OBJECTIVETo ascertain whether a coding mutation (Ile93Met) in ubiquitin carboxy-terminal hydrolase (UCH-L1) gene plays a role in idiopathic Parkinson's disease (IPD).

METHODSPolymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP) was used to distinguish the wild-type (two DNA fragments of 34 and 126 bp) from the variant allele (three fragments of 34, 60 and 66 bp) because the mutation created a new site for restriction endonuclease BsmF1. DNA was isolated from various blood samples using a phenolchloroform extraction.

RESULTSIle93Met substitution was found neither in PD patients nor in controls.

CONCLUSIONSOur study suggested that Ile93Met of UCH-L1 gene did not influence risk of IPD.

Aged ; Amino Acid Substitution ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Parkinson Disease ; genetics ; Thiolester Hydrolases ; genetics ; physiology ; Ubiquitin Thiolesterase

To clone human mitogen-activated protein kinase kinase 6 (MKK6) gene promoter and explore its transcription activity by ubiquitin specific peptidase 22 (USP22).
 Methods: MKK6 gene promoter was amplified by PCR and two bases mutation within USP22 binding site was subsequently introduced. The wild type and mutant MKK6 promoter were inserted into the luciferase report vector pGL3-Basic, respectively. Recombinant plasmids were co-transfected with plasmid pRL-TK into HeLa cells, and the luciferase activities were measured by dual luciferase reporter system. Furthermore, the direct interaction between USP22 and MKK6 promoter was detected by chromatin immunoprecipitation (ChIP) assay. Finally, the MKK6 transcription activity was measured after knockdown of USP22.
 Results: The recombinant luciferase report vectors containing wild or mutant type of MKK6 promoter were successfully constructed. Mutation of USP22 binding site resulted in decrease of MKK6 promoter-driven luciferase activity in HeLa cells (P<0.05). USP22 could interact directly with MKK6 promoter. Down-regulation of USP22 led to the decreased MKK6 mRNA expression (P<0.05).
 Conclusion: USP22 could regulate the transcription activity of MKK6 gene in HeLa cells.
HeLa Cells ; Humans ; Luciferases ; MAP Kinase Kinase 6 ; Promoter Regions, Genetic ; Thiolester Hydrolases ; metabolism ; Transcription, Genetic

Glyoxalase (GLO) II, which is a component of GLO system and catalyze the conversion of S-lactoyl-glutathione to D-lactate, was purified 1488 fold from rat liver by two steps of Affigel blue and carbobenzoxyglutathione-Sepharose 4B affinity chromatography. The molecular weight of the enzyme was estimated to be 29 kDa which is similar to those from other species. The sequence of N-terminal 9 amino acid residues was determined to be MGIRLLPAT. This was then used to synthesize degenerative primers. cDNA clone was isolated by first synthesizing cDNA from RNA and then PCR amplification. The sequence of cDNA clone was determined by serial sequencing analysis.
Amino Acid Sequence ; Animal ; Base Sequence ; Cloning, Molecular ; Comparative Study ; Liver/enzymology* ; Molecular Sequence Data ; Rats ; Rats, Sprague-Dawley ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; Thiolester Hydrolases/isolation & purification ; Thiolester Hydrolases/genetics

Biosynthesis of fatty acid ethyl ester (FAEE) by genetically engineered Escherichia coli has attracted extensive attentions from scientific community. In this study, we evaluated the effects of thioesterase with different origins on FAEE production and the results show that Cc FatB1 from Cinnamomum camphorum is better than tesA' from E. coli for FAEE production. Then, the optimized FAEE-producing strain KC4, with 21.4 mg/(L x OD600) FAEE production under flask condition and 31.16 mg/(L x OD600) under 5 L fermentation condition, was constructed by co-expression of Cc FatB1 and tesA'. Compared with the reported FAEE-producing strain KC3, KC4 possesses the higher FAEE productivity.
Escherichia coli ; genetics ; metabolism ; Esters ; metabolism ; Ethanol ; metabolism ; Fatty Acids ; biosynthesis ; Fermentation ; Genetic Engineering ; Thiolester Hydrolases ; genetics ; metabolism

Thioesterase catalyzes the hydrolysis of acyl-ACP and saturated fatty acyl chain. It plays a key role in the accumulation of medium chain fatty acids in vivo. In this study, to construct an engineering strain to produce MCFAs, the Arabidopsis acyl-ACP thioesterase gene AtFatA was amplified by PCR from cDNA of arabidopsis and double digested by EcoR I/Xba I, then linked to the plasmid digested with same enzymes to get the recombinant plasmid pPICZaA-AtFatA. We transformed the gene into Pichia pastoris GS115 by electroporation and screened positive colonies by YPD medium with Zeocin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the recombinant enzyme had a molecular of 45 kDa band which was consistent with the predicted molecular mass and we constructed the expression system of gene AtFatA in fungus for the first time. Under shake-flask conditions, Gas Chromatograph-Mass Spectrometer-computer results indicated that recombinant strain produced 51% more extracellular free MCFAs than the wild and its yield reached 28.7% of all extracellular fatty acids. This figure is 10% higher than the control group. The result provides a new way to produce MCFAs.
Arabidopsis ; genetics ; Arabidopsis Proteins ; biosynthesis ; DNA, Complementary ; Electrophoresis, Polyacrylamide Gel ; Electroporation ; Pichia ; metabolism ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Thiolester Hydrolases ; biosynthesis ; Transformation, Genetic

A quantitative assessment of the density of the protein gene product 9.5 (PGP9.5), the neural cell adhesion molecule (NCAM), and the low-affinity nerve growth factor receptor (NGFR) expressing nerve fibers in the circular muscle layer in the colon was carried out by morphometric analyses from 13 patients with Hirschsprung's disease (HD). The difference in the nerve fiber density between the ganglionic and aganglionic segments was compared by calculating the ratio of the sum of the areas occupied by positively stained nerve fibers per unit area of the muscle after immunohistochemical staining on paraffin embedded tissue sections using computer software. There was an obvious difference in the density of the PGP9.5 stained nerve fibers between the ganglionic (0.0380 +/- 0.0171) and aganglionic segments (0.0143 +/- 0.01661). The NCAM-positive nerve fibers were fewer in number than those of both the PGP9.5-positive fibers and NCAM-positive fibers, which were also markedly lower in number in the aganglionic segment (0.0066 +/- 0.0076) than in the ganglionic segment (0.0230 +/- 0.0195). Immunostaining for low-affinity NGFR revealed much fainter staining in the ganglionic and aganglionic segment without a statistically significant difference in their density. Considering the fact that PGP9.5 is a very sensitive marker for nerve fibers, the results of this study reaffirm the innervation failure of the proper muscle in HD. The decreased NCAM expression level in the aganglionic segment appears to be caused not by the selective down-regulation of NCAM expression among the nerve fibers but by a markedly reduced number of nerve fibers.
Colon/*innervation ; Hirschsprung Disease/*pathology ; Human ; Muscle, Smooth/*innervation ; Nerve Fibers/*chemistry/pathology ; Neural Cell Adhesion Molecules/*analysis ; Receptor, Nerve Growth Factor/analysis ; Thiolester Hydrolases/*analysis

Merkel cells are thought to function as slowly adapting mechanoreceptors and are known as targets for sensory nerves. However, the nerve-dependency of Merkel cells remains controversial. In this respect, some investigators have found interregional differences between hairy and glabrous skin and others have shown intraregional differences within denervated rat touch domes. Differences between species have also been reported. This study was performed to determine whether Merkel cells proliferate in vitro in the absence of the systemic factors, blood vessels and the intact nerves in human skin. Suspension organ culture was performed using fetal digits to investigate their in vitro proliferation. Merkel cells and cutaneous nerves were identified using antibodies to cytokeratin 20 and protein gene product 9.5 (PGP 9.5), respectively. Fetal digits of 56-82 day gestational age were cultured in serum free medium in a high O2 (45%) environment. Tissues were harvested before starting culture (D0) and 1,4,7,14, 28d after culture. Merkel cells were observed in the volar pads and dorsal nail matrices at D0. After 28d of suspension organ culture, digits looked healthy structurally and the number of Merkel cells had increased. However, PGP 9.5-immunoreactive nerves were markedly diminished after 1 day of culture and almost disappeared after 4 days. Merkel cell proliferation in vitro suggested that Merkel cell development is probably nerve-independent in human fetal glabrous skin.
Cell Division ; Female ; Human ; Intermediate Filament Proteins/analysis ; Merkel Cells/*physiology ; Organ Culture ; Pregnancy ; Skin/cytology/*embryology/*innervation ; Thiolester Hydrolases/analysis

A FatB unigene was obtained from the transcriptome dataset of Lonicera japonica Thunb. Full-length FatB cDNA was cloned from buds of Lonicera japonica Thunb., Lonicera japonica Thunb. var. chinensis (Wats.) Bak., Lonicera hypoglauca Miq. and Lonicera dasystyla Rehd. using RT-PCR technology, and named as LJFatB, LHFatB, LJCFatB and LDFatB. The results of bioinformatic analysis showed that LJFatB, LJCFatB, LHFatB and LDFatB and Arabidopsis thaliana AtFatB had a closely relationship. Nucleotide sequences and protein secondary structure of LJFatB, LJCFatB, LHFatB and LDFatB are different and their proteins had conserved FatB substrate binding sites and catalytic activity sites. Transcriptive level of LJFatB, LJCFatB, LHFatB and LDFatB in bud was not significantly different. Therefore, LJFatB, LJCFatB, LHFatB and LDFatB could have the same biological function as AtFatB.
Amino Acid Sequence ; Cloning, Molecular ; Computational Biology ; DNA, Complementary ; genetics ; Flowers ; chemistry ; genetics ; metabolism ; Lonicera ; chemistry ; classification ; genetics ; metabolism ; Open Reading Frames ; genetics ; Phylogeny ; Plant Proteins ; genetics ; metabolism ; Protein Structure, Secondary ; Thiolester Hydrolases ; genetics ; metabolism ; Transcriptome ; genetics

OBJECTIVETo investigate the clinical features and genetic characteristics of patients with 3-hydroxy-isobutyryl-CoA hydrolase (HIBCH) gene mutations.

METHODThe clinical data of a patient with novel HIBCH mutations were collected, the related literature was searched from China National Knowledge Infrastructure, Wanfang Data Knowledge Service Platform, National Center for Biotechnology Information and PubMed (up to December 2014) by using search terms" HIBCH", "3-hydroxy-isobutyryl-CoA hydrolase" or "beta-Hydroxyisobutyryl CoA Deacylase Deficiency". The clinical features, neuroimage and treatment of the patients with HIBCH gene mutations were studied.

RESULTThe patient was a girl who was born at term after an uneventful pregnancy to non-consanguineous healthy parents, she was hospitalized at 5 years and 5 months of age because of development delay for 5 years and 5 months and abnormal posture on the left of body for more than 10 days. The family history was unremarkable. Her psychomotor development was significantly delayed. Three times brain MRI between 2. 5 years and 5 years of age revealed bilateral symmetrical lesions in basal ganglia. At the age of 5 years and 5 months, she presented with acute encephalopathy and severe extrapyramidal symptoms preceded by fever. At that time, her brain MRI revealed aggravated lesions in bilateral basal ganglia, new lesions in the midbrain cerebral peduncle and pons, and cerebellar atrophy. The results of biochemical tests were normal. A novel compound heterozygous mutation of HIBCH gene, c. 1027C > G, p. H343D and c. 79-1G > T, splicing, were found in the parent. Further study showed that c. 1027 C > G mutation was inherited from her father and c. 79-1 G > T from her mother. Her symptoms were mitigated after "cocktail" therapy and symptomatic treatment. Repeated brain MRI revealed that the lesion in basal ganglia got better, the lesions in brain stem disappeared. Literature relevant to HIBCH published all around the world was reviewed, no Chinese cases with HIBCH gene mutations had been reported, 6 foreign cases with HIBCH gene mutations were reported. Among them, 5 patients were diagnosed as Leigh-like syndrome, with progressive neurodegenerative course, and symmetrical basal ganglia lesions on brain MRI. Another case was reported in 1982, with developmental delay and various physical malformations without data on his brain MRI. HIBCH gene mutational analysis showed that 4 cases had homozygous mutations, which were c. 950G > A (p. G317E) in two brothers, c. 219 _220insTTGAATAG (p. K73fsX86) and c. 1128_1129insT (p. K377X) respectively. Three of them died before 3 years old. Two cases had compound heterozygous mutations: c. 365A > G (p. Y122C) and IVS2-3C > G (p. R27fsX50); c. 517 + 1G > A and c. 410C > T (p. A137V). They were alive at the time of the report.

CONCLUSIONPatients with HIBCH gene mutation mainly presented as Leigh-like syndrome both in clinical manifestation and in neuroimage. HIBCH gene mutational analysis should be performed on children with Leigh-like syndrome, if the mutations of known genes of Leigh syndrome were negative.

Abnormalities, Multiple ; diagnosis ; genetics ; Amino Acid Metabolism, Inborn Errors ; diagnosis ; genetics ; Child ; Child, Preschool ; China ; DNA Mutational Analysis ; Female ; Heterozygote ; Homozygote ; Humans ; Infant ; Leigh Disease ; diagnosis ; genetics ; Magnetic Resonance Imaging ; Male ; Mutation ; Siblings ; Thiolester Hydrolases ; deficiency ; genetics

OBJECTIVETo investigate the effects of YOD1 overexpression on the proliferation and migration of human oral keratinocytes (HOKs), and to clarify whether the mechanisms involve transforming growth factor-β (TGF-β) signaling.

METHODSHOKs were transfected with the plasmid pEGFP-N3-YOD1 containing YOD1. The mRNA levels of YOD1 and TGF-β were determined by qPCR. The protein expressions of YOD1, TGF-β, Smad2/3, Smad4, and phospho-Smad2/3 were determined by western blotting. Cell proliferation and migration were evaluated by Cell Counting Kit-8 assay and wound healing assay, respectively.

RESULTSThe mRNA and protein levels of YOD1 were higher in HOKs transfected with YOD1. YOD1 overexpression significantly enhanced the migration of HOKs. The mRNA and protein levels of TGF-β3 were increased by YOD1 overexpression. HOKs transfected with YOD1 exhibited increased phospho-Smad2/3 levels.

CONCLUSIONYOD1 overexpression enhances cell migration by promoting TGF-β3 signaling which may play an important role in lip and palate formation. YOD1 mutation may contribute to aberrant TGF-β3 signaling associated with decreased cell migration resulting in NSCLP.

Cell Movement ; physiology ; Cell Proliferation ; Cells, Cultured ; Endopeptidases ; genetics ; metabolism ; Humans ; Keratinocytes ; physiology ; Signal Transduction ; physiology ; Smad Proteins ; genetics ; metabolism ; Thiolester Hydrolases ; genetics ; metabolism ; Transforming Growth Factor beta3 ; genetics ; metabolism