Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0040822 (tremor)
 18,428 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) is a member of the tumor necrosis factor (TNF) family of ligands which has been reported in 1995. The TRAIL protein induces apoptosis of certain types of target cells, such as transformed cells that include but are not limited to cancer cells and virus-infected cells but the normal cells. It is a type II transmembrane protein and the extracellular domain of TRAIL is the functional domain in induction of cell apoptosis. A gene fragment encoding for the active domain of TRAIL was modified with oligo-nucleotide directed mutagenesis according to the characters of Pichia pastoris expressing vector. Arginine at the position of 149 corresponding to the amino acid residue 531 which might be a potential Kex2 protease processing sites was substituted with Lysine to prevent the expressed protein from the digestion by the protease. After proved with DNA sequencing. the modified gene fragment coding soluble TRAIL domain was inserted into the Pichia pastoris expression vector pPIC9K in the same reading frame with alpha-factor secreting signal peptide. The recombinant plasmid pPIC9K - TRAIL was transferred into P. pastoris cell by spheroplast transformation. The recombinant yeasts were identified by antibiotic G418 and Southern dot blot. The transformants (His+ Mut(s)) containing multi-copy gene fragment of TRAIL were selected with increasing concentration of G418 and induced with 0.5% methanol in shaking flask to expression the active domain of TRAIL. After inducing for 3 - 4 days, the proteins in the culture supernatant was assayed with SDS-PAGE and Western blot. Two expressed protein bands whose appearant molecular weight were 19kD and 38kD, respectively, could be specifically recognized by polyclonal antibodies against human TRAIL. The 38kD protein might be a dimers of TRAIL in the culture supernatant. The amount of expressed foreign protein made up to 36% of the total proteins in the culture suprenatant. Biological activity assay, in vitro, indicated that the expressed protein could induce tumor cells apoptosis.
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PMID:[Expression of a DNA fragment encoding the active domain of human TNF related apoptosis inducing ligand in pichia pastoris]. 1596 15

(R)-chlorprenaline, a selective activator of beta2 receptor and an effective drug for bronchitis and asthma, is industrially prepared from (R)-2'-chloro-1-phenyl-ethanol. In this communication, we describe (1) the identification of Saccharomyces cerevisiae B5 as an effective host for stereoselective reduction of 2'-chloroacetophenone to (R)-2'-chloro-1-phenyl-ethanol; (2) the presence of ethanol enhances the conversion; and (3) the biochemical factors that effect the yield of the product. Among the four yeast strains capable of reduction 2'-chloroacetophenone to (R)-2'-chloro-1-phenyl-ethanol we screened, Saccharomyces cerevisiae B5 showed the highest activity and stereoselectivity, and was used for the subsequent study. The effect of the presence of methanol, ethanol, 2-propanol, 1-butanol, glucose, glycerol and lactic acid was first investigated, as it was previously reported that they increased the yield and stereoselectivity of the reaction. The addition of the co-substrate methanol, ethanol, 2-propanol, 1-butanol, glucose and glycerol favored the formation of the 2'-chloroacetophenone to (R)-2'-chloro-1-phenyl-ethanol. Lactic acid inhibited the enzyme activity. Ethanol is the best co-substrate among the seven co-substrates and under the optimum concentration of 5% , the yield of (R)-2'-chloro-1-phenyl-ethanol was increased from 17% to 74%. The oxidation of ethanol regenerates NADH required for the reduction. The effects of the reaction time, pH, cell concentration, substrate concentration and temperature on the reduction were investigated next. The enantiometric excess of (R)-2'-chloro-1-phenyl-ethanol reached 100% under the optimal condition: pH8.0, 25 degrees C and 5% ethanol. The product yield went up with the increasing Saccharomyces cerevisiae B5 concentration and reached 100% when the cell dry weight was 10.75 mg/mL and 2'-chloroacetophenone was 6.47 mmol/L. The yield of (R)-2'-chloro-1-phenyl-ethanol decreased sharply with the increase of substrate concentration, as the high concentration of substrates is toxic to the cell and inhibits the activity of reductases. The aerobic cultivation of the yeast and shaking during the reaction increased the yield of (R)-2'-chloro-1-phenyl-ethanol. The yeast can be reused up to 15 times. This research paves the way for economical preparation of chiral 2'-chloroacetophenone to R-2'-chloro-1-phenylethanol.
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PMID:[Saccharomyces cerevisiae B5 efficiently and stereoselectively reduces 2'-chloroacetophenone to R-2'-chloro-1-phenylethanol in the presence of 5% ethanol]. 1596 23

To compare the DAAO expression level in different Pichia pastoris host strains, the gene encoding DAAO from Trigonopsis variabilis was cloned into plasmid pPIC3.5k and then transformed into P. pastoris GS115 and KM71 respectively. The positive transformants PDK13 (MutS) and PD27 (Mut+) were obtained by PCR analysis. Their optimal and different expression conditions were investigated. To compare with PD27, PDK13 was determined to poss a slower consumption of methanol, a longer induction time, a lower oxygen request and apparently higher expression of DAAO. The highest expression levels were reached up to 2700, 2500 IU/L in shaking flask and 10140, 8463.5 IU/L in fermentor respectively. The over-expression of DAAO can meet its large demand for production of 7-ACA, alpha-keto acid and L-amino acid. In addition, the phenylpyruvate and L-phenylalanine were obtained by crude DAAO reacting with DL-phenylalanine at 37 degrees C for 3h.
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PMID:[Compared D-amino acid oxidase expression in different Pichia pastoris host strains]. 1596 91

Because organotin compounds (OTC) are widely used in many fields of activity, they have become an ubiquitous environmental presence. The presence of organotins in the environment impacts upon food safety, making it important to monitor the levels of organotin pesticides in fruits and vegetables. Nevertheless, only a few studies have been published on organotin speciation in plants. The objective of the present study was to evaluate and optimise a specific procedure based on pressurised solvent extraction (PSE) that is suitable for monitoring organotin content in vegetables. In ASE, solvents are used at elevated temperatures and pressures to increase the rate and efficiency of the extraction process. The results from this procedure were compared to those from the technique usually employed, solid/liquid extraction (SLE) performed in an acidic solvent by mechanical shaking. Three extracting solutions were tested-methanol, ethyl acetate and a mixture of methanol and ethyl acetate-and the mixture was found to give the most quantitative results while preserving the speciation. French bean and lettuce leaves as well as potato tubers were used as the plant materials. These vegetables were considered because they are the vegetables consumed in the most quantities in Europe. The study focuses on trisubstituted OTCs, which are the most toxic tin species. The samples were spiked with four trisubstituted organotins: tributyltin (TBT), triphenyltin (TPhT), tricyclohexyltin (TcHexT) and trioctyltin (TOcT). The influence of the pressure and the temperature of the PSE on the quantitativity of the process and on species preservation was evaluated using the experimental design methodology. The optimised PSE allowed detection limits down to 1-2 ng (Sn) g(-1) to be reached. These are higher than those obtained by SLE (0.1-1 ng (Sn) g(-1)). Although the repeatability is similar for both PSE and SLE (2-12% for triorganotin compounds), this appears to be highly time-dependent in the case of SLE. Comparison with SLE confirms that PSE is an interesting tool for vegetable analysis considering the satisfactory OTC preservation and repeatability obtained for a relatively short extraction duration (only 15 min against 2-12 h for SLE).
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PMID:Pressurised solvent extraction for organotin speciation in vegetable matrices. 1600 40

A highly selective immunoaffinity column was obtained by coupling anti-papaverine polyclonal antibodies to CNBr-activated Sepharose 4B. It was found that the coupling efficiency and performance of the immunoaffinity column were greatly improved by prolonging the coupling reaction time from 3 h at 20 degrees C with shaking to incubation overnight at 4 degrees C after the 3 h shaking reaction. The pH and ionic strength were observed to be the most important factors that influence the binding of papaverine to the immunoaffinity column. Using 0.01 mol/L phosphate buffered saline (PBS, pH 8.3) and methanol-water (80:20, v/v) as the loading and eluting solutions, respectively, papaverine was first retained on the column and then quantitatively eluted out with a mean recovery of 86% at a loading concentration of 1 microg/mL. When applied to real samples of pericarpium papaveris and food products, the established immunoaffinity column showed high efficiency in removing the matrix interferences in the samples and satisfactory recovery results were obtained. The method was useful for extraction and purification of papaverine from related samples.
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PMID:An efficient immunoaffinity chromatographic method for extraction and purification of papaverine from samples of pericarpium papaveris and food products. 1611 93

A low-cost thin-layer chromatographic method has been developed for the presumptive measurement of ochratoxin A (OTA) at 5 microg/kg in green coffee beans. The analytical method consisted of extracting OTA by shaking the beans with a mixture of methanol and aqueous sodium bicarbonate solution, which was then purified by liquid-liquid partition into toluene. OTA was separated by normal-phase two-dimensional thin-layer chromatography and detected by visual estimation of fluorescence intensity under a UV lamp at 365 nm. The chromatography solvents were toluene-methanol-formic acid (8:2:0.03) for the first development and petroleum ether-ethyl acetate-formic acid (8:10:1) for the second dimension development. This method was tested with uncontaminated green coffee bean samples spiked with an OTA standard at four different concentrations (5, 10, 20, and 30 microg/kg). The method is rapid, simple, and very easy to implement in coffee-producing countries. It is highly selective and does not involve the use of chlorinated solvents in the sample extraction step. This inexpensive method has been applied to different types of green coffee samples from various countries (Zimbabwe, Brazil, India, Uganda, Colombia, and Indonesia) and different manufacturers, and no OTA below the detection limit of 5 microg/kg was detected in any samples analyzed.
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PMID:Two-dimensional thin-layer chromatographic method for the analysis of ochratoxin A in green coffee. 1616 95

To combine the fibrinolytic with anticoagulant activities for therapy of thrombotic deseases, a fusion protein made of tissue-type plasminogen activator (t-PA) and hirudin was constructed and expressed in chia pastoris. To improve thrombolytic properties of t-PA and reduce bleeding side effect of hirudin, FXa-recognition sequence was introduced between t-PA and hirudin molecules.The anticoagulant activity of hirudin can be target-released through cleavage of FXa at thrombus site. t-PA gene and hirudin gene with FXa-recognition sequence at its 5'-terminal were obtained by RT-PCR and PCR respectively. The fusion protein gene was cloned into plasmid pIC9K and electroporated into the genome of Pichia pastoris GS115. The expression of fusion protein was induced by methanol in shaking flask and secreted into the culture medium. Two forms of the fusion protein, single-chain and double-chain linked by a disulfide bond (due to the cleveage of t-PA at Arg275-Ile276), were obtained. The intact fusion protein retained the fibrinolytic activity but lacked any anticoagulant activity. After cleavage by FXa, the fusion protein liberated intact free hirudin to exert its anticoagulant activity. So, the fusion protein is a bifunctional molecule having good prospect to develop into a new targeted therapeutic agent with reduced bleeding side effect for thrombotic diseases.
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PMID:[Construction and expression of a fusion protein made of tissue-type plasminogen activator and hirudin in Pichia pastoris]. 1617 91

A simple and accurate method for the determination of andrographolide and dehydroandrographolide in andrographis paniculata Nees materials and patent medicines with high performance liquid chromatography (HPLC) has been developed. The two components were extracted from powdered samples by shaking with methanol. The resultant extracts were separated within 15 min on a BECKMAN C18 column (4.6 mm i. d. x 250 mm, 5 microm) and with a gradient elution of acetonitrile-water at a flow rate of 0.5 mL/min. The detection wavelength was 225 nm and the injection volume was 20 microL. In gradient elution program the volume fraction of acetonitrile in mobile phase was as follows: 0 min - 1 min, 40%; 1 min - 5 min, 40% - 50%; 5 min - 15 min, 50% - 70%. Both andrographolide and dehydroandrographolide have good linearity in the range of 10 mg/L to 100 mg/L with the correlation coefficients of 0.997 6 and 0.998 6 respectively. This method has been successfully applied for the analysis of andrographis paniculata Nees materials and related patent medicines.
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PMID:[Determination of andrographolide and dehydroandrographolide in Andrographis paniculata nees materials and related patent medicines by reversed-phase high performance liquid chromatography]. 1635 99

The performance of a commercially available enzyme-linked immunosorbent assay (ELISA) kit containing highly sensitive monoclonal antibodies against fenitrothion was assessed. The experimentally estimated dynamic range (0.087 to 2 ng/g) agreed with that established by the kit manufacturer (0.075 to 1 ng/g). The linearity of the standard curve produced for the kit-assembled standard solutions (slope = -0.3829) agreed with that of the curve produced for the self-made standard solutions (slope = -0.3619). The sensitivity (I50 value) and the limit of detection for the kit were 0.23 and 0.033 ng/g, respectively. Selectivity of the ELISA indicates that the monoclonal antibody can readily distinguish the target pesticide from other structurally related analogs and some metabolites (oxon forms), with the exception of ethyl O-(p-nitrophenyl) phenyl phosphonothionate (EPN), parathion-methyl, and parathion. Methanol was the best organic solvent for the kit, with optimal sensitivity observed at a final concentration in the well of 10% (vol/vol) or less. Matrix interferences were minimized by direct dilution with water (60-fold) of the methanolic extracts fromapple and peach samples. To extract fenitrothion from these two agricultural products as simply and rapidly as possible, three extraction methods were used. The extraction method that involved shaking by hand for 3 min was the best among the three methods. High recovery percentages (116.6% for apple and 110.8% for peach) were obtained. Validation of the ELISA method was carried out using the gas chromatography-mass spectrometry method, resulting in high recovery and close correlation of results (r > 0.95). These findings strongly suggest that the ELISA kit may be routinely employed for on-site fenitrothion screening of fruit samples.
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PMID:Evaluation of performance of a commercial monoclonal antibody-based fenitrothion immunoassay and application to residual analysis in fruit samples. 1641 18

Removal of phthalate esters (PAEs) by chitosan bead in aqueous solution was studied. The adsorption isotherms of PAEs by chitosan bead were well described by Freundlich isotherm equations. Results of kinetic experiments indicated that diheptyl phthalate (DHpP) had the highest adsorption capacity (1.52 mg/g) among six PAEs in our research. PAE adsorption efficiency by chitosan bead was examined in both batch and continuous systems, and DHpP attained 74.9% recovery efficiency from chitosan bead by shaking with an equal volume mixture of methanol and water. The recovered chitosan bead was reusable as an adsorbent. The influences of temperature, pH, Ca+2, and NaCl on PAE adsorption were also evaluated to determine performance in different water environments (e.g., groundwater, surface water, and sea water). The results showed that PAE adsorption decreased as temperature increased. From pH experiments it appeared that pH 8.0 was optimal for adsorption. The effect of Ca+2 showed that adsorption efficiency did not change by increasing the concentrations of Ca+2 until 400 mg/L. NaCl coexistence showed an insignificant effect on PAE adsorption. Furthermore, the chitosan bead was also applied to treating the discharge of a plastics plant, and the treatment results resembled those of a laboratory continuous system. This is the first report to use chitosan bead as an adsorbent to adsorb phthalate esters from aqueous solution. These results indicate that the application of chitosan bead is feasible in the aqueous environments of Taiwan.
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PMID:Removal of phthalate esters from aqueous solutions by chitosan bead. 1642 28


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