No abstract available.
Adult ; Cerebroside-Sulfatase/*metabolism ; Child, Preschool ; *Chromatography, High Pressure Liquid ; Enzyme Assays/instrumentation/*methods ; Female ; Humans ; Kinetics ; Leukocytes/*enzymology ; Leukodystrophy, Metachromatic/diagnosis/enzymology ; Male ; Middle Aged ; Reference Standards ; Substrate Specificity ; Sulfoglycosphingolipids/analysis/metabolism/standards ; *Tandem Mass Spectrometry/standards

To screen potential hamster chymase 2 inhibitors, a high-throughput screening (HTS) model was established. Recombinant hamster chymase 2 with active form was cloned and expressed in E. coli. The HTS model with total volume of 50 microL in 384-well microplate was based on fluorescence analysis and was proved sensitive as well as specific (Z' = 0.84). A total of 40 080 samples (including 28 060 compounds and 12 020 natural products) were screened, and 613 samples with inhibition greater than 90% were selected for further rescreening. Finally, compounds J16647 and J16648 were identified with high inhibitory activity on chymase 2, and whose IC50 values were 0.823 and 0.690 micromol x L(-1), respectively.
Animals ; Chymases ; analysis ; antagonists & inhibitors ; Cricetinae ; Enzyme Inhibitors ; analysis ; pharmacology ; Escherichia coli ; metabolism ; High-Throughput Screening Assays ; methods ; Inhibitory Concentration 50 ; Rats ; Structure-Activity Relationship

Caspases play important roles in cell apoptosis. Measurement of the dynamics of caspase activation in tumor cells not only facilitates understanding of the molecular mechanisms of apoptosis but also contributes to the development, screening, and evaluation of anticancer drugs that target apoptotic pathways. The fluorescence resonance energy transfer (FRET) technique provides a valuable approach for defining the dynamics of apoptosis with high spatio-temporal resolution. However, FRET generally functions in the single-cell level and becomes ineffective when applied in the high throughput detection of caspase activation. In the current study, a FRET sensor was combined with capillary electrophoresis (CE) to achieve a high throughput method for cellular caspase detection. The FRET-based CE system is composed of a homemade CE system and a laser source for detecting the dynamics of caspase-3 in various cells expressing sensors of caspase-3 that have been treated with anticancer drugs, such as cell cycle-independent drug cisplatin and specific cell cycle drugs camptothecin and etoposide, as well as their combination with tumor necrosis factor (TNF). A positive correlation between the caspase-3 activation velocity and drug concentration was observed when the cells were treated with cisplatin, but cells induced by camptothecin and etoposide did not show any apparent correlation with their concentrations. Moreover, different types of cells presented distinct sensitivities under the same drug treatment, and the combination treatment of TNF and anticancer drugs significantly accelerated the caspase-3 activation process. Its high throughput capability and detection sensitivity make the FRET-based CE system a useful tool for investigating the mechanisms of anticancer drugs and anticancer drug screening.
Antineoplastic Agents ; pharmacology ; Camptothecin ; pharmacology ; Caspase 3 ; metabolism ; Cisplatin ; pharmacology ; Drug Screening Assays, Antitumor ; Electrophoresis, Capillary ; methods ; Enzyme Activation ; drug effects ; Etoposide ; pharmacology ; HeLa Cells ; Hep G2 Cells ; Humans ; Neoplasms ; enzymology ; pathology ; Time Factors ; Tumor Necrosis Factor-alpha ; pharmacology

BACKGROUND: We evaluated the performance of multiplex tandem mass spectrometry (MS/MS) in newborn screening for detection of 6 lysosomal storage disorders (LSDs), namely, Niemann-Pick A/B, Krabbe, Gaucher, Fabry, and Pompe diseases and Hurler syndrome. METHODS: We revised the conditions and procedures of multiplex enzyme assay for the MS/MS analysis and determined the precision of our enzyme assay and the effects of sample amounts and incubation time on the results. We also measured the degree of correlation between the enzyme activities in the dried blood spots (DBSs) and those in the leukocytes. DBSs of 211 normal newborns and 13 newborns with various LSDs were analyzed using our revised methods. RESULTS: The intra- and inter-assay precisions were 2.9-18.7% and 8.1-18.1%, respectively. The amount of product obtained was proportional to the DBS eluate volume, but a slight flattening was observed in the product vs. sample volume curve at higher sample volumes. For each enzyme assay, the amount of product obtained increased linearly with the incubation period (range, 0-24 hr). Passing and Bablok regression analysis revealed that the enzyme activities in the DBSs and those in the leukocytes were favorably correlated. The enzyme activities measured in the DBSs were consistently lower in patients with LSDs than in normal newborns. CONCLUSIONS: The performance of our revised techniques for MS/MS detection and enzyme assays was of the generally acceptable standard. To our knowledge, this is the first report on the use of MS/MS for newborn screening of LSDs in an Asian population.
Dried Blood Spot Testing ; Enzyme Assays ; Enzymes/blood ; Humans ; Infant, Newborn ; Leukocytes/enzymology ; Lysosomal Storage Diseases/*diagnosis ; Republic of Korea ; Tandem Mass Spectrometry/*methods ; Time Factors

To screen potential human soluble protein tyrosine phosphatase 1B (PTP1B) inhibitors, a high-throughput screening (HTS) model in 384-well microplate with total volume of 50 microL was established. Recombinant PTP1B was cloned and expressed in E. coli. with its specific substrate 4-nitrophenyl phosphate disodium salt hexahydrate (PNPP). The HTS model was based on enzyme reaction rate with enhanced sensitivity and specificity (Z' = 0.78). A total of 24,240 samples were screened, among them 80 samples with inhibition greater than 70% were selected for further rescreening. Finally, six compounds with high inhibitory activity were identified, whose IC50 values were 21.58, 18.39, 15.37, 11.92, 37.27, and 36.61 microg x mL(-1), separately. The results indicated that the method was stable, sensitive, reproducible and also suitable for high-throughput screening.
Drug Evaluation, Preclinical ; methods ; Enzyme Inhibitors ; analysis ; pharmacology ; Escherichia coli ; metabolism ; High-Throughput Screening Assays ; methods ; Humans ; Inhibitory Concentration 50 ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; antagonists & inhibitors ; metabolism ; Sensitivity and Specificity ; Vanadates ; pharmacology

Angiotensin-I converting enzyme (ACE) is a key regulator of blood pressure, electrolytes and fluid homeostasis through conversion of angiotensin I into angiotensin II. Recently, a genetic polymorphism of the ACE gene, which accounts for 47% of the variation of ACE activity in blood, has been advocated as a biomarker of athletic aptitude. Different methods of analysis and determination of ACE activity in plasma have been used in human and equine research without a consensus of a "gold standard" method. Different methods have often been used interchangeably or cited as being comparable in the existing literature; however, the actual agreement between assays has not been investigated. Therefore, in this study, we evaluated the level of agreement between three different assays using equine plasma obtained from 29 horses. Two spectrophotometric assays using Furylacryloyl-phenylalanyl-glycyl-glycine as substrate and one fluorimetric assay utilizing o-aminobenzoic acid-FRK-(Dnp)P-OH were employed. The results revealed that the measurements from the different assays were not in agreement, indicating that the methods should not be used interchangeably for measurement of equine ACE activity. Rather, a single method of analysis should be adopted to achieve comparable results and critical appraisal of the literature is needed when attempting to compare results obtained from different assays.
Animals ; Enzyme Assays/*methods ; Female ; Fluorometry/*methods ; Horses/blood/genetics/*metabolism ; Male ; Oligopeptides/pharmacology ; Peptidyl-Dipeptidase A/blood/genetics/*metabolism ; Polymorphism, Genetic ; Reference Values ; Spectrophotometry/*methods

We have developed a rapid and high throughput lipase-ANS (8-Anilino-l-naphthalenesulfonic acid) assay to evaluate the thermo-stability of lipases based on the ANS fluorescence signal's increasing and shifting when this small fluorescence probes binds to lipase. The testing lipase samples were incubated at a temperature range of 25 degrees C to 65 degrees C for 30 min before mixed with ANS solution (0.20 mg/mL lipase and 0.05 mmol/L ANS in the buffer of 20 mmol/L Tris-HCl, 100 mmol/L NaCl, pH 7.2) in a cuvette or microplate. Fluorescence signals of the samples were measured at EX 378 nm, EM 465 nm with a fluorescence photometer or a plate reader, and Tm was calculated with the software of GraphPad Prism5.0. The Tm values of several mutants of Penicillium expansum lipase (PEL) were measured with this ANS assay and conventional method simultaneously and the results show that Tm values are comparative and consistent between these methods, suggesting that the lipase-ANS assay is a reliable, rapid and high throughput method for lipase thermo-stability measurement.
Anilino Naphthalenesulfonates ; chemistry ; Enzyme Stability ; High-Throughput Screening Assays ; methods ; Hot Temperature ; Lipase ; metabolism ; Spectrometry, Fluorescence

The aspartate kinase (AK) from Mycobacterium tuberculosis (Mtb) catalyzes the biosynthesis of aspartate family amino acids, including lysine, threonine, isoleucine and methionine. We determined the crystal structures of the regulatory subunit of aspartate kinase from Mtb alone (referred to as MtbAKβ) and in complex with threonine (referred to as MtbAKβ-Thr) at resolutions of 2.6 Å and 2.0 Å, respectively. MtbAKβ is composed of two perpendicular non-equivalent ACT domains [aspartate kinase, chorismate mutase, and TyrA (prephenate dehydrogenase)] per monomer. Each ACT domain contains two α helices and four antiparallel β strands. The structure of MtbAKβ shares high similarity with the regulatory subunit of the aspartate kinase from Corynebacterium glutamicum (referred to as CgAKβ), suggesting similar regulatory mechanisms. Biochemical assays in our study showed that MtbAK is inhibited by threonine. Based on crystal structure analysis, we discuss the regulatory mechanism of MtbAK.
Amino Acid Sequence ; Aspartate Kinase ; chemistry ; genetics ; metabolism ; Binding Sites ; Cloning, Molecular ; Corynebacterium glutamicum ; enzymology ; Crystallization ; methods ; Crystallography, X-Ray ; Enzyme Activation ; Enzyme Assays ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; metabolism ; Lysine ; pharmacology ; Molecular Sequence Data ; Mycobacterium tuberculosis ; drug effects ; enzymology ; Plasmids ; genetics ; metabolism ; Prephenate Dehydrogenase ; metabolism ; Protein Structure, Secondary ; Threonine ; metabolism ; pharmacology

To screen potential human soluble epoxide hydrolase (hsEH) inhibitors, a high-throughput screening model in 384-well microplate with total volume of 50 microL was established. Recombinant hsEH was cloned and expressed in E. coli. and its specific substrate PHOME was synthesized. The HTS model was based on fluorescence analysis with enhanced sensitivity and specificity (Z' = 0.65). A total of 47 360 samples (including 25 040 compounds and 22 320 natural products) were screened, of which 950 samples with inhibition greater than 80% were selected for further rescreening. Finally, two compounds with high inhibitory activity were identified, whose IC50 value were 8.56 and 4.31 micromol x L(-1), separately. The results indicated that the method was stable, sensitive, reproducible and also suitable for high-throughput screening.
Drug Evaluation, Preclinical ; methods ; Enzyme Inhibitors ; analysis ; chemistry ; Epoxide Hydrolases ; antagonists & inhibitors ; chemistry ; metabolism ; Escherichia coli ; metabolism ; High-Throughput Screening Assays ; methods ; Inhibitory Concentration 50 ; Recombinant Proteins ; metabolism ; Reproducibility of Results ; Sensitivity and Specificity ; Spectrometry, Fluorescence ; methods ; Substrate Specificity

As a potent anticoagulant, leech a traditional Chinese medicine, has become increasing topics. Hirudin, which is the primary effective component in leech, is a specific and efficient inhibitor of thrombin, mainly used in prevention and treatment of thrombus on the clinic practice. However, there is still no accurate and convenient method reported about the determination of it's biological activity. This paper reported a method for the determination of the biological activity the of extract from hirudo. The extra thrombin, which was not inhibited by hirudin in the extract from hirudo, reacted with N-benzoyl-L-arginine ethyl ester and was determined. The biological activity of the hirudo extract was determined, indirectly. The linear of calibration curve and accuracy were both perfect, the method was accurate and reliable.
Animals ; Arginine ; analogs & derivatives ; analysis ; Biological Factors ; analysis ; isolation & purification ; pharmacology ; Enzyme Assays ; methods ; Enzyme Inhibitors ; analysis ; isolation & purification ; pharmacology ; Hirudins ; analysis ; isolation & purification ; pharmacology ; Hirudo medicinalis ; chemistry ; Thrombin ; analysis ; antagonists & inhibitors