鲢鱼CPIs的三种电泳鉴定方法的比较研究

本文对Geltain-substrate-SDS-PAGE反相酶谱法(方法 A)、Native-PAGE反相酶谱法(方法 B)和与papain反应后进行SDS-PAGE法(方法 C)三种电泳方法的鉴定效果、灵敏度进行了对比,确定了鲢鱼卵粗提样中小分子CPIs的最适电泳鉴定方法。结果表明,对于在电泳上活性不稳定的鲢鱼卵小分子CPIs而言,方法 A灵敏度较低,且可能出现假阳性现象;方法 B虽能准确鉴定小分子CPIs的存在,但不能判断分子量。方法 C,即小分子CPIs与papain在37℃下反应5h以上,然后进行SDS-PAGE,抑制剂条带清晰可见,鉴定效果好,且能提供分子量分布信息。此外,尽管荧光底物法和Azocasein法都无法检测到鲢鱼肝胰浓缩粗提液中半胱氨酸蛋白酶抑制活性,但是利用C法,则有效地鉴定了其中CPIs的存在和分子量分布情况。综上所述,方法 C或协同方法 B的电泳鉴定方法,适用于鲢鱼下脚料粗提样品中CPIs的鉴定,效果理想,灵敏度高,操作简便。

Decreased blood riboflavin levels are correlated with defective expression of RFT2 gene in gastric cancer

AIM:To investigate the relationship between blood riboflavin levels and riboflavin transporter 2(RFT2) gene expression in gastric carcinoma(GC) development.METHODS:High-performance liquid chromatography was used to detect blood riboflavin levels in patients with GC.Real-time fluorogenic quantitative polymerase chain reaction and immunohistochemistry were used to analyze the expression of RFT2 mRNA and protein in samples from 60 GC patients consisting of both tumor and normal tissue.RESULTS:A significant decrease in the RFT2 mRNA levels was detected in GC samples compared with those in the normal mucous membrane(0.398 ± 0.149 vs 1.479 ± 0.587;P = 0.040).Tumors exhibited low RFT2 protein expression(75%,16.7%,8.3% and 0% for no RFT2 staining,weak staining,medium staining and strong staining,respectively),which was significantly lower than that in the normal mucous membrane(10%,16.7%,26.7% and 46.7% for no RFT2 staining,weak staining,medium staining and strong staining,respectively;P < 0.05).Tumors with low RFT2 expression were significantly associated with tumor stage and histological grade.Moreover,a significantly decrease in Uyghur patients was observed compared with Han patients.However,other parameters-gender,tumor location and lymph node metastasis-showed no significant relationship with RFT2 expression.Blood riboflavin levels were reverse correlated with development of GC(1.2000 ± 0.97 569 ng/mL in high tumor stage patients vs 2.5980 ± 1.31 129 ng/mL in low tumor stage patients;P < 0.05).A positive correlation of plasma riboflavin levels with defective expression of RFT2 protein was found in GC patients(2 = 2.619;P = 0.019).CONCLUSION:Defective expression of RFT2 is associated with the development of GC and this may represent a mechanism underlying the decreased plasma riboflavin levels in GC.

Leptin influences estrogen metabolism and increases DNA adduct formation in breast cancer cells

Objective: The elevated incidence of obesity has been paralleled with higher risks of breast cancer. High adiposity increases leptin secretion from adipose tissue, which in turn increases cancer cell proliferation. The interplay between leptin and estrogen is one of the mechanisms through which leptin influences breast carcinogenesis. An unbalanced estrogen metabolism increases the formations of catechol estrogen quinones, DNA adducts, and cancer mutations. This study aims to investigate the effect of leptin on some estrogen metabolic enzymes and DNA adduction in breast cancer cells.Methods: High performance liquid chromatography(HPLC) was performed to analyze the DNA adducts 4-OHE1[E2]-1-N3 adenine and 4-OHE1[E2]-1-N7 guanine. Reporter gene assay, real time reverse transcription polymerase chain reaction(real time RT-PCR), and Western blot were used to assess the expression of estrogen metabolizing genes and enzymes: Cytochrome P-4501B1(CYP1B1), Nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase1(NQO1), and Catechol-O-methyl transferase(COMT).Results: Leptin significantly increased the DNA adducts 4-OHE1[E2]-1-N3 adenine and 4-OHE1[E2]-1-N7 guanine.Furthermore, leptin significantly upregulated CYP1B1 promoter activity and protein expression. The luciferase promoter activities of NQO1 and m RNA levels were significantly reduced. Moreover, leptin greatly reduced the reporter activities of the COMT-P1 and COMT-P2 promoters and diminished the protein expression of COMT.Conclusions: Leptin increases DNA adduct levels in breast cancer cells partly by affecting key genes and enzymes involved in estrogen metabolism. Thus, increased focus should be directed toward leptin and its effects on the estrogen metabolic pathway as an effective approach against breast cancer.

Baicalin and rutin are major constituents in Shuanghuanglian injection involving anaphylactoid reaction

OBJECTIVE: To identify the constituents in Shuanghuanglian injection(SHLI) that correlate with anaphylactoid reaction.METHODS: Chemical fingerprints of 10 batches SHLI samples were determined by High Performance Liquid Chromatography(HPLC), and further investigated by similarity analysis. Combined with optical microscopy, both anaphylactoid experiments and confirmatory assay were displayed in Rat basophil leukemia cells(RBL-2H3) to obtain the histamine release inducing by SHLI. The content of histamine was tested by Enzyme-Linked Immuno Sorbent As-say method. Partial least squares regression(PLSR)method and HPLC-DAD-ESI-MSntechnology were conducted to analyze constituents in SHLI involving anaphylactoid reaction.RESULTS: The results of spectrum and effect relationships showed that the eight constituents were positively correlated with anaphylactoid reaction.Among which, nearly 90% of them were identified as baicalin and rutin with PLSR and HPLC-DAD-ESI-MSn. This result was in accordance with confirmatory assay on RBL-2H3 cells.CONCLUSION: Baicalin and rutin from SHLI were the main constituents involving anaphylactoid reaction.

A novel organic-inorganic hybrid monolith for trypsin immobilization

In proteomics, attention has focused on various immobilized enzyme reactors (IMERs) for the realization of high throughput digestion. In this report, a novel organic-inorganic hybrid monolith based IMER was prepared in a 100 μm i.d. capillary with 3-glycidoxypropyltrimethoxysilane (GLYMO) as the monomer and tetraethoxysilane (TEOS) as the crosslinker. Trypsin immobilization was achieved via the reaction between vicinal diol groups, which were obtained from hydrolysis of epoxy groups, and the amino groups of trypsin. Bovine serum albumin was digested thoroughly by this IMER in 47 s. After micro-reverse phase liquid chromatography-tandem mass spectrometry (μRPLC-MS/MS) analysis and database searching, beyond 35% sequence coverage was obtained, and the result was comparable to that of 12 h in solution digestion. The present IMER has potential for high throughput digestion.