RESUMEN
The post-translational modifications of conopeptides are the most complicated modifications to date and are well-known and closely related to the activity of conopeptides. The hydroxylation of proline in conopeptides affects folding, structure, and biological activity, and prolyl 4 hydroxylase has been characterized in Conus literatus. However, the hydroxylation machinery of proline in conopeptides is still unclear. In order to address the hydroxylation mechanism of proline in µ-PIIIA, three recombinant plasmids encoding different hybrid precursors of µ-PIIIA were constructed and crossly combined with protein disulfide isomerase, prolyl 4 hydroxylase, and glutaminyl cyclase in a continuous exchange cell-free protein system. The findings showed that prolyl 4 hydroxylase might recognize the propeptide of µ-PIIIA to achieve the hydroxylation of proline, while the cyclization of glutamate was also formed. Additionally, in Escherichia coli, the co-expression plasmid encoding prolyl 4 hydroxylase and the precursor of µ-PIIIA containing pro and mature regions were used to validate the continuous exchange cell-free protein system. Surprisingly, in addition to the two hydroxyproline residues and one pyroglutamyl residue, three disulfide bridges were formed using Trx as a fusion tag, and the yield of the fusion peptide was approximately 20 mg/L. The results of electrophysiology analysis indicated that the recombinant µ-PIIIA without C-terminal amidate inhibited the current of hNaV1.4 with a 939 nM IC50. Our work solved the issue that it was challenging to quickly generate post-translationally modified conopeptides in vitro. This is the first study to demonstrate that prolyl 4 hydroxylase catalyzes the proline hydroxylation through recognition in the propeptide of µ-PIIIA, and it will provide a new way for synthesizing multi-modified conopeptides with pharmacological activity.
Asunto(s)
Prolina , Procesamiento Proteico-Postraduccional , Hidroxilación , Prolil Hidroxilasas , Sistema Libre de Células , Escherichia coli/genéticaRESUMEN
Zishen Pill (ZSP) is a traditional Chinese medicine that is frequently used to treat Benign Prostatic Hyperplasia (BPH), however its specific mechanism of action and active ingredients are yet unknown. We used a combination of serum pharmacochemistry and network pharmacology and a series of biochemical assays to explore the action mechanism of ZSP in the treatment of BPH. The BPH rat model was created using testosterone propionate, and following oral ZSP administration, the components of ZSP in rat serum were detected by UPLC-Q-Exactive Orbitrap/MS method. A "component-target-disease" network and PPI networks were constructed on this foundation. The primary mechanism of ZSP decreasing BPH in rats was studied by KEGG pathway and GO analysis. Finally, the potential pathways and key targets were further verified in vivo by molecular biology and immunological methods. 46 substances were charactered from rat serum, and 164 anti-BPH targets were screened from the database. According to network pharmacology, the primary targets were CASP3, STAT3, JUN, and PTGS2/COX2. Three related pathways (PI3K/Akt signaling pathway, AGE-RAGE signaling pathway and EGFR tyrosine kinase inhibitor resistance) were closely related to the therapeutic effects of ZSP. The findings of molecular biology demonstrated that ZSP may bring Bcl-2, BAX, CASP3, COX2, and 5LOX protein and gene expression in BPH rats appreciably closer to that of normal rats. Additionally, ZSP can lessen the expression of inflammatory cytokines in BPH rats, including VEGF, TNF-α, CCL5, and interleukin. CONCLUSION: The above results suggest that ZSP may reduce BPH through inflammation/immunity and apoptosis/proliferation-related pathways. This study offers a fresh approach to investigate the basic pharmacological effects and mechanism of ZSP in the treatment of BPH.
Asunto(s)
Medicamentos Herbarios Chinos , Hiperplasia Prostática , Animales , Ratas , Masculino , Humanos , Hiperplasia Prostática/tratamiento farmacológico , Farmacología en Red , Caspasa 3 , Ciclooxigenasa 2 , Fosfatidilinositol 3-Quinasas , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Simulación del Acoplamiento MolecularRESUMEN
Cyclosporine A and sirolimus are immunosuppressants that are widely used in many organ transplantation procedures. They exhibit some complementary mechanisms of action and interact synergistically when used together. Howeverï¼ they are critical-dose drugs and have a narrow therapeutic index. They provide the desired therapeutic effect with acceptable tolerability only within a specific range of blood concentrations. Thereforeï¼ the rapid and simultaneous detection of the concentrations of cyclosporine A and sirolimus in whole blood could provide valuable information on planning medicine administration after organ transplantations. In this studyï¼ firstlyï¼ the chromatographic behaviors of cyclosporine A and sirolimus on a biological liquid chromatography ï¼BioLCï¼ column and traditional liquid chromatography ï¼TraLCï¼ columns were investigated systematically under the same chromatographic conditions. The results suggested that the peak height and peak width of cyclosporine A and sirolimus on the BioLC columnï¼ ZORBAX 300SB C8 ï¼250 mm×4.6 mmï¼ 5.0 µmï¼ï¼ were the highest and narrowestï¼ respectively. The number of theoretical plates of cyclosporine A and sirolimus on the ZORBAX 300SB C8 column increased significantly when the volume ratio of acetonitrile in the mobile phases was greater than 70%. Their retention time on the BioLC and TraLC columns was negligibly affected by the use of formic acid and trifluoroacetic acid as the mobile phases. In the range of the experimental column temperatureï¼ the number of theoretical plates of cyclosporine A and sirolimus on the ZORBAX 300SB C8 column was significantly higher than that on the two TraLC columns. Furthermoreï¼ the relationship between the retention factor and column temperature of cyclosporine A on the ZORBAX 300SB C8 column was different from that on the two TraLC columns. Subsequentlyï¼ a high performance liquid chromatography method based on the ZORBAX 300SB C8 column was established for the rapid separation and determination of cyclosporin A and sirolimus in whole blood. A sample of whole blood with a volume of 50 µL was prepared by protein precipitation with 1 mol/L sodium hydroxide and then extracted into 500 µL of ether-methanol ï¼95â¶5ï¼ v/vï¼. After centrifugation at 14000 r/min for 10 minï¼ the organic layer was removed and evaporated under a stream of nitrogen at 50 â. The residue was then reconstituted in 200 µL of methanol for use. Cyclosporin A and sirolimus were separated through isocratic elution on the ZORBAX 300SB C8 column. The column temperature was set at 60 â. The mobile phase was acetonitrile-water ï¼70â¶30ï¼ v/vï¼ and the flow rate was 1.0 mL/min. The detection wavelengths were 205 nm for cyclosporine A and 278 nm for sirolimus. The injection volume was 20 µL. The external standard method was used to quantify cyclosporine A and sirolimus. Under the optimized conditionsï¼ cyclosporine A and sirolimus were well-separated within 6 min with a resolution of 3.7 at 205 nm. In additionï¼ the endogenous substances in whole blood negligibly interfered in the detection of sirolimusï¼ while two endogenous substances slightly affected the detection of cyclosporine A. Cyclosporine A and sirolimus both showed good linear relationships in their respective concentration ï¼r>0.997ï¼. The limits of detection ï¼LODsï¼ of cyclosporine A and sirolimus were respectively calculated to be 10 ng/mL and 1 ng/mL based on a signal-to-noise ratio of 3ï¼ and the limits of quantification ï¼LOQsï¼ were 30 ng/mL and 2 ng/mL based on a signal-to-noise ratio of 10. In the whole blood samplesï¼ the recoveries of cyclosporine A and sirolimus at three spiked levels were in the ranges of 83.5%-89.7% and 95.8%-97.8% with relative standard deviations ï¼RSDsï¼ of 3.2%-9.0% and 3.4%-6.7% ï¼n=5ï¼ï¼ respectively. The established method is simple in operationï¼ can be performed with a simple mobile phaseï¼ has a short analysis timeï¼ and provides a wide linear range and high sensitivityï¼ henceï¼ it can be applied to the determination of cyclosporine A and sirolimus in whole blood.
Asunto(s)
Ciclosporina , Inmunosupresores , Ciclosporina/química , Cromatografía Líquida de Alta Presión , Metanol , SirolimusRESUMEN
Vecuronium, rocuronium, and pancuronium are widely used as non-depolarizing muscle relaxants. There have been occasional cases of allergic reactions and even death when using such muscle relaxants. Rapid determination of the concentration of these muscle relaxants in blood can provide valuable information for early clinical diagnosis. As quaternary ammonium compounds, these muscle relaxants are highly polar. Hence, they cannot be retained effectively on reversed-phase chromatographic columns with conventional mobile phases. These quaternary ammonium muscle relaxants are mainly separated by ion-pair chromatography. Using an ion-pairing reagent can help improve the retention capabilities of quaternary ammonium muscle relaxants. Nevertheless, the sensitivity of MS detection is significantly decreased because of ionic inhibition caused by the ion-pairing reagent in the mobile phase. Furthermore, ion-pairing reagents can pollute the MS system. A method based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established for the simultaneous determination of the three quaternary ammonium muscle relaxants in blood. The blood samples were diluted and subjected to high-speed centrifugation. The supernatant was purified on a Bond Elut AL-N solid phase extraction column and then filtered through a 0.45 µm microporous membrane. The quaternary ammonium muscle relaxants were separated on a ZIC-cHILIC analytical column (50 mm×2.1 mm, 3.0 µm) with gradient elution. Acetonitrile and 0.1% formic acid aqueous solution were used as mobile phases. The separated compounds were analyzed by tandem MS with an electrospray ionization (ESI) source in positive and multiple reaction monitoring (MRM) modes. The matrix effects of vecuronium, rocuronium, and pancuronium in blood were 88.1% to 95.4%. The calibration curves for vecuronium, rocuronium, and pancuronium showed good linear relationships in each range, and all correlation coefficients (R2) were > 0.996. The limits of detection of vecuronium, rocuronium, and pancuronium were 0.2-0.8 ng/mL, with the corresponding limits of quantification being 0.5-2.0 ng/mL. The recoveries of vecuronium, rocuronium, and pancuronium were 92.8% to 110.6%, with relative standard deviations (RSDs) of 3.2%-9.4%. This method is sensitive, accurate, and easy to operate, and it can be used to rapidly determine vecuronium, rocuronium, and pancuronium in blood.
Asunto(s)
Compuestos de Amonio/sangre , Fármacos Neuromusculares/sangre , Cromatografía Líquida de Alta Presión , Humanos , Pancuronio/química , Rocuronio/sangre , Extracción en Fase Sólida , Espectrometría de Masas en Tándem , Bromuro de Vecuronio/sangreRESUMEN
α7 nicotinic acetylcholine receptors (nAChR) is an important nicotinic acetylcholine receptors subtype and closely associated with cognitive disorders, such as Alzheimer's and schizophrenia disease. The mutant ArIB (V11L, V16A) of α-conotoxin ArIB with 17-amino acid residues specifically targets α7 nAChR with no obvious effect on other nAChR subtypes. In the study, the synthetic gene encoding mature peptide of ArIB and mutant ArIB (V11L, V16A) carried a fusion protein Trx and 6 × His-tag was separately inserted in pET-32a (+) vector and transformed into Escherichia coli strain BL21(DE3) pLysS for expression. The expressions of Trx-ArIB-His6 and Trx-ArIB (V11L, V16A)-His6 were soluble in Escherichia coli, which were purified by Ni-NTA affinity chromatography column and cleaved by enterokinase to release rArIB and rArIB (V11L, V16A). Then, rArIB and rArIB (V11L, V16A) were purified by high-performance liquid chromatography (HPLC) and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Bioactivity of rArIB and rArIB (V11L, V16A) was assessed by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing human nAChR subtypes. The results indicated that the yield of the fusion proteins was approximately 50 mg/L and rArIB (V11L, V16A) antagonized the α7 nAChR subtype selectively with 8-nM IC50. In summary, this study provides an efficient method to biosynthesize α-conotoxin ArIB and rArIB (V11L, V16A) in Escherichia coli, which could be economical to obtain massively bioactive disulfide-rich polypeptides at fast speed.
Asunto(s)
Conotoxinas/farmacología , Escherichia coli/metabolismo , Antagonistas Nicotínicos/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/antagonistas & inhibidores , Animales , Conotoxinas/genética , Conotoxinas/metabolismo , Relación Dosis-Respuesta a Droga , Escherichia coli/genética , Histidina/metabolismo , Potenciales de la Membrana , Antagonistas Nicotínicos/metabolismo , Oligopéptidos/metabolismo , Oocitos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Tiorredoxinas/metabolismo , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7/genética , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
(+)-Crambescin A belongs to the polycyclic guanidine natural product family and has been shown to possess various medically important properties. The chiral bicyclic guanidine structure of (+)-crambescin A presents a challenge for chemical synthesis. Here we implement a novel asymmetric Biginelli reaction strategy to achieve the enantiospecific total synthesis of (+)-crambescin A in only 8 steps from the abundant and inexpensive aliphatic aldehyde, urea and methyl 3-oxobutanoate.
RESUMEN
The principal purpose of this study is to set up efficient purification techniques of small DNAs which are suitable for isolation of from tens to three hundred bases of genes. On the bases of the technique, purification methods for big DNA fragments are established. In the experiment, the DNA bands were cut after agarose gel electrophoresis and put into 0.5 mL of tubes with silica wool, glass wood, absorbent cotton and cotton at the bottom. And then 10 000 r/min for 2 min, the liquid was collected. The results indicated that silica wool was the best of the materials. The recovery rate for DNAs below 200bp was over 90%, 85% to approximately 90% for 300bp. And the technique can be applied to purify bigger DNA fragments. The kits for DNA purification hardly recovered DNA below 150bp. The recovery rate for 150bp of DNA was 5%, 60% even for 300bp. The efficiencies of enzymic digestion and enzymic connection for the DNAs purified by the technique were the same as those for the DNAs isolated by the kits. So, the technique is obviously superior to kit purification methods.