Analysis of Antidepressant Quality Markers of Bupleuri Radix Before and After Vinegar-processing Based on UPLC-Q-TOF-MS and Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo analyze the antidepressant quality markers（Q-Marker） of Bupleuri Radix（BP） before and after vinegar-processing by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， multivariate statistical analysis and network pharmacology. MethodUPLC-Q-TOF-MS was used to analyze the chemical basis of raw and vinegar-processed products of BP， and principal component analysis（PCA） orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to identify the differential components in BP that changed significantly before and after vinegar-processing， which were regarded as candidate quality markers（Q-Marker）. Then the disease-drug-component-target network related to antidepressant effect of BP was constructed by network pharmacology， and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined. Rats were randomly divided into blank group， model group， fluoxetine group（2.67 mg·kg-1） and total saponin group（0.72 mg·kg-1）， except the blank group， rats in the other groups were subjected to chronic unpredictable mild stress（CUMS）. Three weeks after the start of modeling， rats in each administration group were given the corresponding dose of drugs once a day for 4 weeks， and rats in the blank and model groups were given normal saline with dose of 10 mL·kg-1. At 1 day before modeling， 21 days and 28 days after administration， body mass weighing， sucrose preference test and open field test were performed on each group . After 28 days of administration， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was used to detect the mRNA expression levels of phosphatidylinositol 3-kinase（PI3K）， protein kinase B（Akt）， mammalian target of rapamycin（mTOR）， glycogen synthase kinase-3β（GSK-3β）， forkhead box transcription factor O3a（FoxO3a） and β-catenin in hippocampal tissues of rats in each group， while protein expression levels of PI3K， Akt， mTOR and FoxO3a in hippocampal tissues of rats in each group were detected by Western blot. ResultThere were 19 components in BP showed significant changes before and after vinegar-processing， and 9 components such as saikosaponin A， saikosaponin B1， saikosaponin B2， saikosaponin C and saikosaponin D were identified as potential Q-Marker through S-plot differential marker screening. Combined with the disease-drug-component-target network， saikosaponin A， saikosaponin B1， saikosaponin B2 and saikosaponin D were identified as antidepressant Q-Marker of raw and vinegar-processed products of BP. According to the results of pharmacodynamic tests， after 28 d of administration， compared with the blank group， the body mass， sucrose preference index and open field total score of rats in model group， fluoxetine group and total saponin group decreased significantly（P<0.01）. Compared with the model group， the body mass， sucrose preference index and open field total score in total saponin group increased significantly（P<0.01）. Compared with the blank group， mRNA expression levels of PI3K， Akt， mTOR and β-catenin in hippocampus of rats in the model group decreased significantly（P<0.05）， while mRNA expression levels of GSK-3β and FoxO3a increased significantly（P<0.05）. Compared with the model group， mRNA expression levels of PI3K， Akt， mTOR and β-catenin in hippocampus of rats in the total saponin group were increased significantly（P<0.05）， while mRNA expression levels of GSK-3β and FoxO3a decreased significantly（P<0.05）. Compared with the blank group， the protein expression levels of Akt and mTOR in hippocampus of the model group decreased significantly（P<0.01）， while the protein expression levels of PI3K and FoxO3a increased significantly（P<0.01）. Compared with the model group， the expression level of Akt in hippocampus of the total saponin group increased significantly（P<0.01）， the mTOR expression level was increased but not statistically significant， while the protein expression levels of PI3K and FoxO3a decreased significantly（P<0.01）. ConclusionThe chemical constituents of BP changed greatly after vinegar-processing， and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined by chemical basis， pharmacodynamics， network pharmacology and signaling pathway， which provided a reference for further research on quality control， pharmacodynamic substance basis and processing mechanism of BP.

Effect of Baihu Jia Renshen Tang on PI3K/Akt Signaling Pathway in Liver of MKR Diabetic Mice / 中国实验方剂学杂志

ObjectiveTo explore the effects of Baihu Jia Renshen Tang （BHRS） on the related molecules on the phosphatidylinositol-3-kinase/protein kinase B（PI3K/Akt）signaling pathway in the liver of MKR diabetic model mice. MethodThirty 6-week-old MKR mice were selected and fed on a high-fat diet for four weeks，followed by intraperitoneal injection of streptozotocin（STZ）for the diabetes model establishment. The model was properly induced in the case of the fasting blood glucose （FBG） of ≥11.1 mmol·L-1. After modeling，the mice were randomly divided into a model group，a BHRS group （12.09 g·kg-1·d-1），and a metformin group （0.065 g·kg-1·d-1），with 10 mice in each group. Ten FVB mice were assigned to the control group. The mice in the groups with drug intervention were continuously administered correspondingly for 28 days. After administration，the mice were sacrificed，followed by the oral glucose tolerance test （OGTT） and FBG detection. Serum very low-density lipoprotein（VLDL）content was determined by semi-quantitative enzyme-linked immunosorbent assay （ELISA）. Four indexes related to blood lipid were determined by the biochemistry analyzer. Liver tissues were subjected to pathological examination by hematoxylin-eosin（HE）staining. Western blot was used to detect the protein expression of PI3K，Akt，phosphorylated（p）-PI3K，p-Akt，forkhead box protein O1 （FoxO1），insulin receptor（InsR），and insulin receptor substrate-2（IRS-2） in liver tissues of mice. Real-time polymerase chain reaction（Real-time PCR） was used to detect the mRNA expression of PI3K，Akt，FoxO1，InsR，and IRS-2 in liver tissues of mice. ResultCompared with the control group，the model group showed poor general conditions，abnormal glucose tolerance （P<0.05），increased FBG （P<0.01），abnormal blood lipid metabolism，increased serum total cholesterol （TC），triglyceride（TG），low-density lipoprotein cholesterol（LDL-C），and VLDL （P<0.05），decreased level of high-density lipoprotein cholesterol（HDL-C）（P<0.05），fatty degeneration and obvious pathological changes of liver cells，reduced protein expression of PI3K，Akt，p-PI3K/PI3K，p-Akt/Akt，IRS-2，and InsR in liver tissues（P<0.05），increased protein expression of FoxO1（P<0.05），decreased mRNA expression of PI3K，Akt，IRS-2，and InsR in liver tissues （P<0.05），and increased FoxO1 mRNA expression（P<0.05）. Compared with the model group，the BHRS group showed improved general conditions and glucose and lipid metabolism （P<0.05），improved pathological state of liver cells，increased protein expression of PI3K，Akt，p-PI3K/PI3K，p-Akt/Akt，IRS-2，and InsR in liver tissues（P<0.05），decreased protein expression of FoxO1（P<0.05），increased mRNA expression of PI3K，Akt，IRS-2，and InsR in liver tissues （P<0.05），and reduced FoxO1 mRNA expression（P<0.05）. ConclusionBHRS can effectively reduce blood glucose，regulate blood lipid metabolism，and improve the pathological state of the liver in MKR diabetic mice，and its mechanism of action may be related to the regulation of the activity of molecules on the PI3K/Akt signaling pathway.

Proteomics Analysis of Mechanism of Shenxiong Glucose Injection in Antagonizing H2O2-Induced Oxidative Damage in H9c2 Cells / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Shenxiong glucose injection （SGI） in inhibiting hydrogen peroxide （H2O2）-induced oxidative damage in H9c2 cells by tandem mass tags （TMT）-labeled quantitative proteomics. MethodH9c2 cells cultured in vitro were exposed to H2O2 for inducing oxidative damage. The cell viability was measured by cell proliferation and cytotoxicity assay （MTS）， followed by peptide fractionation by high performance liquid chromatography （HPLC） and protein expression detection in H9c2 cells by ultrahigh performance liquid chromatography-mass spectrometry. MaxQuant （v1.5.2.8） was utilized for data retrieval， and the high-resolution mass spectrometry was conducted to screen out differentially expressed proteins， which were then subjected to gene ontology （GO） and Kyoto encyclopedia of genes and genomes （KEGG） enrichment analysis. The protein expression levels of perilipin 2 （Plin2） and tropomyosin 1 （Tpm1） in cells were measured by Western blot. ResultThe spectral analysis yielded 48 608 specific peptide fragments and 5 903 quantifiable proteins. Compared with the model group，the SGI group exhibited 82 differentially expressed proteins，of which 22 were up-regulated and 60 were down-regulated. GO analysis results showed that the differentially expressed proteins were mainly involved in biological processes such as programmed cell death regulation，regulation of cell proliferation，cardiovascular system development， and cell migration. As revealed by KEGG analysis， these proteins were mainly related to peroxisome proliferator-activated receptor （PPAR），focal adhesion，phosphatidylinositol 3-kinase/protein kinase B（PI3K/Akt），and Ras-related protein 1 （Rap1） pathways. Western blot results demonstrated that compared with the model group，SGI significantly increased the Plin2 protein expression and decreased the Tpm1 protein expression （P<0.01），consistent with the proteomics results. ConclusionSGI may inhibit cell apoptosis and antagonize H2O2-induced cell oxidative damage by regulating PPAR，focal adhesion，PI3K/Akt and Rap1 pathways，which should be further verified by subsequent experiments.

The Role of PI3K / AKT / FOXO Signaling Pathway-mediated Autophagy in Diabetes-associated Cognitive Dysfunction / 中国生物化学与分子生物学报

Diabetes‚ a metabolic disease characterized by hyperglycemia‚ can cause central nerve system damage‚ lead to alteration of the neuronal structure and function‚ and consequently induce cognitive dysfunction. Recently‚ diabetes-associated cognitive dysfunction (DACD) and its molecular mechanism have become a research frontier. The phospoinositide 3 kinase/ protein kinase B/ Forkhead box O (PI3K/ PKB/ FOXO) signaling pathway is an important upstream regulatory mechanism for autophagy. Here we review the role of the PI3K/ AKT/ FOXO signaling pathway in the regulation of Gs‚ Bnip3 and Spk2 gene expressions. GS regulates the Gln-mTORC1 pathway and thus activates autophagy; BNIP3 enhances LC3 expression and promotes autophagy. Moreover‚ the AMPK-FOXO3a-mTORC1 signaling pathway is also an important pathway that involved in the regulation of autophagy. These studies suggest that FOXO3a may be a key target for the treatment of DACD. This review aims to provide a theoretical basis and molecular target for the clinical treatment of DACD and it related drug development.

Effect of Naringenin on Oxidative Stress and Tau Protein Phosphorylation of Aβ25-35-induced PC12 Cell Injury / 中国实验方剂学杂志

Objective:To investigate the effects of naringenin on oxidative stress and Tau protein phosphorylation of adrenal pheochromocytoma（PC12） cells injured by β-amyloid（Aβ）25-35 and its relationship with estrogen receptor（ER） and phosphatidylinositol -3 kinase/protein kinase B（PI3K/Akt） signaling pathway. Method:The PC12 cells were intervened with Aβ25-35 to prepare the injury model. The experiment was divided into blank group， model group， naringenin（400，40，4，0.4，0.04，4×10-3，4×10-4，4×10-5 μmol·L-1）group， positive drugs estradiol（E2）（1 nmol·L-1）+Aβ25-35 group， naringenin（0.4，0.04，4×10-3，4×10-4，4×10-5 μmol·L-1）+Aβ25-35 group， E2+Aβ25-35+ER antagonist（ICI182780）（1 μmol·L-1） group， naringenin+Aβ25-35+ICI182780 group， E2+Aβ25-35+PI3K blocker（LY294002）（50 μmol·L-1） group， naringenin+Aβ25-35+LY294002 group. Methye thiazolye telrazlium（MTT）method was used to detect the cell proliferation index， 2'，7'-Dichlorodi -hydrofluorescein diacetate（DCFH-DA） was used as a fluorescent probe to detect the content of reactive osygen species（ROS）， the content of malondialdehyde（MDA） and the activity of superoxide dismutase（SOD） were measured by thiobarbituric acid（TBA） and oxidase methods， Western blot was used to detect the expression of phosphorylated Tau protein/total Tau protein（p-Tau/t-Tau）. Result:According to the results of MTT experiment， 0.4 μmol·L-1 was selected as the best effective concentration of naringenin， compared with the blank group， the cell proliferation index of model group decreased significantly （P<0.01）， compared with model group， the cell proliferation index of naringenin+Aβ25-35 group increased significantly （P<0.01）. In addition， compared with blank group， the content of ROS， MDA and the expression of p-Tau/t-Tau in the model group increased significantly （P<0.01）， and the activity of SOD decreased significantly （P<0.01）， compared with model group， the content of ROS， MDA and the expression of p-Tau/t-Tau in naringenin+Aβ25-35 group decreased significantly （P<0.01）， and the activity of SOD increased significantly （P<0.01）， compared with naringenin+Aβ25-35 group， the addition of ICI182780 and LY294002 significantly reversed the role of naringenin in the above indicators （P<0.01）. The effect of naringenin was similar to that of E2. Conclusion:Naringenin can improve the cell proliferation index and protect PC12 cells from Aβ25-35 injury， which may be achieved by activating ER and PI3K/Akt signaling pathway to reduce ROS， MDA content， p-Tau/t-Tau expression and promote SOD activity.

Screening of Antitumor Active Components and Key Targets of Aconiti Lateralis Radix Praeparata-Pinelliae Rhizoma Based on PI3K/Akt Pathway / 中国实验方剂学杂志

Objective:Screen out the antitumor constituents of Aconiti Lateralis Radix Praeparata-Pinelliae Rhizoma base on system pharmacology with chemical constituents of Aconiti Lateralis Radix Praeparata-Pinelliae Rhizoma as study objects, in order to provide the theoretical basis for the development of antitumor and nontoxic activities of Aconiti Lateralis Radix Praeparata-Pinelliae Rhizoma. Method:The small molecule ligand library of Aconiti Lateralis Radix Praeparata-Pinelliae Rhizoma was built based on Traditional Chinese Medicine Systems Pharmacology(TCMSP), energy of Aconiti Lateralis Radix Praeparata-Pinelliae Rhizoma was matched with the key protein targets of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signal pathway by molecular docking (SYBYL2.1, Tripos), the Aconiti Lateralis Radix Praeparata-Pinelliae Rhizoma-targets network model was established based on Cytoscape 3.5.1, and the physicochemical properties of the antitumor activity in Aconiti Lateralis Radix Praeparata-Pinelliae Rhizoma were predicted by using SwissADME and admetSAR. Result:There were 25 small molecule constituents of Aconiti Lateralis Radix Praeparata-Pinelliae Rhizoma. Through the energy match, key antitumor constituents of Pinelliae Rhizoma were gondoic acid, 10,13-eicosadienoic, baicalin, 12,13-epoxy-9-hydroxynonadeca-7,10-dienoic acid. Key antitumor constituents of Aconiti Lateralis Radix Praeparata were deltoin, sitosterol, neokadsuranic acid B, 11,14-eicosadienoic acid. Phosphatidylinositol 3-kinase (PI3Kα), phosphatase and tensin homolog deleted on chromosome ten (PTEN), phosphoinositide dependent protein kinase 1 (PDK1) were key antitumor targets of Aconiti Lateralis Radix Praeparata-Pinelliae Rhizoma. There were 8 key antitumor constituents of Aconiti Lateralis Radix Praeparata-Pinelliae Rhizoma, which had a low CYP450 inhibition and basically followed the Lipinski rule. Conclusion:Antitumor nontoxic constituents of Aconiti Lateralis Radix Praeparata-Pinelliae Rhizoma and key targets are screened out from the molecular level, which provides the new ideas for the effective use of nontoxic traditional Chinese medicine(TCM) and breaks the restrictions in using nontoxic TCM.

Validation Study of Behavior Problems Inventory-01 among Korean Children and Adolescents

OBJECTIVES: The purpose of this study was to examine the reliability and validity of the Korean version of the Behavior Problems Inventory (BPI-01) among children and adolescents aged between 3 and 18. METHODS: The control group consisting of one hundred children and adolescents was recruited from schools and the patient group consisting of forty one children and adolescents with autism spectrum disorder were recruited from a hospital. We compared the measurements of both groups. To assess the concurrent validity of the BPI-01, we compared the problem behavior index of the Korean Scale of Independent Behavior-Revised (K-SIB-R) and, to assess the discriminant validity, we compared the Korean version of the Child Behavior Checklist (K-CBCL). The Cronbach's alpha of the BPI-01 was measured to assess its reliability. Correlation analyses between the BPI-01 and the other scale were carried out to examine the former's concurrent and discriminant validity. RESULTS: The patient group showed a significantly higher score for all three subscales of the BPI than the control group. The Cronbach's alpha was 0.92 for the total severity score of the BPI and ranged between 0.67–0.89 for each subscale in the patient group. All subscales of the BPI-01's, i.e., self injurious behavior, stereotyped behavior and aggressive/destructive behavior, were significantly correlated with the corresponding subscales of the K-SIB-R. The BPI-01 generally did not demonstrate any significant correlation with emotional items such as anxiety/depression in the K-CBCL. Especially, the BPI-01's stereotyped behavior subscale showed little correlation with externalizing behaviors such as social problems and aggressive behaviors. CONCLUSION: This study found that the Korean version of BPI-01 is a reliable and valid behavior rating instrument for problem behavior in developmental disabilities among children and adolescents.

O polietilenoglicol aumenta a penetração do vírus da diarréia viral bovina, do vírus da estomatite vesicular e do vírus sincicial respiratório bovino em células de cultivo / Polyethylene glycol increases the penetration of bovine viral diarrhea virus, vesicular stomatitis virus and bovine respiratory syncytial virus in cultured cells

A baixa eficiência de penetração de alguns vírus em células de cultivo pode representar uma dificuldade para o isolamento e a multiplicação viral in vitro. No presente estudo investigou-se o efeito do polietilenoglicol (PEG) na replicação de sete vírus bovinos em células de linhagem de rim bovino (MDBK). A eficiência de penetração e replicação foi mensurada pela contagem do número de placas virais produzidas em tapetes celulares, após adsorção do inóculo viral (100 DICC50 mL-1) com ou sem a adição de PEG a 5 por cento (peso molecular 6.000). A adição de PEG ao inóculo resultou em aumentos significativos do número de placas para o vírus da diarréia viral bovina (BVDV) (aumento de 3,4 vezes), vírus da estomatite vesicular (VSV) (2,2 vezes) e vírus respiratório sincicial bovino (BRSV) (1,5 vezes). A adição de PEG não produziu aumento significativo no número de placas dos herpesvírus bovinos 1, 2 e 5 (BoHV-1, BoHV-2 e BoHV-5). Por outro lado, o PEG produziu uma redução do número de placas (1,4 vezes) produzidas pelo vírus da parainfluenza bovina (bPI-3V). A adição de PEG a 5 por cento também aumentou a sensibilidade de detecção (entre 10 e 100 vezes) do BVDV no soro de três bezerros persistentemente infectados. Para o BRSV, a adição de PEG aumentou em duas vezes a sensibilidade do isolamento viral de secreções nasais de duas ovelhas infectadas experimentalmente. Esses resultados demonstram que o PEG aumenta a eficiência de infecção do BVDV, VSV e BRSV em células de cultivo, podendo ser utilizado para aumentar a sensibilidade de detecção desses vírus em amostras clínicas (isolamento viral) e/ou, para aumentar os títulos de vírus produzidos em cultivo celular.

The low efficiency of penetration of some viruses in cultured cells may represent an obstacle for viral isolation and/or viral multiplication in tissue culture. This study investigated the effect of polyethylene glycol (PEG) on the penetration and replication of seven bovine enveloped viruses in culture cells. Penetration efficiency was measured by counting the number of viral plaques produced in bovine kidney cells (MDBK). The addition of 5 percent PEG (molecular weight 6.000) to the viral inoculum containing 100 TCID50 mL-1 (tissue culture median infectious dosis) of each virus, during adsorption for 2h at 37ºC, resulted in a significant increase in the number of plaques for bovine viral diarrhea virus (BVDV) (increase of 3.4-fold), vesicular stomatitis virus (VSV) (2.2-fold) and bovine respiratory syncytial virus (BRSV) (1.5-fold). The addition of 5 percent PEG to the inoculum of bovine herpesviruses 1, 2 and 5 (BoHV-1, BoHV-2 and BoHV-5) did not increase the number of viral plaques. On the other hand, PEG produced a reduction in the number of plaques by bovine parainfluenza virus (bPI-3V) (1.4-fold). Furthermore, the addition of 5 percent PEG produced a 10- to 1000-fold increase in the sensitivity of BVDV detection in the serum of three persistently infected calves; and doubled the sensitivity of detection of BRSV in nasal secretions of two experimentally infected sheep. These results demonstrate that PEG enhances the efficiency of infection by BVDV, VSV and BRSV in cultured bovine cells and therefore may be used to increase the sensitivity of virus detection in clinical samples (viral isolation), and/or to increase virus titers in cell cultures.

Change of lipopolysaccharide activity-modulating protein in serum of cirrhosis patients and its clinical significance / 中国康复理论与实践

@#ObjectiveTo explore changes of lipopolysaccharide (LPS), lipopolysaccharide-binding protein (LBP), bactericidal/permeability-increasing protein (BPI), soluble CD14 (sCD14) in blood of cirrhosis patients and their clinical significance.MethodsSerum samples of 45 cirrhosis patients were detected with chromogenic limulus amebocyte lysate assay for LPS and detected with ELISA for LBP, BPI and sCD14. While, serum samples of 15 normal subjects were used as controls.ResultsLevels of LPS, LBP, BPI and sCD14 in blood of cirrhosis patients with liver function being grade A, B and C were significantly higher than that in normal subjects. Also, those indexes fore mentioned were obviously higher in died cirrhosis patients than that in survived cirrhosis patients.Conclusion High levels of LBP, LPS and relative deficiency of BPI in cirrhosis patients accompanied with intestinal endotoxemia (IETM) may significantly increase the sensitivity of body to endotoxin.

The Diagnostic Value of MRI in Traumatic Brachial Plexus Injury / 대한정형외과학회잡지

Exploration of the injured brachial plexus is very hard due to the close approximation with other vital structures and the anatomic complexity. It is essential to identify the exact level and type of traumatic brachial plexus injury (BPI) to decide the appropriate surgical approach for the injury and to infer the postoperative prognosis. However, it can be difficult to image the brachial plexus because of the anatomic properties. The purpose of this study is to analyze the diagnostic value of MRI according to various planes of the level and the type of the traumatic BPI. In sixty patients with traumatic BPI, whose diagnosis was confirmed by clinicopathological and surgical findings, the preoperative MRI films were reread retrospectively. Brachial plexus injuries were divided into two groups of preganglionic BPI and postganglionic BPI, and then postganglionic BPI was divided into 3 subgroups of Zone I, Zone II and Zone III by major adjacent structures such as scalenus anterior muscle and pectoralis minor muscle. The accuracy of MRI was investigated with the confirmed diagnosis on axial, sagittal and coronal planes. In preganglionic BPI, the accuracy of MRI was 96% on axial plane and it was statistically significant compared to sagittal and coronal planes. In postganglionic BPI, the accuracy of MRI was 100% on sagittal plane and 86% on coronal plane in Zone I, but it was not significant statistically. In Zone II and III the accuracy of MRI were 89% and 80% on sagittal plane, and 61% and 60% on coronal plane, but it was not significant statistically. In conclusion, the MRI can provide useful guidance to diagnose preganglionic and postganglionic BPI. Axial imaging is considered better for preganglionic lesion and sagittal imaging for postganglionic BPI, but it demands further study on larger number of subjects with traumatic RPI.

Secretory expression of BPI_(m23) recombinant bactericidal protein in Pichia pastoris / 中国免疫学杂志

Objective:To express and secrete rBPI m23 bactericidal protein in Pichia pastoris expression system. Methods:pPICZ? synBPI m600 recombinant expression vector was constructed with prepared vectors (pUC18 synBPI 414 and PBV220 BPI m420 ) according to Molecular Cloning Laboratory Manual. The Pichia pastoris GS115 were electroporated with linearized pPICZ? synBPI m600 vectors , positive clones were screened on low salt LB medium with Zeocin , then the positive clones were identified by PCR and Mut phenotype analysis. rBPI m23 was induced to express in BMMY ,purified by SP Sepharose beads , identified by SDS PAGE and Western blot and measured with BCA method. Results:①pPICZ? synBPI m600 expression vector was successfully constructed.②Pichia pastoris GS115 strains integrated with synBPI m600 stably were obtained.③SDS PAGE showed the molecular weight of the recombinant protein was approximate 23 kD,and Western blot confirmed that the protein could bind to the anti BPI antibody specially.④The supernate protein concentration was about 2 9 mg/L.Conclusion:rBPI m23 was expressed and secreted effectively in Pichia pastoris GS115.

AFFINITIES OF PEPTIDE (BNEP) MIMICKING BACTERICIDALITY/PERMEABILITY-INCREASING PROTEIN FOR LIPOPOLYSACCHARIDE AND Lipid A BY BIOSENSOR TECHNOLOGY / 解放军医学杂志

To explore the mechamisms of bactericidal neutralizing endotoxin peptide(BNEP), a synthetic peptide mimicking bactericidality/permeability-increasing protein (BPI). The affinities of BNEP for LPS and Lipid A were determined with biosensor technology, and the ability of BNEP neutralizing LPS in vitro was tested by quantitative limulus amoebocyte lysate assay. The results showed that BNEP had high affinities for both LPS and Lipid A. The Kd value for LPS was at the level between 25.8 and 48.8nmol/L and for Lipid A from 11.8 to 21.8nmol/L. When 8?g/ml of BNEP was used, it could completely neutralize the concentration of 2ng/ml of LPS in vitro. It is concluded that BNEP has high binding affinities for both LPS and Lipid A. Our results also suggest that the binding site of LPS is at the glucosaminyl-?1'-6-glucosamine disaccharide of Lipid A. The binding activity of BNEP for LPS is in accord with its neutralizing activity for LPS.