Calmodulins and calmodulin-like proteins-mediated plant organellar calcium signaling networks under abiotic stress

Plant calmodulins(CaMs)and calmodulin-like proteins(CMLs)mediate Ca~(2+)signaling in response to abiotic stresses.Manipulation of this signaling in crops could increase stress tolerance.We review methods for detecting Ca~(2+)signals,regulatory roles of Ca Ms and CMLs,binding targets,and Ca~(2+)networks under abiotic stress in organelles.

Calcium-binding ability of soy protein hydrolysates

This present study investigated the ability of various soy protein hydrolysates (SPHs) in binding calcium. It was demonstrated that the amount of Ca-bound depended greatly on the SPHs obtained using different proteases, which included: neutrase, flavourzyme, protease M and pepsin. The maximum level of Ca-bound (66.9 mg/g) occurred when protease M was used to hydrolyze soy protein. Peptide fragments exhibiting high Ca-binding capacity had molecular weights of either 14.4 or 8–9 kDa. The level of Ca-bound increased linearly with the increment of carboxyl content in SPHs, and further deamidation on SPHs from protease M improved Ca-binding of the hydrolysate.

Interaction of calcium- and integrin-binding protein 1 with integrin <i>α</i>11 and its possible involvement in pulmonary fibrosis

Integrin α11 (ITGA11) is one of the collagen-binding integrin α chains;however, its biological significance remains unknown. To determine the functions of ITGA11, we performed a yeast two-hybrid screen using the cytoplasmic domain of ITGA11 as bait and transformed an EGY48 yeast strain with the bait-containing plasmid using the plasmid from a human lung fibroblast cDNA library. This screen identified calcium- and integrin-binding protein 1 (CIB1) as prey. Recombinant ITGA11 and CIB1 were expressed in mammalian cells and used in coimmunoprecipitation experiments, which showed that full-length ITGA11 and CIB1 are also associated in vivo. Over-expression of CIB1 in the human lung myofibroblast MRC-5 cells decreased the expression of α-smooth muscle actin and fibronectin. Using a mouse model of pulmonary fibrosis (bleomycin-treatment), we detected elevated expression of CIB1 in lung tissues compared with controls. These data suggest that CIB1 may regulate pulmonary fibrosis in concert with IT-GA11.

Serum Proteins Stabilized Calcium Phosphate Nanoparticles and Its Effect on Bel-7402 Cells

Hydroxyapatite has a high affinity to biological macromolecules, especially to proteins. Bovine serum proteins were extracted to be used as stablizer to prepare calcium phosphate nanoparticles . 167.7 nm and 87.7 nm particles were respectively prepared by using bovine serum protein fractions at the concentration of 0. 5 mg/mL and 1.0 mg/mL. As the polysaccharide stabilized hydroxyapatite nanoparticles, the protein-stablized nanoparticles also inhibited the proliferation rate of Bel-7402 cells. It suggested that proteins could be applied to prepare calcium phosphate nanoparticles and it also has the anticaneer effect.

A new size and shape controlling method for producing calcium alginate beads with immobilized proteins

A method for producing size- and shape-con-trolled calcium alginate beads with immobilized proteins was developed. Unlike previous cal-cium alginate bead production methods, pro-tein-immobilized alginate beads with uniform shape and sizes less then 20 micrometers in diameter could successfully be produced by using sonic vibration. BSA and FITC-conjugated anti-BSA antibodies were used to confirm pro-tein immobilization in the alginate beads. Pro-tein diffusion from the beads could be reduced to less than 10% by cross-linking the proteins to the alginate with 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) and N-hydroxysul-fosuccinimide (NHSS). The calcium alginate beads could also be arranged freely on a slide glass by using a femtosecond laser.

Gene Expression of Atrial Calcium - Handling Proteins in Patients with Rheumatic Heart Disease and Atrial Fibrillation

Objectives To investigate the gene expression of calcium - handling proteins in patients with rheumatic heart disease (RHD) and atrial fibrillation (AF) . Methods A total of 50 patients with rheumatic mitral valve disease were included. According to cardiac rhythm and duration of episode of AF, patients were divided into four groups: sinus rhythm group, paroxysmal AF group, persistent AF for less than 6 months group and persistent AF for more than 6 months group. Atrial tissue was obtained from the right atrial appendage, the right atrial free wall and the left atrial appendage respectively during open heart surgery. Total RNA was isolated and reversly transcribed into cDNA. In a semi - quantitative polymerase chain reaction the cDNA of interest and of glyceralde-hyde3 - phosphate dehydrogenase (GAPDH) were amplified and separated by ethidium bromide - stained gel electrophoresis. Multiple liner regress was used for correlation between the mRNA amount and age, sex, right atrial diameter (RAd) and left atrial diameter (LAd) . Results The mRNA of L - type calcium channeled subunit, of Ca2+ - ATPase and of ryanodine receptor in patients with persistent AF for more than 6 months were significantly decreased ( P all < 0. 01) . But no alterations of the mRNA levels for SR phos-pholamban and calsequestrin were observed in patients with persistent AF for more than 6 months compared with patients with sinus rhythm, paroxysmal AF and persistent AF for less than 6 months ( P all > 0. 05) . There was no difference of the gene expression among the three atrial tissue sampling sites (P all > 0. 05) . Age, gender, RAd and LAd had no significant effects on the gene expression of calcium - handling proteins (P all>0. 05). Conclusions The mRNA expression of calcium - handling proteins is down - regulated only in patients with RHD and long - term persistent AF. Such abnormalities may be related to the initiation and/or perpetuation of AF in the patients with RHD.

Involvement of Calcium dependent Protein Kinases in ABA regulation of Stomatal Movement

Patch clamp techniques were employed to investigate if calcium dependent protein kinases (CDPKs) be involved in the signal transduction pathways of stomatal movement regulation by the phytohormone abscisic acid (ABA) in Vicia faba. Stomatal opening was completely inhibited by external application of 1 μmol/L ABA, and such ABA inhibition was significantly reversed by the addition of CDPK inhibitor trifluoperazine (TFP). The inward whole cell K + currents were inhibited by 60% in the presence of 1 μmol/L intracellular ABA, and this inhibition was completely abolished by the addition of CDPK competitive substrate histone Ⅲ S. The results suggest that CDPKs may be involved in the signal transduction cascades of ABA regulated stomatal movements.

CIPK9: a calcium sensor-interacting protein kinase required for low-potassium tolerance in Arabidopsis

Potassium is one of the major macro-nutrients essential for a number of cellular processes in plants. Low potassium level in the soil represents a limiting factor for crop production. Recent studies have identified potassium transporters that are involved in potassium acquisition, and some of them are critical for potassium nutrition under low potassium conditions. However, little is understood on the molecular components involved in low potassium signaling and responses. We report here the identification ofa calcineurin B-like protein-interacting protein kinase （CIPK9） as a critical regulator of low potassium response in ,Arabidopsis. The CIPK9 gene was responsive to abiotic stress conditions, and its transcript was inducible in both roots and shoots by potassium deprivation. Disruption of CIPK9 function rendered the mutant plants hypersensitive to low potassium media. Further analysis indicated that K^＋ uptake and content were not affected in the mutant plants, implying CIPK9 in the regulation of potassium utilization or sensing processes.

Altered expression of stromal interaction molecule(STIM)-calcium release-activated calcium channel protein(ORAI) and inositol1,4,5-trisphosphate receptors(IP_3Rs)in cancer:will they become a new battlefield for oncotherapy?

The stromal interaction molecule(STIM)-calcium release-activated calcium channel protein(ORAI) and inositol1,4,5-trisphosphate receptors(IP_3Rs) play pivotal roles in the modulation of Ca^(2+)-regulated pathways from gene transcription to cell apoptosis by driving calcium-dependent signaling processes.Increasing evidence has implicated the dysregulation of STIM-ORAI and IP_3Rs in tumorigenesis and tumor progression.By controlling the activities,structure,and/or expression levels of these Ca^(2+)-transporting proteins,malignant cancer cells can hijack them to drive essential biological functions for tumor development.However,the molecular mechanisms underlying the participation of STIM-ORAI and IP_3Rs in the biological behavior of cancer remain elusive.In this review,we summarize recent advances regarding STIM-ORAI and IP_3Rs and discuss how they promote cell proliferation,apoptosis evasion,and cell migration through temporal and spatial rearrangements in certain types of malignant cells.An understanding of the essential roles of STIM-ORAI and IP_3Rs may provide new pharmacologic targets that achieve a better therapeutic effect by inhibiting their actions in key intracellular signaling pathways.

Regulation of Microstructure of Calcium Carbonate Crystals by Egg White Protein

Crystal growth of calcium carbonate in biological simulation was investigated via egg white protein with different volume fractions,during which calcium carbonate was synthesized by calcium chloride and sodium carbonate.The morphology,thermal properties and microstructure of the calcium carbonate micro-to-nanoscale crystals were characterized by scanning electron microscopy（SEM）,transmission electron microscopy（TEM）,Fourier transform infrared spectroscopy（FTIR）,thermogravimetric analysis（TG） and X-ray diffraction（XRD） analysis.The results show that the volume fraction of egg white protein has great influence on the shape,size and morphology of calcium carbonate crystals.The calcium carbonate crystals were the mixtures of calcite-vaterite-like crystals including spherical and rough surface,which are different from that formed in pure water.With the increase of egg white protein concentration,the diameter of calcium carbonate crystals changed,the amount of formed spherical calcium carbonate particles decreased and that of vaterite increased.These results indicate that the coordination and electrostatic interaction between egg white protein and Ca2＋ significantly affect the calcium carbonate crystalization.

Hepatitis B Virus X Protein Upregulates Intracellular Calcium Signaling by Binding C-terminal of Orai1 Protein

Hepatitis B virus X(HBx)protein plays a pivotal role in the development of hepatitis B virus(HBV)-associated hepatocellular carcinoma.Although regulation of cytosolic calcium is essential for HBV replication and is mediated by HBx protein,the mechanism of HBx protein regulating intracellular calcium level remains poorly understood.The present study examined whether HBx protein elevated the intracellular calcium through interacting with storeoperated calcium entry(SOCE)components,Orai1 and stromal interaction molecule 1,and then identified the targets of HBx protein,with an attempt to understand the mechanism of HBx protein upsetting intracellular calcium homeostasis.By employing co-immunoprecipitation and GST-pull-down assay,we found that Orai1 protein interacted with HBx protein,and the C-terminus of Orai1 was implicated in the interaction.Confocal microscopy also revealed that HBx protein could co-localize with full-length Orai1 protein in HEK293 cells.Moreover,live cell calcium imaging exhibited that HBx protein elevated intracellular calcium,possibly by binding to SOCE components.Our results suggest that HBx protein binds to STIM1-Orai1 complexes to positively regulate the activity of plasma membrane store-operated calcium channels.

Dietary branched-chain amino acids modulate the dynamics of calcium absorption and reabsorption in proteinrestricted pigs

Background:Very low-protein(VLP)diets negatively impact calcium(Ca)metabolism and absorption.The objective of this study was to investigate the effect of supplemental branched-chain amino acids(BCAA)and limiting amino acids(LAA)on Ca digestibility,absorption and reabsorption in pigs fed with VLP diets.Forty-eight piglets were assigned to six treatments:positive control(PC),negative control(NC),and NC containing LAA 25%,LAA 50%,LAA+BCAA 25%(LB25)and LAA+BCAA 50%(LB50)more than recommendations.Results:Relative to PC or NC,LB25 and LB50 had higher digestibility of Ca and plasma Ca and phosphorus(P),but lower plasma vitamin D3.LB50 tended to increase vitamin D receptor transcript and protein in the gut,but decreased mRNA or protein abundance of parathyroid hormone 1 receptor(PTH1R),calbindin 1(CALB1),cytochrome P450 family 27 subfamily B member 1 and occludin in small intestine.LB50 increased the transcript of cytochrome P450 family 24 subfamily A member 1 and PTH1R but decreased the transcript of transient receptor potential cation channel subfamily V member 5,CALB1 and solute carrier family 17 member 4 in kidney.Conclusion:Overall,BCAA increased Ca digestibility through regulating the transcellular and paracellular Ca absorption in the gut and reabsorption in kidney during protein restriction.

Effect of Low Dose Radiation on Intracellular Calcium and Protein Kinase C in Lymphocytes

It is first reported in the present paper that whole-body irradiation (WBI) with low dose X-rays could increase intracellular calcium ions ([Ca2+]i) and stimulate protein kinase C (PKC) activity of mouse lymphocytes. Following WBI of male Kunming micc With 75 mGy X-rays at a dose rate of 12.5 mGy/min the mobilization of [Ca2+]i with Con A in CD4+ and CD8+ Cells in the thymus and spleen was potentiated and the amplitude of [Ca2+], mobilization in thymocytes in response to anti-CD3 monoclonal antibody increased with time from 4 to 24 h following low dose radiation. The PKC activity in the homogenate of spleen was markedly stimulated 12 h after WBl with 75 mGy, reaching its peak value at 24-48 h and coming down to lower than normal on day 7. However, the PKC activity in the separated T lymphocytes reached its peak value at 12 h and that in the B lymphocytes reached its peak value on day 4, both coming down to below control on day 7. The implications of this facilitation of signal transduction in T lymphocytes in the mechanism of immunoenhancement after low dose radiation were discussed

S100 calcium binding protein A6 and associated long noncoding ribonucleic acids as biomarkers in the diagnosis and staging of primary biliary cholangitis

BACKGROUND Primary biliary cholangitis(PBC)is a chronic and slowly progressing cholestatic disease,which causes damage to the small intrahepatic bile duct by immunoregulation,and may lead to cholestasis,liver fibrosis,cirrhosis and,eventually,liver failure.AIM To explore the potential diagnosis and staging value of plasma S100 calcium binding protein A6(S100A6)messenger ribonucleic acid(mRNA),LINC00312,LINC00472,and LINC01257 in primary biliary cholangitis.METHODS A total of 145 PBC patients and 110 healthy controls(HCs)were enrolled.Among them,80 PBC patients and 60 HCs were used as the training set,and 65 PBC patients and 50 HCs were used as the validation set.The relative expression levels of plasma S100A6 mRNA,long noncoding ribonucleic acids LINC00312,LINC00472 and LINC01257 were analyzed using quantitative reverse transcription-polymerase chain reaction.The bile duct ligation(BDL)mouse model was used to simulate PBC.Then double immunofluorescence was conducted to verify the overexpression of S100A6 protein in intrahepatic bile duct cells of BDL mice.Human intrahepatic biliary epithelial cells were treated with glycochenodeoxycholate to simulate the cholestatic environment of intrahepatic biliary epithelial cells in PBC.RESULTS The expression of S100A6 protein in intrahepatic bile duct cells was up-regulated in the BDL mouse model compared with sham mice.The relative expression levels of plasma S100A6 mRNA,log10 LINC00472 and LINC01257 were upregulated while LINC00312 was down-regulated in plasma of PBC patients compared with HCs(3.01±1.04 vs 2.09±0.87,P<0.0001;2.46±1.03 vs 1.77±0.84,P<0.0001;3.49±1.64 vs 2.37±0.96,P<0.0001;1.70±0.33 vs 2.07±0.53,P<0.0001,respectively).The relative expression levels of S100A6 mRNA,LINC00472 and LINC01257 were up-regulated and LINC00312 was down-regulated in human intrahepatic biliary epithelial cells treated with glycochenodeoxycholate compared with control(2.97±0.43 vs 1.09±0.08,P=0.0018;2.70±0.26 vs 1.10±0.10,P=0.0006;2.23±0.21 vs 1.10±0.10,P=0.0011;1.20±0.04 vs 3.03±0.15,P<0.0001,respectively).The mean expression of S100A6 in the advanced stage(III and IV)of PBC was up-regulated compared to that in HCs and the early stage(II)(3.38±0.71 vs 2.09±0.87,P<0.0001;3.38±0.71 vs 2.57±1.21,P=0.0003,respectively);and in the early stage(II),it was higher than that in HCs(2.57±1.21 vs 2.09±0.87,P=0.03).The mean expression of LINC00312 in the advanced stage was lower than that in the early stage and HCs(1.39±0.29 vs 1.56±0.33,P=0.01;1.39±0.29 vs 2.07±0.53,P<0.0001,respectively);in addition,the mean expression of LINC00312 in the early stage was lower than that in HCs(1.56±0.33 vs 2.07±0.53,P<0.0001).The mean expression of log10 LINC00472 in the advanced stage was higher than those in the early stage and HCs(2.99±0.87 vs 1.81±0.83,P<0.0001;2.99±0.87 vs 1.77±0.84,P<0.0001,respectively).The mean expression of LINC01257 in both the early stage and advanced stage were up-regulated compared with HCs(3.88±1.55 vs 2.37±0.96,P<0.0001;3.57±1.79 vs 2.37±0.96,P<0.0001,respectively).The areas under the curves(AUC)for S100A6,LINC00312,log10 LINC00472 and LINC01257 in PBC diagnosis were 0.759,0.7292,0.6942 and 0.7158,respectively.Furthermore,the AUC for these four genes in PBC staging were 0.666,0.661,0.839 and 0.5549,respectively.The expression levels of S100A6 mRNA,log10 LINC00472,and LINC01257 in plasma of PBC patients were decreased(2.35±1.02 vs 3.06±1.04,P=0.0018;1.99±0.83 vs 2.33±0.96,P=0.036;2.84±0.92 vs 3.69±1.54,P=0.0006),and the expression level of LINC00312 was increased(1.95±0.35 vs 1.73±0.32,P=0.0007)after treatment compared with before treatment using the paired t-test.Relative expression of S100A6 mRNA was positively correlated with log10 LINC00472(r=0.683,P<0.0001);serum level of collagen type IV was positively correlated with the relative expression of log10 LINC00472(r=0.482,P<0.0001);relative expression of S100A6 mRNA was positively correlated with the serum level of collagen type IV(r=0.732,P<0.0001).The AUC for the four biomarkers obtained in the validation set were close to the training set.CONCLUSION These four genes may potentially act as novel biomarkers for the diagnosis of PBC.Moreover,LINC00472 acts as a potential biomarker for staging in PBC.

Metabolomic changes in fatty liver can be modified by dietary protein and calcium during energy restriction

AIM： To characterise the effect of energy restriction （ER） on liver lipid and primary metabolite profile by using metabolomic approach. We also investigated whether the effect of energy restriction can be further enhanced by modification of dietary protein source and calcium. METHODS： Liver metabolomic profile of lean and obese C57BI/6J mice （n = 10/group） were compared with two groups of weight-reduced mice. ER was performed on control diet and whey protein-based high-calcium diet （whey ＋ Ca）. The metabolomic analyses were performed using the UPLC/MS based lipidomic platform and the HPLC/MS/MS based primary metabolite platform.RESULTS： ER on both diets significantly reduced hepatic lipid accumulation and lipid droplet size, while only whey ＋ Ca diet significantly decreased blood glucose （P 〈 0.001） and serum insulin （P 〈 0.01）. In hepatic lipid species the biggest reduction was in the level of triacylglycerols and cerarnides while the level of cholesterol esters was significantly increased during ER. Interestingly, diacylglycerol to phospholipid ratio, an indicator of relative amount of diabetogenic diglyceride species, was increased in the control ER group, but decreased in the whey ＋ Ca ER group （P 〈 0.001, vs obese）. ER on whey ＋ Ca diet also totally reversed the obesity induced increase in the relative level of lipotoxic cerarnides （P 〈 0.001, vs obese; P 〉 0.05, vs lean）. These changes were accompanied with up-regulated TCA cycle and pentose phosphate pathway rnetabolites. CONCLUSION： ER-induced changes on hepatic rnetabolornic profile can be significantly affected by dietary protein source. The therapeutic potential of whey protein and calcium should be further studied.

Effect of Wheat Middlings, Microbial Phytase, and Citric Acid on Phytate-Phosphorus, Calcium, and Protein Utilization of Broilers

A corn-soybean meal diet （CSB） （or Diet 1） containing 23% crude protein （CP） was used as the positive control, and another corn-soybean meal diet containing 21% CP and 15% wheat middlings （WM） （or Diet 2） was used as the basal diet, which was treated with four different treatments. Digestibility experiment was employed to discuss the collective effect of citric acid, and intrinsic and microbial phytase. By comparing and analyzing effects of them in the low-nutrient broiler diets, the results showed five treatments had similar effects on Tibia ash （%） （mg） （P〉0.05）. Under the supplementation of bacterial phytase or citric acid, the daily body weight gain （ADG）, gain：feed （G：F） ratio, and calcium （Ca） utilization were similar to that of standard-nutrient CSB diet （Diet 1） （P 〉 0.05）. And, fecal phosphorus （P） and CP utilization were lower than （P〈0.05） that of Diet 1. But P utilization was significantly higher than （P〈0.01） that of Diet 1. However, the ADG, G：F, and CP utilization produced by supplementation of intrinsic phytase were lower than those of Diet 1, but other aspects were similar to those produced by Diet 1 （P〉0.05）. In Diet 5, citric acid, intrinsic and bacterial phytase were added to the diet, which produced a 1.4% decrease on fecal P, a 7.2% increase on Ca utilization, which was significantly higher than （P〈0.01） those of the other four Diets, a 3.9% increase on G：F, which was similar to that of Diet 1, and a 2.3% increase on CP utilization, which was higher than （P〈0.05） that of the other three diets. In summary, the results of this study indicated that citric acid, intrinsic and bacterial phytase might have some additive or synergistic effects, and low-nutrient CSB diets with 15% wheat middlings, 750 U kg^-1 phytase, and 3% citric acid might substitute completely for standard CSB in broilers.

Phase Transformation of Amorphous Calcium Carbonate to Single-Crystalline Aragonite with Macroscopic Layered Structure in the Presence of Egg White Protein and Zinc Ion

Highly oriented calcium carbonate lamellas are exquisite structure produced by biomineralization. Strategies mimicking nature have been developed to synthesize inorganic materials with excellent structures and optimal properties. In our strategy, egg white protein and zinc ion were employed in the solution to induce the crystallization of calcium carbonate, resulting in the macroscopic aragonite laminate with an average length of 1.5 mm, which was comprised of single-crystalline tablets. During the crystallization at initial stage, it was found that the particles displayed the characteristics of amorphous calcium carbonate, which was then transformed into the sophisticated structured aragonite through a multistage assembly process. The rebuilt nacre structure in vitro was achieved owing to the synergistic effects of egg white protein and zinc ion.

Mutant amyloid precursor protein and presenilin-1 genes effect on ischemia vulnerability via calcium homeostasis disturbance

BACKGROUND： Previous studies have demonstrated that mutant amyloid precursor protein （APP） or presenilin-1 （PS1） genes increase susceptibility to ischemic brain damage induced by middle cerebral artery occlusion. Possible mechanisms include over-production of beta-amyloid peptide （Aβ）. OBJECTIVE： Because Aβ is over-produced in the APP/PS1 double-transgenic mouse, the present study focused on mechanisms of increased ischemic damage due to mutant APP and PS1 genes by measuring oxidative stress, mitochondrial function, and calcium homeostasis. DESIGN, TIME AND SETTING： The non-randomized, controlled, in vivo and in vitro experiments were performed at the Medical Research Center, Second Clinical College, Jinan University between May and October 2008. MATERIALS： Male APP transgenic mice carrying the mutant 695swe gene and female PS1 transgenic mice carrying the mutant Leu235Pro gene were donated from the University of Hong Kong. SHSY5Y human neureblastoma cells were purchased from ATCC （Manassas, VA, USA）, and Aβ1-42 was obtained from Sigma-Aldrich （St. Louis, MO, USA）. METHODS： APP transgenic mice were mated with PS1 transgenic mice to produce APP/PS1 double-transgenic mice and wildtype littermates mice. The photothrombotic stroke model was induced in six APP/PS1 double-transgenic and 6 wildtype littermates mice. SHSY5Y human neuroblastoma cells were cultured in vitro, and were divided into 4 groups： Aβ group, cells were exposed to 5 pmol/L Aβ for 24 hours; oxygen-glucose deprivation （OGD） group, cells were exposed to OGD for 1 hour after treatment with sterile, ultra-pure water for 24 hours; OGD＋Aβ group, cells were exposed to OGD and Aβfor 1 hour after treatment with 5 pmol/L Aβ for 24 hours; sham control group： cells were exposed to sterile, ultra-pure water for 25 hours. OGD was achieved by exposing the cells to glucose-free DMEM and placing the cells in an anaerobic chamber flushed with 5% CO2 and 95% N2 （v/v） at 37 ℃ for 1 hour. MAIN OUTCOME MEASURES： TTC staining was used to measure infarct volume 7 days after photothrombotic stroke. Cell viability was evaluated using the MTT kit. Opening of the mitochondrial permeability transition pore, intracellular concentration of superoxide anion, and calcium after OGD were detected with fluorescence intensity of calcein-AM, hydroethidine, and fluo-3/AM. RESULTS： At 7 days after stroke, total infarct volume and cortical infarct volume were significantly greater in the APP/PS1 transgenic mice compared with the wildtype littermates mice （P 〈 0.01）. Aβ, OGD, and Aβ ＋ OGD significantly decreased cell viability and increased fluorescence intensity of hydroethidine and fluo-3/AM （P 〈 0.01）. Compared with the Aβ or OGD group, Aβ ＋ OGD significantly decreased cell viability （P 〈 0.01） and significantly increased fluorescence intensity of calcein-AM, hydroethidine, and fluo-3/AM （P 〈 0.01 or P 〈 0.05）. CONCLUSION： The APP/PS1 double-transgenic mice were more vulnerable to ischemia. The possible mechanisms included enhanced opening of the mitochondrial permeability transition pore, overproduction of superoxide anion due to pore opening, and disturbed calcium homeostasis induced by excess superoxide anion.

Protein Adsorption of Calcium Phosphate Ceramics in vitro

In order to provide valuable information for the design of new calcium phosphate bone repair materials,bone tissue engineering scaffold materials, and other dinical application, the interaction between calcium phosphate materials and proteins were investigated. The adsorption of the calcium phosphate ceramic to the protein was investigated by using FT-IR, XPS, SEM, and SDS-PAGE. As the results shown, the proteins were strongly adsorbed adsorbed the CPC, and a shift of the feature peak of the protein and also a chemical shift in the Ca2p and O1s bind energy of CPC was observed. This indicated that the acidic amino-group and alkaline amino- residue on the proteins＇ surface bonded to the Ca^2＋ in the β- TCP crystal by ionic bond and the proteins＇ alkaline amino groups to the oxygen in PO4^3＋ by hydrogen bond and electrostatic attraction. The adsorption mechanism of the protein in the CPC can be described as three adsorption layers ： irreversible chemical adsorption layer, physical adsorption layer and biomineralized adsorption layer.

Ribotrap Analysis of Proteins Associated with FHL3 3'Untranslated Region in Glioma Cells

Objective To screen the proteins associated with four-and-a-half LIM domains 3(FHL3) 3' untranslated region(3'UTR) in glioma cells. Methods Western blot was adopted to detect the regulatory effect of poly(C)-binding protein 2(PCBP2) on FHL3. Biotin pull-down and sliver staining were employed to screen and verify the candidate binding proteins of FHL3 3'UTR. Then liquid chromatography-tandem mass spectrometry(LC-MS/MS) and molecule annotation system were used to identify and analyze the candidate binding proteins. Immunoprecipitation was conducted to study the interaction between PCBP2 and polypyrimidine tract-binding protein 1(PTBP1), a binding protein identified by LC-MS/MS. Results PCBP2 could bind to FHL3 mRNA 3'UTR-A and inhibited the expression of FHL3 in T98 G glioms cells. 22 candidate binding proteins were identified. Among them, there were 11 RNA binding proteins, including PCBP2. PTBP1 associated with FHL3 mRNA 3'UTR and interacted with PCBP2 protein. Conclusion PCBP2 and PTBP1 can both associate with FHL3 mRNA 3'UTR through forming a protein complex.