According to the amino acids sequence of OC-IdeltaD86 gene and Escherichia coli codon usage, we synthesized this gene by overlap extension PCR method with 7 oligonucleotides DNA fragments. The PCR fragment was inserted into pGEM-T-easy vector and the recombined plasmid was named pGEM-T-OC-IdeltaD86. Two oligonucleotides into which the BamH I and Xho I sites were introduced were designed and synthesized based on pGEM-T-OC-IdeltaD86 and pet21b, and the PCR fragment into which the BamH I and Xho I sites were introduced was obtained. After digesting it with BamH I and Xho I, OC-IdeltaD86 gene was cloned into the corresponding sites of pet21b and obtained prokaryotic expression vector pet21b-OC-IdeltaD86. OC-IdeltaD86 gene was expressed in E. coli (BL21(DE3)plysS) after IPTG(Isopropyl beta-D-1-thiogalactopyranoside) inducement for 5 hours. The fusion protein of OC-IdeltaD86:6His gene accounted for 11.4% of total protein and 16.4% of soluble protein, which had been successfully purified by Ni-NTA and concentrated by PEG20000. This protein can effectively inhibit papain activity in vitro and may be used in anti-nematode research in vivo.
Cloning, Molecular ; Cystatins ; biosynthesis ; genetics ; Cysteine Endopeptidases ; metabolism ; Cysteine Proteinase Inhibitors ; genetics ; Escherichia coli ; genetics ; metabolism ; Genes, Plant ; genetics ; Mutation ; Oligonucleotides ; chemical synthesis ; genetics ; Oryza ; genetics ; Papain ; antagonists & inhibitors ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; metabolism

Objective The purpose of this study is to investigate the factors associated with incisional surgical site infection (SSI) in colorectal cancer. Methods We retrospectively analyzed impacting factors of SSI including age, gender, BMI, diabetes mellitus, operation, albumin levels, hemoglobin levels, tumor staging, and length of stay post-operation in 327 colorectal patients from 2001 to 2005. Results The incidences of SSI and fat liquefaction were 2. 4%(8/327) and 12. 2%(40/327) respectively. The incidences of SSI in patients with obesity and diabetes mellitus increased significantly (16. 8% vs. 10. 0% ,28. 6% vs. 13. 7%). The length of stay in patients with SSI prolonged significantly (29.0 d vs. 15.8 d). Conclusion Obesity and diabetes mellitus are the most important factors associated with SSI in colorectal cancer patients.

We constructed and identified cardiac-specific a-myosin heavy chain (alpha-MHC) promoter-driven expression vector. alpha-MHC promoter was amplified by PCR by using mouse genomic DNA as template and inserted into pGEM-T Easy vector. The inserted fragment was released by enzyme digestion, and then the cytomegalovirus (CMV) promoter in pcDNA3.1(+)-EGFP-hygro vector was replaced by the alpha-MHC promoter to construct alpha-MHC-EGFP expression vector. After identification with enzyme digestion, alpha-MHC-EGFP was transfected into mouse primary cardiomyocytes by electroporation. Green fluorescence could be observed in transfected cardiomyocytes, but not in transfected non-cardiomyocytes. Alpha-MHC-EGFP expression vector was specifically expressed in cardiomyocytes, and could be used to purify embryonic stem cell-derived cardiomyocytes.
Animals ; Cell Differentiation ; Cytomegalovirus ; genetics ; metabolism ; DNA, Complementary ; genetics ; Electroporation ; Embryonic Stem Cells ; cytology ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Mice ; Myocardium ; cytology ; metabolism ; Myosin Heavy Chains ; biosynthesis ; genetics ; Promoter Regions, Genetic ; genetics ; Transfection

A chimeric protein called Wallerian degeneration slow (Wld(S)) was first discovered in a spontaneous mutant strain of mice that exhibited delayed Wallerian degeneration. This provides a useful tool in elucidating the mechanisms of axon degeneration. Over-expression of Wld(S) attenuates the axon degeneration that is associated with several neurodegenerative disease models, suggesting a new logic for developing a potential protective strategy. At molecular level, although Wld(S) is a fusion protein, the nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) is required and sufficient for the protective effects of Wld(S), indicating a critical role of NAD biosynthesis and perhaps energy metabolism in axon degeneration. These findings challenge the proposed model in which axon degeneration is operated by an active programmed process and thus may have important implication in understanding the mechanisms of neurodegeneration. In this review, we will summarize these recent findings and discuss their relevance to the mechanisms of axon degeneration.
Animals ; Axons ; physiology ; Humans ; Mice ; Mice, Mutant Strains ; Models, Neurological ; Mutant Proteins ; genetics ; physiology ; Mutation ; NAD ; biosynthesis ; Nerve Degeneration ; etiology ; genetics ; physiopathology ; Nerve Tissue Proteins ; genetics ; physiology ; Nicotinamide-Nucleotide Adenylyltransferase ; genetics ; physiology

Glutamate-ammonia ligase (GLUL, also known as glutamine synthetase) is a crucial enzyme that catalyzes ammonium and glutamate into glutamine in the ATP-dependent condensation. Although GLUL plays a critical role in multiple cancers, the expression and function of GLUL in gastric cancer remain unclear. In the present study, we have found that the expression level of GLUL was significantly lower in gastric cancer tissues compared with adjacent normal tissues, and correlated with N stage and TNM stage, and low GLUL expression predicted poor survival for gastric cancer patients. Knockdown of GLUL promoted the growth, migration, invasion and metastasis of gastric cancer cells in vitro and in vivo, and vice versa, which was independent of its enzyme activity. Mechanistically, GLUL competed with β-Catenin to bind to N-Cadherin, increased the stability of N-Cadherin and decreased the stability of β-Catenin by alerting their ubiquitination. Furthermore, there were lower N-Cadherin and higher β-Catenin expression levels in gastric cancer tissues compared with adjacent normal tissues. GLUL protein expression was correlated with that of N-Cadherin, and could be the independent prognostic factor in gastric cancer. Our findings reveal that GLUL stabilizes N-Cadherin by antagonizing β-Catenin to inhibit the progress of gastric cancer.