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The α-amino acid ester acyltransferase (SAET) from Sphingobacterium siyangensis is one of the enzymes with the highest catalytic ability for the biosynthesis of l-alanyl-l-glutamine (Ala-Gln) with unprotected l-alanine methylester and l-glutamine. To improve the catalytic performance of SAET, a one-step method was used to rapidly prepare the immobilized cells (SAET@ZIF-8) in the aqueous system. The engineered Escherichia coli (E. coli) expressing SAET was encapsulated into the imidazole framework structure of metal organic zeolite (ZIF-8). Subsequently, the obtained SAET@ZIF-8 was characterized, and the catalytic activity, reusability and storage stability were also investigated. Results showed that the morphology of the prepared SAET@ZIF-8 nanoparticles was basically the same as that of the standard ZIF-8 materials reported in literature, and the introduction of cells did not significantly change the morphology of ZIF-8. After repeated use for 7 times, SAET@ZIF-8 could still retain 67% of the initial catalytic activity. Maintained at room temperature for 4 days, 50% of the original catalytic activity of SAET@ZIF-8 could be retained, indicating that SAET@ZIF-8 has good stability for reuse and storage. When used in the biosynthesis of Ala-Gln, the final concentration of Ala-Gln reached 62.83 mmol/L (13.65 g/L) after 30 min, the yield reached 0.455 g/(L·min), and the conversion rate relative to glutamine was 62.83%. All these results suggested that the preparation of SAET@ZIF-8 is an efficient strategy for the biosynthesis of Ala-Gln.
Subject(s)
Escherichia coli/genetics , Glutamine , Zeolites/chemistry , Amino AcidsABSTRACT
Hyperthyroidism is a clinically common endocrine disease. It often has no specific clinical symptoms in the early stage and is easily overlooked. The long-term effects of excessive thyroid hormones in the body can alter cardiovascular hemodynamics, which may lead to heart enlargement, atrial fibrillation, and heart failure. Cardiovascular disease is one of the common complications of hyperthyroidism, but it is the main cause of death. This article focuses on the related cardiovascular diseases of hyperthyroidism, and summarizes the molecular mechanism of thyroid hormone on the heart, the mechanism of hyperthyroidism induced heart failure, atrial fibrillation, pulmonary hypertension, and the treatment and prognosis of hyperthyroidism. In addition, we also analyzed the association between subclinical hyperthyroidism and the occurrence of cardiovascular disease. When combined with risk factors, subclinical hyperthyroidism patients need early treatment. It should be noted that long-term use of amiodarone can cause secondary hyperthyroidism, which should be used with caution in clinical use.
ABSTRACT
Hyperthyroidism is a clinically common endocrine disease.It often has no specific clinical symptoms in the early stage and is easily overlooked.The long-term effects of excessive thyroid hormones in the body can alter cardiovascular hemodynamics,which may lead to heart enlargement,atrial fibrillation,and heart failure.Cardiovascular disease is one of the common complications of hyperthyroidism,but it is the main cause of death.This article focuses on the related cardiovascular diseases of hyperthyroidism,and summarizes the molecular mechanism of thyroid hormone on the heart,the mechanism of hyperthyroidism induced heart failure,atrial fibrillation,pulmonary hypertension,and the treatment and prognosis of hyperthyroidism.In addition,we also analyzed the association between subclinical hyperthyroidism and the occurrence of cardiovascular disease.When combined with risk factors,subclinical hyperthyroidism patients need early treatment.It should be noted that long-term use of amiodarone can cause secondary hyperthyroidism,which should be used with caution in clinical use.
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Objective To explore the impact of a“one-week”staged multivessel percutaneous coronary intervention (PCI) versus culprit-only PCI on deaths and major adverse cardiac events (MACE). Methods We retrospectively analyzed 447 patients with multivessel disease who experienced a ST-segment elevation myocardial infarction (STEMI) within 12 h before undergoing PCI between July 26, 2008 and Septem-ber 25, 2011. After completion of PCI in the infarct artery, 201 patients still in the hospital agreed to undergo PCI in non-infarct arteries with more than 70%stenosis for a“one-week”staged multivessel PCI. A total of 246 patients only received intervention for the culprit vessel. Follow-up ended on September 9, 2014. This study examined the differences in deaths from any cause (i.e., cardiac and noncardiac) and MACE between the two treatment groups. Results Compared to a culprit-only PCI treatment approach, the“one-week”staged multivessel PCI was strongly associated with greater benefits for 55-month all cause death [41 (16.7%) vs.13 (6.5%), P=0.004] and MACE [82 (33.3%) vs. 40 (19.9%), P=0.002] rates. In addition, there were significant differences in the number of myocardial infarctions [43 (17.5%) vs. 20 (10.0%), P=0.023], coronary-artery bypass grafting [CABG;20 (8.1%) vs. 6 (3.0%), P=0.021], and PCI [31 (12.6%) vs. 12 (6.0%), P=0.018]. Patients undergoing culprit-only PCI compared to“one-week”PCI had the same number of stent thrombosis events [7 (2.8%) vs. 3 (1.5%), P=0.522]. Conclusions Compared to a culprit-only PCI treatment approach,“one-week”staged multi-vessel PCI was a safe and effective selection for STEMI and multi-vessel PCI.
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Objective To investigate apoptosis of HepG2 ceils after transfecfion with LIGHT gene and interferon-γ. Methods LIGHT gene and interferon-γ were transfected into HepG2 cells by liposome mediated method. The HepG2 cells were divided into group A (transfected with LIGHT gene or interferon-γ), group B (transfeeted with LIGHT gene and interferon-γ) and group C (non-transfection group). The apoptosis rate of the HepG2 cells and the expression of Bcl-2 and Caspase-8 were detected 12, 24, 48 hours after transfeetion. Results (1) The apoptosis rates of HepG2 cells at hour 12, 24 and 48 after transfeetion were 18.8% ± 3.5%, 25.7%± 2.8% and 36.4% ±3.6% in group A, 23.8% ±2.4%, 31.1% ±2.1% and42.5% ±4.5% in group B, and 8.7% ± 2.1%, 9.3% ± 1.6% and 10.9% ± 1.2% in group C. There was a significant difference in apoptosis rate among the 3 groups (F = 15.69, 53.33, 48.28, P < 0.01). (2) The expression of Bcl-2 in HepG2 cells at hour 12, 24 and 48 after transfection was 16.4% ± 5.0%, 13.4% ± 3.5% and 8.6% ± 2.3% in group A, 14.7%±3.8%, 9.1% ±2.0% and 4.6% ±2.0% in group B, and 25.3% ±6. 3%, 19.8% ±4.4% and 10.1% ±3.8% in group C. There was a significant difference in the expression of Bcl-2 among the 3 groups (F = 6.19, 12.29, 5.81, P <0.05). (3) The expression of Caspase-8 at hour 12, 24 and48 after transfection were 19.3% ±2.4%, 27.2% ±1.9% and 33.7% ±3.0% in group A, 22.7% ±2.2%, 30.9% ±3.1% and 38.2% ±3.2% in group B, and 1.2% ±0.8%, 1.8% ±0.6% and 3.2% ±1.5% in group C. There was a significant difference in the expression of Caspase-8 among the 3 groups (F =71.54, 112. 78, I01.61, P < 0.01). Condusions LIGHT gene can signiticanfly promote cell apoptosis through regulating the expression of Bcl-2 and Caspase-8. Interferon-γ enhanced the effect of LIGHT gene on the apoptosis of HepG2 cells.
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Objective To construct an efficient eukaryotic expression recombinant vector of human interleukin-1O(hIL-lO),and observe its expression in rabbit synoviocytes(RSCs).Methods Total RNA was extracted from peripheral blood mononuclear cells(PBMCs)of a patient with drug allergy.Specific Drimers for full-length open reading frames(ORFs)of hIL-10 were designed according to GeneBank(NM 000572).Withtotal RNA as the template,full-length ORFs of hIL-10 were amplified by reverse transcription Dolymerase chain reaction(RT-PCR).RT-PCR products were digested by restrictive endonucleotidase.then inserted into plasmid pcDNA4/HisMaxA.Both restrictive endonucleotidase analysis and DNA sequencing Were carried out for inserts verification.RSCs were transfected with recombinant plasmid expression vector PcDNA4 HisMaxA-hiL10 by liposome-mediated gene transfer methods,then cultured in vitro.The supernatants were collected af-ter transfection for 12 hours,24 hours,48 hours,72 hours,7 days,14 days respectively for IL-10 measure-ment by enzyme linked immunosorbent assay(ELISA).Results Full-length ORFs of hIL-1o(0.54 kb)had been successfully cloned from PBMCs through RT-PCR.The inserts and insert location of pcDNA4 HisMaxA were in a fight way verified by enzyme analysis and DNA sequencing.ELISA results showed that exogenous hIL-10 gene had expressed in the transfected RSCs from 12 hours to 7 days after transfection,and hIL-10level of transfection group significantly higher than that of the control group.Conclusion pcDNA4 HisMaxA-hiL10,the hIL-10 efficient eukaryotic expression vectors,has been suecessfully constructed.
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BACKGROUND: It has been successful to repair articular cartilage defects by using solid carrier as cytoskeleton. We tried to transplant liquid or gel carrier materials combined cells into the body of animals, and investigated its feasibility.OBJECTIVE: To investigate the feasibility of homo-transplatation with liquid or gel carrier materials of Pluronic F-127-recombinant human bone morphogenetic protein-2 (rhBMP-2) engineered chondrocytes for the repair of full-thickness rabbit articular cartilage defect.DESIGN: A controlled experiment.SETTINGS: Department of Orthopaedics, Weihai Municipal Hospital;Shandong Institute of Orthopaedics and Traumaology.MATERIALS: The experiments were carried out in the laboratory of Shandong Institute of Orthopaedics and Traumaology from November 2001 to September 2003. Thirty-six healthy adult New Zealand rabbits of 2.5-4.5 kg, either male or female, were divided into four groups according to the method of random number table: Pluronic F-127-rhBMP-2 engineered chondrocytes group, Pluronic F-127-rhBMP group, Pluronic F127 engineered chondrocytes group and blank control group, with 9 rabbits in each group.METHODS: After grouping, the 36 rabbits were made into models of articular cartilage defects. Pluronic F-127-rhBMP-2 was used as a vector of chondrocytes which were obtained from New Zealand rabbits after cultured and amplified in vitro. The mixture of Pluronic F-127, Pluronic F-127-rhBMP-2 and cultured chondrocytes was transplanted into the defects of articular cartilage that had been made previously with φb3.5 mm drill.There was not any treatment in the blank control group. At 4, 8 and 12 weeks postoperatively, the repairing conditions of the defects were evaluated with gross observation and histological observation under light microscope and under electron microscope. The repaire quality was assessed blindly according to the Wakitani scoring standard.MAIN OUTCOME MEASURES: ① Healing of cartilage defects; ② Property and morphology of the chondrocytes, characteristics, number and arrangement of collagens in matrix.RESULTS: ① In the Pluronic F-127-rhBMP-2 engineered chondrocytes group, the transplanted chondrocytes could grow better than those in other groups, the defected areas were completely filled at 4 weeks. The regenerated tissues at 8 and 12 weeks had similar appearance with the surrounding normal cartilage tissue, but vague. Delimitation. The histological examination showed that transparent cartilages formed, and the defects were healed. ② Under electron microscope at 8 and 12 weeks, there were mature transparent cartilages in the repaired tissues, and there were irregularly arranged slight, even and non-periodical collagen Ⅱ in surrounding. In the blank control group, only fibrous repair was observed, the regenerated tissue lacked elasticity with rough surface. ③ Repairing quality score: The scores at each time point in the Pluronic F-127-rhBMP-2 engineered chondrocytes group were significantly different from those in the other groups.Those in the Pluronic F-127-rhBMP-2 engineered chondrocytes group and Pluronic F-127-rhBMP-2 group and Pluronic F-127 engineered chondrocytes group were significantly different from those in the blank control group [4 weeks: (3.93±1.91), (4.56±1.07), (4.78±1.09), (8.44±1.13) points:8 weeks: (2.80±1.45), (3.24±1.00), (3.33±1.00), (8.44±1.13) points; 12 weeks (2.22±1.10), (3.01±0.69), (3.00±0.71), (9.00±0.87) points, P ＜ 0.001],but there were no significant differences between the two groups (P ＞ 0.05).CONCLUSION: The mixture of Pluronic F-127-rhBMP-2 and cultured chondrocytes can repair successfully the cartilage defects of femoral condyle of rabbit knees by means of hyaline cartilage than simple application of Pluronic F-127-rhBMP-2 or Pluronic F-127 engineered chondrocytes.
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Objective To investigate the protective effect of recombinant mouse OP 1 (rmOP 1) on renal ischemia reperfusion injury. Methods The full length open reading frame of mouse OP 1 was cloned by RT PCR. The rmOP 1 was prepared and its osteogenesis was assayed in vitro. Thirty Wistar rat models of renal ischemia reperfusion were randomly divided into 3 groups: preventive group, therapeutic group and control group. The former two groups were administered with rmOP 1 (250 ?g/kg) via the superior mesenteric vein at different time points. The renal function and structure were assessed by serum BUN, Cr, histological findings and the modified Miller's scoring. Results rmOP 1 showed osteoinductive activities in vitro. The level of serum BUN, Cr and Miller's scoring were higher in control group than the others ( P 0.05 ). Histological finding showed that rmOP 1 minimized the cell necrosis. Conclusion rmOP 1 has an protective effect on renal function and structure.
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<p><b>OBJECTIVE</b>To generate an HPV16 prophylactic vaccine candidate for cervical cancer.</p><p><b>METHODS</b>HPV16 major capsid protein L1 gene and minor capsid protein L2 gene were amplified using PCR. These genes were mutated by PCR site-directed mutagenesis for removal of sequence motifs (TTTTTNT) which would cause transcription termination when expressed from a vaccinia virus early promoter, then inserted into a vaccinia virus expression vector. A strain replication-deficient recombinant vaccinia virus containing the mutant sequences was obtained through a homologous recombination and identified.</p><p><b>RESULTS</b>The nucleotide sequence remained the correct amino acid sequence of the L1 and L2 proteins after mutated. Full-length L1 and L2 proteins were generated in cells infected with the recombinant virus. The virus strain propagated at very low titer or could not reproduce in some kinds of cell derived from different human tissues.</p><p><b>CONCLUSIONS</b>The authors have generated a strain replication-deficient recombinant vaccinia virus expressing HPV16 L1 plus L2 proteins as an HPV16 prophylactic vaccine candidate for cervical cancer.</p>