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1.
Amino-terminated dendrimers are well-defined synthetic hyperbranched polymers and have previously been shown to destabilize aggregates of the misfolded, pathogenic, and partially protease-resistant form of the prion protein (PrPSc), transforming it into a partially dissociated, protease-sensitive form with strongly reduced infectivity. The mechanism behind this is not known, but a low pH, creating multiple positively charged primary amines on the dendrimer surface, increases the efficiency of the reaction. In the present study, surface amines of the dendrimers were modified to yield either guanidino surface groups (being positively charged at neutral pH) or urea groups (uncharged). The ability of several generations of modified dendrimers and unmodified amino-terminated dendrimers to deplete PrPSc from persistently PrPSc-infected cells in culture (SMB cells) was studied. It was found that destabilization correlated with both the generation number of the dendrimer, with higher generations being more efficient, and the charge density of the surface groups. Urea-decorated dendrimers having an uncharged surface were less efficient than positively charged unmodified- (amino) and guanidino-modified dendrimers. The most efficient dendrimers (generation 4 (G4) and G5-unmodified and guanidino dendrimers) cleared PrPSc completely by incubation for 4 days at less than 50 nM. In contrast to both unmodified and guanidine-modified dendrimers, the uncharged urea dendrimers showed much lower cytotoxicity toward noninfected SMB cells. Therapeutic uses of modified dendrimers are indicated by the low concentrations of dendrimers needed. 相似文献
2.
In this research, a capillary electrophoretic method for the fast enantiomeric resolution of (R,S)-naproxen was investigated. Method development involved variation of applied potential, buffer concentration, buffer pH, and cyclodextrin concentration. The optimum electrophoretic separation conditions were 110 mM sodium acetate run buffer (pH 6.0), 30 mM methyl-beta-cyclodextrin, 20% (v/v) acetonitrile, 25 degrees C. The total length of capillary was 48 cm, (50 microm I.D.) with ultra violet (UV) detection at 232 nm. Using these conditions, the number of theoretical plates was close to one million (896,000/m). The possibility of achieving a fast chiral separation of (R,S)-naproxen on a microchip of 2.5 cm in length was investigated. Complete enantiomeric resolution of naproxen was achieved in less than 1 min, on this microchip platform, with linear imaging UV detection. This system had the advantage of real-time separation monitoring, so that enantiomeric resolution could be visually observed, and high-speed chiral analysis was realized. The microchip electrophoresis (MCE) separation was compared with the capillary electrophoresis (CE) separation with regards to speed, efficiency, separation platform, and precision. This work highlights the potential of CE and MCE in future chiral separations. 相似文献
3.
Dendrimers are highly branched macromolecules with the potential in biomedical applications. Due to positively charged surfaces, several dendrimers reveal toxicity. Coating peripheral cationic groups with carbohydrate residues can reduce it. In this study, the cytotoxicity and genotoxicity of three types of 4th generation poly(propylene imine) dendrimers were investigated. Peripheral blood mononuclear cells (PBMCs) were treated with uncoated (PPI-g4) dendrimers, and dendrimers in which approximately 40% or 90% of peripheral amino groups were coated with maltotriose (PPI-g4-OS or PPI-g4-DS) at concentration of 0.05, 0.5, 5 mg/ml. Abbreviations OS and DS stand for open shell and dense shell respectively, that describes the structure of carbohydrate modified dendrimers. After 1 h of cell incubation at 37°C, the MTT and comet assays were performed. PPI dendrimers demonstrated surface-modification-degree dependent toxicity, although genotoxicity of PPI-g4 and PPI-g4-OS measured by the comet assay was concentration dependent up to 0.5 mg/ml and at 5 mg/ml the amount of DNA that left comet's head decreased. Results may suggest a strong interaction between dendrimers and DNA, and furthermore, that coating PPI dendrimers by maltoriose is an efficient method to reduce their genotoxicity what opens the possibilities to use them as therapeutic agents or drug carriers. 相似文献
4.
Zunić G Jelić-Ivanović Z Colić M Spasić S 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,772(1):19-33
This report describes a rapid, single-run procedure, based on the optimization of capillary electrophoresis (CE) and indirect absorbance detection capabilities, which was developed for the separation and quantification of 30 underivatized physiological amino acids and peptides, usually present in biological fluids. p-Aminosalicylic acid buffered with sodium carbonate at pH 10.2+/-0.1 was used as the running electrolyte. Electrophoresis, carried out in a capillary (87 cm x 75 microm) at 15 kV potential (normal polarity), separated the examined compounds within 30 min. Limits of detection ranged from 1.93 to 20.08 micromol/l (median 6.71 micromol/l). The method was linear within the 50-200 micromol/l concentration range (r ranged from 0.684 to 0.989, median r=0.934). Within run migration times precision was good (median C.V.=0.7%). Less favorable within run peak area precision (median C.V.=6.6%) was obtained. The analytical procedure presented was successfully tested for separation and quantification of amino acids in physiological fluids, such as plasma or supernatant of macrophage cultures. Sample preparations require only a protein precipitation and dilution step. 相似文献
5.
Bellis E Hajba L Kovács B Sándor K Kollár L Kokotos G 《Journal of biochemical and biophysical methods》2006,69(1-2):151-161
Three generations of alpha,gamma-diaminobutyric acid modified poly(propyleneimine) dendrimers [DAB(AM)n, n = 4, 8, 16] containing 4, 8, 16 free amino groups were coupled with Boc-protected alpha,gamma-diaminobutyric acid (DABA) moieties in high yields. These modified dendrimers were deprotected and the chiral dendritic amines with 8, 16 and 32 amino groups on the surface were isolated in excellent yields. Dendrimers with cisplatin moieties at the periphery were obtained in the reaction of the free amine dendrimers and potassium tetrachloroplatinate(II). The highly insoluble complexes were isolated as hydrates and characterized by means of IR, TGA and elemental analysis. 相似文献
6.
Powerful capillary electrophoresis (CE) methods were developed for monitoring the reaction of ecto-5'-nucleotidase (ecto-5'-NT, CD73), a (patho)biochemically important enzyme that hydrolyzes nucleoside-5'-monophosphates to the corresponding nucleosides. The enzymatic reaction was performed either before injection into the capillary (method A) or directly within the capillary (method B). In method A, separation of substrates and products was achieved within 8 min using an eCAP fused-silica capillary (20 cm effective length, 75 microM i.d., UV detection at 260 nm), 40 mM sodium borate buffer (pH 9.1), normal polarity, and a constant voltage of 15 kV. In method B, the sandwich technique was applied; substrate dissolved in reaction buffer (10mM Hepes [pH 7.4], 2mM MgCl2, and 1mM CaCl2) was hydrodynamically injected into a fused-silica capillary (30 cm, 75 microM i.d.), followed by enzyme (recombinant rat ecto-5'-NT) and subsequent injection of substrate solution. The reaction was initiated by the application of 1 kV voltage for 1 min. The voltage was turned off for 1 min and again turned on at a constant voltage of 15 kV to elute products (nucleosides) within 4 min using borate buffer (40 mM, pH 9.1). Thus, assays could be performed within 6 min, including enzymatic reaction, separation, and quantification of the formed nucleoside. The CE methods were used for measuring enzyme kinetics and for assaying inhibitors and substrates. In addition, the online assay was successfully applied to melanoma cell membrane preparations natively expressing the human ecto-5'-NT. 相似文献
7.
Vávrová K Lorencová K Klimentová J Novotný J Hrabálek A 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2007,853(1-2):198-203
A simple HPLC/UV method for the determination of the transdermal permeation and dermal penetration of a broad-spectrum antiviral drug adefovir (PMEA) was developed. The separation was achieved on a C18 column with the mobile phase composed of 10 mM KH2PO4 and 2 mM Bu4NHSO4 at pH 6.0 and 7% acetonitrile. The method was validated with respect to selectivity, linearity (0.1-50 microg/ml), precision, accuracy, and stability. Transdermal permeation of 2% PMEA was studied in vitro using the Franz diffusion cell and porcine skin. The flux values were 1.8, 3.0, and 0.6 microg/cm2/h from aqueous donor samples at pH 3.4 and 7.4, and isopropyl myristate, respectively. The respective skin concentrations at 48 h were 294, 263, and 971 microg/g from these vehicles. These results will serve as a lead for further studies on transdermal and topical delivery of antivirals from the group of acyclic nucleoside phosphonates. 相似文献
8.
Poly(amidoamine) (PAMAM) dendrimer derivatives have been investigated for their biological applications, especially for delivery of drugs, including antimicrobial drugs to eukaryotic cells, but their effects on bacterial cells are largely unexplored. Herein we report that amino-terminated PAMAM dendrimers are highly toxic to the common Gram-negative pathogen Pseudomonas aeruginosa. The concentration that kills 50% of the bacteria (EC50) was in the range of approximately 0.9-1.5 microg/mL for the generation 5, amino-terminated dendrimers with or without partial (43%) coating of poly(ethylene glycol) (PEG). These EC50 values were lower than that ( approximately 1.9-2.8 microg/mL) for LL-37, a potent antimicrobial peptide expressed in a variety of epithelia. On the contrary, the dendrimers were far less toxic (EC50 > 21 microg/mL) to the Gram-positive pathogen Staphylococcus aureus than LL-37 (EC50 = approximately 1.9 microg/mL). In agreement with the previous studies on other cell types, the dendrimers were not cytotoxic to human corneal epithelial cells at the concentrations that were toxic to P. aeruginosa. Our findings indicate that amino-terminated PAMAM dendrimers and their partially PEG-coated derivatives possess attractive antimicrobial properties, particularly against Gram-negative bacteria, thus expanding the potential biological application of the dendrimers. 相似文献
9.
Polyether dendritic compounds bearing folate residues on their surface were prepared as model drug carriers with potential tumor cell specificity. Starting from ester-terminated polyether dendrimers, hydrazide groups were easily introduced to the surface of the dendrimers by reaction with hydrazine. Folate residues were then conjugated to the hydrazide chain ends of the dendrimers by direct condensation with folic acid in the presence of a condensing agent or by reaction with an active ester derivative of folic acid. Essentially complete functionalization of the terminal hydrazide groups was achieved for both the first and the second generation dendrimers with four and eight hydrazide groups. For the G-2 dendrimer with 16 hydrazide groups, an average number of only 12.6 folate residues were attached to each dendrimer. The conjugates are soluble in aqueous medium above pH 7.4. In addition, a similar conjugation of the antitumor drug methotrexate to the dendrimer was also investigated. Once optimized, these molecules may form the basis for a novel family of multivalent drug carriers. 相似文献
10.
Jicun Ren Zheng-Fa Fang 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,761(2):3037
A short chain poly(acrylamide–dimethylacrylamide) (PADMA) was synthesized in aqueous phase using isopropanol as a chain transfer agent, and was characterized according to the chemical composition and molecular mass. This polymer can form a stable dynamic coating on the inner surface of the capillary, thereby suppressing the electroosmotic flow and DNA–capillary wall interaction. The sieving medium has low viscosity and capillary filling with this medium and medium replacement were conveniently carried out by commercial capillary electrophoresis instruments. The effects of components and concentration of copolymers on the separation of DNA fragments were investigated. Highly efficient separation of DNA fragments, successful single strand conformation polymorphism (SSCP) analysis and good reproducibility of the migration time were obtained in bare capillaries using these copolymers as sieving media. Our preliminary results demonstrate that PADMA will become an alternative matrix for DNA separation by capillary electrophoresis. 相似文献
11.
Angelika Küng Thomas Pieper Rene Wissiack Erwin Rosenberg Bernhard K. Keppler 《Journal of biological inorganic chemistry》2001,6(3):292-299
High performance capillary electrophoresis (HPCE) as well as high performance liquid chromatography-mass spectrometry (HPLC-MS) have been applied to the separation, identification and quantification of the tumor-inhibiting ruthenium compounds HIm trans-[RuCl4(im)2] (im = imidazole) and HInd trans-[RuCl4(ind)2] (ind = indazole) and their hydrolysis products. The half-lives for the hydrolytic decomposition of the Ru(III) compounds were determined by monitoring the relative decrease of the original complex anion under different conditions by means of capillary electrophoresis. The decomposition follows pseudo-first-order kinetics. The rate constants in water at 25 degrees C are 1.102 +/- 0.091 x 10(-5) s-1 for HIm trans-[RuCl4(im)2] and 0.395 +/- 0.014 x 10(-5) s-1 for HInd trans-[RuCl4(ind)2]. About 8% of HIm trans-[RuCl4(im)2] but only about 2% of HInd trans-[RuCl4(ind)2] were hydrolyzed after 1 h at room temperature. Whereas the hydrolysis rate of the imidazole complex is independent of the pH value, the indazole complex hydrolyzes much faster at higher pH. The half-life of HInd trans-[RuCl4(ind)2] in phosphate buffer at pH 6.0 and 37 degrees C is 5.4 h, whereas it is less than 0.5 h at pH 7.4. In contrast to the imidazole complex, where no dependence on the buffer system was observed, hydrolysis of the indazole complex is even faster if a buffer containing hydrogen carbonate is used. The formation of [RuCl2(H2O)2(im)2]+ could be demonstrated by HPLC-MS measurements. In the case of the indazole complex, a release of the indazole ligands results in the formation of [RuCl4(H2O)2]-. 相似文献
12.
The high-resolution separation achievable with capillary electrophoresis has been applied successfully to the analysis of
glycoproteins. Inherent in the implementation of this technology for glycoprotein analysis is the use of specific buffer additives.
Bifunctional cationic reagents, such as simple alkyl chains bearing terminal amino or quaternary ammonium groups, have been
particularly useful for the analysis of ovalbumin, an excellent model glycoprotein. Although dynamic coating of the capillary
wall and the subsequent decrease in protein-wall interactions is known to be key in the effectiveness of these additives,
much remains to be learned regarding the mechanism through which they function. 相似文献
13.
Yu‐Yu He Jun‐Hui Zhang Qing Pu Sheng‐Ming Xie Yan‐Xia Li Lan Luo Xue‐Xian Chen Li‐Ming Yuan 《Chirality》2019,31(12):1053-1059
Chiral mesoporous silica (CMS) has attracted widespread attention because of some unique properties, such as high surface area, uniformly structured nanoscale cavities, and excellent chemical and thermal stability. In this work, we report the utilization of a CMS as the stationary phase for the separation of racemates in gas chromatography (GC). A CMS‐coated capillary column was fabricated by a dynamic coating method. Eighteen racemates belonging to different classes of organic compounds were separated on this column, including chiral alcohols, aldehydes, ketones, organic acids, halohydrocarbons, alkenes, alcohol amines, epoxides, and amino acid derivatives. In addition, linear alkanes, alcohols, and aromatic hydrocarbons have also been resolved. 相似文献
14.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,750(1):129-135
A simple, rapid and sensitive method has been developed and validated for the analysis of a mixed-backbone oligonucleotide (GEM 231) in tumor tissues. The analysis was performed using a capillary electrophoresis (CE) system with UV detection. An extended light path (bubble cell) capillary column of 64.5 cm (effective length 56 cm)×50 μm I.D. is used as the separation column. The optimized chromatographic conditions were background electrolyte: sodium borate buffer (60 mM, pH 9.1), electrokinetic injection: 10 s, applied voltage: 30 kV, detection at λ=210 nm. A linear relationship was observed between the peak area and the amount of GEM 231 in the range of 1.0–1000 μg/ml. The lower detection limit of the drug was 100 pg with an average recovery of about 75±5%. The inter-day and intra-day relative standard deviations were <10%. Assay validation studies revealed that CE method is reproducible and specific for the determination of GEM 231 in tissue homogenates with a run time of less than 5 min. 相似文献
15.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,681(1):77-82
Collagen type-I CNBr peptides were separated under acidic conditions by capillary electrophoresis. Separation conditions were: 100 mM phosphate buffer pH 2.5, 50 cm × 50 μm capillary (placed in a cartridge), 8 kV, running time 30–45 min, detection by UV at 200 nm. The peptides were separated strictly by their molecular mass and the overall pattern was well comparable to RP-HPLC separations of these analytes. It is proposed that the separation mechanism may involve hydrophobic sorptions to the capillary wall. 相似文献
16.
E. Renbutsu S. Okabe Y. Omura F. Nakatsubo S. Minami H. Saimoto Y. Shigemasa 《Carbohydrate polymers》2007,69(4):697-706
Novel chitosan derivatives with UV-curable functional groups, such as 3-methoxy-4-(2-hydroxy-3-methacryloyloxypropoxy)benzyl, 3,4-bis(2-hydroxy-3-methacryloyloxypropoxy)benzyl, 3-methoxy-4-methacryloyloxybenzyl, and 3,5-dimethacryloyloxybenzyl groups, were prepared. Introduction of photosensitive functional groups to chitosan was accomplished by reductive N-alkylation via Schiff’s bases using corresponding photosensitive aldehydes. Compared to starting chitosan, UV-curable chitosan derivatives showed better solubility in several organic solvents, such as DMSO and 70% methacrylic acid. The solubility of these compounds increased with an increase in the degree of substitution of the N-alkyl side chains. After UV irradiation for 20 s under a high-pressure mercury lamp at a distance of 15 cm from the samples, acidic methanol solutions of these derivatives were transformed to gels in the presence of photo-initiator, and their dried films adsorbed palladium (II) at pH 1.1 and pH 5.3. The UV-curable chitosan derivatives were successfully used as coating materials for electroless plating on non-conductive substances. 相似文献
17.
Detection of d-Serine in rat brain by capillary electrophoresis with laser induced fluorescence detection 总被引:2,自引:0,他引:2
Zhao S Yuan H Xiao D 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,822(1-2):334-338
A capillary zone electrophoresis method with laser induced fluorescence detection for the chiral separation of highly fluorescent enantiomeric derivatives of d/l-Serine from 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-d/l-Serine) was developed and optimized. Enantiomeric separation of NBD-d/l-Serine was accomplished by using 40 mM hydroxypropyl-beta-cyclodextrin (HP-beta-CD) contained in 100 mM borate buffer, pH 10.0. A 70 cm (effective length of 50 cm) uncoated fused-silica capillary at a voltage of 15 kV was used for the separation. The optimized electrophoretic conditions were subsequently applied to the analysis of d-Serine in rat brain, and satisfactory analytical results with respect to accuracy were obtained. This assay showed acceptable precision, with linearity in the d-Serine concentration range of 0.2-20.0 microM. The limit of detection for d-Serine was 3.0 x 10(-7)M. 相似文献
18.
Polyamidoamine (PAMAM) dendrimer represents one of the most efficient polymeric gene carriers. To investigate the effect of the core structure and generation of dendrimers on the complex formation and transfection efficiency, a series of PAMAM dendrimers with a trimesyl core (DT) at different generations (DT4 to DT8) were developed as gene carriers and compared with the PAMAM dendrimers derived from pentaerythritol (DP) and inositol (DI). The minimal generation number of DTs at which the dendrimer has enough amino group density to effectively condense DNA was higher (generation 6) than those of DPs and DIs (generation 5). DTs of generation 6 or higher condensed DNA into complexes with an average diameter ranging from 100 to 300 nm, but the 4th and 5th generations of DT (DT4 and DT5) formed only a severe aggregate with DNA. Interestingly, the DT6/pDNA complex was determined to be much smaller (100-300 nm) than those prepared with DP5 or DI5 (>600 nm) at N/P ratios higher than 15. The optimal generation numbers at which the dendrimers showed the highest transgene expression in COS-7 cells were 5 for DPs and DIs but 6 for DTs. The DT6/pDNAcomplex with smaller size mediated higher transgene expression in COS-7 cells than those prepared with DP5 or DI5. The in vitro transfection efficiency of the DT dendrimers as evaluated in HeLa cells, COS-7 cells, and primary hepatocytes decreased in the order of DT6 > DT7 > DT8 > DT5 > DT4. The transfection mediated by DT6 was significantly inhibited by bafilomycin A1. The acid-base titration curve for DT6 showed high buffer capacity in the pH range from 5.5 to 6.4 (pK(a) approximately 6). This permits dendrimers to buffer the pH change in the endosomal compartment. However, the transfection efficiency mediated by DT6 decreased significantly in the presence of serum in both HeLa cells and COS-7 cells. The cytotoxicity of DTs evaluated in HeLa cells using the 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide assay showed a trend of increasing toxicity with the polymer generations. The LD50 values of DT4 through DT8 were 628, 236, 79, 82, and 77 microg/mL, respectively, which were higher than that of poly(ethyleneimide) (18 microg/mL) and poly(L-lysine) (28 microg/mL) in the same assay. With a lower cytotoxicity and versatility for chemical conjugation, these PAMAM dendrimers with a DT core warrant further investigation for nonviral gene delivery. 相似文献
19.
Covalent-bonded immobilization of lipase from burkholderia cepacia onto two poly(phenylene sulfide) (PPS) dendrimers with different generations (two and three) was achieved using carbodiimide as a coupling reagent. The hydrolysis activity of olive oil to fatty acid was studied on enzyme-immobilized PPS dendrimers. Enzyme activity was proportional to the enzyme loading, and highest recovered activity was obtained at the medium enzyme loading for both G2 and G3 dendrimers. The immobilization improved the optimum pH and caused the temperature range to widen. Immobilization of enzyme has enhanced the thermal stability of enzyme activity in comparison with free enzyme. The immobilized enzyme as a biocatalyst for batch hydrolysis of olive oil retained 80 approximately 90% activity even after 20 times of recycling. This retention of activity after recycle is very valuable and powerful in enzyme technology. The present noteworthy and vital availability on enzyme reaction of the covalently bonded immobilized lipase on dendrimer came from the structure of dendrimer with a large number of functional terminal groups, which are easily available for immobilization of many lipases at the situation keeping reactive enzymes on the surface of dendrimer. 相似文献
20.
When lysozyme was treated with Cu(II) and H2O2 at pH 7.4, the protein underwent polymerization as well as changes in its fluorescent characteristics. Upon prolonged incubation, most of the protein aggregates were degraded into smaller peptides. Amino acid analysis indicated that the basic amino acid residues were most susceptible to the oxidation. Tryptophan residues were converted to N-formylkynurenine and kynurenine, and lysine residues were deaminated to form α-aminoadipic acid δ-semi- aldehyde. During Cu(II)H2O2 treatment, the formation of carbonyl groups was accompanied by the loss of free amino groups in the protein. Succinylation of free amino groups protected lysine residues from oxidation by Cu(II)H2O2, but failed to prevent polymerization. The studies with the modified lysozyme suggest that Cu(II)H2O2 can oxidize various amino acid residues in addition to lysine to generate different types of carbonyl compounds and these carbonyl compounds may be responsible for the formation of crosslinks in the polymerization process. 相似文献