Analysis of the human APC mutation spectrum in a saccharomyces cerevisiae strain with a mismatch repair defect

Somatic APC mutations in colorectal tumors with an RER phenotype reflect excessive frameshift mutations, especially in simple repetition tracts within the coding sequence. Because this type of mutation is characteristic of cells with a deficient DNA MMR system, the APC mutation signature of RER tumors may be attributable to a defect in the MMR system. However, there is little experimental evidence to prove that the spectrum of mutations and the APC gene distribution are directly influenced by MMR system defects. We therefore examined the mutation spectrum of the MCR of the APC gene after transfection into both MMR-proficient and MMR-deficient yeast strains and compared it with a previously reported human APC mutation database. Small insertions or deletions in mono- or dinucleotide repeats were more common in the MMR-deficient than in the MMR-proficient strain (91.2% vs. 38.1%, Fisher's exact test p < 0.0001). Furthermore, the 2 mutation hot spots, 4385-4394(AG)(5) and 4661-4666(A)(6), found in the yeast system corresponded with those in human tumors. Combining our data with those from human tumors, there appears to be hypermutable mutations in specific simple repetitive sequences within the MCR, which are more prevalent in MMR-deficient cells and RER tumors than in MMR-proficient cells and non-RER tumors. We therefore consider that the differences in the spectra of RER and non-RER tumors are attributable at least in part to the MMR system of the host cells.     

Response to activated protein C during normal menstrual cycle and ovarian stimulation

Oestrogen has been pointed out as a pre-thrombotic factor. Protein C is a key enzyme in the down-regulation of blood coagulation. Recent data suggest that activated protein C (APC) resistance which is not due to the factor V:Q 506 Leiden mutation and appears to be acquired, is also a risk factor for thrombosis. In this study, we evaluated the endogenous oestradiol production and its possible influence on APC. Eighteen normally menstruating women were studied during one ovulatory cycle. Furthermore, 20 women undergoing controlled ovarian stimulation, and achieving extremely high oestradiol concentrations, were investigated. Normalized APC (nAPC) ratio (clotting time of tested sample/clotting time of pooled control plasma) was measured. Samples collected on menstrual cycle days 1-3, 6-8, 13-14, 20-24 corresponded to nAPC ratios 1.02 +/- 0.19 (mean +/- SD), 1.05 +/- 0. 15, 1.02 +/- 0.16 and 1.03 +/- 0.21 respectively. During ovarian stimulation, the nAPC ratios were 0.99 +/- 0.12, 1.03 +/- 0.18, 1.01 +/- 0.16 and 0.97 +/- 0.13 at oestradiol minimum, days 5-8 pre-oocyte retrieval, oestradiol maximum and at oocyte retrieval respectively. In spite of the great difference in the concentrations of oestradiol between women in normal menstrual cycle and women undergoing ovarian stimulation, no difference in nAPC ratios was observed.     

IL-12基因转染B细胞的抗原提呈功能研究

目的 以IL-12双亚基共表达载体pL35P40SN转染B细胞,并检测其对Th细胞发育分化和CTL功能的影响。方法 尼龙毛法分离T,B细胞,以pL35P40SN转染B细胞,并以ELISA检测其IL-12的分泌,以IFN-γ的合成分泌检测其对Th1细胞发育分化的影响,以^3H-TdR法检测其对CTL特异杀伤HepG2细胞功能的影响。结果 以pL35P40SN转染B细胞后,pL35P40SN基因转染的B细胞IL-12分泌量、Th应答细胞上清IFN-γ分泌量和CTL的杀伤功能显著高于pLXPXSN和未转染的细胞。结论 pL35P40SN转染B细胞后,可刺激Th1细胞发育分化和CTL的特异性杀伤功能。     

Piroxicam and Acarbose as Chemopreventive Agents for Spontaneous Intestinal Adenomas in APC Gene 1309 Knockout Mice

The use of nonsteroidal anti-inflammatory drugs has been suggested to have a chemopreventive effect against colon carcinoma, through the inhibition of cyclooxygenases 1 and 2, in patients with familial adenomatous polyposis and in animal models. Acarbose, an alpha-glycosidase inhibitor, may also be chemopreventive. In order to examine the effects of these drugs we employed APC gene knockout mice randomized into 3 groups, one for treatment with piroxicam (0.05% concentration in drinking water), one for acarbose (0.04% concentration in food) and another for the control. After 14 weeks of treatment, mice were killed for quantitation of gastric and intestinal adenomas. Tumor multiplicity in the whole gastrointestinal tract decreased from 33.89±13.07 tumors/mouse in the control group to 17.05±7 tumors/mouse in the piroxicam-treated group ( P <0.001). The decrease in the acarbose-treated group (29.68±12.86 tumors/mouse) was not significant ( P >0.05). The number of tumors ≥3 mm in diameter was also quantified in all gastrointestinal segments. The number of such tumors in the piroxicam group was decreased to 0.56±1.2 tumors/mouse from the control value of 3.78±1.17 tumors/mouse ( P <0.001), while in the acarbose-treated group the number decreased to 2.36±1.7 tumors/mouse ( P <0.01). Thus, piroxicam decreases the size and number of gastrointestinal adenomas in APC 1309 knockout mice, while acarbose decreases only the size.     

肝细胞癌中SFRP1和APC基因启动子甲基化与其mRNA的表达

目的探讨肝细胞癌（HCC）中SFRP1和APC基因启动子甲基化状态及其mRNA表达的关系。方法应用甲基化特异性聚合酶链反应（MSP）和逆转录-聚合酶链反应（RT—PCR）技术，检测30例肝细胞癌（HCC组）及相应的癌旁组织（癌旁组）和10例正常肝组织（正常对照组）中SFRP1及APC基因启动子甲基化状态和mRNA的表达水平，分析甲基化与某些临床参数与mRNA表达的关系。结果HCC组、癌旁组及正常对照组中的SFRP1和APC基因启动子甲基化率分别是11／30，4／30，0／10和14／30，5／30，0／10；HCC组明显高于其余两组（P〈0．05）。SFRP1基因启动子甲基化与临床资料无关（P〉0．05）；APC基因启动子甲基化与年龄（〈60）岁和癌肿无假包膜有关（P〈0．05）。HCC组SFRP1基因mRNA表达明显低于其余两组（P〈0．05），各组APC基因mRNA表达差异无显著性。两基因甲基化之间无相关性。结论SFRP1和APC基因启动子甲基化与HCC的形成和进展有关，但HCC组织中基因甲基化与mRNA表达的关系尚不明确。     

Novel APC-cleavage sites in FVa provide insights into mechanisms of action of APC and its cofactor protein S

Summary.  Background: Activated protein C (APC) inhibits factor Va (FVa) by cleaving at Arg306, Arg506 and Arg679. Protein S serves as cofactor, in particular for the Arg306 site, and a protein S-mediated relocation of the active site of APC closer to the membrane has been proposed as a mechanism. Recently, it was demonstrated that FVa, which was mutated at all three APC-cleavage sites (FVa-306Q/506Q/679Q), could still be cleaved by APC. These sites were close to Arg306 and Arg506 but not further defined. Objective: To identify and characterize the additional APC-cleavage sites in FVa. Methods: The cDNA for FV-306Q/506Q/679Q was used as a template to create FV variants with one or more possible cleavage sites being mutated. The FV variants were expressed and their sensitivity for APC characterized functionally and with Western blotting. Results: The additional APC-cleavage sites were located at Lys309, Arg313, Arg316, Arg317 and Arg505. FVa-306Q/309Q/313Q/316Q/317Q/505Q/506Q/679Q (denoted 8M-FVa) was APC resistant. To investigate individual sites, they were mutated back using 8M-FV as a template. The kinetics of APC-degradation of these variants demonstrated that protein S was equally efficient in enhancing the APC effect for all the novel sites. Conclusions: Multiple APC-cleavage sites close to Arg306 and a single site close to Arg506 were identified. Protein S was equally efficient as APC cofactor for all novel sites. The stimulation by protein S of the Arg505 cleavage argues against a specific protein S-mediated stimulation of cleavage at Arg306 due to relocation of the APC active site closer to the membrane.     

PLS—紫外分光光度法同时测定复方阿斯匹林片中三组分含量

本文研究了复方阿斯匹林片中阿斯匹林、非那西丁和咖啡因含量同时测定的紫外分光光度法的最佳实验条件,并简述偏最小二乘法(PLS)在多组分同时测定中的基本原理和应用。三组分模拟试样回收率平均值的置信区间分别为100.1±0.23%,100.0±0.25%和100.1±0.33%(置信度95%)。PLS法是一种理想的多组分测定方法,计算速度较快,结果更准确可靠,尤其适用于成批试样的分析,为微机控制的紫外可见分光光度计提供了一种新方法。     

Differential effects of high prothrombin levels on thrombin generation depending on the cause of the hyperprothrombinemia

BACKGROUND: Hyperprothrombinemia, resulting from the prothrombin G20210A mutation or other causes, is associated with activated protein C (APC) resistance and increased thrombosis risk. When high prothrombin levels are a result of increased hepatic biosynthesis, these effects may be counteracted by concomitantly increased levels of the anticoagulant factors (particularly protein S). Differently, in prothrombin G20210A carriers only prothrombin levels are elevated. OBJECTIVE: To investigate whether prothrombin G20210A carriers have a more severe hypercoagulable state than non-carriers with comparable prothrombin levels. PATIENTS/METHODS: Coagulation factor levels, thrombin generation (Calibrated Automated Thrombogram in the presence and absence of APC) and APC resistance were measured in normal (n = 132), heterozygous (n = 167) and homozygous (n = 3) individuals. RESULTS: Prothrombin levels, thrombin generation and APC resistance were higher in carriers of the prothrombin G20210A mutation (especially those who had experienced venous thrombosis) than in non-carriers, whereas protein S and antithrombin levels were similar among genotype groups. Because individuals with high prothrombin levels in the absence of the prothrombin G20210A mutation tend to have all liver-synthesized factors elevated, carriers of the mutation had lower protein S and antithrombin levels than non-carriers with equally high prothrombin levels. Accordingly, they also generated more thrombin and showed a tendency toward higher APC resistance. Analogous effects, but less pronounced, were observed in homozygotes for the prothrombin A19911G polymorphism, which also upregulates prothrombin levels. CONCLUSIONS: Individuals with hyperprothrombinemia as a result of prothrombin gene mutations generate more thrombin and tend to be more APC-resistant than individuals with comparable prothrombin levels because of other causes.