OBJECTIVETo investigate the mechanisms of Yinxing Pingchan recipe (YXPC) and its components, i.e. the components for detoxicating (A), for calming liver (B) and for dissolving blood stasis(C), in preventing and treating Parkinson's disease, and the path of its inhibition on nigrostriatal dopaminergic neuron (DAn) apoptosis in model mice of Parkinson's disease.

METHODSMale C57BL/6J mice were divided into the normal group, the model group and four Chinese medicinal groups, that is, the YXPC group, and Group A, B and C, treated with YXPC and its components A, B and C respectively. Mouse model of Parkinson's disease was established by intraperitoneal injection with 1-methl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP). All mice were sacrificed in 2 batches at the 14th and the 28th day respectively. The activity of mitochondrial enzyme complex I, II and IV (MEC I, II and IV) in the brain of mice were measured, respectively.

RESULTSAs compared with the normal group, the activity of MEC I and IV in brain was significantly lower (P < 0.05 or P < 0.01), and that of MEC II had no obvious change in the model group. As compared with the model group, the activity of MEC I was significantly higher in YXPC group and Group C at the 14th day (P < 0.05), while the activity of MECII in Group A at the 14th day, Group B at the 28th day and Group C at both 14th and 28th day was significantly lower (P<0.05 or P<0.01). Activity of MEC IV in the four Chinese medicinal groups at the 14th day all significantly increased (P<0.05 or P<0.01), and retained at high level in Group B and Group C at the 28th day (P<0.05).

CONCLUSIONYXPC and its components can maintain the mitochondrial function by partial inhibiting the activity of its enzyme complex, preventing DAn apoptosis to slow down the progress of Parkinson's disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Brain ; enzymology ; Drugs, Chinese Herbal ; pharmacology ; Electron Transport Complex I ; metabolism ; Electron Transport Complex II ; metabolism ; Electron Transport Complex III ; metabolism ; Electron Transport Complex IV ; metabolism ; Enzyme Activation ; drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Mitochondria ; enzymology ; Parkinson Disease ; drug therapy ; enzymology ; etiology ; Random Allocation

OBJECTIVETo study the clinical and enzymological characteristics of the children with mitochondrial respiratory chain complex III deficiency.

METHODThe clinical manifestations of five patients (3 males, 2 females) were summarized. Spectrophotometric assay was used for the analysis of respiratory chain complex I to V enzyme activity in peripheral blood leukocytes, after obtaining venous blood.

RESULT(1) Five patients were hospitalized at the age of 1 month to 15 years. Three patients had Leigh syndrome with progressive motor developmental delay or regression and weakness. One had severe liver damage and intrahepatic cholestasis. One presented muscle weakness. (2) Deficient complex I + III activity was identified in five patients. Their complex I + III activities in peripheral blood leukocytes were 3.0 to 14.2 nmol/min per mg mitochondrial protein (control: 84.4 ± 28.5 nmol/min per mg mitochondrial protein). The ratio of complex I + III to citrate synthase decreased to 3.5 to 22.9% (normal control 66.1 ± 14.7%). The activities of complex III decreased to 10.4 to 49.3% of the lowest control value, while complex I, II, IV and V activities were normal. The results supported the diagnosis of isolated respiratory chain complex III deficiency.

CONCLUSIONComplex III deficiency is a kind of disorder of energy metabolism with various manifestations. The complex I + III activities and the ratio of complex I + III to citrate synthase were lower than those of the control. The activities of complex I, II, IV and V were normal.

Adolescent ; Child ; Child, Preschool ; Electron Transport Complex I ; metabolism ; Electron Transport Complex II ; metabolism ; Electron Transport Complex III ; metabolism ; Female ; Humans ; Infant ; Leigh Disease ; Leukocytes, Mononuclear ; enzymology ; Male ; Mitochondrial Diseases ; diagnosis ; metabolism ; physiopathology

In vitro multi-enzyme molecular machines that follow the designed multi-enzyme pathways, require the rational optimization and adaptation of several purified or partially purified enzyme components, in order to convert certain substrates into target compounds in vitro in an efficient manner. This type of molecular machine is component-based and modularized, so that its design, assembly, and regulation processes are highly flexible. Recently, the advantages of in vitro multi-enzyme molecular machines on the precise control of reaction process and the enhancement of product yield have suggested their great application potential in biomanufacturing. Studies on in vitro multi-enzyme molecular machines have become an important branch of synthetic biology, and are gaining increasing attentions. This article systematically reviews the enzyme component-/module-based construction strategy of in vitro multi-enzyme molecular machines, as well as the research progress on the improvement of compatibility among enzyme components/modules. The current challenges and future prospects of in vitro multi-enzyme molecular machines are also discussed.
Biotechnology ; Enzymes ; chemistry ; metabolism ; Multienzyme Complexes ; chemistry ; metabolism ; Synthetic Biology

Besides the catalytic domain, some xylanases contained a non-catalytic domain which is named as carbohydrate binding module (CBM). CBM can be used to improve their binding-ability to insoluble substrates. We illustrated the importance of CBM by reviewing the source of CBMs, type of families, features of binding to insoluble substrates, specific amino acids involved in substrate-binding, linker peptides connecting the catalytic domain, and the effect of CBMs on xylanase thermostability. CBM is important for xylanase to break down complicate carbohydrates. Perspectives on engineering xylanase activity according to the characteristics of CBMs were given.
Binding Sites ; Carbohydrate Metabolism ; Catalysis ; Endo-1,4-beta Xylanases ; metabolism ; Multienzyme Complexes ; chemistry ; Substrate Specificity

The mitochondrial respiratory chain consists of 5 enzyme complexes that are responsible for ATP generation. The paradigm of the electron transport chain as discrete enzymes diffused in the inner mitochondrial membrane has been replaced by the solid state supercomplex model wherein the respiratory complexes associate with each other to form supramolecular complexes. Defects in these supercomplexes, which have been shown to be functionally active and required for forming stable respiratory complexes, have been associated with many genetic and neurodegenerative disorders demonstrating their biomedical significance. In this review, we will summarize the functional and structural significance of supercomplexes and provide a comprehensive review of their assembly and the assembly factors currently known to play a role in this process.
Adenosine Triphosphate ; metabolism ; Arylamine N-Acetyltransferase ; metabolism ; Cardiolipins ; metabolism ; Electron Transport ; Humans ; Mitochondria ; enzymology ; metabolism ; Multienzyme Complexes ; chemistry ; metabolism

OBJECTIVESTo investigate the relationships between proteasome active center LMP7 subunit and the occurrence and development of alcoholic liver disease.

METHODSEighty male Wistar rats, 170 to 190 g, were randomly divided into two groups: a model group (60 rats) and a control group (20 rats). The model group was given alcoholic intragastric administration plus an olive oil diet. Gavage, twice a day, was used to administer ethanol (30%) in a dose of 4 g/kg/d to the model group rats in the first 4 weeks. In the next 4 weeks, 40% ethanol in a dose of 5 g/kg/d was used, and then in the last 4 weeks, 50% ethanol in a dose of 6 g/kg/d was used. After infusion for 12 weeks, 15 rats (fatty liver group) were sacrificed. Others were divided into two groups; one was the hepatitis group with continued alcohol intragastric administration, the other was the hepatitis control group, receiving equal amounts of normal saline. Both groups were sacrificed after 4 weeks. By HE staining, histological pathology of the rat livers was analyzed. The expression of proteasome LMP7 subunit mRNA was examined by reverse transcription and real-time PCR. The content of LMP7 subunit protein was determined by Western blot.

RESULTSThe LMP7 mRNA level of the fatty liver group was 36% of the control group. The level of the hepatitis control group was 51% of the control group. The level of the hepatitis group was the lowest, which was only 26% of the control group. Western blot results showed that the level of the LMP7 protein content of the control group was 0.50+/-0.01; the level was 0.39+/-0.02 of the fatty liver group; 0.30+/-0.04 of the hepatitis group, and 0.38+/-0.02 of the hepatitis control group. The differences of the LMP7 protein content and mRNA expression correlated with the severity of the pathological alterations of the livers.

CONCLUSIONSThe proteasome LMP7 mRNA expression and protein content decreased in the alcoholic liver group. It may be one of the factors responsible for the decreased activity of proteasome and may play an important role in the pathogenesis of alcoholic liver disease.

Animals ; Liver ; pathology ; Liver Diseases, Alcoholic ; metabolism ; pathology ; Male ; Multienzyme Complexes ; metabolism ; Proteasome Endopeptidase Complex ; Rats ; Rats, Wistar

Preparation of a pure autoantigen by way of recombinant DNA technology has an important value in an accurate diagnosis or prognosis of an autoimmune disease. BCOADC-E2 subunit, a mitochondrial protein, has been known to be the autoantigen of primary biliary cirrhosis (PBC), a chronic autoimmune liver disease, as well as idiopathic dilated cardiomypathy (IDCM), a chronic autoimmune heart disease. Recombinant form of this molecule had been expressed in E. coli but with low yield and severe degradation. Furthermore, sera from IDCM patients failed to recognized BCOADC-E2 molecule produced in prokaryotic expression system. In this study, a recombinant bovine BCOADC-E2 fusion protein has been expressed in insect cells using baculovirus expression system and analyzed anti-BCOADC-E2 reactivity in sera from patients with PBC or with IDCM. Optimal production of the recombinant fusion protein has been achieved at 20 multiplicity of infection (MOI), and the protein was affinity-purified using metal-binding resins. The affinity-purified BCOADC-E2 protein was successfully recognized by sera from PBC patients, but not by sera from IDCM patients suggesting that the different auto-immune response against BCOADC-E2 is needed to be elucidated in terms of epitope recognition.
Acetyltransferases/metabolism ; Acetyltransferases/immunology ; Acetyltransferases/genetics* ; Animal ; Baculoviridae/genetics ; Cardiomyopathy, Congestive/immunology ; Cattle ; Human ; Immune Sera ; Insects/cytology* ; Ketone Oxidoreductases/metabolism ; Ketone Oxidoreductases/immunology* ; Ketone Oxidoreductases/genetics* ; Liver Cirrhosis, Biliary/immunology ; Multienzyme Complexes/metabolism ; Multienzyme Complexes/immunology* ; Multienzyme Complexes/genetics* ; Protein Engineering/methods ; Recombinant Proteins/isolation & purification ; Recombinant Proteins/immunology ; Recombinant Proteins/genetics*

PURPOSE: Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) myopathy is an autosomal recessive neuromuscular disorder characterized by early adult-onset weakness of the distal muscles of the lower limbs. The clinical spectrum of GNE myopathy varies, and it is not clear how the same GNE gene mutations can result in different phenotypes. Here, we present clinical, pathological and genetic characteristics of twenty-one Korean patients with GNE myopathy. MATERIALS AND METHODS: Twenty-one GNE myopathy patients were included in this study, conducted from 2004 to 2011. Based on medical records, patients' gender, onset age, family history, clinical history, serum creatine kinase (CK) level, neurologic examination, findings of muscle biopsy, muscle imaging findings and electrophysiologic features were extensively reviewed. Mutation of the GNE gene (9p13.3) was confirmed by DNA direct sequencing analysis in all patients. RESULTS: The mean onset age was 23.8+/-8.8 years (mean+/-SD). Patient serum CK levels were slightly to moderately elevated, ranging from 41 to 2610 IU. Among the patients, twelve patients were female and nine patients were male. Except for eight patients, all of the patients presented initially with only distal muscle weakness in the lower extremities. The most common mutation was V572L, followed by C13S. CONCLUSION: The clinical manifestations of our patients with GNE mutations varied. Among twenty-one patients, thirteen patients showed the typical GNE myopathy phenotype. There was no relationship between clinical features and site of mutation. Therefore, we suggest that neither homozygous nor compound heterozygous models are correlated with disease phenotype or disease severity.
Adolescent ; Adult ; Creatine Kinase/blood ; Distal Myopathies/diagnosis/*genetics/pathology ; Female ; Humans ; Male ; Multienzyme Complexes/*genetics ; Republic of Korea ; Sequence Analysis, DNA

Genetic engineering on macrolide antibiotics was a new field in recent years and more than 100 novel polyketides had been produced until then. Using genomic DNA of S. erythraea A226 as a template, about 3.2 kb DNA fragment without KR6 domain was amplified by overlapping PCR technique and cloned into pWHM3 carrier, which resulted in the construction of homologous recombinant plasmid pWHM2201. Plasmid pWHM2201 was introduced into protoplasts of S. erythraea A226 by PEG-mediated transformation and integrated into the gene locus for erythromycin biosynthesis. After integrants grew for two generations on R3M media without Tsr, they were protoplasted and grown on R3M plates. By PCR identification, 8 mutants without KR6 domain were selected out and named S. erythraea M(1-8). With the identification of mass spectrometry, it was proved that S. erythraea M1 synthesized a novel ketolide compound 3-deoxy-3-oxo-erythronolide B.
Anti-Bacterial Agents ; biosynthesis ; chemistry ; Chromosomes, Bacterial ; Erythromycin ; analogs & derivatives ; biosynthesis ; chemistry ; Genetic Engineering ; Ketolides ; Molecular Structure ; Multienzyme Complexes ; genetics ; Saccharopolyspora ; enzymology ; genetics ; metabolism

The pre-diagnostic test for preimplantation genetic diagnosis (PGD) of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency was performed by polymerase chain reaction (PCR) and direct sequencing for hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (HADHA) gene. We obtained unexpected genotyping results of HADHA gene by allele drop-out in the analysis of patients' genomic DNA samples with a referred PCR primer set. Upon further analysis with a re-designed primer set, we found a novel single nucleotide polymorphism (SNP) at the referred primer-binding site in the normal allele of HADHA gene (NT_022184, 5233296 a>t). We found that the frequency of this novel SNP was 0.064 in Korean population. Pre-diagnostic test using single lymphocytes and clinical PGD were successfully performed with the re-designed primer set. Nineteen embryos (95.0%) among 20 were successfully diagnosed to 5 homozygous mutated, 8 heterozygous carrier and 6 wild type. Among 6 normal embryos, well developed and selected 4 embryos were transferred into the mother's uterus, but a pregnancy was not achieved. We proposed that an unknown SNP at primer-binding sites would be a major cause of allele drop-out in the PGD for single gene dis-order.
*Preimplantation Diagnosis ; *Polymorphism, Single Nucleotide ; Polymerase Chain Reaction ; Mutation ; Multienzyme Complexes/*genetics ; Male ; Humans ; Female ; Binding Sites ; Adult ; 3-Hydroxyacyl CoA Dehydrogenases/deficiency