A new biosynthesis route for production of 5-aminovalanoic acid, a biobased plastic monomer / 生物工程学报

5-aminovalanoic acid (5AVA) can be used as the precursor of new plastics nylon 5 and nylon 56, and is a promising platform compound for the synthesis of polyimides. At present, the biosynthesis of 5-aminovalanoic acid generally is of low yield, complex synthesis process and high cost, which hampers large-scale industrial production. In order to achieve efficient biosynthesis of 5AVA, we developed a new pathway mediated by 2-keto-6-aminohexanoate. By combinatory expression of L-lysine α-oxidase from Scomber japonicus, α-ketoacid decarcarboxylase from Lactococcus lactis and aldehyde dehydrogenase from Escherichia coli, the synthesis of 5AVA from L-lysine in Escherichia coli was achieved. Under the initial conditions of glucose concentration of 55 g/L and lysine hydrochloride of 40 g/L, the final consumption of 158 g/L glucose and 144 g/L lysine hydrochloride, feeding batch fermentation to produce 57.52 g/L of 5AVA, and the molar yield is 0.62 mol/mol. The new 5AVA biosynthetic pathway does not require ethanol and H2O2, and achieved a higher production efficiency as compared to the previously reported Bio-Chem hybrid pathway mediated by 2-keto-6-aminohexanoate.

The Influence of Agitation Rates, pH and Calcium Carbonate on L-lysine Production by Bacillus subtilis using Agricultural Products as Carbon and Nitrogen Sources

L-Lysine is an essential amino acid that is required in the diet of humans and animals. It is utilized in human medicine, cosmetics and pharmaceutical industry. ’The influence of agitation rates, pH and calcium carbonate on L-lysine production by Bacillus subtilis using agricultural products as carbon and nitrogen sources was studied. The L-lysine-producing bacteria had already been isolated from Nigerian soil. They were purified and Identified as B. subtilis PR13 and B. subtilis PR9, using cultural, biochemical and molecular characteristics. Optimization of some parameters which included agitation rates, pH values and CaCO3 concentrations, on L-lysine production by the Bacillus species was carried out. The L-lysine was produced in 250 ml flasks containing fermentation media (FM1 and FM2). The findings revealed that, enhanced L-lysine yield of 2.10 and 1.33 mg/ml was observed at agitation rate of 180 rpm for B. subtilis PR13 and PR9 respectively. There was a positive correlation between agitation rates and L- lysine production by B. subtilis PR13 and PR9 (r = 0.96 and 0.83 respectively). The pH of 7.5, stimulated optimum L- lysine yield of 2.27 mg/ml for PR13 and 1.38 mg/ml for PR9. There was a positive correlation between pH values and L-lysine production by B. subtilis PR13 and PR 9 (r = 0.63 and 0.50 respectively). The supplementation of 40g/l of CaCO3, enhanced optimum L-lysine yield of 2.18 mg/ml for B. subtilis PR 13 and 1.30 mg/ml for B. subtilis PR9. There was a positive correlation between varying concentrations of calcium carbonate and L-lysine production by the B. subtilis PR13 (r =0.35), while negative correlation was observed for B. subtilis PR 9 (r = -0.10). The results obtained in the study illustrated that the optimization of process parameters could increase the L-lysine yield from agricultural products by B. subtilis PR13 and B. subtilis PR9.

Effect of poly (L-lysine) modified silk fibroin film on the growth and differentiation of neural stem cells / 生物工程学报

In order to provide a basic theory for the materials of repairing central nervous system injury, we have studied the growth and differentiation of neural stem cells (NSCs) on poly (L-lysine) modified silk fibroin film. First, we used poly (L-lysine) to modify silk fibroin film and confirmed by UV-vis and 1H NMR spectra. Then NSCs were isolated and seeded on the silk fibroin film (Silk group), poly (L-lysine) (PLL group) and poly (L-lysine) modified Silk fibroin film (Silk-PIL group). The proliferation of NSCs was evaluated by Cell Counting Kit-8 (CCK-8) assay on days 1, 3, 5 and 7 after seeding. Immunofluorescence was used to analyze the differentiation of NSCs at the 7th day. The levels of apoptosis were detected by Western blotting and TUNEL. The mRNA level of brain derived neurotrophic factor (BDNF) was identified by real-time PCR. UV-vis and 1H NMR spectra confirmed that poly (L-lysine) was successfully grafted onto the silk fibroin film. From the 3rd day after seeding to the 7th day, the CCK-8 test showed that proliferation rate of NSCs in the Silk-PIL was significantly higher than Silk group (P<0.05) but had no significant difference compared with PLL group (P>0.05). Immunofluorescence staining displayed that more NSCs in Silk-PIL group were differentiated into neuron compared with Silk group (P<0.05), however, there was no significant difference compared with PLL group (P>0.05). The number of NSCs differentiated into astrocytes was not significantly different between the three groups. Western blotting and TUNEL test presented that the degree of apoptosis of NSCs in the Silk-PIL group was significantly lower than Silk group (P<0.05). RT-PCR exhibited that mRNA level of brain derived neurotrophic factor (BDNF) of NSCs was higher in Silk-PIL group compared with Silk group (P<0.05) but had no significant difference compared with PLL group (P>0.05). Thus, poly (L-lysine) modified silk fibroin film could promote the proliferation of NSCs and reduce NSCs apoptosis. Furthermore, it also can enhance the differentiation of NSCs into neurons. It is expected to become a new type of tissue engineering scaffold carrying NSCs to repair central nervous system injury.

In vitro targeting efficiency evaluation of reduction-responsive co-loaded doxorubicin/siRNA nanoparticles / 药学学报

In this study a reduction-responsive nanoparticles (NPs) modified with hyaluronic acid (HA) was prepared for the co-delivery of doxorubicin (DOX) and siRNA and then evaluated as a lung cancer targeting delivery system in vitro. The amphiphilic polymer of poly-L-lysine-lipoic acid (PLA) based on poly-L-lysine (PLL) with lipoic acid (LA) was synthesized via amidation reaction and characterized by 1H NMR. The DOX loaded PLA NPs were prepared via dialysis method, and siRNA was loaded via electrostatic attraction to prepare the co-delivery NPs system (PLA/DOX-siRNA-NPs). Then PLA/DOX-siRNA-NPs were coated with HA to obtain HA-PLA/DOX-siRNA-NPs. The tumor microenvironment-responsive properties under different pH or reduction condition of HA-PLA/DOX-siRNA-NPs were evaluated by investigating the particle size and zeta potential. Cellular uptake of HA-PLA/DOX-siRNAFAM-NPs by A549 cells and endosomal escape of siRNA were studied using confocal laser scanning microscope (CLSM). 1H NMR spectrum demonstrated that PLA was successfully synthesized with LA grafting rate of 25.1%. The encapsulation efficiency (EE) and drug loading (DL) of HA-PLA/DOX-NPs was (86.93±8.91)% and (4.17±0.68)%, respectively, and siRNA was loaded at an N/P of 6:1 in carrier. HA-PLA/DOX-siRNA-NPs exhibited a suitable size of (167.3±9.9) nm and negative charge of (-15.5±1.4) mV with the optimal ratio of PLA and HA of 1:3. Additionally, the zeta potential of HA-PLA/DOX-siRNA-NPs significantly increased with charge reversal from negative to positive after the treatment with HAase, and the particle size of HA-PLA/DOX-siRNA-NPs changed significantly under the condition of 10 mmol·L-1 glutathione (GSH). The release profiles in vitro demonstrated that HA-PLA/DOX-NPs exhibited a maintained release behavior at pH 7.4 and the adding of GSH (10 mmol·L-1) led to rapid release of DOX from NPs. In vitro cellular uptake and subcellular distribution study demonstrated that themodification of HA enhanced the affinity of NPs to A549 cells and targeting ability, and the cellular uptake of HA-PLA/DOX-siRNAFAM-NPs significantly increased after the treatment with HAase. It was observed that HA-PLA/DOX-siRNAFAM-NPs could escape from endo-lysosomes followed by sharp payloads release to their relative targets. All these results demonstrated that the co-loaded NPs have a high entrapment efficiency of DOX and siRNA. And they also exhibited an active tumor targeting efficiency and tumor microenvironment-responsive properties, which were beneficial to cellular uptake and intracellular release of DOX and siRNA. In conclusion, these reduction-responsive NPs modified with HA have great potential as co-delivery systems for antitumor agents and siRNA.

Maillard Reaction Products in Powder Based Food for Infants and Toddlers.

Aim: To investigate the formation of Maillard reaction products (MRPs) during 28 days storage of the two most consumed brands (B1, B2) of gruel products on the Swedish market. Methodology: The MRPs; furfural, 5-hydroxymethyl furfural (HMF), N-(1-Carboxyethyl)-L-Lysine (CEL), N-(1-Carboxymethyl)-L-Lysine (CML), fluorescent advanced glycation end products (AGEs) and melanoidins (brown colour) were selected for analysis. High performance liquid chromatography coupled to UV spectrophotometry, fluorescence spectrophotometry or tandem mass spectrometry was used for analysis. Results: In general, MRPs were higher in B2 than in B1 at the time of opening the package. The initial content of MRPs in B1 and B2 respectively was as follows: 4.39 and 13.74 μg/g of furfural; 1.11 and 1.47 μg/g of HMF; 73.64 and 134.3 μg/g of total CML; 19.79 and 30.42 μg/g of total CEL; 51.11 and 73.01 AU/g of fluorescent AGEs; 0.52 and 1.45 AU/g of MRPs that absorb light at 420 nm and 1.40 and 3.22 AU/g of MRPs that absorb light at 360 nm. During storage for 28 days, furfural, HMF, MRPs that absorb light at 360 nm and at 420 nm as well as fluorescent MRPs increased significantly by respectively 7, 30, 60, 83 and 21% in B2. In B1, only the fluorescent MRPs (21%) increased during storage. Conclusion: A higher initial content and more pronounced increase of MRPs during 28 days storage time was observed in B2. Consequently, children consuming gruel from B2 are exposed to 1.3-3.1 times more MRPs compared to B1. Considering that a child often sticks to one gruel brand throughout the first years of life and that some MRPs are inflammatory drivers, more studies are required to understand the role of food-process induced chemicals at an early age for future health of the children.

Synthesis of barbell-like peptide dendrimer / 国际药学研究杂志

Objective To synthesize barbell-like peptide dendrimer. Methods Through the introduction of small molecule Fmoc-Gly-OH as the linker,NH2-CH2-COO-PEG2000-OOC-CH2-NH2 was obtained efficiently. And then N-α-N-ε-di-Fmoc-L-lysine and N-α-Fmoc-N-ε-Boc-L-lysine were used as branching agents,and barbell-like poly(ethylene glycol)-block-poly(L-lysine)dendrimer with a large number of surface amino groups was synthesized by the liquid-phase peptide synthesis method and divergent approach. Re?sults and Conclusion The structure and relative molecular mass of the final products and intermediates were characterized and con?firmed by 1H NMR and MS. The results revealed that barbell-like peptide dendrimer can be obtained by this method,which lays the foundation of its application in biological areas.

Synthesis of barbell-like peptide dendrimer / 国际药学研究杂志

Objective To synthesize barbell-like peptide dendrimer. Methods Through the introduction of small molecule Fmoc-Gly-OH as the linker, NH2-CH2-COO-PEG2000-OOC-CH2-NH2 was obtained efficiently. And then N-α-N-ε-di-Fmoc-L-lysine and N-α-Fmoc-N-ε-Boc-L-lysine were used as branching agents, and barbell-like poly(ethylene glycol)-block-poly(L-lysine)dendrimer with a large number of surface amino groups was synthesized by the liquid-phase peptide synthesis method and divergent approach. Results and Conclusion The structure and relative molecular mass of the final products and intermediates were characterized and confirmed by 1H NMR and MS. The results revealed that barbell-like peptide dendrimer can be obtained by this method, which lays the foundation of its application in biological areas.

Development of an L-Lysine Enriched Bran for Animal Nutrition via Submerged Fermentation by Corynebacterium glutamicum using Agroindustrial Substrates

L-Lysine is an essential aminoacid added as supplement for animal feed. The aim of this work was to produce an L-Lysine enriched bran using Brazilian agroindustrial byproducts. Both the raw material costs and purification steps were minimized. Firstly, medium composition for the growth of Corynebacterium glutamicum ATCC 21799 was optimized targeting enhanced L-Lysine production - salt, vitamins and nitrogen sources concentrations were tested and selected. Next, UV mutant strains were generated and the best producers were used in formulated media using sugarcane molasses. It was reached a production of 9.3 g/L of L-Lysine with the optimized formulated media. This L-Lyisne rich broth was then impregnated and cyclically reimpregnated in pre-treated solid matrixes (sugarcane bagasse, citrus pulp, brewer spent grain, soybean husk and wheat bran). After processing, it was generated enriched brans with significant amounts of L-Lysine (13.8%, 7.0%, 8.9%, 5.9% and 8.4%, respectively), which has an interesting market potential for animal feed.

Study on the preparation of ligand of hepatic asialoglycoprotein receptor / 国际生物医学工程杂志

Objective To optimize the preparation of high-efficiency galactocylated poly-L-lysine (Gal-PLL) ligand of the asialoglycoprotein receptor in liver, providing premise and foundation for upper preparation of ultrasound contrast agent of liver targeted nanoscale perfluorocarbon microballoon and the liver targeted molecular imaging. Methods Chemical reactions of reductive amination were carried out on group A and group B according to different proportions of reaction component. Each group was subdivided into three subgroups. In group A, three different molar ratios of D-galactose and poly-L-lysine (PLL) were compounded respectively with equivalent and sufficient reductant borohydride. In group B, identical molar ratios of D-galactose and PLL were compounded respectively with three unequal reductants borohydride. Products of each group were separated and purified by sephadex column to acquire different molecular weight distributions and the results were analyzed. Results In the condition of identical reductant, the peak curve of compound's molecular weight appeared earlier when D-galactose decreased properly. In the condition of identical molar ratio of D-galactose and PLL,the peak curve of compound's molecular weight appeared also earlier when reductant decreased properly. When the molar ratio of D-galactose and reductant was 1∶1, the peak curve of compound Gal-PLL and free components was more obvious, and the quantity of compound Gal-PLL increased to maximum. Conclusions In the condition of identical reductant, coupling effect of D-galactose and PLL increased when D-galactose decreased properly. In the condition of identical molar ratio of D-galactose and PLL, coupling effect was better when reductant decreased properly. When the molar ratio of D-galactose and reductant was 1∶1, coupling effect of them was the best. The coupling of D-galactose and PLL was related to not only the proportion of D-galactose and PLL, but also the proportion of D-galactose and reductant.

PRGD composite nerve conduit in repair of human peripheral nerve defect in upper extremity / 中华显微外科杂志

Objective To explore the clinical efficacy of PRGD composite nerve conduit in the treatment of human large-diameter,critical peripheral nerve defect in upper extremity.Methods From December,2011 to August,2014,19 patients with large-diameter,critical peripheral nerve defect in upper extremity were treated with PRGD composite nerve conduit.The patients were followeded-up periodically.The sensory and motor function recovery,high frequency ultrasound,and EMG were employed to assess the efficacy.Results The patients were followed up for an average time of 12-32 months(mean 21.75 ± 6.86 months),sensory and motor function recovered excellent in 7 patients,satisfactory in 7 patients,tolerable in 3 patients and no improvement in 2 patients were obtained according to the peripheral nerve function assessment standard built by British medical research council,the rate excellent and satisfactory results was 73.7％.Conclusion It is clinically promising to use PRGD composite nerve conduit to repair large-diameter,critical peripheral nerve defect in upper extremity,thus laying a foundation for its further application in clinical practice.

Cloning and sequence analysis of L-lysine decarboxylase gene in Huperzia serrata var. longipetiolata / 中草药

Objective: To obtain and analyze the full-length L-lysine decarboxylase (LDC) gene sequence of Huperzia serrata var. longipetiolata and predictively analyze its protein structure on the basis of cloning the coding region of LDC gene from four species of Huperziaceae. The species are Huperzia serrata var. longipetiolata, Phlegariurus minchegensis, Phlegariurus austrosinicus, and Phlegariurus petiolatus. Methods: The LDC coding region sequences were cloned by RT-PCR strategy with the template of total RNA extracted from the leaves. Then the sequences were analyzed by means of BLAST and MEGA 5.0. The full-length of LDC gene sequence of H. serrata var. longipetiolata was obtained by RACE technology. And then the secondary structure and three-dimensional structure of LDC protein were predictively analyzed. Results: The coding region sequences were highly similar to the lysine decarboxylase in the database. And the encoding protein of H. serrata var. longipetiolata was highly similar to the amino acid sequences of H. serrata in NCBI, and with high homology to Selaginella moellendorffii. The full-length LDC gene sequence of H. serrata var. longipetiolata contained 1 266 bp open reading frame and encodes a predicted protein of 403 amino acids. The GenBank accession number for this gene is KF040056. Conclusion: The LDC genes of the four species of Huperziaceae are cloned in this study. The full-length LDC gene sequence of H. serrata var. longipetiolata is obtained and analyzed, and its protein structure is predictively analyzed. The result will provide a foundation for exploring the mechanism of huperzine A biosynthesis in the plants of Huperziaceae.

Study on Electrochemical Behaviors and Determination of Xanthine and Uric Acid on Modified Glassy Carbon Electrode / 分析化学

The silver doped poly ( L-lysine ) modified glassy carbon electrode was fabricated by cyclic voltammetry, the surface of the electrode was characterized by scanning electron microscopy and electrochemical impedance spectroscopy. The electrochemical behaviors and the simultaneous detection of xanthine and uric acid were studied by cyclic voltammetry and differential pulse voltammetry. The results indicated that this modified electrode exhibited excellent electrocatalytic activity towards the oxidation of xanthine and uric acid. The stable oxidation peaks of xanthine and uric acid appeared with the peak potential of 0. 980V and 0. 600V respectively at the modified electrode in pH 3. 0 phosphate buffer solution. The oxidation peaks of xanthine and uric acid were separated at 380 mV. Under the optimum conditions, the linear ranges for the determination of xanthine and uric acid were 1 . 00 × 10-6-2 . 50 × 10-4 mol/L respectively by differential pulse voltammetry. The detection limits were 5. 0×10-7mol/L. The method has been applied to the simultaneous detection of xanthine and uric acid in healthy human urine with satisfactory results.

Biocompatibility of olfactory ensheathing cells and poly-L-lactic acid reinforced by chitosan in vitro / 中国组织工程研究

BACKGROUND:Previous studies have shown that composite scaffold of chitosan and poly-L-lactic acid has good biocompatibility with some cells. OBJECTIVE:To study the biocompatibility of poly-L-lactic acid reinforced by chitosan and olfactory ensheathing cells. METHODS:In experimental group, olfactory ensheathing cells from Sprague-Dawley rats aged 1-3 days were incubated onto chitosan-reinforced poly-L-lactic acid film. And in control group, olfactory ensheathing cells were co-cultured with poly-L-lysine. The proliferative ability of olfactory ensheathing cells was detected and the cells were observed with immunofluorescence histochemical staining at 1, 3, 5, 7 days after culture. RESULTS AND CONCLUSION:Olfactory ensheathing cells could survive on the chitosan-reinforced poly-L-lactic acid film, and the cytotoxic grade wasⅠ. Morphology of the cells in the experimental group was round or oval, with little processes and the cells aggregated into groups. One day after implantation, the periphery cells of the mass extended short projections and gradual y spread outward;3 days after implantation, the cells spread and most of the cells generated projections, most of which were bipolar or tri-polar;5 days after implantation, cel processes significantly extended, most cells were bipolar and tri-polar cells, while some were oval cells and irregular triangular cells;7 days after implantation, the cel density increased, and cel processes extended. Cel morphology of the control group had similar characteristics as the experimental group. There was no obvious difference between the control and the experimental group in number, perimeter or area of the cells (P>0.05). It showed that chitosan-reinforced poly-L-lactic acid had good biocompatibility with olfactory ensheathing cells.

Evaluation of umbilical cord mesenchymal stem cells labeling with superparamagnetic iron oxide nanoparticles coated with dextran and complexed with Poly-L-Lysine / Avaliação da marcação de células-tronco mesenquimais de cordão umbilical com nanopartículas superparamagnéticas de óxido de ferro recobertas com Dextran e complexadas a Poli-L-Lisina

Objetivo: O objetivo deste estudo foi avaliar o efeito da marcação de células-tronco mesenquimais obtidas da parede da veia do cordão umbilical com nanopartículas de óxido de ferro superparamagnéticas recobertas com dextran e complexadas a um agente transfector não viral denominado de Poli-L-Lisina. Métodos: A marcação das células-tronco mesenquimais foi realizada utilizando as nanopartículas de óxido de ferro superparamagnéticas recobertas com dextran complexadas e não complexadas a Poli-L-Lisina. As nanopartículas de óxido de ferro superparamagnéticas recobertas com dextran foram incubadas com o Poli-L-Lisina em um sonicador ultrassonico a 37ºC por 10 minutos, para a formação do complexo através de interação eletrostática. Em seguida, as células-tronco mesenquimais foram incubadas overnight com as nanopartículas de óxido de ferro superparamagnéticas complexadas e não com Poli-L-Lisina. Após o período de incubação as células-tronco mesenquimais foram avaliadas quanto à internalização do complexo nanopartícula de óxido de ferro superparamagnéticas /dextran/Poli-L-Lisina e nanopartícula de óxido de ferro superparamagnéticas /dextran através de ensaio citoquímico com azul de prússia. A viabilidade celular das célulastronco mesenquimais marcadas foi avaliada através do ensaio de proliferação celular utilizando o método de 5,6-carboxy-fluoresceinsuccinimidyl-ester e de morte celular através do método de anexinaiodeto de propídeo, ambos utilizando o recurso de citometria de fluxo. Resultados: Observamos nos ensaios citoquímicos que as célulastronco mesenquimais que foram marcadas com as nanopartícula de óxido de ferro superparamagnéticas /dextran sem a Poli-L-Lisina, não internalizaram com eficiência as nanopartículas devido pouca detecção de sua presença no interior das células. As células-tronco mesenquimais marcadas com o complexo nanopartícula de óxido de ferro superparamagnéticas /dextran/Poli-L-Lisina internalizaram com eficiência as nanopartículas devido à maior presença destas no interior das células. Os ensaios de viabilidade e morte celular demonstraram respectivamente que as células-tronco mesenquimais marcadas com as nanopartícula de óxido de ferro superparamagnéticas /dextran/Poli-L-Lisina continuam proliferando ao longo de sete dias e a porcentagem de células em apoptose inicial e tardia é baixa em relação à porcentagem de células vivas ao longo de três dias. Conclusão: Evidenciamos através de nossos resultados a necessidade da utilização da Poli-L-Lisina complexada com a nanopartícula de óxido de ferro superparamagnéticas /dextran para melhor internalização nas célulastronco mesenquimais. Paralelamente, demonstramos que este tipo de marcação não é citotóxico para as células-tronco mesenquimais já que os testes de morte e viabilidade celular mostraram que as células continuam vivas e proliferando.

Inhibition of polylysine-EDTA on rabbit lens epithelial cell growth in vitro / 中华实验眼科杂志

BackgroundPosterior capsule opacification(PCO) is the main cause inducing low vision after extacapsular cataract extraction. Our previous study determined that polylysine-ethylene diamine tetraacetic acid (EDTA) (PLE) can suppress the incidence of PCO. ObjectiveThe goal of this experiment was to investigate the inhibition of polylysine-EDTA on rabbit lens epithelial cells (LECs)proliferation in vitro and the effective concentrations of polylysine-EDTA. MethodsThe anterior capsular membranes from 10 3-month-old clean New Zealand white rabbits were digested and then cultured to obtain the LECs. The second and third generation of LECs were inoculated on the 96-hole culture plate with the cell density of the 1 × 105/ml. 12.5,25.0,50. 0,100. 0 μmol/Lof PLE were added into the culture medium for 48 hours respectively,and the DMSO medium was used at the same way as the control group. The proliferation of the LECs was then detected by MTT method and the inhibitory rate of PLE on LECs growth was calculated. ResultsLECs grew at a near normal state in ≤25.0 μmol/L PLE groups,however,cultured LECs were out of shape and the numbers decreased with the weakened adhesion ability in ≥50.0 μ mol/L PLE groups. The A490 values of LECs were 0. 278±0. 013,0. 266±0. 028,0. 260±0. 022 and 0. 247±0. 012 in 12. 5,25.0,50. 0, 100. 0 μmol/L polylysine-EDTA groups respectively and were lower than 0. 311 ±0. 038 of DMSO control group( P=0. 035,0. 011,0. 009,0.013 ). The inhibitory rates of 12. 5,25.0,50. 0, 100.0 μmoL/L PLE on LECs proliferation were 10.61％ , 14.47％ , 16.40％ and 20. 58％ respectively. ConclusionsPolylysine-EDTA can inhibit the growth and proliferation of LECs in vitro at a dose-dependent manner.

Nanoliposomes of L-lysine-conjugated poly(aspartic acid) Increase the Generation and Function of Bone Marrow-derived Dendritic Cells

BACKGROUND: Biodegradable polymers have increasingly been recognized for various biological applications in recent years. Here we examined the immunostimulatory activities of the novel poly(aspartic acid) conjugated with L-lysine (PLA). METHODS: PLA was synthesized by conjugating L-lysine to aspartic acid polymer. PLA-nanoliposomes (PLA-NLs) were prepared from PLA using a microfluidizer. The immunostimulatory activities of PLA-NLs were examined in mouse bone marrow-derived dendritic cells (BM-DCs). RESULTS: PLA-NLs increased the number of BM-DCs when added to cultures of GM-CSF-induced DC generation on day 4 after the initiation of cultures. Examination of the phenotypic properties showed that BM-DCs generated in the presence of PLA-NLs are more mature in terms of the expression of MHC class II molecules and major co-stimulatory molecules than BM-DCs generated in the absence of PLA-NLs. In addition, the BM-DCs exhibited enhanced capability to produce cytokines, such as IL-6, IL-12, TNF-alpha and IL-1beta. Allogeneic mixed lymphocyte reactions also confirmed that the BMDCs were more stimulatory on allogeneic T cells. PLA- NL also induced further growth of immature BM-DCs that were harvested on day 8. CONCLUSION: These results show that PLA-NLs induce the generation and functional activities of BM-DCs, and suggest that PLA-NLs could be immunostimulating agents that target DCs.

Nanoliposomes of L-lysine-conjugated poly(aspartic acid) Increase the Generation and Function of Bone Marrow-derived Dendritic Cells

BACKGROUND: Biodegradable polymers have increasingly been recognized for various biological applications in recent years. Here we examined the immunostimulatory activities of the novel poly(aspartic acid) conjugated with L-lysine (PLA). METHODS: PLA was synthesized by conjugating L-lysine to aspartic acid polymer. PLA-nanoliposomes (PLA-NLs) were prepared from PLA using a microfluidizer. The immunostimulatory activities of PLA-NLs were examined in mouse bone marrow-derived dendritic cells (BM-DCs). RESULTS: PLA-NLs increased the number of BM-DCs when added to cultures of GM-CSF-induced DC generation on day 4 after the initiation of cultures. Examination of the phenotypic properties showed that BM-DCs generated in the presence of PLA-NLs are more mature in terms of the expression of MHC class II molecules and major co-stimulatory molecules than BM-DCs generated in the absence of PLA-NLs. In addition, the BM-DCs exhibited enhanced capability to produce cytokines, such as IL-6, IL-12, TNF-alpha and IL-1beta. Allogeneic mixed lymphocyte reactions also confirmed that the BMDCs were more stimulatory on allogeneic T cells. PLA- NL also induced further growth of immature BM-DCs that were harvested on day 8. CONCLUSION: These results show that PLA-NLs induce the generation and functional activities of BM-DCs, and suggest that PLA-NLs could be immunostimulating agents that target DCs.

Detoxification of Glutaraldehyde Treated Porcine Pericardium Using L-arginine & NABH4

BACKGROUND: Calcification is the most frequent cause of clinical failure of bioprosthetic tissues fabricated from GA-fixed porcine valves or bovine pericardium. A multi-factorial approach using different mechanisms was recently developed to reduce the calcification of bioprosthetic tissues. The purpose of the present study was to evaluate the synchronized synergism of using L-arginine and NaBH4, compared with ethanol and L-lysine, in glutaraldehyde treated porcine pericardium from the standpoint of calcification and tissue elasticity. MATERIALS AND METHODS: Porcine pericardium was fixed at 0.625% GA (7 days at room temperature after 2 days at 4degrees C). An interim step of ethanol (80%; 1 day at room temperature) or L-lysine (0.1 M; 2 days at 37degrees C) or L-arginine (0.1 M; 2 days at 37degrees C) was followed by completion of the GA fixation. A final step of NaBH4 (0.1 M; 2 days at room temperature) was followed. Their tensile strength, thickness, and thermal stability were measured. Treated pericardia were implanted subcutaneously into three-week-old Sprague-Dawley rats for 8 weeks. Calcium content was assessed by atomic absorption spectroscopy and histology. RESULTS: L-arginine and NaBH4 pretreatment (1.81+/-0.39 kgf/5 mm p=0.001, 0.30+/-0.08 mm p<0.001) significantly increased tensile strength and thickness compared with the control (0.53+/-0.34 kgf/5 mm, 0.10+/-0.02 mm). In a thermal stability test, L-arginine and NaBH4 pretreatment (84.25+/-1.12degrees C, p=0.023) caused a significant difference from the control (86.25+/-0.00degrees C). L-lysine and NaBH4 pretreatment (183.8+/-42.6 ug/mg, p=0.804), and L-arginine and NaBH4 pretreatment (163.3+/-27.5 ug/mg, p=0.621) did not significantly inhibit calcification compared to the control (175.5+/-45.3 ug/mg), but ethanol and NaBH4 pretreatment did (38.5+/-37.3 ug/mg, p=0.003). CONCLUSION: The combined pretreatment using L-arginine and NaBH4 after GA fixation seemed to increase the tensile strength and thickness of porcine pericardium, fixed with GA. Additionally, it seemed to keep thermal stability. However it could not decrease the calcification of porcine pericardium fixed with GA. NaBH4 pretreatment seemed to decrease the calcification of porcine pericardium fixed with GA, but only with ethanol.

Physical Properties and Biocompatibility of Ba-Alginate-Poly-L-Ornithine-Alginate microcapsules / 中国康复理论与实践

@#ObjectiveTo test and verify whether Ba-alginate-Poly-L-Ornithine-Alginate microcapsules(B-PLO-A) can improve the physical properties and biocompatibility of the traditional BPA microcapsules.MethodsThe B-PLO-A and Ba-alginate-Poly-L-lysine-alginate（B-PLL-A） microcapsules were made by the static generator. The physical property of the microcapsules was evaluated by observing the morphological changes of the microcapsules in the hypotonic environment, changes in diameter of microcapsules in vitro culture and calculating broken microcapsules ratio by shaking method. The biocompatibility was observed by transplanting into peritoneal cavity of rat.ResultsB-PLO-A microcapsules are stronger and more stable in a hypotonic environment than B-PLL-A microcapsules. After 96 h mechanism shaking, the unbroken microcapsules ratio of B-PLO-A and B-PLL-A microcapsules were (99.3±1.0)% and (96.2±1.5)% respectively. The microcapsules were retrieved from peritoneal cavity of rat at 2, 4 and 8 weeks after transplantation, most of the microcapsules were of integrity, rotundity, and surface smooth without obviously bundled by connective tissue. 8 weeks after transplantation the intact microcapsules ratio of B-PLO-A and B-PLL-A microcapsules were (97.3±2.1)% and (95.4±2.4)% respectively.ConclusionB-PLO-A microcapsules as a whole have bettermechanical strength compared with B-PLL-A microcapsules, while maintaining a good biocompatibility.

Superparamagnetic iron oxide labeled mesenchymal stem cells and their magnetic resonance imaging in vitro / 第三军医大学学报

Objective To explore the optimal situation of labeling bone mesenchymal stem cells (BMSCs) with superparamagnetic iron oxides (SPIO) mediated by poly-L-lysine (PLL), and determine the most optimal protocol of magnetic resonance imaging according to the patterns of MR in vitro. Methods BMSCs were isolated from white rat and purified, incubated with SPIO-PLL complexes at the range of concentrations (0, 4.2, 8.4, 21, 42, 84 ?g Fe per ml medium). The labeling ratio and distribution of SPIO particles in BMSCs, and the morphological evidence of abnormal visualization were evaluated by Prussian blue staining, fluorescent microscope and electron microscopy. MTT growth curves and magnetic resonance imagings were obtained at the range of concentrations. Trypan blue exclusion test was performed to elevate the viability of BMSCs labeled with PLL at the range of concentrations (0, 0.05, 0.25, 0.5, 1.0, 5.0 ?g PLL per ml medium). Results The cellular labeling ratio was strongly correlated to the concentrations of SPIO (P