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Polyhydroxyalkanoates (PHAs) have obtained much attention in biomaterial fields due to their similar physicochemical properties to those of the petroleum-derived plastics. Poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] is one member of the PHAs family, and has better toughness and transparency compared to existing polylactic acid (PLA) and poly[(R)-3-hydroxybutyrate] [P(3HB)]. First, we confirmed the one-step biosynthesis of P(LA-co-3HB) with the lactate fraction of 23.8 mol% by introducing P(3HB-co-LA) production module into Escherichia coli MG1655. Then, the lactate fraction was increased to 37.2 mol% in the dld deficient strain WXJ01-03. The genes encoding the thioesterases, ydiI and yciA, were further knocked out, and the lactate fraction in the P(3HB-co-LA) was improved to 42.3 mol% and 41.1 mol% respectively. Strain WXJ03-03 with dld, ydiI and yciA deficient was used for the production of the LA-enriched polymer, and the lactate fraction was improved to 46.1 mol%. Notably, the lactate fraction in P(3HB-co-LA) from xylose was remarkably higher than from glucose, indicating xylose as a potent carbon source for P(3HB-co-LA) production. Therefore, the deficiency of thioesterase may be considered as an effective strategy to improve the lactate fraction in P(3HB-co-LA) in xylose fermentation.
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Escherichia coli/genetics , Hydroxybutyrates , Lactic Acid , Polyesters , Polyhydroxyalkanoates , XyloseABSTRACT
Objective To summarize the clinical and electroencephalogram features of neuronal ceroid lipofus-cinosis (NCL). Methods A retrospective analysis of the clinical phenotypes and electroencephalogram features of pa-tients diagnosed with NCL in Department of Pediatrics,Peking University First Hospital from February 2000 to August 2015 were conducted. Results Among the 30 patients,18 were male and 12 were female. The age of onset was between 9 months to 7 years old. The first symptoms included seizure in 22 patients,psychomotor developmental delay or regre-ssion in 7 cases,and visual loss in 1 case. Clinical manifestations included psychomotor regression in 29 cases,epilepsy in 28 cases,visual impairment in 19 cases,ataxia in 20 patients,and positive pyramidal tract sign in 13 cases. Twenty-one patients accepted fundus oculi examination. Seven patients were found with macular degeneration,8 cases with optic nerve atrophy,2 cases with retinal pigment degeneration,and 8 patients were normal. Brain atrophy were found in all 30 cases,including diffuse brain atrophy in 14 cases,only cerebellar atrophy in 6 cases,and cerebral atrophy with periven-tricular T2W high signal in 10 cases. Video electroencephalogram(EEG)examination was performed in 27 patients and their backgrounds were diffuse slow waves. Seven patients didn't have physiological vertex sharp waves or sleep spin-dles. Generalized epileptiform discharges were captured in 6 cases,focal epileptiform discharges in 15 cases. Both of generalized and focal epileptiform discharges were captured in 6 cases. Generalized slow wave burst in 4 cases,and in-termittent photic stimulation evoked epileptiform discharges in 3 cases. Ten patients were observed with clinical sei-zures,including 4 cases of myoclonic episodes,3 cases of atypical absences,3 cases of focal seizures,1 case of atonic and one of tonic spasms. Peripheral blood enzyme examination was taken in 13 patients,among whom 8 patients were identified with tripeptidyl peptidase 1 (TPP1)deficiency and 1 patient with palmitoyl protein thioesterase 1 (PPT1) deficiency. Twenty-eight patients accepted skin and/or muscle electron microscope examination. Osmiophilic granular was found in 2 cases,curvilinear bodies in 15 cases,fingerprint profiles in 2 cases,curvilinear and linear bodies in 1 case,fingerprint profiles and osmiophilic granular in 1 case. NCL-related gene detection was conducted in 3 patients, with 1 patient identified with CLN6 compound heterozygous mutations and 2 patients with TPP1 homozygous mutations. Thirty patients were classified into 3 groups based on the onset age,enzymatic examination results and pathological examination of skin and muscle,including 5 cases of infantile NCL,20 cases of late-infantile NCL,and 5 cases of juvenile NCL. Conclusions The clinical features of NCL included multiple types of epileptic seizures (among which myoclonus was the most common type),psychomotor developmental delay or regression,vision loss,ataxia,and positive pyramidal tract sign. Its MRI was characterized with brain atrophy. EEG showed diffuse slow wave activity,with focal and/or generalized epileptiform discharges. Specific enzyme examination,and skin or muscle pathology or gene test could help to make diagnosis.
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Purpose To investigate the relations of THEM4/Akt expression and extracellular matrix deposit in kidney of diabetic mice. Methods Diabetic mice models were successfully established by intraperitoneally injected STZ. Both normal control mice and diabetic mice were raised for 8 week until they were sacrificed. Western blot, immunohistochemistry and realtime PCR were used to detect the expression of THEM4, phospho-Akt (Ser 473), TGF-β1, a-SMA, Col Ш, FN protein and THEM4 mRNA in the kidneys of normal mice and diabetic mice. Results Compared to normal control mice, THEM4 expression decreased by 37.7％ followed by 3.66, 1.29 2.33, 1.99 and 2.82 times increased of phospho-Akt (Ser473), TGF-β1, a-SMA, Col Ш and FN in kidney of diabetes mellitus. Extracellular matrix accumulation was found in renal interstitial region of diabetic mice. Conclusion The decreased THEM4 might cause extracellular matrix deposit in kidney of diabetic mice by upregulating the phosphorylation of Akt and TGF-β1, α-SMA expression in diabetic mice.
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Aim To observe the effect of thioesterase superfamily member 4 ( THEM4 ) expression on extra-cellular matrix ( ECM ) accumulation in the kidney of diabetic mice .Methods For in vivo vector delivery experiment , male CD1 mice were randomly divided in-to four groups: normal control mice ( Control group ) , diabetic mice ( DM group ) , diabetic mice receiving pYr-ads-4-THEM4 vector ( DM+THEM4 vector ) and diabetic mice receiving pYr-adshuttle-4 vector ( DM+V vector ) .pYr-ads-4-THEM4 vector or pYr-adshuttle-4 vector ( 1 mg · kg -1 ) were mixed with TransIT-EE Hydrodynamic Delivery Solution from Mirus Co .and injected into tail vein once a week for four weeks after STZ injection.Four weeks later, mice were sacrificed and Western blot , immunohistochemistry and real-time PCR were used to detect the expression of phospho-Akt(Ser 473), THEM4, TGF-β1, α-SMA, Col Ⅲ, FN proteins and THEM4 mRNA in kidneys respectively . Results THEM4 decreased in kidney of diabetes mel-litus accompanied with increased phospho-Akt ( Ser 473), TGF-β1, α-SMA and ECM.The delivery of pYr-ads-4-THEM4 vector increased THEM4 expression and decreased phospho-Akt (Ser 473), TGF-β1, α-SMA and ECM deposit in kidneys of diabetic mice . Conclusion The up-regulation of THEM4 may pre-vent ECM deposit by inhibiting the phosphorylation of Akt and down-regulating the expression of TGF-β1 andα-SMA in kidneys of diabetic mice .
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Aim To investigate the effect of thioester-ase superfamily member 4(THEM4)expression on col-lagen secretion in human renal proximal tubular epithe-lial cells (HKC)treated with high glucose. Methods In order to examine the direct effect of THEM4 ex-pression vector on PI3K/ Akt pathway and collagen se-cretion,pYr-ads-4-THEM4 expression vector was con-structed and transfected into the HKC with lipo-fectamine 2000 in vitro. HKC cells were randomly di-vided into four groups:normal glucose group (Con-trol),high glucose group (HG),high glucose plus pYr-ads-4-THEM4 vector group (HG + THEM4 vec-tor) and high glucose plus pYr-adshuttle-4 vector group (HG + V vector). After 48 h with HG stimula-tion,the cells were collected for extraction of protein and phospho-Akt (Ser 473),THEM4,TGF-β1 andα-SMA protein expression were examined by Western blot and immunofluorescence staining respectively. Col Ⅰ and Col Ⅲ were detected using the competitive sandwich ELISA kit according to the manufacturer's instructions. Results High glucose inhibited THEM4 expression,and induced increased phospho-Akt (Ser 473),TGF-β1,α-SMA and secreted ColⅠand secre-ted Col Ⅲ in HKC cells. Up-regulation of THEM4 re-versed high glucose-induced decreased THEM4,in-creased phospho-Akt (Ser 473),TGF-β1,α-SMA, secreted Col Ⅰ and secreted Col Ⅲ in HKC cells. Conclusion The up-regulation of THEM4 may de-crease Col Ⅰ and Col Ⅲ secretion by inhibiting the phosphorylation of Akt and down-regulating the expres-sion of TGF-β1 and α-SMA in high glucose-induced HKC cells.
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Objective To investigate the trans‐regulative effect of hepatitis B virus (HBV) preS2 on the promoter of human acyl protein thioesterase 1 (APT1) gene .Methods The promoter sequence of human APT1 gene was identified applying the soft‐ware of bioinformatics .The APT1 promoter and HBV preS2 gene were amplified with PCR and cloned into pGL3 and pcDNA3 .1 (-) plasmids to construct the luciferase reporter gene plasmid of human APT1 gene promoter pGL3‐APT1 and the preS2 eukary‐otic expression plasmid pcDNA3 .1(-)‐preS2 ,respectively .The effect of the preS2 on the human APT1 gene promoter was exam‐ined by cotransfecting hepatocellular carcinoma cell HepG2 with pGL3‐APT1 and pcDNA3 .1(-)‐preS2 and measuring luciferase activities of the HepG2 cells .The statistical data were analyzed with independent‐samples t test .Results Both plasmids of pGL3‐APT1 and pcDNA3 .1(-)‐preS2 were confirmed by DNA sequencing to be accurately constructed as design .The luciferase activity of the pGL3‐APT1 was 1 .2 times (P<0 .01) that of the positive control plasmid pGL3‐Control .And the luciferase activity of the HepG2 cells cotransfected with pcDNA3 .1(-)‐preS2 and pGL3‐APT1 was 2 .6 times (P<0 .01) that of the HepG2 cells cotrans‐fected with the plasmid without preS2 gene pcDNA3 .1(-) and pGL3‐APT1 .Conclusion The human APT1 promoter cloned in the study has high promoter activity ;HBV preS2 activates human APT1 promoter .
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BACKGROUND: Acyl protein thioesterase-1 (APT1) is a cytosolic protein that may function in the depalmitoylation of numerous proteins, including the Ras family. However, the clinical role of depalmitoyl thioesterase in human cancer is not known. We evaluated the APT1 expression in lung cancer tissue and its clinicopathological findings according APT1 expression pattern. METHODS: APT1 expression was examined by immunohistochemistry in the tumor tissue from 79 patients, who had undergone curative surgical removal of the primary lesion; all patients had been diagnosed with stage I non-small cell lung cancer between 1993 and 2004, at Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea. RESULTS: The APT1 expression was seen in 50 out of 79 (63.3%) cases. The positive APT1 expression was significantly related with histologic subtype and T stage, but was not influenced by differentiation. The positive APT1 expression was not significantly related to patient age, gender, or smoking history. The median follow-up duration was 10.0 years; the 5-year survival rate was 71.0%. The positive APT1 expression group showed significantly worse overall survival and worse disease-free survival without statistical significance. CONCLUSION: We conclude that positive APT1 expression in stage I lung cancer after surgery is closely associated with overall survival. To evaluate APT1 as a prognostic marker in lung cancer, comprehensive studies on advanced stage cases are needed.