Production of chiral amines and unnatural amino-acid using ω-transaminase can be achieved by kinetic resolution and asymmetric synthesis, thus ω-transaminase is of great importance in the synthesis of pharmaceutical intermediates. By genomic data mining, a putative ω-transaminase gene hbp was found in Burkholderia phytofirmans PsJN. The gene was cloned and over-expressed in Escherichia coli BL21 (DE3). The recombinant enzyme (HBP) was purified by Ni-NTA column and its catalytic properties and substrate profile were studied. HBP showed high relative activity (33.80 U/mg) and enantioselectivity toward β-phenylalanine (β-Phe). The optimal reaction temperature and pH were 40 ℃ and 8.0-8.5, respectively. We also established a simpler and more effective method to detect the deamination reaction of β-Phe by UV absorption method using microplate reader, and demonstrated the thermodynamic property of this reaction. The substrate profiling showed that HBP was specific to β-Phe and its derivatives as the amino donor. HBP catalyzed the resolution of rac-β-Phe and its derivatives, the products (R)-amino acids were obtained with about 50% conversions and 99% ee.
Bacterial Proteins ; biosynthesis ; genetics ; Burkholderia ; enzymology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Transaminases ; biosynthesis ; genetics

ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t_1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T¹⁰₅₀) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.
Transaminases/chemistry* ; Molecular Docking Simulation ; Amines/chemistry* ; Pyruvic Acid/metabolism* ; Enzyme Stability

OBJECTIVETo evaluate the influence of Chinese drug Dahuang Zhechong pill on the hepatocellular function.

METHODThirty-seven patients with hepatocirrhosis and twelve normal controls were performed the hepatobiliary scintgraphy with Tc-99m labeled ethylene hepatobiliary iminodiacetic acid (99 mTc-EHIDA), and the biochemical examination of hepatic function. There was 19 cases repeated the imaging after 6 months treated with chineses drug. By the three compartmental model configurations, the function parameters of hepatocellular extraction and excretion were calculated.

RESULTIn the hepatocirrhosis groups, the hepatocellular uptake peak time and mean residence index were higher than those in normal controls (P < 0.01). Compared to normal controls, the uptake index, uptake speed index and descendent speed index were decreased markedly (P < 0.05). After treatment for 6 months with Chinese drug, the level of serum transaminase, globulin and bilirubin was lower than that before treatment. The uptake peak time and mean residence index decreased notably after treatment for 6 months (P < 0.01), and the uptake index increased, (P < 0.05).

CONCLUSIONChinese drug Dahuang Zhechong pill may improve the hepatocellular function and liver function status in patients with hepatocirrhosis.

Adult ; Bilirubin ; blood ; Drugs, Chinese Herbal ; pharmacology ; Globulins ; metabolism ; Hepatocytes ; drug effects ; metabolism ; Humans ; Liver Cirrhosis ; blood ; drug therapy ; metabolism ; Liver Function Tests ; Male ; Middle Aged ; Transaminases ; blood

Aromatic L-Amino acids are important chiral building blocks for the synthesis of many drugs, pesticides, fine chemicals and food additives. Due to the high activity and steroselectivity, enzymatic synthesis of chiral building blocks has become the main research direction in asymmetric synthesis field. Guided by the phylogenetic analysis of transaminases from different sources, two representative aromatic transaminases TyrB and Aro8 in type I subfamily, from the prokaryote Escherichia coli and eukaryote Saccharomyces cerevisia, respectively, were applied for the comparative study of asymmetric transamination reaction process and catalytic efficiency of reversely converting keto acids to the corresponding aromatic L-amino acid. Both TyrB and Aro8 could efficiently synthesize the natural aromatic amino acids phenylalanine and tyrosine as well as non-natural amino acid phenylglycine. The chiral HPLC analysis showed the produced amino acids were L-configuration and the e.e value was 100%. L-alanine was the optimal amino donor, and the transaminase TyrB and Aro8 could not use D-amino acids as amino donor. The optimal molar ratio of amino donor (L-alanine) and amino acceptor (aromatic alpha-keto acids) was 4:1. Both of the substituted group on the aromatic ring and the length of fatty acid carbon chain part in the molecular structure of aromatic substrate alpha-keto acid have the significant impact on the enzyme-catalyzed transamination efficiency. In the experiments of preparative-scale transamination synthesis of L-phenylglycine, L-phenylalanine and L-tyrosine, the specific production rate catalyzed by TryB were 0.28 g/(g x h), 0.31 g/(g x h) and 0.60 g/(g x h) and the specific production rate catalyzed by Aro8 were 0.61 g/(g x h), 0.48 g/(g x h) and 0.59 g/(g x h). The results obtained here were useful for applying the transaminases to asymmetric synthesis of L-amino acids by reversing the reaction balance in industry.
Amino Acids, Aromatic ; biosynthesis ; genetics ; Catalysis ; Escherichia coli ; enzymology ; Phenylalanine ; biosynthesis ; genetics ; Protein Engineering ; methods ; Saccharomyces cerevisiae ; enzymology ; Stereoisomerism ; Transaminases ; genetics ; metabolism ; Tyrosine ; biosynthesis ; genetics

OBJECTIVETo retrospectively investigate the incidence of severe neutropenia and elevation of transaminase during neoadjuvant chemotherapy using epirubicin, cyclophosphamide and fluorouracil in breast cancer patients.

METHODSFrom January 2011 to December 2012, 303 consecutive breast cancer patients with complete treatment data treated in our department were included in this analysis. All patients received neoadjuvant chemotherapy with equal dose of EPI (100 mg/m(2)) administered every 3 weeks for 4 cycles before surgery.

RESULTS200 patients (66.0%) experienced at least one episode of grade 3/4 neutropenia/leukopenia, among them 176 patients experienced their first episode after the first cycle. Febrile neutropenia (FN) occurred in 13 patients for 14 episodes. Elevation of transaminase occurred in a total of 46 patients (15.2%), among them, grade 2 or higher elevation occurred in 15 patients (5.0%). Three blood test plans were adopted to monitor the patients during chemotherapy: (1) Routine blood count repeated every week; (2) Routine blood count before and on day 10 of each chemotherapy episode; (3) Routine blood count before and on day 7, 10 and 14 of each chemotherapy episode. The number of patients whose chemotherapy was delayed due to 3/4 neutropenia/leucopenia in each blood test plan was 3 (5.0%), 7 (3.9%) and 2 (3.2%), respectively. The number of patients with febrile neutropenia (FN) in each blood test plan was 2 (3.3%), 8 (4.4%) and 3 (4.8%), respectively. No statistically significant difference in treatment delay or the incidence of FN was observed among different blood test plans. No statistically significant difference in the incidence of grade 3/4 neutropenia/leukopenia or grade 2 or higher transaminase elevation was observed among different 5-Fu regimens.

CONCLUSIONSDuring neoadjuvant chemotherapy using FE100 C, Fci E100 C or E100 C for breast cancer patients without routine prophylactic G-CSF, the incidence of grade 3/4 neutropenia/leukopenia is 66.0%. With the patient management plan we adopted, 4.3% of patients developed febrile neutropenia. Prophylactic medication may not be necessary for patients without evident liver dysfunction.

Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Breast Neoplasms ; drug therapy ; Cyclophosphamide ; therapeutic use ; Epirubicin ; therapeutic use ; Female ; Fluorouracil ; therapeutic use ; Granulocyte Colony-Stimulating Factor ; Humans ; Incidence ; Neoadjuvant Therapy ; Neutropenia ; metabolism ; Retrospective Studies ; Transaminases ; metabolism

The aim of this study was to assess changes of Hsp70 and HSF-1 protein and mRNA expression in stress-sensitive organs of pigs during transportation for various periods of time. Twenty pigs were randomly divided into four groups (0 h, 1 h, 2 h, and 4 h of transportation). A significant increased activity of AST and CK was observed after 1 h and 2 h of transportation. Histopathological changes in the heart, liver, and stomach indicated that these organs sustained different degrees of injury. Hsp70 protein expression in the heart and liver of transported pigs did not change significantly while it increased significantly (p < 0.05) in the stomach. Hsp70 mRNA levels decreased significantly (p < 0.05) in the heart after 4 h of transportation. However, mRNA expression increased significantly in the liver after 1 (p < 0.05) and 4 h (p < 0.01) of transportation, and increased significantly in the stomach of the transported pigs after 1, 4 (p < 0.01), and 2 h (p < 0.05). HSF-1 levels were reduced at 1 and 4 h (p < 0.05) only in the hearts of transported pigs. These results indicate that Hsp70 mediates distinct stress-related functions in different tissues during transportation.
Animals ; Creatine Kinase/blood ; DNA-Binding Proteins/*metabolism ; Enzyme-Linked Immunosorbent Assay/veterinary ; HSP70 Heat-Shock Proteins/*metabolism ; Liver/*metabolism ; Myocardium/*metabolism ; RNA, Messenger/metabolism ; Random Allocation ; Real-Time Polymerase Chain Reaction/veterinary ; Stomach/*metabolism ; Stress, Physiological ; Swine/blood/*metabolism ; Time Factors ; Transaminases/blood ; Transcription Factors/*metabolism ; *Transportation

Black Bengal bucks (Capra hircus) were supplemented with 0, 0.2 or 0.4 mg elemental chromium (Cr) as chromium chloride hexahydrate per day for 70 days. Intake of dry matter (p<0.001), crude protein (p<0.001) and neutral detergent fiber (p<0.01) increased due to Cr supplementation. The apparent total tract digestibility of dry matter (p<0.01), organic matter (p<0.05), crude protein (p<0.001) and acid detergent fiber (p<0.01) improved and the total body weight gain and the live weight gain to feed intake ratio also increased (p<0.001) due to supplemental Cr feeding. The intake and apparent absorption (p<0.001) of Cr was enhanced due to its supplementation. The intake of copper, zinc, manganese and iron was also more (p<0.001) in the Cr supplemented bucks. As supplementation progressed, plasma glucose concentration was elevated particularly in 0.4 mg Cr supplemented bucks and a significant day x dose interaction effect (p<0.001) with this parameter. The activity of plasma alkaline phosphatase increased (p<0.001) and that of glutamate pyruvate transaminase in plasma decreased (p<0.01) in the Cr supplemented bucks. Supplemental Cr had minimal (p>0.05) effect on the plasma half life (k) and clearance rate of glucose (T1/2) during an intravenous glucose tolerance test. Area under the response curve from 0 to 180 minutes after glucose loading was lower (p<0.001) in the control group of bucks. The study revealed that Cr supplementation might promote growth and nutrient utilization in black Bengal bucks. However, little difference between the 0.2 and 0.4 mg Cr supplemented bucks suggested limited benefit of increasing the level of supplementation beyond 0.2 mg per day under the normal management regimes.
Alkaline Phosphatase/blood ; Animal Feed ; Animals ; Blood Glucose/drug effects ; Chromium Compounds/administration&dosage/*pharmacology ; Dietary Supplements ; Dose-Response Relationship, Drug ; Goats/*growth&development/metabolism ; Male ; Trace Elements/blood ; Transaminases/blood ; Weight Gain/*drug effects

Carrier woman of Duchenne muscular dystrophy (DMD) can mimic the inflammatory myositis in presenting symptoms. Two diseases should be differentiated by the clinical history, muscle biopsy and genetic study. There are few reports in which both histochemical and genetic study showed the possible link of overlapping inflammatory pathophysiology with dystrophinopathy. We report a 40-yr-old woman who presented with subacute proximal muscle weakness and high serum level of creatine kinase. She had a history of Graves' disease and fluctuation of serum liver aminotransferase without definite cause. MRI, EMG and NCV were compatible with proximal muscle myopathy. Muscle biopsy on vastus lateralis showed suspicious perifascicular atrophy and infiltration of mono-macrophage lineage cells complicating the diagnosis. Dystrophin staining showed heterogeneous diverse findings from normal to interrupted mosaic pattern. Multiple ligation probe amplification and X chromosome inactivation test confirmed DMD gene deletion mutation in exon 44 and highly skewed X inactivation.
Adult ; Creatine Kinase/blood ; Diagnosis, Differential ; Dystrophin/metabolism ; Echocardiography ; Exons ; Female ; Heterozygote ; Humans ; Magnetic Resonance Imaging ; Muscle Weakness ; Muscular Dystrophy, Duchenne/*diagnosis/genetics/pathology ; Myositis/diagnosis/genetics/pathology ; Transaminases/blood

Paracetamol (PCM) hepatotoxicity is related to reactive oxygen species (ROS) formation and excessive oxidative stress; natural antioxidant compounds have been tested as an alternative therapy. This study evaluated the hepatoprotective activity of an alcoholic extract of Boswellia ovalifoliolata (BO) bark against PCM-induced hepatotoxicity. BO extract also demonstrated antioxidant activity in vitro, as well as scavenger activity against 2, 2-diphenyl-1-picrylhydrazyl. Administration of PCM caused a significant increase in the release of transaminases, alkaline phosphatase, and lactate dehydrogenase in serum. Significant enhancement in hepatic lipid peroxidation and marked depletion in reduced glutathione were observed after parac intoxication with severe alterations in liver histology. BO treatment was able to mitigate hepatic damage induced by acute intoxication of PCM and showed a pronounced protective effect against lipid peroxidation, deviated serum enzymatic variables, and maintained glutathione status toward control. The results clearly demonstrate the hepatoprotective effect of BO against the toxicity induced by PCM.
Acetaminophen ; adverse effects ; Alkaline Phosphatase ; blood ; Animals ; Antioxidants ; metabolism ; pharmacology ; therapeutic use ; Biphenyl Compounds ; metabolism ; Boswellia ; Chemical and Drug Induced Liver Injury ; drug therapy ; metabolism ; pathology ; Glutathione ; metabolism ; L-Lactate Dehydrogenase ; blood ; Lipid Peroxidation ; drug effects ; Liver ; drug effects ; metabolism ; pathology ; Liver Function Tests ; Male ; Oxidative Stress ; drug effects ; Phytotherapy ; Picrates ; metabolism ; Plant Bark ; Plant Extracts ; pharmacology ; therapeutic use ; Rats, Wistar ; Transaminases ; blood

OBJECTIVETo investigate the possible protection provided by oral quercetin pretreatment against hepatic ischemia-reperfusion injury in rats.

METHODSThe quercetin (0.13 mmol/kg) was orally administrated in 50 min prior to hepatic ischemia-reperfusion injury. Ascorbic acid was also similarly administered. The hepatic content of quercetin was assayed by high performance liquid chromatography (HPLC). Plasma glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) activities and malondialdehyde (MDA) concentration were measured as markers of hepatic ischemia-reperfusion injury. Meanwhile, hepatic content of glutathione (GSH), activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and xanthine oxidase (XO), total antioxidant capacity (TAOC), contents of reactive oxygen species (ROS) and MDA, DNA fragmentation were also determined.

RESULTSHepatic content of quercetin after intragastric administration of quercetin was increased significantly. The increases in plasma GPT, GOT activities and MDA concentration after hepatic ischemia-reperfusion injury were reduced significantly by pretreatment with quercetin. Hepatic content of GSH and activities of SOD, GSH-Px and TAOC were restored remarkably while the ROS and MDA contents were significantly diminished by quercetin pretreatment after ischemia-reperfusion injury. However, quercetin pretreatment did not reduce significantly hepatic XO activity and DNA fragmentation. Ascorbic acid pretreatment had also protective effects against hepatic ischemia-reperfusion injury by restoring hepatic content of GSH, TAOC and diminishing ROS and MDA formation and DNA fragmentation.

CONCLUSIONIt is indicated that quercetin can protect the liver against ischemia-reperfusion injury after oral pretreatment and the underlying mechanism is associated with improved hepatic antioxidant capacity.

Administration, Oral ; Animals ; Antioxidants ; pharmacokinetics ; therapeutic use ; Ascorbic Acid ; pharmacokinetics ; therapeutic use ; Biological Availability ; Biomarkers ; blood ; DNA Fragmentation ; Glutathione Peroxidase ; metabolism ; Liver ; blood supply ; drug effects ; enzymology ; Male ; Malondialdehyde ; blood ; Quercetin ; pharmacokinetics ; therapeutic use ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism ; Reperfusion Injury ; blood ; enzymology ; metabolism ; prevention & control ; Superoxide Dismutase ; metabolism ; Transaminases ; blood ; Xanthine Oxidase ; metabolism