Total synthesis of bis-（2,3-dibromo-4,5-dihydroxyphenyl）-methane as potent PTP1B inhibitor

Protein tyrosine phosphatase 1B （PTP1B） plays an important role as a negative regulator and has been proved to be an effective target for the treatment of type 2 diabetes mellitus. Bis-（2,3-dibromo-4,5-dihydroxyphenyl）-methane 7 was first reported as a natural bromophenol with significant inhibition against PTP1B which was isolated from red algae Rhodomela conrervoides. Intrigued by its astonishing activity （IC50 = 2.4 μmol/L）, compound 7 was synthesized with the overall yield of 24% and evaluated for its PTPIB inhibitory activity compared with natural compound.     

Purification and Characterization of the Catalytic Domain of Protein Tyrosine Phosphatase SHP-1 and the Preparation of Anti-ΔSHP-1 Antibodies

This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1（designated ΔSHP-1） and the preparation of its polyclonal antibodies. A cDNA fragment encoding ΔSHP-1 was amplified by PCR and then cloned into the pT7 expression vector. The recombinant pT7-ΔSHP-1 plasmid was used to transform Rosetta（DE3） E. coli cells. ΔSHP-1 was distributed in the exclusion body of E. coli cell extracts and was purified through a two-column chromatographic procedure. The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns. It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases. To generate polyclonal anti-ΔSHP-1 antibodies, purified recombinant ΔSHP-1 was used to immunize a rabbit. The resultant anti-serum was subjected to purification on ΔSHP-1 antigen affinity chromatography. The purified polyclonal antibody displayed a high sensitivity and specificity toward ΔSHP-1. This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.     

Expression of Catalytic Domain of Protein Tyrosine Phosphatase 1B and Preparation of Its Polyclonal Antibody

This study focuses on the expression of human protein tyrosine phosphatase 1B(PTP1B) catalytic domain (△PTP1B) and preparation of polyclonal antibody against △PTP1B. △PTP1B gene was PCR amplified with the cDNA of human PTP1B as the template, and cloned into the pT7 expression vector. The recombinant pT7-△PTP1B was expressed in E. coli Rosetta( DE3 ) host cells and purified. The antiserum was prepared by immunizing rabbit with purified recombinant △PTP1B. The polyclonal antibody against △PTP1B was purified by PVDF immobilized antigen affinity chromatography. △PTP1B was correctly cloned, expressed, and purified as confirmed by PCR, DNA sequence ratio) and 0. 1 ng, respectively. This study provides an important basis for further studying the biological function of PTP1B and its relationship with human diseases.     

Purification and Characterization of the Catalytic Domain of Protein Tyrosine Phosphatase SHP-1 and the Preparation of Anti-△SHP-1 Antibodies

This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1(designated △SHP-1) and the preparation of its polyelonal antibodies.A cDNA fragment encoding △SHP-1 was amplified by PCR and then cloned into the pT7 expression vector.The recombinant pT7-△SHP-1 plasmid was used to transform Rosetta(DE3) E.coll cells.△SHP-1 was distributed in the exclusion body of E.coll cell extracts and was purified through a two-column chromatographic procedure.The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns.It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases.To generate polyclonal anti-△SHP-1 antibodies,purified recombinant △SHP-1 was used to immunize a rabbit.The resultant anti-serum was subjected to purification on △SHP-1 antigen affinity chromatography.The purified polyclonal antibody displayed a high sensitivity and specificity toward △SHP-1.This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.     

Expression and Characterization of Catalytic Domain of T Cell Protein Tyrosine Phosphatase（△TC-PTP） —— Immunohistochemical Study of △TC-PTP Expression in Non-small Cell Lung Carcinomas

This study objective was to express and characterize the catalytic domain of the human T cell protein tyrosine phosphatase（△TC-PTP） and to study immunohistochemically the expression of △TC-PTP in human non-small cell lung cancers. △TC-PTP gene was PCR amplified with the cDNA of human TC-PTP as template, and cloned into the pT7 expression vector. The recombinant pT7-△TC-PTP was expressed in E. coli Rosetta （ DE3 ） host cells and puri- fied. The enzymatic characteristics of △TC-PTP including enzyme activity and kinetics assay were measured. The antiserum was prepared by immunizing rabbit with the purified recombinant △TC-PTP. Rabbit polyclonal antibody against △TC-PTP was purified by PVDF immobilized antigen affinity chromatography. Immunohistochemical staining of lung cancer tissues was performed with antibody against △TC-PTP protein. △TC-PTP gene was correctly cloned, expressed, and purified. The recombinant △TC-PTP had a highly catalytic activity of PTPase. Squamous cell lung carcinoma showed a significantly higher expression rate of △TC-PTP （76. 92%, 10/13 ） than adenocarcinoma （57.14%, 4/7） and normal lung tissue（20%, 1/5 ）. This study represents the first demonstration that △TC-PTP is highly expressed in human squamous cell lung carcinomas. In addition, this study provides an important basis for further studying the biological function of TC-PTP and its relationship with lung carcinomas and other diseases.     

Purification and Characterization of Protein Tyrosine Phosphatase MEG1 and Preparation of Anti-PTPMEG1 Antibody

<正>PTPMEG1 is an intracellular protein tyrosine phosphatase(PTP),which contains FERM and PDZ domains. This study focuses our attention on the expression,purification and characterization of catalytic domain of PTPMEG1(△MEG1) and preparation of its polyclonal antibody.A cDNA fragment encoding△MEG1 protein(amino acid residues 643—926) was amplified by PCR and then cloned into the pT7-7 vector.Both soluble and insoluble recombinant△MEG1 proteins were observed after induction by IPTG.Soluble△MEG1 was purified via two chromatographic steps,and the purified enzyme was characterized.With para-nitrophenylphosphate(pNPP) as a substrate,△MEG1 exhibited typical enzymatic characteristics of classic PTPs and classical Michaelis-Menten kinetics.Insoluble△MEG1,which was mainly distributed in the inclusion body of E.coli cells extracts,was purified by preparative electrophoresis gel for the preparation of the polyclonal antibody.A rabbit was immunized with△MEG1 purified by preparative electrophoresis to generate anti-△MEG1 antibody.Anti-serum was collected on 28th day after initial injection and purified via affinity chromatography.The purified polyconal antibody displayed a satisfactory titer and sensitivity.     

A new tetrastilbene from Caragana sinica

A new tetrastilbene named carasinol D （1） was isolated from the roots of Caragana sinica. Its structure was elucidated by spectroscopic analysis. Compound 1 was obtained from carasinol B （2） by an acid-catalyzed conversion.     

Structural Analysis of an Oligosaccharide and Glycopeptide Mixture from Panax Ginseng Root with Inhibition SHP-1 Function

A mixture of oligosaccharide and glycopeptide was isolated from the aqueous extract of Panax ginseng roots. The mixture inhibits protein tyrosine phosphatase(SHP-1) function, implying it enhances immune activity. The peak molecular mass of the oligosaccharide portion is 1800 calculated via GPC software after separation by HPLC. And the structure of the oligosaccharide portion is the backbone of (1→3)-and (1→4)-linked arabinopyranoside, and (1→4)-and (1→6)-linked glucopyranoside, with non-reducing terminals ...     

Expression and Characterization of the Intracellular Part of Receptor Protein Tyrosine Phosphatase PTPRU

PTPRU is an MAM domain-containing receptor-like protein tyrosine phosphatase. Previous studies have demonstrated an important role of the enzyme in the maintenance of epithelial integrity and in the regulation of the Wnt/β-catenin signaling pathway. To better understand the function of PTPRU, we cloned and expressed the intracellular portion of PTPRU as a GST fusion protein in E. coli cells. We purified the protein to homogeneity and used it to immunize mice for antibody production. The resultant antibody s...     

Anti-diabetes Agents---Ⅰ: Tetralone Derivative from Juglans regia

A new compound, 4-hydroxy-α-tetralone-4-O-β-D-[6′-O-(3″,4″,5″-trihydroxybenzoyl) glucopyranoside (1), together with a known compound, 4-hydroxy-(-tetralone (2), has been isolated from the roots of Juglans regia.2 showed moderate bioactivity against protein tyrosine phosphatase 1B (PTP1B).     

Binding Model and 3D-QSAR of 3-（2-Chloropyrid-5- ylmethylamino）-2-cyanoacrylates as PSⅡ Electron Transport Inhibitor

The binding model of 3-（2-chloropyrid-5-ylmethylamino）-2-cyanoacrylate photosystem Ⅱ （PSⅡ） electron transport inhibitors with the D 1 protein of PSII was built. The high herbicidal activity of this kind of inhibitors was explained by docking studies： in addition to usual factors, the N atom on the pyridine ring could form an H-bond with the backbone amide of Phe265 on the D1 protein. 3D-QSAR analysis on sixteen 3-（2-chloropyrid-5-yl- methylamino）-2-cyanoacrylate compounds was performed using CoMFA method to explain the nature of interactions between the compounds and D1 protein. These studies may provide useful insights for designing new PSII electron transport inhibitors.     

Expression and Characterization of Catalytic Domain of T Cell Protein Tyrosine Phosphatase(ΔTC-PTP)——Immunohistochemical Study of ΔTC-PTP Expression in Non-small Cell Lung Carcinomas

This study objective was to express and characterize the catalytic domain of the human T cell protein tyrosine phosphatase(ΔTC-PTP) and to study immunohistochemically the expression of ΔTC-PTP in human non-small cell lung cancers. ΔTC-PTP gene was PCR amplified with the cDNA of human TC-PTP as template, and cloned into the pT7 expression vector. The recombinant pT7-ΔTC-PTP was expressed in E.coli Rosetta(DE3) host cells and purified. The enzymatic characteristics of ΔTC-PTP including enzyme activity and kinetics assay were measured. The antiserum was prepared by immunizing rabbit with the purified recombinant ΔTC-PTP. Rabbit polyclonal antibody against ΔTC-PTP was purified by PVDF immobilized antigen affinity chromatography. Immunohistochemical staining of lung cancer tissues was performed with antibody against ΔTC-PTP protein. ΔTC-PTP gene was correctly cloned, expressed, and purified. The recombinant ΔTC-PTP had a highly catalytic activity of PTPase. Squamous cell lung carcinoma showed a significantly higher expression rate of ΔTC-PTP(76.92%, 10/13) than adenocarcinoma(57.14%, 4/7) and normal lung tissue(20%, 1/5). This study represents the first demonstration that ΔTC-PTP is highly expressed in human squamous cell lung carcinomas. In addition, this study provides an important basis for further studying the biological function of TC-PTP and its relationship with lung carcinomas and other diseases.     

Expression and Characterization of Catalytic Domain of T Cell Protein Tyrosine Phosphatase(ΔTC-PTP)——Immunohistochemical Study of ΔTC-PTP Expression in Non-small Cell Lung Carcinomas

This study objective was to express and characterize the catalytic domain of the human T cell protein tyrosine phosphatase(ΔTC-PTP) and to study immunohistochemically the expression of ΔTC-PTP in human non-small cell lung cancers. ΔTC-PTP gene was PCR amplified with the cDNA of human TC-PTP as template, and cloned into the pT7 expression vector. The recombinant pT7-ΔTC-PTP was expressed in E.coli Rosetta(DE3) host cells and purified. The enzymatic characteristics of ΔTC-PTP including enzyme activity and kinetics assay were measured. The antiserum was prepared by immunizing rabbit with the purified recombinant ΔTC-PTP. Rabbit polyclonal antibody against ΔTC-PTP was purified by PVDF immobilized antigen affinity chromatography. Immunohistochemical staining of lung cancer tissues was performed with antibody against ΔTC-PTP protein. ΔTC-PTP gene was correctly cloned, expressed, and purified. The recombinant ΔTC-PTP had a highly catalytic activity of PTPase. Squamous cell lung carcinoma showed a significantly higher expression rate of ΔTC-PTP(76.92%, 10/13) than adenocarcinoma(57.14%, 4/7) and normal lung tissue(20%, 1/5). This study represents the first demonstration that ΔTC-PTP is highly expressed in human squamous cell lung carcinomas. In addition, this study provides an important basis for further studying the biological function of TC-PTP and its relationship with lung carcinomas and other diseases.     

Anti—diabetes Agents—I：Tetralone Derivative from Juglans regia

A new compound,4-hydroxy-α-tetralone-4-O-β-D-[6‘‘‘‘‘‘‘‘-O-(3“,4“,5“-trihydroxybenzoyl)glucopyranoside(1),together with a known compound,4-hydroxy-α-tetralone(2),has been isolated from the roots of Juglans regia.2 showed moderate bioactivity against protein tyrosine phosphatase 1B(PTP1B).     

用多维NMR谱和圆二色CD光谱研究蛋白质亲环素A的结构稳定性

The structural stability of cyclophilin A （CypA） was investigated using H/D exchange and temperature coefficients of chemical shifts of amide protons, monitored by 213 heteronuclear NMR spectroscopy. Amide proton exchange rates were measured by H/D exchange experiments for slow-exchange protons and measured by SEA （Solvent Exposed Amides）-HSQC experiments for fast-exchange protons. Temperature coefficients of chemical shifts and hydrogen exchange rates of amide protons show reasonably good correlation with the protein structure. Totally, 44 out of 153 non-proline assigned residues still exist in 86 d of hydrogen-deuterium exchange at 4 ℃, suggesting that CypA structure should be highly stable. Residues in secondary structures of α2, β1, β2, β5, β6 and β7 might constitute the hydrophobic core of the protein. The change in free energy of unfolding （ △Gu^H2O ） of CypA was estimated to be （21.99± 1.53） kJ·mol^-1 by circular dichroism （CD）. The large free energy change is also an indicator of the high structural stability.     

Characterization of Hapten-Protein Conjugates for Immunoassay of Polycyclic Aromatic Hydrocarbons（PAHs）

Preparation and characterization of the hapten-protein conjugates are fundamental to developing environmental immunoassays. As a hapten, 1-pyrenebutyric acid（PBA） was conjugated to the carrier protein of bovine serum albumin（BSA） or ovalbumin（OVA） by active ester method. Infrared spectra（IR） showed that PBA-BSA and PBA-OVA conjugates were successfully prepared. The number of the haptens conjugated to the carrier protein was determined by ultraviolet spectra（UV） and matrix-assisted laser desorption ionization time-of-flight mass spectrometry（MALDI-TOF-MS）. The calculated average binding ratios of PBA/BSA and PBA/OVA were 18：1 and 10：1 by UV, and 31：1 and 22：1 by MALDI-TOF-MS, respectively. Although there was a discrepancy between the results determined by the two methods, both of them were useful for the characterization of the hapten-protein conjugates. The antibody was produced against the antigen of PBA-BSA, and the affinity was tested by the double agar diffusion method The conjugates and the antibody could be used for developing a sensitive and selective immunoassay of polycyclic aromatic hydrocarbons（PAHs）.     

Using capillary electrophoresis mobility shift assay to study the interaction of CdTe quantum dots with bovine serum albumin

In this work, the capillary electrophoresis mobility shift assay （CEMSA） was first adopted to study the interaction of protein with quantum dots （QDs）. In this study, bovine serum albumin （BSA） and CdTe QDs were used as model samples. We observed that BSA was facilely adsorbed to CdTe QDs surface, and the QD-BSA complex was formed by a 1：1 stoichiometric ratio. A value of 2.17 4-0.27 × 10^6 mol^-1 L^-1 （at 25 ℃） for the association constant was obtained by CEMSA.     

CoMFA Study on Anti-proliferative Activity of Fluoroquinolone Amide Derivatives

The in vitro anti-proliferative activity （pICi,i=hp,ca,hl） of fluoroquinolone （rhodanineα,β-unsaturated ketone）amide compounds,referred to as“fluoroquinolone amide derivatives （FQADs）”towards Hep-3B,Capan-1 and HL60 cells,was studied by the 3D-QSAR method of comparative molecular field analysis （Co MFA）.Based on the training set of 14 compounds,the prediction model was established,which was further verified by the test set of 5 compounds with template molecule included.It is found that steric an...     

CHEMICAL DERIVATION OF HUMAN INSULIN SUPERAGONISM

The role of three highly conserved insulin residues Tyr^B26 was studied to better understand the relationship between insulin and receptor from rat adipose tissue plasma membranes, lnsulin analogues with a single amino acid substitution or single N-methylation of the peptide bond in the position B26 were all shortened in the C-terminus of the B-chain by four amino acids. The effect of modifications was followed by the binding to the insulin receptor. From our results, we can deduce several conclusions： （1） the replacement of tyrosine in the position B26 by histidine, [N-MeHis^B26]-des-tetrapeptide-（B27-B30）-insulin-B26-amide and [N-MeGlu^B26]-des-tetrapeptide- （B2-B30）-insulin-B26-amide, have no significant effect on the binding affinity and they show binding affinity 105%, 190% and 208%, respectively, of that of human insulin; （2） [Aad^B26] -des-tetrapeptide-（B27-B30）-insulin-B26-amide and [Phe（4-carboxy^B26）]-des-tetrapeptide- （B27~B30）-insulin-B26-amide affect the potency highly positively in vitro studies; they show binding affinity 529 and 289 %, respectively, of that of human insulin.     

A rapid protein sample preparation method based on organic-aqueous microwave irradiation technique

Fast and efficient sample preparation methods are a prerequisite for protein identification in bottom-up proteomics. Here, an innovative microwave irradiation sample preparation method was developed based on an optimized organic-aqueous solvent system for protein identification. Specifically, protein solutions containing high-concentration acetonitrile were subjected to 5 min microwave irradiation. After cooling down, trypsin was added and the digestion was performed with 30 s microwave irradiation, and the resulting peptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS). A shortened processing time of only 5.5 min is needed with this method(more than 12 h is necessary in the traditional overnight protein sample preparation). Moreover, due to the absence of urea and other chaotropic reagents, the digests can be readily identified by MALDI-TOF MS. When an assessment of this method was performed by digesting a model protein BSA, 69% ± 3% sequence coverage corresponding to 47 ± 3 peptides was obtained, which shows better protein identification than that from the standard overnight protein sample preparation method(51% ± 2% sequence coverage and 23 ± 1 peptides). Another model protein α-casein was used for the analysis of protein phosphorylation with the newly developed method that yielded 4 phosphopeptides with 8 phosphorylation sites, whereas 3 phosphopeptides with 2 phosphorylation sites were obtained from the traditional overnight approach. Moreover, the organic-aqueous microwave irradiation method provides effective digestion for proteins down to fmol.