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Carboxyl CoA ligases(CCLs) is an important branch of adenylate synthetase gene family, which mainly has two-step catalytic reactions. Firstly, in the presence of adenosine triphosphate, it can catalyze the pyrophosphorylation of carboxylateswith diffe-rent structures to form corresponding acyl adenosine monophosphate intermediates. Secondly, adenosine monophosphate was replaced by free electrons in the mercaptan group of enzyme A or other acyl receptors by nucleophilic attack to form thioesters. In this study, on the basis of the transcriptome database of Arnebia euchroma, two genes were selected, named AeCCL5(XP_019237476.1) and AeCCL7(XP_019237476.1). Bioinformatics analysis showed that their relative molecular weights were 60.569 kDa and 60.928 kDa, theoretical PI were 8.59 and 8.92, respectively. They both have transmembrane domains but without signal peptide. By multiple sequence alignment and phylogenetic tree analysis, we found that the similarity between AeCCLs and other plant homologous proteins was not high, and the substrate binding sites of AeCCLs were not highly conserved. The reasons might be that the sequence and structure need to adapt to the changes of new substrates in the process of evolution. In this study, the full-length of AeCCL5 and AecCCL7 were cloned into the expression vector pCDFDuet-1. The proteins of AeCCL5 and AeCCL7 with His-tag were expressed in Escherichia coli. The proteins of AeCCL5 and AeCCL7 were purified by nickel column. In vitro enzymatic reactions proved that both AeCCL5 and AeCCL7 can participate in the upstream phenylpropane pathway of shikonin biosynthesisby catalyzing 4-coumaric acid to produce 4-coumarin-CoA, and then to synthesis p-hydroxybenzoic acid, which is an important precursor of shikonin biosynthesis in A. euchroma.
Subject(s)
Boraginaceae/genetics , Cloning, Molecular , Coenzyme A , Coenzyme A Ligases/genetics , Ligases , PhylogenyABSTRACT
Colorectal cancer has become one of the most frequent malignancy and the leading cause of death from cancer globally. Global Burden Cancer in 2013 had placed colorectal cancer in third place morbidity level and forth most lethal cancer globally. Understanding prognostic marker is essential to increase prognosis prediction in patients with colorectal cancer, especially in patient with the later stages. Several studies have reported the prognostic value of Ki67 expression in patient with colorectal cancer. Ki67 protein expression is associated with proliferative activation from intrinsic cell population in malignant tumor cells, where Ki67 can be used as a marker for tumor aggressivity. Expressions from protein Ki67 are associated with proliferative activities from intrinsic cell population in malignant tumor cells, where Ki67 can be used as marker from tumor cell’s aggressivity. Several diagnostic applications for pKi67 has already been explained, where Ki67 is significantly higher in malignancy compared to normal tissue. pKi67 is also increased along with the decrease of tissue differentiation, and it correlates with the presence of metastasis that can not be seen and tumor clinical phase. Prognostic value from Ki67 had been investigated in many studies with their potential as reliable marker in breasts, soft tissue, lungs, prostate, cervix, and central nervous system. Positive expression from Ki67 in colorectal cancer shows a better prognosis in patient who received surgical treatment and adjuvant radiochemotherapy, but not in a patient who received only a surgical treatment.
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Objective To provide more valuable information for diagnosis of dilated cardiomyopathy (DCM),we detected differentially expressed proteins in serum from patients with DCM and healthy people and protein biomarkers were selected.Methods During the period from march 2011 to may,a total of 29 samples of resident patients with DCM from Qilu Hospital of Shandong University,Jinan Central Hospital and Jinan First Peoples Hospital and 30 local healthy people in Jinan were selected as DCM and control groups,respectively.Serum samples from these patients with DCM and controls were detected by ClinProt MALDII-TOF-MS.ClinProTools 2.2 software was used to get mass spectrometric data.The ClinPrott discrimination model was established to screen out differentially expressed proteins as potential biomarkers.Results Via comparing proteins/polypeptides peaks of DCM patients and healthy controls,57 of all 73 peaks were found to be significantly different between the two groups.Compared with the control group,35 peaks were up-expressed while the other 22 peaks were downexpressed.Five peaks were screened out as protein biomarkers.They were mass-to-charge ratio (m/z):4 247.95,4 209.37,1 058.69,1 074.78,and 2 364.71.The sensitivity and specificity of ClinPrott discrimination model was 99.14％ and 99.16％,respectively.Conclusion Patients with DCM have expressed serum proteins differently and we have found five protein markers which might have some value for diagnosis of DCM.
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Objective To study the expression of RhoA and C-myc in gastric carcinoma , and to discuss the action in infiltration and metastasis of gastric carcinoma as well as their dependability. Methods RhoA protein expression and C-myc protein expression were detected by immunohistochemical method in 46 cases of gastric carcinoma and 20 cases of normal tissues..RhoA protein expression was compared with clinical pathologic parameters as related to C-myc. Results The positive rate of RhoA,C-myc were significantly higher in gastric carcinoma than in normal tissue (P < 0.05). The expression of RhoA and C-myc were associated with the loss of tumor differentiation, lymph node metastasis and deep invasion (P<0.05). There was a significant correlation between RhoA and C-myc (r=0.62, P<0.05). Conclusions The data suggests that the expression level of RhoA protein was closely related to biological behavior of gastric carcinoma. RhoA and C-myc were possibly both correlated with the infiltration and metastasis of gastric carcinoma.
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0.05). The PCNA and Ki-67 expression of blood stasis syndrome is more evident than of Qi stagnation (P
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Objective To reconstruct the proteinic sequence of human cathelicidin LL-37 to increase the bactericidal activity of LL-37 and to express the reconstructed LL-37 (rLL-37) in bacterium. Methods The two dimensional structure, three dimensional structure and chemical characteristic of LL-37 were analyzed by Soft Ware Anthepro 5.0 and SWISS-MODEL. Without the three dimensional changes of LL-37, some negative amino acids of human cathelicidin LL-37 were replaced by positive amino acids and the positive charge of LL-37 was increased. According to the proteinic sequence changes of rLL-37, the DNA sequence of rLL-37 was reconstructed by Touch-Down PCR and recombined with vector pET-28a (+), thus rLL-37 was expressed in E.coli. BL21 (DE3) by the induction of IPTG and was purified by chromatography. Results Glu~ 16 , Asp~ 26 , Glu~ 36 of LL-37 were replaced by Gln~ 16 , Asn~ 26 , Gln~ 36 and the static charge of LL-37 was increased from +5.8 to +9.0 at pH 7.4. The DNA sequence of rLL-37 was reconstructed and inserted into vector pET-28a (+), the rLL-37 was expressed in E.coli. BL21 (DE3) and purified by strong cation exchange supports Macro-Prep High S successfully. The rLL-37 was proved by the means of inhibitory zone to be able to kill Gram-negative bacteria and Gram-positive bacteria. Conclusion It is feasible to reconstruct human cathelicidin LL-37 and express the protein in bacteria by fusion, which make it possible to produce more rLL-37 and study its biological function deeply.
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Objective To study the effects of localized thermochemotherapy on cellular proliferation and the expression of PCNA and IGF-I protein of C6 gliomas in rat, and explore the possibility of developing new therapeutic method of gliomas. Methods C6 glioma cells were injectcd subcutaneously to rats. The rats were randomly assigned to 4 groups,and treatment were initiated on day 22 after tumor inoculation. Prior to treatment, the subcutaneous gliomas were examined to verify tumor size, which were ranged 1.5 to 2.0 cm. Then localized thermochemotherapy, hyperthermia and chemotherapy were given, respectively. The size and weight of subcutaneous tumors were measured. PCNA and IGF-I protein expression were detected by the technique of S-P immunohistochemistry. Glioma cells proliferation was detected by HE staining method and electron-microscopic observation. Results Compared with the control group, the tumor size of rats in the hyperthermia and chemotherapy as well as thermochemotherapy groups was decreased,the tumor weight was also decreased significantly (2.95g vs 1.95g vs 1.86g vs 1.09g, P
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Objective To investigate the relation between expression of P73 , P53, P21 waf1 ,PCNA protein in the different diseases of gastric mucosa and the malignant transformation of gastric mucosa epithelium and their correlation in gastric carcinoma and the relation to histological type , lymph node metastases, depth of invasion of gastric mucosa ,TNM stage in gastric carcinoma. Methods Immunohistochemical staining(S-P method) was used to detect the expression of P73, P53, P21 waf1 , PCNA protein in 39 cases of gastric carcinoma, 17 cases of chronic atrophic gastritis and 40 cases of chronic inflammatory polyp and a statistical analysis was made according to clinicopathogical data. Results The positive rate of P73 protein expression in gastric carcinoma was remarkably higher than that in chronic inflammatory polyp and that in chronic atrophic gastritis( P 0.05). there was negative correlation between expression of P21 waf1 protein and TNM stage, lymph node metastases( P 0.05). Conclusion Probably high expression of P73 gene is one hot spot gene alteration of gastric carcinoma and expression of P73 protein in gastric carcinoma may play an important role in the course of cancerous development. As one tumor suppressor gene, P73 has its tissular specificity. Cooperative expression of P73 and PCNA,and the correlation of expression to P73 and P53 、P21 waf1 protein could be an important index to judge the malignant transformation and clinicopathological process and prognosis. P21 waf1 may be an early signal of gastric carcinoma or one of indexes to judge prognosis.
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Objective To study the implication of phosphoinositide-specific phospholipase C?2(PLC-?2) in gastric cancer MGC80-3 cells treated by TPA.Methods The effect of 12-O-tetradecanoylphorbol-1,3-acetate(TPA) on gastric cancer MGC80-3 cells was detected and analyzed by DAPI staining under fluorescence microscope.The effect of TPA on the expression level of PLC-?2 protein was detected by Western blotting,when nuclear and cytoplasmic protein fractions were prepared through lysis of cell and centrifugation.Localization and translocation of PLC-?2 were observed under laser scanning confocal microscope with immune-fluorescence technique.After MGC80-3 cells were pretreated by U73122(PLC-?2 inhibitor),the effect of TPA on PLC-?2 was detected by Western blotting and laser scanning confocal microscope with immunefluorescence technique,and the effect of U73122 on MGC80-3 cells followed by TPA was observed and analyzed by DAPI staining under fluorescence microscope. Results TPA induced the apoptosis of gastric cancer MGC80-3 cells.Meanwhile,TPA increased the expression level of PLC-?2 protein and induced its translocation from cytoplasm to nucleus.U73122 inhibited the effect of TPA on the expression level of PLC-?2 protein,but it did not affect on the cell apoptosis induced by TPA.However,the translocation of PLC-?2 protein induced by TPA was not inhibited by its inhibitor(U73122).Conclusion Although TPA could increase the expression level of PLC-?2 protein,it is not directly related to the cell apoptosis induced by TPA.However,its translocation might be related with the apoptosis of gastric cancer.
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Objective It was designed to investigate the relationship between the productions of p16 and Rb genes expression and gastric carcinogenesis and evaluate the correlation between the expression of p16 and Rb genes in gastric carcinoma.Methods The expression of P16 and Rb proteins was examined in gastric carcinoma and precancerous lesion and normal gastric mucosa by streptavidin-peroxidase immunohistochemical method (S-P).Results The positive rates of P16 and Rb proteins expression showed as below: There were 96 25%(77/80) and 98 75%(79/80) in normal gastric mucosa, 92 00%(46/50) and 80 00%(40/50) in atypical hyperplasia gastric mucosa and 47 54%(58/122) and 59 84%(73/122) in gastric carcinoma respectively. The positive rates of P16 and Rb proteins expression in gastric carcinoma were significantly lower than that in normal gastric mucosa and atypical hyperplasia gastric mucosa (P