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.     

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 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.     

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.     

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.     

Osmotic Regulation of Betaine Content in Leymus chinensis Under Saline-alkali Stress and Cloning and Expression of Betaine Aldehyde Dehydrogenase（BADH） Gene

The potted Leymus chinensis seedlings were treated with saline-alkali solution of six different（from Ⅰ to Ⅵ） concentrations. The results demonstrate that the betaine content and Betaine-aldehyde dehydrogenase（BADH： EC 1.2.1.8） activities have a direct relation with increased stressing time in the same treatment; both exhibit a single peak with increasing the concentration of saline-alkali solution, and number V shows the highest value. The BADH gene of Leyrnus chinensis was cloned by RT-PCR and RACE technology and was designated as LcBADH. The cDNA sequence of LcBADH was 1774bp including the open reading frame（ORF） of 1521bp（coding 506 amino acids）. The vector of prokaryotic expression was constructed by inserting the LcBADH gene fragmcnt into pET30a（＋） and transformed into E. coli BL21（DE3）. The result of SDS-PAGE shows that the idio-protein with a molecular mass of 56.78 kDa was effectively expressed in the recombinant bacteria induced by isopropyl fl-D-thiogalactoside（IPTG）.     

Toxicity Assessment of Cyanide and Tetramethylene Disulfotetramine （Tetramine） Using Luminescent Bacteria Vibrio-qinghaiensis and PbO= Electrochemical Sensor

The toxicities of cyanide and tetramethylene disulfotetramine （tetramine） were evaluated by two methods of luminescent bacteria and PbO2 electrochemical sensor. Vibrio-qinghaiensis, a kind of luminescent bacteria, can produce bioluminescence and the bioluminescence was decreased with the addition of toxicants. The toxicities of cyanide and tetrarnine were expressed as 10 min-EC50 value, which was the concentration of chemical that reduces the light output by 50% after contact for 10 min. Nano PbO2 modified electrode, a rapid toxicity determination method was also described in this work. By the PbO2 modified electrode, the current responses of Escherichia coli （E. coli） were changed with the addition of toxicants. The value of 10 min-EC50 was also provided with the PbO2 electrochemical sensor. Compared with the 10 min-EC50 and detection limits （38.38 and 0.60 μg/mL for cyanide, 0.24 and 0.02 μg/mL for tetramine） with luminescent bacteria, the PbO2 sensor provided a simple and convenient method with lower 10 min-EC50 and detection limits （26.37 and 0.52 μg/mL for cyanide, 0.21 and 0.01 μg/mL for tetramine） and fast response time.     

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.     

Microcalorimetric Evaluation of the Effect of Kanamycin： An Analysis Based on the Median-Effect Principle

A study of the effect of drug, kanamycin, on the growth metabolism of recombinant Escherichia coli B 1 was carded out by microcalorimeter monitoring of the metabolic activity of treated cells. Power-time curves of growing recombinant Escherichia coli cell suspensions, treated with different kanamycin doses, were recorded. The extent of the effect was evaluated by changes in the slopes of the microcalorimetric curves and the kinetics of the drug action was interpreted from the time at which these changes reached their maximum values and maintained their maximum values. Experimental dose-effect relationships conform to the median-effect principle of the mass-action law： fa/（1-fa）=（D/D50）^m. A plot of y=lg[（fa）^1-1]^-1 versus x=lg D gives the slope m, D50 and R∞. The experimental results revealed that high concentration of kanamycin had an inhibitory effect on the growth of recombinant Escherichia coli B 1 in the lg phase, and had a promoting effect in the stationary period. Moreover, it was demonstrated that microcalorimetry was a reliable method for the detection of modulatory effects in biology.     

Synthesis and Antibacterial Activity of Selenium-functionalized Poly(ε-caprolactone)

A selenium-functionalizedε-caprolactone was synthesized by introducing a phenyl selenide group at the 7-position.A polymer was obtained through the ring-opening polymerization of this monomer in a base/thiourea binary organocatalytic system.A living polymerization process was achieved under mild conditions.The resulting polymers had a controlled molecular weight with a narrow molecular weight distributions and high end-group fidelity.Random copolymers could be obtained by copolymerizing this monomer withε-caprolactone.The thermal degradation temperature of the obtained copolymers decreased with the increasing molar ratio of selenide functionalized monomer in copolymers,while the glass transition temperature increased.In addition,the phenyl selenide side group could be further modified to a polyselenonium salt,which resulted in a polymer with good antibacterial properties.The survival rate of E.coli and S.aureus was only 9%with a polymer concentration of 62.5μg/mL.     

Synthesis and Expression in Escherichia coli of a Human Neutrophil Activating Protein-1/Interleukin-8 Gene

The complete gene coding for human neutrophilactivating protein-1/interleukin-8 was synthesized using a semi-chemical semi-enzymatic method. The synthetic gene was then overexpressed in Escherichia coli under the temperature-regulated control of the P_RP_L tandem promoters. As determined by SDS-PAGE and densitometry, the overexpressed protein comprised up to 18.5% and 10.9% of the total soluble protein in E. coli cells grown in shake flasks and in batch fermentation, respectively. The recombinant NAP-1/IL-8 was then purified to>95% homogeneity by gel filtration and cation exchange chromatography. The purified protein appeared as a single band on the SDS-PAGE gel and possessed potent chemotactic activity in the concentration of <10 ng/ml, as assayed by the agarose plate method. An early skin reactivity was also observed when the pure NAP-1/IL-8 was injected subcutaneously into the rabbits. The N-terminal 36 amino acid sequence of the recombinant NAP1/IL-8 was determined using the Edman method and was sho     

Fluorescence detection of Escherichia coli on mannose modified ZnTe quantum dots

Rapid detection and identification of Escherichia coli(E.coli) is essential to prevent its quickly spread.In this study,a novel fluorescence probe based on ZnTe quantum dots(QDs) modified by mannose(MAN)had been prepared for the determination of E.coli.The results showed that the obtained QDs showed excellent selectivity toward E.coli,and presented a good linearity in range of 1.0×10~5～1.0×10~8 CFU/mL.The optimum fluorescence intensity for detecting E. coli was found to be at pH 7.0 with a temperature of25℃ and incubation time of 20 min.Under these optimum conditions,the detection limit of E.coli was4.6×10~4 CFU/mL.The quenching was discussed to be a static quenching procedure,which was proved by the quenching efficiency of QDs decreased with the temperature increasing.     

Two Eremophilane-type Sesquiterpenoids from the Rhizomes of Ligularia veitchiana（Hemsl.） Greenm

Two eremophilane-type sesquiterpenoids from the rhizomes of Ligularia veitchiana(Hemsl.) Greenm were isolated and determined as 10α,11-endoperoxide-1(2)-en-7α-hydroeremophilane(1) and 1(10)-en-2-oxo-7α-isopropanol-eremophilane(2).Compound 1 was a new compound and compound 2 was a new natural product.The structures of the two compounds were established by means of spectral methods including 1H NMR,13C NMR,2D NMR and MS.     

Cloning and Sequencing of Trichosanthin Gene and Its Expression in Escherichia coli and Tobacco Plant

A Trichosanthin gene was cloned from Trichosanthes kirilowii genomic DNA by polymerase chain reaction (PCR). Nucleotide sequence data indicated that we obtained the coding region of the mature Trichosanthin peptide as well as its signal peptide at the N-terminus. Comparisons of our sequence with the previously reported nucleotide sequences of this gene showed 99.25% homology, yet there were notable differences between the previously reported amino acid sequence and our deduced result. This gene was subcloned into a highlevel expression plasmid (pJLA502) of E. coli under the control of a P_RP_L promoter, and we observed the gene product after temperature induction. The gene was further cloned into plant intermediate vector pE3 under the control of a CaMV 35S promoter, and transferred into a tobacco genome using the agrobacterium-mediated gene transfer system. Western blotting analysis of the protein extracted from Escherichia coli and transgenic tobacco plants proved that the Trichosanthin gene has been     

Prokaryotic Expression, Purification, Antibody Production of a NH2 -Terminal Fragment of mCLCA3 Protein and Analysis of mClCA3 Protein Expression in Asthmatic Mouse Lung

mCLCA3 is a member of calcium activated chloride channel(CACC)family that may play an important role in mucin packaging and secretion in asthmatic and cystic fibrosis lung.To study the protein structure and expression of mCLCA3 in asthmatic mouse lung,an N-terminal 269 amino acid peptide of mCLCA3 was expressed in E.coli,purified to homogeneity and rabbit polyclonal antibodies against this peptide were generated.Immunohistochemistry of asthmatic mouse lung using the antibody indicated exclusive mCLCA3 expression in mucin granules of goblet cells in airway surface and lumen.Immunoblot analysis of lavage fluid from asthmatic mouse lung revealed a single 90 kDa protein form of mClCA3.The results demonstrate that the 90 kDa N-terminal peptide,neither the full-length protein nor the reported N-terminal 35 kDa cleaved form of mClCA3 is the major functional form involved in the packaging and exocytosis of mucin granules in asthmatic goblet cells.     

Isolation and Structure of 1-Deoxybaccatin VI from the Root of Taxus chinensis, Rehd. var. mairei

Deoxybaccatin Ⅵ (4α,7β,9α,10β,13α-penta-acetoxy-2α-benzoyloxy-5β,20-epoxytax-11-ene) was isolated from the roots of Taxus chinensis, Rehd. var. mairei. The structural assignments of the compound were based on their spectral data, including 2D NMR experiments and chemical correlation. The X-ray crystallographic analysis of 1-deoxybaccatin Ⅵ provided unambiguous characterization for the structures. In the structure, the six-membered A ring exhibits boat conformation, the eight-membered B ring adopts boat-chair conformation, and the six-membered C ring exhibits a sofa conformation.     

Electrochemical formation and phase control of Mg-Li alloys

The electrochemical formation of Mg-Li alloys was investigated in a molten LiCl-KCl(58-42 mol%)eutectic melt at 723 K. The cyclic voltammogram for a Mo electrode showed that the electroreduction of Li~ proceeds in a single step and the deposition potential of Li metal was -2.40 V(vs.Ag/AgCl).For Mg electrode,the electroreduction of Li~ takes place at less cathodic potential than that at the Mo electrode which was caused by the formation of Mg-Li alloys.Phase of the deposited Mg-Li alloys could be controlled by the electrolysis potential,and the samples were characterized by X-ray diffraction and scanning electron microscopy. The results showed thatα-Mg andβ-Li phases were obtained at -2.35 and -2.55 V,respectively.     

A new triterpenoid saponin from the roots of Platycodon grandiflorum

A new triterpenoid saponin, 3-O-β-D-glucopyranosyl 16-oxo-platycodigenin 28-O-β-D-apiofuranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyra-nosyl ester, was isolated from the roots of Platycodon grandiflorum, together with three known saponins, including platycodin D, deapio platycoside E and platycoside E. The structure of the new compound, named 16-oxo-platycodin D, was elucidated on the basis of spectroscopic data.     

Analysis of local structure of Arg10 domain in apo-α- lactalbumin with a polarity-sensitive arginine-specific fluorescent probe

The polarity-sensitive fluorescent probe, 3-(4-chloro-6-p-glyoxalphenoxy-1,3,5-triazinylamino)-7- (dimethylamino)-2-methylphenazine, was used to analyze the local structure of apo-α-lactalbumin by detecting the polarity and conformational changes of the arginine residue (Arg10) domain. The polarity of the Arg10 domain in both native and heat-denatured apo-α-lactalbumin was determined, which corresponds to a dielectric constant of 16, and the hydrophobic core near the Arg10 was found to be conservative for h...     

Synthesis and Expression of a Gene From Kringle-2 Domain of Tissue Plasminogen Activator in E. coli

The DNA fragment corresponding to the tissue plasminogen activator (tPA) sequence 174-262 (Kringle-2 domain) has been synthesized by using the solid phase phosphotriester method. The Kringle-2 domain of human tPA was expressed in Escherichia colt by secretion into the periplasmic space using the Lpp-Lac promoter and PIN-Ⅲ OmpA2 signal sequence. About two thirds of the expression product was secreted into the periplasmic space , and purified with ammonium sulfate fractionation, affinity chro-matography on Lysine-Sepharose, and FPLC-Mono Q exchange chromatography. The amino acid composition observed from the Kringle-2 purified from E. coli is identical with that expected for the 174-262 fragment of human tPA. Radio binding assay shows that the recombinant Kringle-2 domain possesses the activity of fibrin binding.