PM cytotoxicity assessment.

Effect of ionic liquids as mobile phase additives on retention behaviors of G-quadruplexes in reversed-phase high performance liquid chromatography.

G-quadruplexes (G4s) play an important role in a variety of biological processes and have extensive application prospects. Due to the significance of G4s in physiology and biosensing, studies on G4s have attracted much attention, stimulating the development or improvement of methods for G4 structures and polymorphism analysis. In this work, ionic liquids (ILs) were involved as mobile phase additives in reversed-phase high performance liquid chromatography (RP-HPLC) to analyse G4s with various conformations for the first time. How ILs affected the retention behaviors of G4s was investigated comprehensively. It was found that the addition of ILs markedly enhanced G4 retention, along with obvious amelioration on chromatographic peak shapes and separation. The influence of pH of mobile phase and types of ILs were also included in order to acquire an in-depth understanding. It appeared that the effect of ILs on G4 retention behaviors was the result of a combination of various interactions between G4s with the hydrophobic stationary phase and with the IL-containing mobile phase, where ion pair mechanism and enhanced hydrophobic interaction dominated. The findings of this work revealed that ILs could effectively improve the separation of G4s in RP-HPLC, which was conducive to G4 structural analysis, especially for G4s polymorphism elucidation.

Study on the Interaction of a Peptide Targeting Specific G-Quadruplex Structures Based on Chromatographic Retention Behavior.

G-quadruplexes (G4s) are of vital biological significance and G4-specific ligands with conformational selectivity show great application potential in disease treatment and biosensing. RHAU, a RNA helicase associated with AU-rich element, exerts biological functions through the mediation of G4s and has been identified to be a G4 binder. Here, we investigated the interactions between the RHAU peptide and G4s with different secondary structures using size exclusion chromatography (SEC) in association with circular dichroism (CD), ultraviolet-visible (UV-Vis) absorption, and native polyacrylamide gel electrophoresis (Native-PAGE). Spectral results demonstrated that the RHAU peptide did not break the main structure of G4s, making it more reliable for G4 structural analysis. The RHAU peptide was found to display a structural selectivity for a preferential binding to parallel G4s as reflected by the distinct chromatographic retention behaviors. In addition, the RHAU peptide exhibited different interactions with intermolecular parallel G4s and intramolecular parallel G4s, providing a novel recognition approach to G4 structures. The findings of this study enriched the insight into the binding of RHAU to G4s with various conformations. It is noteworthy that SEC technology can be easy and reliable for elucidating G4-peptide interactions, especially for a multiple G4 coexisting system, which supplied an alternative strategy to screen novel specific ligands for G4s.

A novel particulate matter sampling and cell exposure strategy based on agar membrane for cytotoxicity study.

Laboratories use different strategies to sample and extract atmospheric particulate matter (PM), some of which can be very complicated. Due to the absence of a standard protocol, it is difficult to compare the results of PM toxicity assessment across different laboratories. Here, we proposed a novel PM sampling and cell exposure strategy based on agar membrane. The agar membrane, prepared by a simple freeze-drying method, has a relatively flat surface and porous interior. We demonstrated that the agar membrane was a reliable substitute material for PM sampling. Then the PM on the agar membranes was directly extracted with the culture medium by vortex method, and the PM on the polytetrafluoroethylene (PTFE) filters was extracted with water by the traditional ultrasonic method for comparison. The extraction efficiency was evaluated and in vitro cytotoxicity assays were carried out to investigate the toxic effects of PM extracted with two strategies on macrophage cells. The results showed that the PM extracted from agar membranes induced higher cytotoxicity and more differentially expressed proteins. Overall, the novel PM sampling-cell exposure strategy based on the agar membrane is easy to operate, biocompatible and comparable, and has low disturbance, could be an alternative sampling and extraction method for PM toxicity assessment.

Transport of environmental natural organic matter coated silver nanoparticle across cell membrane based on membrane etching treatment and inhibitors.

Environmental natural organic matters (NOMs) have great effects on the physicochemical properties of engineering nanoparticles, which may impact the transport of nanoparticles across plasma membrane and the cytotoxicity. Therefore, the kinetics, uptake pathway and mass of transporting into A549 cell membrane of silver nanoparticles (AgNPs) coated with citric acid (CA), tartaric acid (TA) and fulvic acid (FA) were investigated, respectively. CA, FA and TA enhanced the colloidal stability of AgNPs in culture medium and have greatly changed the surface plasmon resonance spectrum of AgNPs due to the absorption of CA, FA and TA on surface of AgNPs. Internalizing model showed that velocity of CA-, TA- and FA-nAg transporting into A549 cell were 5.82-, 1.69- and 0.29-fold higher than those of the control group, respectively. Intracellular mass of Ag was dependent on mass of AgNPs delivered to cell from suspension, which obeyed Logistic model and was affected by NOMs that CA- and TA-nAg showed a large promotion on intracellular mass of Ag. The lipid raft/caveolae-mediated endocytosis (LME) of A549 cell uptake of AgNPs were susceptible to CA, TA and FA that uptake of CA-, TA- and FA-nAg showed lower degree of dependent on LME than that of the control (uncoated AgNPs). Actin-involved uptake pathway and macropinocytosis would have less contribution to uptake of FA-nAg. Overall, transmembrane transport of NOMs-coated AgNPs differs greatly from that of the pristine AgNPs.

Cytotoxicity and toxicoproteomic analyses of human lung epithelial cells exposed to extracts of atmospheric particulate matters on PTFE filters using acetone and water.

Differences of cytotoxicity associated with exposure to different extracts of atmospheric particulate matters (PMs) are still not well characterized by in vitro toxicoproteomics. In this study, in vitro cytotoxicity assays and toxicoproteomic analyses were carried out to investigate toxic effects of PM collected using polytetrafluoroethylene (PTFE) filters extracted with acetone for PM2.1 and water for PM2.1 and PM10 on A549 human lung epithelial cells. The cytotoxicity assays based on cell viability, cell apoptosis and reactive oxygen species generation indicated that PM2.1 extracted with acetone had the highest toxicity. iTRAQ labeling and LC-MS/MS analyses indicated that the number of differentially expressed proteins in A549 cells affected by PM2.1 extracted with acetone was noticeably higher than that of the other two groups. Hierarchical cluster analyses showed that the influences of the extracts of PM2.1 and PM10 using water on the proteome of A549 cells were similar, whereas significantly different from the effect of PM2.1 extracted with acetone. Pathways analyses indicated that PM2.1 extracted with acetone influenced the expression of proteins involved in 14 pathways including glycolysis/gluconeogenesis, pentose phosphate pathway, proteasome, etc. PM2.1 extracted with water affected the expression of proteins involved in 3 pathways including non-homologous end-joining, ribosome and endocytosis. However, PM10 extracted with water affected the expression of proteins involved in only spliceosome pathway. The extracts of PM using different extractants to detach PM from PTFE filters influenced the cytotoxic effects of PM and the proteome of A549 cells. Therefore, extractants should be assessed carefully before the investigations on cytotoxicity to improve the compatibility of experimental results among research teams.

Monolithic alkylsilane column: A promising separation medium for oligonucleotides by ion-pair reversed-phase liquid chromatography.

In this paper, a monolithic octadecylsilane column and particle-packed octadecylsilane columns were used to investigate the retention behaviors of oligonucleotides by ion-pair reversed-phase liquid chromatography (IP-RPLC). Results showed that, with same base composition, hairpin oligonucleotides always had weaker retention than corresponding random coil oligonucleotides on the monolithic column, but not on the particle-packed columns. In addition, the linear correlation between the retention factor k of oligonucleotides and the reciprocal of temperature (1/T), especially for hairpins, was relatively weaker on the particle-packed columns, as compared to the correlation on the monolithic column. The correlation between k and 1/T became weaker with decreasing particle size of the particle-packed columns. Moreover, results revealed that the overall retention order on the particle-packed column with small particles (3 µm) differed greatly from that on the monolithic column. In contrast, the retention order on the 10 µm particle-packed column was very close to that on the monolithic column. From the above, we inferred that oligonucleotides could keep their primary conformations unchanged when passing through the monolithic column, attributed to the special pore structures of the monolith. However, the conformations of oligonucleotides were suppressed or even destroyed when oligonucleotides passed through the particle-packed columns. This because the narrow and tortuous channels created by the stacked stationary phase particles could lead to more complex and unequable retention behaviors. Therefore, the monolithic column exhibited better retention regularity for oligonucleotides of secondary structure especially for hairpins than the particle-packed columns. It is noteworthy that the monolith-based IP-RPLC opens an intriguing prospect in accurately elucidating the retention behaviors of oligonucleotides.

Time-dependent response of A549 cells upon exposure to cadmium.

Cadmium is considered one of the most harmful carcinogenic heavy metals in the human body. Although many scientists have performed research on cadmium toxicity mechanism, the toxicokinetic process of cadmium toxicity remains unclear. In the present study, the kinetic response of proteome in/and A549 cells to exposure of exogenous cadmium was profiled. A549 cells were treated with cadmium sulfate (CdSO4 ) for different periods and expressions of proteins in cells were detected by two-dimensional gel electrophoresis. The kinetic expressions of proteins related to cadmium toxicity were further investigated by reverse transcription-polymerase chain reaction and western blotting. Intracellular cadmium accumulation and content fluctuation of several essential metals were observed after 0-24 hours of exposure by inductively coupled plasma mass spectrometry. Fifty-four protein spots showed significantly differential responses to CdSO4 exposure at both 4.5 and 24 hours. From these proteins, four expression patterns were concluded. Their expressions always exhibited a maximum abundance ratio after CdSO4 exposure for 24 hours. The expression of metallothionein-1 and ZIP-8, concentration of total protein, and contents of cadmium, zinc, copper, cobalt and manganese in cells also showed regular change. In synthesis, the replacement of the essential metals, the inhibition of the expression of metal storing protein and the activation of metal efflux system are involved in cadmium toxicity.

Study on the polymorphism of G-quadruplexes by reversed-phase HPLC and LC-MS.

Polymorphism is inherent for G-quadruplexes (G4s), and the different structural forms are important for the participation in different biological functions of telomeres. A lot of progress has been made in the exploration of G4 polymorphism. However, quick separation and reliable assessment methods for different conformations of G4 are still very few. In this work, the polymorphism of three sequences d[(G3T)3G3], d[(G3C)3G3] and d[(G3T)4] annealed in six different solutions were investigated by means of reversed-phase high performance liquid chromatography (RP-HPLC), liquid chromatography-mass spectrometry (LC-MS), fluorescence spectroscopy, circular dichroism spectroscopy, together with native-polyacrylamide gel electrophoresis. Different G4 conformations of these three sequences can be separated clearly by RP-HPLC, and further characterized by on-line LC-MS analysis. It is revealed that high-order structures other than intramolecular quadruplexes were favored for d[(G3T)3G3] and d[(G3C)3G3] under the annealing conditions. However, flanking loop impeded d[(G3T)4] to form higher-order structures than dimer. In addition, the nature and concentration of cation, as well as the annealing solution component, all have decent influence on the stability and relative ratios of various G4 building blocks. Based on the above findings, RP-HPLC and LC-MS combined with spectroscopic techniques can be used as a facile and powerful tool for quick separation and identification of G4s in solutions, and for effective assessment of DNA sequences and annealing environments on G4 polymorphism. The established protocol provides a novel strategy for evaluating G4 polymorphism, which will facilitate studies on quadruplex structures and their biophysical properties.

Expression and purification of RHC-EGFP fusion protein and its application in hyaluronic acid assay.

Hyaluronan is a widely distributed glycosaminoglycan which has multiple functions. Hyaluronic acid (HA) accumulation has been reported in many human diseases. Understanding the role of hyaluronan and its binding proteins in the pathobiology of disease will facilitate the development of novel therapeutics for many critical diseases. Current techniques described for the analysis of HA are mainly for HA quantification in solutions, not for the direct detection of HA in tissues or on cell surfaces. In our study, a fusion protein, named C-terminal domain of RHAMM-enhanced green fluorescence protein (RHC-EGFP), combined the HA-binding domain, C-terminal of receptor for hyaluronan-mediated motility, with EGFP, a widely used enhanced green fluorescence protein, was expressed and purified from Escherichia coli with high purity. Based on the sensitivity and convenience of fluorescence detection, methods for direct assay of HA in solutions, on cell surface or in tissues were established using RHC-EGFP. The binding specificity was also confirmed by competitive binding experiment and hyaluronidase degradation experiment. Our results provide an alternative choice for the specific and convenient assay of HA in various samples, and maybe helpful for further understanding of the fundamental and comprehensive functions of HA.

Effects of Different Zinc Species on Cellar Zinc Distribution, Cell Cycle, Apoptosis and Viability in MDAMB231 Cells.

Intracellular metal elements exist in mammalian cells with the concentration range from picomoles per litre to micromoles per litre and play a considerable role in various biological procedures. Element provided by different species can influence the availability and distribution of the element in a cell and could lead to different biological effects on the cell's growth and function. Zinc as an abundant and widely distributed essential trace element, is involved in numerous and relevant physiological functions. Zinc homeostasis in cells, which is regulated by metallothioneins, zinc transporter/SLC30A, Zrt-/Irt-like proteins/SLC39A and metal-response element-binding transcription factor-1 (MTF-1), is crucial for normal cellular functioning. In this study, we investigated the influences of different zinc species, zinc sulphate, zinc gluconate and bacitracin zinc, which represented inorganic, organic and biological zinc species, respectively, on cell cycle, viability and apoptosis in MDAMB231 cells. It was found that the responses of cell cycle, apoptosis and death to different zinc species in MDAMB231 cells are different. Western blot analysis of the expression of several key proteins in regulating zinc-related transcription, cell cycle, apoptosis, including MTF-1, cyclin B1, cyclin D1, caspase-8 and caspase-9 in treated cells further confirmed the observed results on cell level.

The Kinetic Response of the Proteome in A549 Cells Exposed to ZnSO4 Stress.

Zinc, an essential trace element, is involved in many important physiological processes. Cell responses to zinc stress show time-dependent effects besides concentration-dependence and tissue-specificity. Herein, we investigated the time-dependent differential expression of the proteome in A549 cells after administered with ZnSO4 for both 9 and 24 h using 2DE. 123 differentially expressed protein spots were detected, most of which were up-regulated by Zn2+ treatment. Interestingly, 49 proteins exhibited significant differential expression repeatedly during these two treatment periods, and moreover showed a conserved change with different ratios and four time-dependent expression patterns. Pattern 1 (up-regulated with rapid initial induction and subsequent repression) and pattern 4 (down-regulated with steady repression) were the predominant expression patterns. The abundances of the proteins in patterns 1 and 4 after 24 h of zinc treatment are always lower than that after 9 h, indicating that exogenous zinc reduced the expression of proteins in cells after 24 h or longer. Importantly, these findings could also reflect the central challenge in detecting zinc homeostasis proteins by 2DE or other high throughput analytical methods resulting from slight variation in protein expression after certain durations of exogenous zinc treatment and/or low inherent protein content in cells. These time-dependent proteome expression patterns were further validated by measuring dynamic changes in protein content in cells and in expression of two proteins using the Bradford method and western blotting, respectively. The time-dependent changes in total zinc and free Zn2+ ion contents in cells were measured using ICP-MS and confocal microscopy, respectively. The kinetic process of zinc homeostasis regulated by muffling was further revealed. In addition, we identified 50 differentially expressed proteins which are predominantly involved in metabolic process, cellular process or developmental process, and function as binding, catalytic activity or structural molecule activity. This study further elucidates our understanding of dynamic nature of the cellular response to zinc stress and the mechanism of zinc homeostasis.

Zn-responsive proteome profiling and time-dependent expression of proteins regulated by MTF-1 in A549 cells.

Zinc plays a critical role in many biological processes. However, it is toxic at high concentrations and its homeostasis is strictly regulated by metal-responsive transcription factor 1 (MTF-1) together with many other proteins to protect cells against metal toxicity and oxidative stresses. In this paper, we used high-resolution two-dimensional gel electrophoresis (2DE) to profile global changes of the whole soluble proteome in human lung adenocarcinoma (A549) cells in response to exogenous zinc treatment for 24 h. Eighteen differentially expressed proteins were identified by MALDI TOF/TOF and MASCOT search. In addition, we used Western blotting and RT-PCR to examine the time-dependent changes in expression of proteins regulated by MTF-1 in response to Zn treatment, including the metal binding protein MT-1, the zinc efflux protein ZnT-1, and the zinc influx regulator ZIP-1. The results indicated that variations in their mRNA and protein levels were consistent with their functions in maintaining the homeostasis of zinc. However, the accumulation of ZIP-1 transcripts was down-regulated while the protein level was up-regulated during the same time period. This may be due to the complex regulatory mechanism of ZIP-1, which is involved in multiple signaling pathways. Maximal changes in protein abundance were observed at 10 h following Zn treatment, but only slight changes in protein or mRNA levels were observed at 24 h, which was the time-point frequently used for 2DE analyses. Therefore, further study of the time-dependent Zn-response of A549 cells would help to understand the dynamic nature of the cellular response to Zn stress. Our findings provide the basis for further study into zinc-regulated cellular signaling pathways.

Herbal compound triptolide synergistically enhanced antitumor activity of vasostatin120-180.

Angiogenesis is essential for the survival and growth of most tumors. As such, targeting the tumor neovasculature is an attractive strategy for effective cancer therapy. Angiogenesis inhibitors have strong therapeutic potential as antitumor agents in suppressing tumor growth and metastatic progression. The functional domain within amino acid residues 120-180 of vasostatin (VAS) has been confirmed to be effective in inhibiting the proliferation, migration, and invasiveness of cancer cells by its suppressive capacity against angiogenesis. Triptolide (TPL) is an active component extracted from the traditional Chinese herbal medicine Tripterygium wilfordii Hook F that has shown antitumor activities in various cancer cell types. However, its therapeutic application is limited by its toxicity in normal tissues and complications caused in patients. In this study, we attempted to investigate the synergistic antitumor activity of TPL and VAS in solid tumor cells. Our results showed that the sensitivity of combined therapy using TPL and VAS was higher than that of monotherapy using TPL or VAS. Apoptosis induced by the combined treatment was accompanied by activation of caspase-9, caspase-8, and caspase-3. Upregulation of proapoptotic protein (Bax, Bak, and Bad) expression and suppression of NF-κB transcriptional activity and its targeting antiapoptotic genes (c-FLIP, cIAP, Bcl-2, Bcl-xl, and Mcl-1) may contribute to the synergistic effects of this combination therapy. Further, using a mouse xenograft model, we demonstrated that combined treatment completely suppressed tumor growth as compared with treatment with TPL or VAS alone. These results suggest that the combination of TPL and VAS at lower concentrations may produce a synergistic antitumor effect that warrants further investigation for its potential clinical applications.

Effects of exogenous zinc on cell cycle, apoptosis and viability of MDAMB231, HepG2 and 293 T cells.

As a non-toxic metal to humans, zinc is essential for cell proliferation, differentiation, regulation of DNA synthesis, genomic stability and mitosis. Zinc homeostasis in cells, which is crucial for normal cellular functioning, is maintained by various protein families including ZnT (zinc transporter/SLC30A) and ZIP (Zrt-, Irt-like proteins/SLC39A) that decrease and increase cytosolic zinc availability, respectively. In this study, we investigated the influences of a specific concentration range of ZnSO4 on cell cycle and apoptosis by flow cytometry, and cell viability by MTT method in MDAMB231, HepG2 and 293 T cell lines. Fluorescent sensors NBD-TPEA and the counterstain for nuclei Hoechst 33342 were used to stain the treated cells for observing the localisation and amount of Zn(2+) via laser scanning confocal microscope. It was found that the influence manners of ZnSO4 on cell cycle, apoptosis and cell viability in various cell lines were different and corresponding to the changes of Zn(2+) content of the three cell lines, respectively. The significant increase on intracelluar zinc content of MDAMB231 cells resulted in cell death, G1 and G2/M cell cycle arrest and increased apoptotic fraction. Additionally, the mRNA expression levels of ZnT and ZIP families in the three cell lines, when treated with high concentration of ZnSO4, increased and decreased corresponding to their functions, respectively.

Effects of exogenous zinc on the cellular zinc distribution and cell cycle of A549 cells.

As the second most abundant transition metal in humans, zinc plays essential roles in normal cellular biological functions, including metabolism, signalling, proliferation, gene expression and apoptosis. We use ZnSO(4) as a stressor in this study to investigate for the first time the effects of exogenous Zn(2+) on both the cellular distribution of zinc and zinc-related proteins and the cell cycle of human lung adenocarcinoma (A549) cells. The cellular distribution of zinc and soluble proteins was determined in the whole cell as well as in the cytoplasmic and nuclear fractions. Exogenous zinc in the tested exposure range (0-100 µM) resulted in an altered cellular distribution of both zinc and the soluble proteins, together with total glutathione (GSx), the ratio of glutathione (GSH) to glutathione disulfide (GSSG) and non-protein sulphydryl (NPSH). Surprisingly, a turning point was observed in the re-distribution trend at a concentration of approximately 50 µM ZnSO(4). It is concluded that there exists a regulatory system in A549 cells that maintains the cellular zinc content stable in the presence of a certain range of extracellular zinc concentration. In addition, an MTT assay and flow cytometric analysis showed that the ZnSO(4) treatment led to a bi-phasic variation in viability and a slight fluctuation in the apoptosis of A549 cells. Our results will help to further elucidate zinc-related cell biology and biochemistry.

Regulation of protein kinase C inactivation by Fas-associated protein with death domain.

Protein kinase C (PKC) plays important roles in diverse cellular processes. PKC has been implicated in regulating Fas-associated protein with death domain (FADD), an important adaptor protein involved in regulating death receptor-mediated apoptosis. FADD also plays an important role in non-apoptosis processes. The functional interaction of PKC and FADD in non-apoptotic processes has not been examined. In this study, we show that FADD is involved in maintaining the phosphorylation of the turn motif and hydrophobic motif in the activated conventional PKC (cPKC). A phosphoryl-mimicking mutation (S191D) in FADD (FADD-D) abolished the function of FADD in the facilitation of the turn motif and hydrophobic motif dephosphorylation of cPKC, suggesting that phosphorylation of Ser-191 negatively regulates FADD. We show that FADD interacts with PP2A, which is a major phosphatase involved in dephosphorylation of activated cPKC and FADD deficiency abolished PP2A mediated dephosphorylation of cPKC. We show that FADD deficiency leads to increased stability and activity of cPKC, which, in turn, promotes cytoskeleton reorganization, cell motility, and chemotaxis. Collectively, these results reveal a novel function of FADD in a non-apoptotic process by modulating cPKC dephosphorylation, stability, and signaling termination.

Codon usage biases in Alzheimer's disease and other neurodegenerative diseases.

Establishing codon usage biases are crucial for understanding the etiology of central nervous system neurodegenerative diseases (CNSNDD) especially Alzheimer's disease (AD) as well as genetic factors. G and C ending codons are strongly biased in the coding sequences of these proteins as a result of genomic GC composition constraints. On the other hand, codons that identified as translationally optimal in the major trend all end in C or G, suggesting translational selection should also be taken into consideration additional to compositional constraints. Furthermore, this investigation reveals that three common codons, CGC (Arg), AGC (Ser), and GGC (Gly), are also critical in affecting codon usage bias. They not only can offer an insight into the codon usage bias of AD and its mechanism, but also may help in the possible cures for these diseases.

Expression, purification and characterization of C-FADD.

FADD is an important proapoptotic adaptor in death receptor-induced apoptosis. Recently, FADD has been found to participate in a variety of non-apoptotic processes, such as development, cell cycle progression and survival. Its non-apoptotic activities were regulated by the phosphorylated status of the serine residue located at the C-terminal region, a domain distinct from the proapoptotic function related DED and DD domains. However, due to the difficulties in expression and crystallization of natural FADD, by far the molecular structures of all FADD variants did not contain the C-terminal region. To elucidate the structure-function relationship of C-terminal region, we need to obtain an FADD variant that containing C-terminal region. In this study, mouse FADD (80-205) containing DD domain and C-terminal region, designated as C-FADD, was expressed in E. coli with His-tag at the N-terminus and purified by Ni2+ affinity chromatography. The purified protein existed as a homogenous monomer in glutaraldehyde cross-linking analysis and exhibited a typical alpha-helix spectrum in CD (circular dichroism) assay. In vitro His-tag pull-down assay demonstrated that the purified C-FADD possessed the CK Ialpha-binding activity which was important for its non-apoptotic function.

Model and molecular dynamic simulations of active and inactive endo-beta-1,4-mannanase in tomato fruit.

Endo-beta-mannanase is a hemicellulase that is present in tomato fruit, and plays a role in its ripening. This enzyme protein is detectable in the cultivar Walter, but it is inactive due to the absence of the terminal four amino acids from its carboxyl-end. To elucidate why this deletion eliminates the activity of endo-beta-mannanase, a molecular dynamics (MD) study was conducted on the conformation of the enzyme at normal and elevated temperatures. The root mean square deviations, root mean square fluctuations per residue, and secondary structural evolution during MD simulations were analyzed. Differences in stability and dynamics between the active and inactive endo-beta-mannanases were documented; the inactive form has a lower stability than the active one. The loss of key amino acids from the C-terminal end of the protein indirectly affects the conformation of the catalytic Glu318 and stability of active site because of interactions between residues at the C-terminus and the rest of protein.