Identification of New Snake Venom Metalloproteinase Inhibitors Using Compound Screening and Rational Peptide Design

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MODERN TREATMENT OF LYMPHOEDEMA II. THE BENZOPYRONES.

The benzo-pyrones reduce all high-protein oedemas, including lymphoedema and elephantiasis, by increasing the numbers of macrophages and their normal proteolysis. Thus they remove the excess protein, and thereby the oedema which is caused by it. They also remove the stimulus it provides for chronic inflammation and fibrosis, and its action as a culture medium for bacteria. Coumarin (5,6 benzo-[alpha]-pyrone, 56 BaP) and oxerutins (HR, O(beta-hydroxy-ethyl)-rutosides) have been used in many clinical trials on a variety of high-protein oedemas. Four such trials are summarised here: on lymphoedema and elephantiasis (from many causes in Australia, and filaritic in India and China). The drugs reduced these much more slowly than adequate physical therapy, but they did reduce them. About half the excess volume was removed over six months in the Australian trials. In India and China similar rates were achieved with lymphoedema, but elephantiasis reduced at a slower rate. The benzo-pyrones convert a slowly worsening condition into a slowly improving one. No compression garments are necessary. In addition, the drugs considerably reduce the number of attacks of secondary acute infection, reduce the deformities of elephantiasis and considerably improve the patients' comfort and mobility. They may be taken orally, or applied topically, have very low toxicities and only few, minor side-effects. They are useful in many other forms of high-protein oedema, and improve the results of physical therapy for lymphoedema.     

A novel in vitro pancreatic carcinogenesis model

Environmental factors (e.g., BaP) have been pointed out as one of the etiologies of pancreatic cancer. However, very limited experimental assays are available to identify pancreatic specific environmental mutagens or susceptibility genes. In this study, we have developed a simple in vitro cell culture model system that can be used to study the molecular and biochemical aspects of carcinogenesis in a near-normal immortalized pancreatic ductal epithelial cell lines. In order to demonstrate that xenobiotic stress response is intact in these cells, we employed standard molecular biology techniques. For examples, luciferase reporter and/or real-time quantitative PCR assays were used to determine stress-induced CYP1A1 and CYP1B1 gene expression. Western blotting and immunocytochemistry assays were used to demonstrate that TCDD or BaP could activate AhR signaling. For exploring the carcinogenesis mechanism, we incubated cells with [³H]BaP and determined BaP-DNA binding activity by measuring its radioactivity. BaP-DNA adduct formation was further confirmed by [³²P]-postlabeling assay. Finally, we demonstrated the effects of endogenous AhR or BRCA1 in BaP-DNA adduct accumulation in our cell system. As results, no apparent BaP-DNA adduct accumulation by [³²P]-postlabeling assay was found in either control-siRNA or AhR-siRNA pretreated cells. On the other hand, a significant increase of BaP-DNA adduct accumulation was found in BRCA1 knockdown cells. In conclusion, we suggest that this in vitro model may provide the feasibility for future studies on the molecular basis of pancreatic ductal cell carcinogenesis caused by dietary mutagens.     

某大学及周边烤炸食品中亚硝酸盐和苯并芘监测

[目的]了解某大学及周边摊点烧烤和油炸食品中亚硝酸盐和苯并芘(Benzo(a)pyrene,BaP)的含量。[方法]采集西南大学食堂及周边8个点共26个烧烤和油炸食品,由西南大学食品科学学院测定其亚硝酸盐和BaP的含量。[结果]摊点4的烤羊肉串的亚硝酸盐35.585mg/kg,超过国家规定的30mg/kg;食堂3的烤豆干样本中BaP的含量为5.08μg/kg,食堂4的炸土豆中BaP的含量为5.98μg/kg,摊点4的全部样本中BaP的含量超过国家规定的5μg/kg水平。[结论]烧烤和油炸食品中存在亚硝酸盐和BaP超标情况,摊点超标情况高于食堂,BaP高于亚硝酸盐,建议尽量避免食用烤炸食品。     

Exposure to and deposition of fine and ultrafine particles in smokers of menthol and nonmenthol cigarettes

Introduction: Research on the deposition of mainstream smoke particulate in the respiratory tract of smokers is needed to understand how exposure may vary based on cigarette menthol content.

Methods: We conducted a nine-participant crossover study in which smokers were randomly assigned to cigarettes differing primarily in menthol content. Participants smoked the test cigarettes ad libitum for one week, provided spot urine samples, and then smoked four test cigarettes in a laboratory session; this was repeated for the other test cigarette in week two. Fine and ultrafine particulate matter in exhaled breath were characterized, and smoking behavior was monitored. Participant-specific mainstream smoke, generated using each participant’s topography data, was characterized. During home smoking, participants collected their spent test cigarette butts for estimates of mouth-level exposures (MLE) to mainstream nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

Results: Participant-specific mainstream smoke NNK was higher (39%) and daily MLE to NNK was also higher (52%) when participants smoked the menthol cigarette. Nicotine was not significantly different. Participants retained more ultrafine particulate (43%) and fine particulate benzo(a)pyrene (43%) when smoking the menthol cigarette. There were no significant differences in the levels of urinary biomarkers for nicotine, NNK, or pyrene.

Conclusion: This study demonstrates the use of noninvasive real-time techniques to measure exposure differences between cigarettes differing primarily in menthol content. Differences between NNK exposure, ultrafine particle and benzo(a)pyrene deposition, and smoking behavior were observed. Additional research using these techniques with cigarettes that differ only in menthol content is required to unequivocally attribute the exposure differences to presence or absence of menthol.     

The analysis of DNA adducts: The transition from P-postlabeling to mass spectrometry

The technique of ³²P-postlabeling, which was introduced in 1982 for the analysis of DNA adducts, has long been the method of choice for in vivo studies because of its high sensitivity as it requires only <10 μg DNA to achieve the detection of 1 adduct in 10¹⁰ normal bases. ³²P-postlabeling has therefore been utilized in numerous human and animal studies of DNA adduct formation. Like all techniques ³²P-postlabeling does have several disadvantages including the use of radioactive phosphorus, lack of internal standards, and perhaps most significantly does not provide any structural information for positive identification of unknown adducts, a shortcoming that could significantly hamper progress in the field. Structural methods have since been developed to allow for positive identification of DNA adducts, but to this day, the same level of sensitivity and low sample requirements provided by ³²P-postlabeling have not been matched. In this mini review we will discuss the ³²P-postlabeling method and chronicle the transition to mass spectrometry via the hyphenation of gas chromatography, capillary electrophoresis, and ultimately liquid chromatography which, some 30 years later, is only just starting to approach the sensitivity and low sample requirements of ³²P-postlabeling.     

Glycine N-methyltransferase affects the metabolism of aflatoxin B1 and blocks its carcinogenic effect

Previously, we reported that glycine N-methyltransferase (GNMT) knockout mice develop chronic hepatitis and hepatocellular carcinoma (HCC) spontaneously. For this study we used a phosphoenolpyruvate carboxykinase promoter to establish a GNMT transgenic (TG) mouse model. Animals were intraperitoneally inoculated with aflatoxin B₁ (AFB₁) and monitored for 11 months, during which neither male nor female GNMT-TG mice developed HCC. In contrast, 4 of 6 (67%) male wild-type mice developed HCC. Immunofluorescent antibody test showed that GNMT was translocated into nuclei after AFB₁ treatment. Competitive enzyme immunoassays indicated that after AFB₁ treatment, the AFB₁-DNA adducts formed in stable clones expressing GNMT reduced 51.4% compared to the vector control clones. Experiments using recombinant adenoviruses carrying GNMT cDNA (Ad-GNMT) further demonstrated that the GNMT-related inhibition of AFB₁-DNA adducts formation is dose-dependent. HPLC analysis of the metabolites of AFB₁ in the cultural supernatants of cells exposed to AFB₁ showed that the AFM₁ level in the GNMT group was significantly higher than the control group, indicating the presence of GNMT can enhance the detoxification pathway of AFB₁. Cytotoxicity assay showed that the GNMT group had higher survival rate than the control group after they were treated with AFB₁. Automated docking experiments showed that AFB₁ binds to the S-adenosylmethionine binding domain of GNMT. Affinity sensor assay demonstrated that the dissociation constant for GNMT-AFB₁ interaction is 44.9 μM. Therefore, GNMT is a tumor suppressor for HCC and it exerts protective effects in hepatocytes via direct interaction with AFB₁, resulting in reduced AFB₁-DNA adducts formation and cell death.     

苯并a芘致人k-ras基因DNA损伤位点检测

目的 探索苯并a芘对k-ras基因的DNA损伤位点.方法 苯并a芘(AaP)染毒细胞提取基因DNA,采用依赖随机化末端连接物PCR(PCPCR)进行核酸杂交及产物测序.结果 BaP染毒剂量200 μmol/L检测到比-kras外显子2短的一条清晰的杂交条带;测序结果表明,linker与k-ras外显于2的第67位碱基C发生连接.结论 BaP可引起k-ras外显子2发生损伤,损伤位点在第66位碱基T,该损伤位点可能是其致癌作用靶点.