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A natural amino acid, aspartic acid, was modified to bind to silica microspheres with a click chemistry method (-Click Asp).The modified Click Asp microspheres were further modified by incorporating Fe 3+ ions, resulting in Fe 3+ and Click Asp based immobilized ion affinity chromatography (IMAC)(Fe 3+ -Click Asp).The prepared materials were characterized using Fourier transform infrared spectrometry, X-ray photoelectron spectrometry, and scanning electron microscopy, and it was confirmed that Fe 3+ -Click Asp was successfully prepared.Subsequently, the Fe 3+ -Click Asp microspheres were used to enrich phosphopeptides from tryptic digests of proteins and milk.It was found that the phosphopeptides could be selectively enriched with this material.This study describes a novel material and enrichment method for phosphoproteomics.

In this study, a method was developed for the determination of dichloroacetonitrile (DCAN) in drinking water by liquid- liquid micro-extraction and gas chromatography/mass spectrometry ( LLE-GC/MS), which used 1,2-dibromopropane as the internal standard and methyl tertiary butyl ether (MTBE) as the extractant for high accuracy. The aspartic acid was used as the precursor of the DCAN formation during chlorination and the influencing factors were evaluated. The formation mechanism of DCAN was also discussed. The results showed that the DCAN amount increased with the increase of pH value under the neutral and acidic conditions, however, the amount of DCAN decreased with the increase of pH value under the alkali condition. And the final amount of DCAN under the alkali condition was much less than that under the neutral and acidic conditions. It was also found that the DCAN amount increased with the increase of chlorine addition, while the temperature in the range of 10-30°C had little influence on the DCAN formation. The formation process of the DCAN from aspartic acid by chlorination included seven steps, such as substitution, decarboxylation, oxidation, etc and ultimately formed DCAN.

The aim of this paper is to compare the cytotoxicity and cellular uptake efficiency of three kinds of poly(b-benzyl-L-amino) block-poly(ethylene glycol) nanoparticles (PXA-PEG-NPs) using Calu-3 cells, and select one as a nasal drug delivery vector for curcumin (Cur). Poly(gamma-benzyl-L-glutamate) block-poly(ethylene glycol) nanoparticles (PBLG-PEG-NPs), poly(gamma-benzyl-L-lysine) block-poly(ethyleneglycol) nanoparticles (PZLL-PEG-NPs) and poly(gamma-benzyl-L-aspartate) block-poly(ethylene glycol) nanoparticles (PBLA-PEG-NPs) were prepared by emulsion-solvent evaporation method. MTT assays were used to evaluate the cytotoxicity of PXA-PEG-NPs against Calu-3 cells. The cellular uptake of nanoparticles was visualized by an inverted fluorescence microscope and quantified by a flow cytometer. The results indicated that even at high concentration of 2 mg x mL(-1) the three nanoparticles had no cytotoxicity on Calu-3 cells. Compared to the curcumin solution, the three curcumin-loaded PXA-PEG-NPs showed significantly higher cellular uptake efficiency on Calu-3 cells (at equal concentration of curcumin with 5 microg x mL(-1) Cur solution), PBLG-PEG-NPs group was the highest. The cellular uptake increased with incubation time, and has positive correlation with nanoparticle concentration. In brief, PXA-PEG-NPs are conducive to delivery Cur into cells, and PBLG-PEG-NPs might be provided as a good nasal drug delivery carrier.

This paper was aimed to study conserved motifs of voltage sensing proteins (VSPs) and establish a voltage sensing model. All VSPs were collected from the Uniprot database using a comprehensive keyword search followed by manual curation, and the results indicated that there are only two types of known VSPs, voltage gated ion channels and voltage dependent phosphatases. All the VSPs have a common domain of four helical transmembrane segments (TMS, S1-S4), which constitute the voltage sensing module of the VSPs. The S1 segment was shown to be responsible for membrane targeting and insertion of these proteins, while S2-S4 segments, which can sense membrane potential, for protein properties. Conserved motifs/residues and their functional significance of each TMS were identified using profile-to-profile sequence alignments. Conserved motifs in these four segments are strikingly similar for all VSPs, especially, the conserved motif [RK]-X(2)-R-X(2)-R-X(2)-[RK] was presented in all the S4 segments, with positively charged arginine (R) alternating with two hydrophobic or uncharged residues. Movement of these arginines across the membrane electric field is the core mechanism by which the VSPs detect changes in membrane potential. The negatively charged aspartate (D) in the S3 segment is universally conserved in all the VSPs, suggesting that the aspartate residue may be involved in voltage sensing properties of VSPs as well as the electrostatic interactions with the positively charged residues in the S4 segment, which may enhance the thermodynamic stability of the S4 segments in plasma membrane.

A novel stationary phase was synthesized for chiral ligand-exchange chromatography via atom transfer radical polymerization (ATRP). Glycidyl methacrylate (GMA) was grafted onto the surface of the silica by ATRP using bromoisobutyryl bromide as an initiator, and the organic metal compound formed in the CuCl/2,2'-bipyridine(Bpy) system as a catalyst at room temperature. The chiral stationary phase was then synthesized by grafting L-phenylalanine on the surface of the silica. The stationary phase was characterized by means of elementary analysis and evaluated in detail to determine its separability. The amount of L-phenylalanine on the surface of silica was calculated to be 4.32 mg/m2. The results showed that the good enantioseparations of some DL-amino acids were obtained using ligand-exchange chromatography on the synthesized chiral stationary phase (50 degrees C) with 0.05 mol/L KH2PO4 and 0.1 mmol/L Cu(Ac)2 solution (pH 4.5) as the mobile phase at a flow rate of 1.0 mL/min and a wavelength of 223 nm. The influences of the mobile phase pH, concentration of Cu (II), and temperature of column on the resolution of DL-amino acids by ligand-exchange chromatography were investigated. The results showed that these conditions could affect the resolution of racemates. Compared with the column prepared by radical method using L-phenylalanine directly bonded onto the surface of the silica, the synthesized stationary phase showed a better separation ability, and the DL-aspartic acids and DL-asparagines could be separated at baseline.

Titanium dioxide (TiO2) is one of metal oxides widely used for phosphopeptide enrichment in phosphoproteomic research nowadays. However it can bind to some non-phosphorylated peptides containing one or more aspartic acid residues and/or glutamic acid residues. These non-phosphorylated peptides can be eluted along with phosphorylated peptides and cause the reduction of the selectivity. Conventional inhibitors for the non-specific binding of non-phosphorylated peptides can often contaminate the ion source of mass spectrometry and therefore their applications are limited in liquid chromatography-mass spectrometry (LC-MS). In this study, aspartic acid was reported as a novel non-specific binding inhibitor for phosphopeptide enrichment by titanium dioxide. Firstly, the tryptic peptide mixtures of 3 and 9 standard proteins were used for the comparison of the enrichment efficiency of titanium dioxide. The effects with the presence of aspartic acid, glutamic acid and no-inhibitor in the enrichment systems were compared separately. The results showed that aspartic acid can greatly improve the selectivity of titanium dioxide for phosphopeptide enrichment. Then, aspartic acid was used for the enrichment of tryptic peptide mixture of C57BL/6J mouse liver lysate and good results were also obtained which demonstrated that aspartic acid was a promising non-specific binding inhibitor for complex biological samples. Besides, no contamination in the ion source occurred during the mass spectrometric analysis.

Arg-Gly-Asp-(RGD) containing peptide characterized as the non-viral gene vector was synthesized to modify the surface of PLGA-(ASP-PEG). The Peptide (K16-GRGDSPC) was synthesized. PLGA-(ASP-PEG) was executed into chips A, B and C. Chip C was regarded as control. Chips A and B reacted with the cross-linker, then Chip A reacted with peptide. Mass spectrometry (MS) and high performance liquid chromatography (HPLC) detected the molecular weight and the purity of peptide. Sulphur in the surface of materials was detected by X-ray photoelectron spectrometry (XPS). The peptide content in the residual solution was detected by Spectrometer. HPLC showed the peptide purity was 94.13%; MS showed the molecular weight was 2741.26. XPS revealed the binding energy of the sulphur in reacted Chip A was 164 eV in reacted Chip B, 164eV and 162 eV; the ratios of carbon to sulphur in reacted Chip A and B were 99.746:0.1014 and 99.574:0.4255, respectively. There was no sulphur in Chip C. The optical density value (OD) of the resident solution was 0.069. The peptide density of reacted Chip A was 0.04 mg/mm2. The peptide was manufactured and linked to the surface of the biomimetic PLGA-(ASP-PEG) with the cross-linker.

Objective: To explore the adhesion,proliferation and osteodifferentiation of bone mesenchymal stem cells (BMSCs)on the prepared lactic acid/glycolic acid/asparagic acid-co-polyethylene glycol(PLGA-[ASP-PEG])tri-block polymer scaffolds., Methods: Modified PLGA with polyethylene glycol (PEG) and asparagic acid(ASP)that has many liga nds,and then the synthesis PLGA-[ASP-PEG] tri-block polymer material was prepared. BMSCs were cultured in PLGA-[ASP-PEG] polymer material and poly lactic acid-co-glycolic acid(PLGA)were used as control group. Precipitation method, MUT assay and total cellular protein detection were used to test the adhersion and proliferation of BMSCs. After the third generation of BMSCs was cultured on PLGA-[ASP-PEG] tri-block polymer scaffolds for 14 day and 28 day with osteogenic supplements,the osteodifferentiation of MSCs were observed through alkaline phosphatase(ALP) staining and calcium tubercle staining., Results: BMSCs grew adherent to the surface of PLGA-[ASP-PEG] polymer scaffolds and the number of BMSCs was much higher than that of PLGA. The precipitation method suggested that adhesion and proliferation of BMSCs on the surface of PLGA-[ASP-PEG] was much higher than the control group (P < 0.05). MTU assay showed that after BMSCs were cultured for 20 days,the absorbance A of PLGA-[ASP-PEG] polymer scaffolds and PLGA were 1.336 and 0.780 respectively. Total cellular protein could image the adhersion and proliferation of BMSCs indirectly. After BMSCs were cultured for 12 days,the total cellular protein of PLGA-[ASP-PEG] and PLGA were 66.44 microg/pore and 41.23 microg/pore respectively. PLGA-[ASP-PEG] polymer scaffolds had well biocompatibility and cell adhersion. The positive results with ALP staining and calcium tubercle staining in both groups indicated tri-block polymer scaffold and its degradations had no effect on osteodifferentiation., Conclusion: PLGA-[ASP-PEG]could improve the adhesion and proliferation of seed cells on bone-matrixmaterial, maintain the morphous of seed cells and had no obvious effect on cell osteodifferentiation.

Aspartic acid, as a biodegradable natural amino acid, was used to separate and remove the heavy metals from the sewage sludge based on chemical extraction technology. Under various conditions, the extraction processes were carried out for the sewage sludge from Shanghai Taopu Municipal Wastewater Plant. The comparison of extraction between aspartic acid and citric acid was also discussed for the separation of three heavy metals from sewage sludge. The results showed that pH and the dosage of aspartic acid or citric acid had a significant effect on the extraction efficiency. Zn, Ni and Cu can be apart extracted for more than 85% by aspartic acid at low pH. With the increment of pH value, the extraction ration decreased gradually for both two systems. Within the whole pH range, aspartic acid showed higher extraction efficiency for Ni, Cu than citric acid and the extraction efficiencies of aspartic acid for Ni, Cu were found to respectively be more than 50%, 40%. For the situation of Zn, citric acid had a higher extraction efficiency at pH > or = 3.0.

To characterize the roles played by surface-charged residue Asp44 in the structure stability of horse heart myoglobin, the code of Asp44, GAT, in the gene of horse heart myoglobin was changed into AAA for Lys by PCR site-directed mutagenesis. The mutant gene was ligated into PstI/BamHI-cut pGYM and the resulting plasmid was transformed into E. coli BL21. The mutant protein (D44K) was expressed in BL21 successfully. The bacteria containing mutant myoglobin were treated with lysozyme. Then the mutant protein was purified by ammonium sulfate precipitation, ion-exchange chromatography and gel filtration. Circular dichroism spectra were employed to monitor the kinetic behaviors of wild-type and mutant myoglobins' denaturation at different pHs or upon heating, and the "two-state" model was used to simulate the kinetic process of wild-type and mutant myoglobins' denaturation upon heating to determine the unfolding thermodynamic parameters of Mb and its mutant (D44K). The results show that the mutation of the surface-charged residue Asp44 to Lys44 can increase the protein's stability on its resistance to heat, resulting in the increase in the protein's denaturing mid-temperature by about 4 degrees C, from 71.9 to 75.1 degrees C, but shows little effect on its resistance to acid denaturation.

Using Sepharose CL-6B as support, 3-Chloro-1, 2-epoxypropane as activated agent, carboxymethylated aspartate (CM-Asp) as chelating ligand, A chelate affinity chromatographic medium based on Co2+, named Co-CM-Asp-Sepharose, was prepared and used to purify 6 x His-tagged fusion proteins. The amount of Co-CM-Asp-Sepharose reacted with 200 microL of lysate, the incubation time, wash condition and the imidazole concentration in the elution buffer were optimized. The purification results using Co-CM-Asp-Sepharose and Ni-NTA-Agarose (product of Qiagen) were compared. The CD155D1 fusion protein was also purified from 5mL of lysate and the amount of protein was determined by Bradford method. The results show that 60 microL of Co-CM-Asp-Sepharose (50% suspension) was suitable for the protein purification from 200 microL of lysate, the optimal incubation time of medium and lysate was 30 min, the optimal imidazole concentration in the eluting buffer was 200 mmol/L, and 200 microg of fusion protein was obtained. In a big scale experiment, 4.6 mg of fusion protein was obtained from 5 mL of lysate using 1.5 mL of Co-CM-Asp-Sepharose (50% suspension). Compared with Ni-NTA-Agarose, the Co-CM-Asp-Sepharose medium exhibits higher selectivity and the protein possesses higher purity.

In this study we examined the in vitro characteristics of MSCs adhesion to polypeptides modified surface of polymer PLGA-[ASP-PEG]. We study the adhesion of marrow stromal cells in biomaterials at diferrent times using a micropipette aspiration technique. Comparison the adhesion of PLGA-[ASP-PEG] combinating GRGDSPC polypeptides versus PLGA-[ASP-PEG]. The adhesive conditions of MSCs on the materials were observed by scanning electron microscope. Four hours after MSCs inoculating in biomerials, the cell adhesion force of PLGA-[ASP-PEG] is 172.78 +/- 15. 23N and the force of PLGA-[ASP-PEG] combinating GRGDSPC polypeptides is 209.47 +/- 92.59N. There are no difference between two biomaterials. After 12 hours,the adhesion force of PLGA-[ASP-PEG] combinating GRGDSPC polypeptides is 576.23 +/- 165.74N, and the cell force of PLGA-[ASP-PEG] is 261.84 +/- 100.09 N. There are very significant difference between the two biomaterials. However, after 24 hours,the adhesion forces of the two biomaterials have no difference. The density of MSCs on PLGA-[ASP-PEG]-GRGDSPC surface was much higher than that of PLGA-[ASP-PEG]. Combination polypeptides in the surface of biomaterials can enhance the adhesion of MSCs.

A series of Poly[aspartic acid-co-L-lysine](PAL) are copolycondensed by DL-aspartice acid and L-lysine with different ratios. Their constructions are identified by the spectra of 1H-NMR, FT-IR, X-Ray). These spectra are proved to have good regularity of these copolymers. alpha,beta-Poly[(N-hydroxypropyl/aminoethyl)-DL-Aspartamide-co-L-lysine] (PHAAL) is synthesized by ring-opening poly [aspartic acid-co-lysine] (PAL). PHAAL has good degradability in the phosphoric acid buffer solution (0.01 M, pH = 7.4) in the enzyme solution (Papain, Trypsine). PHAAL appeared tobe low cytotoxicity in Hela, ECV-304, Bcap37 cell lines, which was quantified by MTT assay. The combination ability of PHAAL with plasmid DNA was evaluated by agarose gel electrophoresis with agarose gel (1.0% w/v) containing ethidium bromide (0.25 microg/ml). The PHAAL with higher ratios of lysine in the copolymers have higher ability of condensing DNA. In summary, PHAAL, the polyaminoacid materials, could be one kind of macromolecule materials tobeused as the non-viral gene vehicle.

Objective: To explore the biocompatibility of poly (lactic acid/glycolic acid/asparagic acid-copolyethylene glycol) biomaterials (PLGA-ASP-PEG) and biological behaviors of cultured marrow stroml stem cells (MSCs) combined with this new type of scaffold in tissue engineering., Methods: The PLGA-ASP-PEG tri-block copolymers were obtained through bulk ring-opening copolymerization method. MSCs were isolated from the bone marrow of 4-week-old New Zealand rabbits. The 3rd-generation MSCs were cultured combining with PLGA-ASP-PEG in vitro, while cells cultured in PLGA as control group. The cell adhesion rate and the adhesive power were examined by conventional precipitation method and micropipette aspiration technique respectively. The morphological features were studied by scanning electron microscope. The proliferation behavior of the cells was analyzed by MTT assay. The cell cycle, proliferation index, DNA index and apoptosis of the cells were detected by flow cytometry. The synthesis of protein and collagen were examined by Coomassie Brilliant Blue dyes and 3H-Proline incorporation test., Results: The MSCs adhered and grew well on the surface of the biomaterial PLGA-ASP-PEG. The powers of cell adhesion, proliferation and protein and collagen synthesis of the cells were all significantly higher than those of PLGA group (P< 0.05), but the apoptosis rate was significantly lower than that of PLGA group (P < 0.05). The DNA indexes showed the cells of both PLGA-ASP-PEG group and PLGA group were normal diploid cells., Conclusion: PLGA-ASP-PEG showed good biocompatibility and the biological properties improved greatly compared with the PLGA scaffold materials. These results demonstrated that the promise of PLGA-ASP-PEG can be used as an ideal scaffold material for construction of tissue engineered bone to restore bone defects in bone tissue engineering.

To prepare poly(lactic acid/glycolic acid/ asparagic acid-co- polyethylene glycol) (PLGA-[ASP-PEG]) and examine the cellular biocompatibility. PLGA-[ASP-PEG] was obtained by bulk ring-opening copolymerization method, examined by infrared spectrometry (IR) and 1H nuclear magnetic resonance spectroscopy (1H NMR). Bone marrow stromal cells(BMSCs) were cultured with PLGA-[ASP-PEG] (experiment gruop) and PLGA (control group) in vitro respectively, and were observed by phase-contrast microscopy and scanning electron microscopy. The resuls showed that PLGA-[ASP-PEG] was obtained and proved by IR and 1H NMR. The BMSCs of the experiment group could well attach to and extend on the surface of the PLGA-[ASP-PEG], and could proliferate and secrete better extracellular matrix, compared with control. The PLGA-[ASP-PEG] has good cellular a biocompatibility. It can be used as a biomaterial for bone tissue engineering.

This study was aimed at investigating the effect of glutamate on motor neurons in organotypic cultured spinal cord slices treated by threohydroxyaspartate (THA), an inhibitor of glutamate transporter. The spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentration of THA(50 micromol/L,100 micromol/L,500 micromol/L) was added into the culture medium respectively. Ventral alpha-motor neurons survival was evaluated by immunohistochemistry staining monoclonal antibody SMI-32, a nonphosphorylated neurofilament marker, and interneurons in dorsal horn were identified by monoclonal anti-calretinin antibody staining. Lactate dehydrogenase (LDH) level in the culture medium was also measured. The spinal cord slices in the control group could maintain excellent organotypic cellular organization and a stable population of ventral alpha-motor neurons. THA caused a slow dose-dependent loss of alpha-motor neurons and an increase in LDH enzyme activity in the culture medium while dorsal interneurons were less damaged. 100 micromol/L THA resulted in a significant decrease in (alpha-motor neurons after cultured for 4 weeks. On the contrary, the interneurons in the dorsal horn were less affected. It was also observed in patients with amyotrophic lateral sclerosis (ALS). Excellular Glu mainly caused selective alpha-motor neuron death, and motoneurons were more sensitive to glutamate excitotoxicity than sensory neurons in the spinal cord.

The adsorption state and the characteristics of L-aspartic acid adsorbed on silver sol were studied by the Surface-Enhanced Raman Scattering (SERS) method. Strong Raman signals were detected in the experiments. The results suggested that L-aspartic acid adsorbed on the silver surfaces through carboxyl and nitrogen atoms since the signals were mainly due to the carboxyl and the nitrogen of the molecule of L-aspartic acid. The adsorption of carboxyl on the silver surfaces is chemical adsorption, which is based on the mechanism of charge-transfer, while the adsorption of nitrogen on the silver surfaces is physical type, which is due to the electromagnetic mechanism. The intensity of surface-enhanced Raman scattering of L-aspartic acid adsorbed on silver surface was also analyzed, and it was found that the intensity of surface-enhance Raman scattering will change with different concentrations of L-aspartic acid adsorbed on the silver surfaces. The intensity will reach the top value when the concentration of L-aspartic acid was 10(-3) mol x L(-1). When the concentration of L-aspartic acid decreased to 10(-4) mol x L(-1), the intensity of surface-enhanced Raman scattering became a little weaker than that with 10(-3) mol x L(-1). With further decrease in the concentration of L-aspartic acid, the intensity of surface-enhanced Raman scattering also decreased gradually. When the concentration of L-aspartic acid decreased to 10(-6) mol x L(-1), the intensity of surface-enhanced Raman scattering was very low. The above study will be helpful to the further study of peptide and other complex biological systems.

For the purpose of increasing the hydrophilicity of poly aspartic acid, a series of polymer of L-aspartic acid and 4-aminobutanoic acid with different ratios (mol/mol) were prepared. The copolymers were characterized by 13CNMR, DSC and x-ray. The confirmed the structures of the polymers. In-vitro tests of release at phosphate buffer saline, enzyme solution of trypsin and papain (37.0 degrees C, pH = 7.4) were carried out. The result indicated that the polymers could be degraded in some degree, and that 4-aminobutanoic acid segments accelerated the degradation rate of the polymers. Skin irritation test and systemic acute toxicity test were carried out, which showed that the polymer was a nontoxic biomedical material.

Two polyasparamide derivants having different hydrophilicities were selected to be degraded in vivo and in vitro. The results demonstrate that the degradation process of polyasparamide is enzymatic hydrolysis. Materials with different hydrophobicities are different in degradation rate and the same material has different metabolic rates in the planting position, kidney and liver. The release results of two polymeric drugs indicate that protease is of advantage to release of drug, and hydrophobicity of material is also an advantage to the release.

A biodegradable condensation polymer alpha,beta-poly (3-hydroxypropyl, propyl)-DL-asparamide has been synthesized. Its monomer is DL-aspartic-acid and its spacers are mixture of 3-amino-propanol-1 and propylamine. The polymers thus synthesized are characterized by 1H-NMR, water sorption and intrinsic viscosity. For the purpose of applicability, we have synthesized the polymers with different properties in reference to the molar ratios of propyl amine to 3-amino-hydroxypropyl in polymer synthesis.