PCR快速鉴定actinobacteria三种模板制备方法的比较

目的 本研究旨在建立准确、简便、快速的放线细菌鉴定技术，为普通和极端环境放线细菌资源的调查和开发利用创造条件。方法 从放线细菌固体培养基上挑取少量菌体，用微波炉法快速制备基因组DNA作为PCR模板，与液体培养法得到的菌体以超声波法或冻融法制备的模板进行了PCR扩增效果的比较研究。结果P CR检测结果表明微波炉法制备的模板可进行有效的体外扩增，目的条带特异，而超声波法或冻融法并不对所有菌株有效，并有非特异扩增产物产生。结论 组合微波炉法快速制备放线细菌基因组DNA技术和23S rRNA特异插入序列PCR扩增技术建立了准确、简便、快速的actinobacteria鉴别体系。     

Prognostic significance of c-kit mutation in localized gastrointestinal stromal tumors.

PURPOSE: Constitutive mutational activation of c-kit has been found to be associated with the pathogenesis of gastrointestinal stromal tumors (GISTs). The prognostic significance of c-kit mutations, however, is still controversial. EXPERIMENTAL DESIGN: We examined 86 patients curatively resected for localized GIST. Genomic DNA was extracted from paraffin-embedded tumor tissues. Exons 9, 11, 13, and 17 of the c-kit gene were amplified by PCR and sequenced. RESULTS: Mutations in exon 11 were detected in 61 tumors, and mutations in exon 9 were observed in three tumors, whereas no mutations were detected in exons 13 or 17. The overall c-kit mutation frequency was 74%. Amino acid alterations in the 61 tumors with exon 11 mutations were deletion in 33 tumors, substitution in 20, both deletion and substitution in 4, insertion in 1, and duplication in 3. Histologically, tumors with c-kit mutations showed higher mitotic counts and higher cellularity. The 5-year relapse-free survival (RFS) in patients having GISTs with c-kit mutations was 21%, compared with 60% in those without c-kit mutations. Significantly higher RFS rates were observed in patients with tumors having mitotic counts < 5 mitoses/50 high power field, spindle-cell histology, tumor size < 5 cm, or gastric GISTs. Multivariate analyses indicated association of poorer RFS with a higher mitotic count > or = 5 of 50 high power fields; odds ratio (OR) = 3.0], presence of c-kit mutations (OR = 5.6), and a larger tumor size (> or = 5 cm; OR = 4.2). CONCLUSIONS: The presence of c-kit mutation, along with high mitotic count and larger tumor size, was an independent factor for poor prognosis in patients with localized GISTs.     

HPLC法测定复方联苯双酯颗粒中肌苷的含量

目的:应用HPLC色谱法测定复方联苯双酯颗粒中肌苷的含量.方法:采用C8柱以甲醇-水(60:40)为流动相,检测波长为248 nm.结果:肌苷线性范围为2.50～25.00μg·ml-1,r=0.999 97(n=6).肌苷平均回收率100.1%,RSD 0.4%(n=9).结论:方法简便,结果准确,重复性好.     

Kinetic analysis shows that iron deficiency decreases liver vitamin A mobilization in rats

In view of evidence that nutritional status of iron and vitamin A may affect the other nutrient's metabolism, we used model-based compartmental analysis to examine effects of iron deficiency on whole-body vitamin A dynamics in rats. Weanling male Sprague-Dawley rats were fed the AIN93G diet with 2.5 nmol retinyl palmitate/g and either 45 [control (CN)] or 4 microg/g Fe [iron-deficient (ID)] for 8 wk. ID rats consumed food ad libitum; CN rats were food-restricted so that their body weights were the same as ID rats. Two rats/group were killed; liver vitamin A was determined and used for vitamin A balance calculations. [(3)H]Retinol-labeled plasma was administered intravenously to remaining rats, and 27 serial blood samples were collected for 7 wk. At killing, plasma vitamin A was 0.52+/-0.12 (ID, n = 5) vs. 1.34+/-0.12 micromol/L (CN, n = 6; P<0.001), and liver vitamin A was 809+/-94 (ID) vs. 112+/-24 nmol (CN, P<0.001). Plasma tracer data were fit to a three- or four-compartment model using the Simulation, Analysis and Modeling computer program and kinetic parameters were calculated. Vitamin A transfer rate between the retinyl ester storage pool [14+/-3 (ID) vs. 24+/-4 nmol/d (CN), P<0.05] and plasma was lower in ID rats. Vitamin A remained longer in the body [44+/-11 (ID) vs. 22+/-3 d (CN), P<0.05]. Adjusted mean disposal rate was lower in ID (10.0) than CN rats (19.9 nmol/d), as was estimated vitamin A absorption efficiency [58% (ID) vs. 76% (CN)]. Our results suggest that iron deficiency inhibits mobilization of vitamin A stores and may decrease the absorption and irreversible utilization of vitamin A.     

Sequential production and activation of matrix-metalloproteinase-9 (MMP-9) with breast cancer progression

The degradation of the basement membrane by matrix-metalloproteinase(MMP) and serine protease is a critical pointin tumor invasion and metastasis. We measured theactivity of MMP-9 from 28 normal, 12 benignand 126 breast cancer tissues using gelatin zymographywith an image analysis system. ProMMP-9 was expressedin 17.5% of the cancer patients compared to2.5% in 40 non-cancerous tissues (p=0.014).The mature form of MMP-9 (82 kD) wasexpressed only in T2–T4 stages. During the earlyphase of breast cancer (DCIS and T1 stage)progression, only production of proMMP-9 increased. However, asthe cancer grew or invaded skin (T2–T4), orwith lymphovascular permeation, both production and activation ofMMP-9 increased. In conclusion, proMMP-9 production was themain cause of increased MMP-9 activity during theearly phase, while both production and activation increasedin the late phase of breast cancer.     

N-Ethylmaleimide modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons

The effects of N-ethylmaleimide (NEM), an alkylating reagent to protein sulfhydryl groups, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. When currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV NEM decreased the amplitude of TTX-S sodium current, but exerted little or no effect on that of TTX-R sodium current. The inhibitory effect of NEM on TTX-S sodium channel was mainly due to the shift of the steady-state inactivation curve in the hyperpolarizing direction. NEM did not affect the voltage-dependence of the activation of TTX-S sodium channel. The steady-state inactivation curve for TTX-R sodium channel was shifted by NEM in the hyperpolarizing direction as that for TTX-S sodium channel. NEM caused a change in the voltage-dependence of the activation of TTX-R sodium channel unlike TTX-S sodium channel. After NEM treatment, the amplitudes of TTX-R sodium currents at test voltages below -10 mV were increased, but those at more positive voltages were not affected. This was explained by the shift in the conductance-voltage curve for TTX-R sodium channels in the hyperpolarizing direction after NEM treatment.     

Vascular endothelial growth factor gene polymorphisms and risk of primary lung cancer.

Angiogenesis is an essential process in the development, growth, and metastasis of malignant tumors including lung cancer. DNA sequence variations in the vascular endothelial growth factor (VEGF) gene may lead to altered VEGF production and/or activity, thereby causing interindividual differences in the susceptibility to lung cancer via their actions on the pathways of tumor angiogenesis. To test this hypothesis, we investigated the potential association between three VEGF polymorphisms (-460T > C, +405C > G, and 936C > T)/haplotypes and the risk of lung cancer in a Korean population. VEGF genotypes were determined in 432 lung cancer patients and 432 healthy controls that were frequency matched for age and sex. VEGF haplotypes were predicted using Bayesian algorithm in the phase program. Compared with the combined +405 CC and CG genotype, the +405 GG genotype found associated with a significantly decreased risk of small cell carcinoma [SCC; adjusted odds ratio (OR), 0.36; 95% confidence interval (95% CI), 0.17-0.78]. The 936 CT genotype and the combined 936 CT and TT genotype were also associated with a significantly decreased risk of SCC compared with the 936 CC genotype (adjusted OR, 0.47; 95% CI, 0.26-0.85 and adjusted OR, 0.44; 95% CI, 0.24-0.80, respectively). Haplotype CGT was associated with a significantly decreased risk of SCC (adjusted OR, 0.39; 95% CI, 0.18-0.87), whereas haplotype TCC conferred a significantly increased risk of SCC (adjusted OR, 1.63; 95% CI, 1.14-2.33). None of the VEGF polymorphisms studied significantly influenced the susceptibility to lung cancer except SCC. However, haplotypes TCT and TGT were significantly associated with the risk of overall lung cancer, respectively (adjusted OR, 0.38; 95% CI, 0.25-0.60 and adjusted OR, 3.94; 95% CI, 2.00-7.76, respectively). These effects of haplotypes TCT and TGT on lung cancer risk were observed in three major histologic types of lung cancer. These results suggest that the VEGF gene may be contribute to an inherited predisposition to lung cancer.