Recombinant E.coli JM109, containing pHZ1818 plasmid which included the fused gene encoding human interleukin 6(IL 6), expressed a fusion protein with glutathion S transferase(GST). The fusion protein existed both...Recombinant E.coli JM109, containing pHZ1818 plasmid which included the fused gene encoding human interleukin 6(IL 6), expressed a fusion protein with glutathion S transferase(GST). The fusion protein existed both in the supernatant and inside the bacterial cell,but the insoluble protein had no biological activity and could not be refolded. The rotative speed of the shaker and the temperature of induction were optimized to maximize the expression of the soluble fusion protein. From the supernatant of the cell sonicates Glutathion Sephrose 4B affinity column chromatography was employed to isolate the fusion protein which could be purified to >80 0 0 in a single step. The yield of soluble GST IL 6 was about 10 mg per liter culture. The GST was site specifically cloven by 6 hours of treatment with thrombin and from the thrombin digest mixture IL 6 was purified by Q high performance ion exchange chromatography. From 1 liter of E.coli culture 2 mg refined IL 6 was obtained. The purified IL 6 had a purity of more than 95 0 0 and a biological activity of 1.02×10 8 IU/mg.展开更多
The stigmas of five species, Populus euphratica Oliv., P. alba L., P. simonii Carr., P. lasiocarpa Oliv. and P. nigra L. have been studied. Scanning electron microscopy re veals that exudates are present in the ...The stigmas of five species, Populus euphratica Oliv., P. alba L., P. simonii Carr., P. lasiocarpa Oliv. and P. nigra L. have been studied. Scanning electron microscopy re veals that exudates are present in the intercellular spaces, in the clefts between the multicel lular papillae and on the receptive surface. Release and movement of exudates can be visual ized when the fresh stigmas are stained with sudan Ⅲ and auramine O. Paraffin and semi thin resin sections of stigmas after glutaraldehyde osmium fixation evidence the lipidic nature of the exudates. Transmission electron microscopy reveals the glandular features of the stigmatic papillae cells, such as abundance of rough and smooth endoplasmic reticulum, polyribosomes, and well developed dictyosomes with secretory vesicles. Pellicle and epicuticular lamellate layers which have been considered as typical features of the dry type stigmas are also present in the species where stigmas appear extremely wet. It is concluded that stigmas in all of the five species are secretory at the receptive stage. Well developed generative and sperm cells were observed in the pollen tubes penetrating through the deep layers of the stigmatic tissue in the reciprocal crosses between P. euphratica and P. simonii, which indicated that there is no significant barrier in the stigma.展开更多
AIM:To investigate whether human acyl-CoA synthetase 5(ACSL5) is sensitive to the ACSL inhibitor triacsin C.METHODS:The ACSL isoforms ACSL1 and ACSL5 from rat as well as human ACSL5 were cloned and recombinantly expre...AIM:To investigate whether human acyl-CoA synthetase 5(ACSL5) is sensitive to the ACSL inhibitor triacsin C.METHODS:The ACSL isoforms ACSL1 and ACSL5 from rat as well as human ACSL5 were cloned and recombinantly expressed as 6xHis-tagged enzymes.Ni 2+-affinity purified recombinant enzymes were assayed at pH 7.5 or pH 9.5 in the presence or absence of triacsin C.In addition,ACSL5 transfected CaCo2 cells and intestinal human mucosa were monitored.ACSL5 expression in cellular systems was verified using Western blot and immunofluorescence.The ACSL assay mix included TrisHCl(pH 7.4),ATP,CoA,EDTA,DTT,MgCl 2,[9,103 H] palmitic acid,and triton X-100.The 200 μL reaction was initiated with the addition of solubilized,purified recombinant proteins or cellular lysates.Reactions were terminated after 10,30 or 60 min of incubation with Doles medium.RESULTS:Expression of soluble recombinant ACSL proteins was found after incubation with isopropyl betaD-1-thiogalactopyranoside and after ultracentrifugation these were further purified to near homogeneity with Ni 2+-affinity chromatography.Triacsin C selectively and strongly inhibited recombinant human ACSL5 protein at pH 7.5 and pH 9.5,as well as recombinant rat ACSL1(sensitive control),but not recombinant rat ACSL5(insensitive control).The IC50 for human ACSL5 was about 10 μmol/L.The inhibitory triacsin C effect was similar for different incubation times(10,30 and 60 min) and was not modified by the N-or C-terminal location of the 6xHis-tag.In order to evaluate ACSL5 sensitivity to triacsin C in a cellular environment,stable human ACSL5 CaCo2 transfectants and mechanically dissected normal human intestinal mucosa with high physiological expression of ACSL5 were analyzed.In both models,ACSL5 peak activity was found at pH 7.5 and pH 9.5,corresponding to the properties of recombinant human ACSL5 protein.In the presence of triacsin C(25 μmol/L),total ACSL activity was dramatically diminished in human ACSL5 transfectants as well as in ACSL5-rich human intestinal mucosa.CONCLUSION:The data strongly indicate that human ACSL5 is sensitive to triacsin C and does not compensate for other triacsin C-sensitive ACSL isoforms.展开更多
The synthesis route was investigated and optimized for the preparation of iminodiacetic acid-polyethylene glycol (IDA-PEG) for immobilized metal ion affinity partitioning in aqueous two-phase systems. IDA-PEG was synt...The synthesis route was investigated and optimized for the preparation of iminodiacetic acid-polyethylene glycol (IDA-PEG) for immobilized metal ion affinity partitioning in aqueous two-phase systems. IDA-PEG was synthesized from PEG in two steps by the reaction of iminodiacetic acid with a monosubstituted derivative of epichlorohydrin-activated PEG. The Cu2+ content combined with IDA-PEG was determined by atomic absorption spectrometry as 0.5 mol·mol^-1 (PEG). Furthermore, the affinity partitioning behavior of lactate dehydrogenase in polyethylene glycol/hydroxypropyl starch aqueous two-phase systems was studied to clarify the affinity effect of the Cu(Ⅱ)-IDA-PEG.展开更多
Objectives To study the expression patterns of two Eph family molecules, the receptor EphA5, and the ligand ephrin-A5, during spinal cord development. Methods The receptor expression was analyzed using beta-galactosid...Objectives To study the expression patterns of two Eph family molecules, the receptor EphA5, and the ligand ephrin-A5, during spinal cord development. Methods The receptor expression was analyzed using beta-galactosidase knockin mice, and affinity ligand probe binding. The ligand expression was assessed using two different affinity probes, and knockout mouse tissues as controls. Results EphA5 was expressed in the ventral spinal cord, while ephrin-A5 was located in the dorsolateral regions of the spinal cord throughout development. Conclusions These results show that EphA5 and ephrin-A5 are expressed over broad developmental stages and may play important roles in establishing the dorsoventral organization of the spinal cord.展开更多
It is a new strategy to immobilize cells on the inner wall of a capillary column and use affinity capillary electrophoresis（ACE） to study receptor-ligand interactions or to screen natural products and compounds synt...It is a new strategy to immobilize cells on the inner wall of a capillary column and use affinity capillary electrophoresis（ACE） to study receptor-ligand interactions or to screen natural products and compounds synthesized by combinatorial chemistry. In this paper, we developed a new method of immobilizing HEK293 cells on the inner wall of a capillary column. Four important experimental conditions were optimized, including cell injection density, PLL concentration, cell culturing time and sterile processing method. Immobilized cell-coated capillary columns prepared under the optimized experimental conditions exhibited good uniformity, stability and durability, which were suitable for capillary electrophoresis. The method could also be used to immobilize HEK293 cells over-expressing certain membrane receptors on the inner wall of a capillary. In this way, cell-coated capillary columns could be applied to ACE drug screening targeting certain membrane proteins.展开更多
Since aptamer and its in vitro selection process called SELEX were independently described by Ellington and Gold in 1990, extensive research has been undertaken and numerous isolated aptamers for various targets have ...Since aptamer and its in vitro selection process called SELEX were independently described by Ellington and Gold in 1990, extensive research has been undertaken and numerous isolated aptamers for various targets have been applied. Aptamers can bind to a wide range of targets that include small organic molecules, inorganic compounds, haptens and even whole cells with high binding affinity and specificity. Aptamers for a wide range of targets have been selected currently. In addition, aptamers are thermo stable and can also be regenerated easily within a few minutes denaturation, which makes them easy to store or handle. These advantages make aptamers extremely suitable for applications based on molecular recognition as analytical, diagnostic and therapeutic tools. In this review, the recent applications of aptamers for chemistry analysis, medicine and food security, along with the future trend will be discussed.展开更多
Infections of patients from consumption of contaminated pharmaceutical products constituted major health risk problems. Medicinal products are liable to microbial intrusion during in-use application. The current study...Infections of patients from consumption of contaminated pharmaceutical products constituted major health risk problems. Medicinal products are liable to microbial intrusion during in-use application. The current study focused on repeated contamination with constant level of microbiological burden by two bacteria viz. Staphylococcus aureus and Pseudomonas aeruginosa were used as dose-response models for infection through two different routes of administration. Nine different forms of insulin vials were subjected to this type of simulation study at constant assumed level of contaminations, preservative efficacy test(PET) and dose potency. Multi-spot contamination imitation study showed that initial fast rise in contamination, followed shortly by longer but steeper slope which finally turned into higher rate of contamination during the few last doses of the unit dosage forms, where the volume of the product became increasingly and progressively very small. When the probability of infection curves was constructed, both S. aureus and P. aeruginosa showed same pattern, with notably higher risk from septicemia route of the latter rather than subcutaneous route of the former. The present simulation study showed that continuous use of the same contaminated syringe progressively increased the risk of infection, especially at final few doses(between 3th and 10 th last doses depending on the dosage form sizes in the vials and the administration volumes) of the product. Small volume parenterals(SVP) are especially products at higher risk than the larger volume ones.展开更多
文摘Recombinant E.coli JM109, containing pHZ1818 plasmid which included the fused gene encoding human interleukin 6(IL 6), expressed a fusion protein with glutathion S transferase(GST). The fusion protein existed both in the supernatant and inside the bacterial cell,but the insoluble protein had no biological activity and could not be refolded. The rotative speed of the shaker and the temperature of induction were optimized to maximize the expression of the soluble fusion protein. From the supernatant of the cell sonicates Glutathion Sephrose 4B affinity column chromatography was employed to isolate the fusion protein which could be purified to >80 0 0 in a single step. The yield of soluble GST IL 6 was about 10 mg per liter culture. The GST was site specifically cloven by 6 hours of treatment with thrombin and from the thrombin digest mixture IL 6 was purified by Q high performance ion exchange chromatography. From 1 liter of E.coli culture 2 mg refined IL 6 was obtained. The purified IL 6 had a purity of more than 95 0 0 and a biological activity of 1.02×10 8 IU/mg.
基金the NationalNaturalScience Foundation ofChina (NNSFC)
文摘The stigmas of five species, Populus euphratica Oliv., P. alba L., P. simonii Carr., P. lasiocarpa Oliv. and P. nigra L. have been studied. Scanning electron microscopy re veals that exudates are present in the intercellular spaces, in the clefts between the multicel lular papillae and on the receptive surface. Release and movement of exudates can be visual ized when the fresh stigmas are stained with sudan Ⅲ and auramine O. Paraffin and semi thin resin sections of stigmas after glutaraldehyde osmium fixation evidence the lipidic nature of the exudates. Transmission electron microscopy reveals the glandular features of the stigmatic papillae cells, such as abundance of rough and smooth endoplasmic reticulum, polyribosomes, and well developed dictyosomes with secretory vesicles. Pellicle and epicuticular lamellate layers which have been considered as typical features of the dry type stigmas are also present in the species where stigmas appear extremely wet. It is concluded that stigmas in all of the five species are secretory at the receptive stage. Well developed generative and sperm cells were observed in the pollen tubes penetrating through the deep layers of the stigmatic tissue in the reciprocal crosses between P. euphratica and P. simonii, which indicated that there is no significant barrier in the stigma.
基金Supported by Deutsche Forschungsgemeinschaft, No. GA785/6-1Deutsche Krebshilfe, No. 109313the Rotationsprogramm of the Medical Faculty RWTH Aachen University (to Kaemmerer E)
文摘AIM:To investigate whether human acyl-CoA synthetase 5(ACSL5) is sensitive to the ACSL inhibitor triacsin C.METHODS:The ACSL isoforms ACSL1 and ACSL5 from rat as well as human ACSL5 were cloned and recombinantly expressed as 6xHis-tagged enzymes.Ni 2+-affinity purified recombinant enzymes were assayed at pH 7.5 or pH 9.5 in the presence or absence of triacsin C.In addition,ACSL5 transfected CaCo2 cells and intestinal human mucosa were monitored.ACSL5 expression in cellular systems was verified using Western blot and immunofluorescence.The ACSL assay mix included TrisHCl(pH 7.4),ATP,CoA,EDTA,DTT,MgCl 2,[9,103 H] palmitic acid,and triton X-100.The 200 μL reaction was initiated with the addition of solubilized,purified recombinant proteins or cellular lysates.Reactions were terminated after 10,30 or 60 min of incubation with Doles medium.RESULTS:Expression of soluble recombinant ACSL proteins was found after incubation with isopropyl betaD-1-thiogalactopyranoside and after ultracentrifugation these were further purified to near homogeneity with Ni 2+-affinity chromatography.Triacsin C selectively and strongly inhibited recombinant human ACSL5 protein at pH 7.5 and pH 9.5,as well as recombinant rat ACSL1(sensitive control),but not recombinant rat ACSL5(insensitive control).The IC50 for human ACSL5 was about 10 μmol/L.The inhibitory triacsin C effect was similar for different incubation times(10,30 and 60 min) and was not modified by the N-or C-terminal location of the 6xHis-tag.In order to evaluate ACSL5 sensitivity to triacsin C in a cellular environment,stable human ACSL5 CaCo2 transfectants and mechanically dissected normal human intestinal mucosa with high physiological expression of ACSL5 were analyzed.In both models,ACSL5 peak activity was found at pH 7.5 and pH 9.5,corresponding to the properties of recombinant human ACSL5 protein.In the presence of triacsin C(25 μmol/L),total ACSL activity was dramatically diminished in human ACSL5 transfectants as well as in ACSL5-rich human intestinal mucosa.CONCLUSION:The data strongly indicate that human ACSL5 is sensitive to triacsin C and does not compensate for other triacsin C-sensitive ACSL isoforms.
基金Supported by the National Natural Science Foundation of China(No.29736180).
文摘The synthesis route was investigated and optimized for the preparation of iminodiacetic acid-polyethylene glycol (IDA-PEG) for immobilized metal ion affinity partitioning in aqueous two-phase systems. IDA-PEG was synthesized from PEG in two steps by the reaction of iminodiacetic acid with a monosubstituted derivative of epichlorohydrin-activated PEG. The Cu2+ content combined with IDA-PEG was determined by atomic absorption spectrometry as 0.5 mol·mol^-1 (PEG). Furthermore, the affinity partitioning behavior of lactate dehydrogenase in polyethylene glycol/hydroxypropyl starch aqueous two-phase systems was studied to clarify the affinity effect of the Cu(Ⅱ)-IDA-PEG.
基金This work was supported in part by grants from New Jersey Commission on Spinal Cord Research and the National Science Foundation （No. 0548561,USA）.
文摘Objectives To study the expression patterns of two Eph family molecules, the receptor EphA5, and the ligand ephrin-A5, during spinal cord development. Methods The receptor expression was analyzed using beta-galactosidase knockin mice, and affinity ligand probe binding. The ligand expression was assessed using two different affinity probes, and knockout mouse tissues as controls. Results EphA5 was expressed in the ventral spinal cord, while ephrin-A5 was located in the dorsolateral regions of the spinal cord throughout development. Conclusions These results show that EphA5 and ephrin-A5 are expressed over broad developmental stages and may play important roles in establishing the dorsoventral organization of the spinal cord.
基金The National Natural Science Foundation(Grant No.81373372)Specialized Research Fund for the Doctoral Program of Higher Education of China(Grant No.20130001110059)
文摘It is a new strategy to immobilize cells on the inner wall of a capillary column and use affinity capillary electrophoresis（ACE） to study receptor-ligand interactions or to screen natural products and compounds synthesized by combinatorial chemistry. In this paper, we developed a new method of immobilizing HEK293 cells on the inner wall of a capillary column. Four important experimental conditions were optimized, including cell injection density, PLL concentration, cell culturing time and sterile processing method. Immobilized cell-coated capillary columns prepared under the optimized experimental conditions exhibited good uniformity, stability and durability, which were suitable for capillary electrophoresis. The method could also be used to immobilize HEK293 cells over-expressing certain membrane receptors on the inner wall of a capillary. In this way, cell-coated capillary columns could be applied to ACE drug screening targeting certain membrane proteins.
基金supported by the 863 Project(2012AA022703,2015AA020502)the National Natural Science Foundation of China(61271056)
文摘Since aptamer and its in vitro selection process called SELEX were independently described by Ellington and Gold in 1990, extensive research has been undertaken and numerous isolated aptamers for various targets have been applied. Aptamers can bind to a wide range of targets that include small organic molecules, inorganic compounds, haptens and even whole cells with high binding affinity and specificity. Aptamers for a wide range of targets have been selected currently. In addition, aptamers are thermo stable and can also be regenerated easily within a few minutes denaturation, which makes them easy to store or handle. These advantages make aptamers extremely suitable for applications based on molecular recognition as analytical, diagnostic and therapeutic tools. In this review, the recent applications of aptamers for chemistry analysis, medicine and food security, along with the future trend will be discussed.
基金supported and partially financially by HIKMA Pharma Pharmaceutical Company-2nd Industrial Zone-6th of October city,Egypt
文摘Infections of patients from consumption of contaminated pharmaceutical products constituted major health risk problems. Medicinal products are liable to microbial intrusion during in-use application. The current study focused on repeated contamination with constant level of microbiological burden by two bacteria viz. Staphylococcus aureus and Pseudomonas aeruginosa were used as dose-response models for infection through two different routes of administration. Nine different forms of insulin vials were subjected to this type of simulation study at constant assumed level of contaminations, preservative efficacy test(PET) and dose potency. Multi-spot contamination imitation study showed that initial fast rise in contamination, followed shortly by longer but steeper slope which finally turned into higher rate of contamination during the few last doses of the unit dosage forms, where the volume of the product became increasingly and progressively very small. When the probability of infection curves was constructed, both S. aureus and P. aeruginosa showed same pattern, with notably higher risk from septicemia route of the latter rather than subcutaneous route of the former. The present simulation study showed that continuous use of the same contaminated syringe progressively increased the risk of infection, especially at final few doses(between 3th and 10 th last doses depending on the dosage form sizes in the vials and the administration volumes) of the product. Small volume parenterals(SVP) are especially products at higher risk than the larger volume ones.
