Cellular distribution of phosphofructokinase activity and implications to metabolic regulation in human breast cancer

Neoplastic cells generally present profound changes in glucose metabolism. The mechanisms underlying such process are numerous and all may involve altered cellular hormonal responses. Here we report the first evidence that cellular location of phosphofructokinase activity in human breast cancer tissues is different from the one observed in control tissues and that this phenomenon may be involved in the increased glycolytic flux observed in those cells. Through co-sedimentation techniques, we observed that 60% of phosphofructokinase activity in neoplastic tissues is located in an actin-enriched fraction, against 36% in control tissues. Additionally, metastatic tumor tissues presented a two fold increase in this particulate activity when compared to non-metastatic tumor samples. We propose that the alteration in cellular distribution of phosphofructokinase activity in human breast cancer tissues is a mechanism associated to the process of cell transformation and may be a consequence of the altered hormonal milieu observed in several types of cancer.     

巢式PCR检测龈下菌斑中HCMV、EBV、HSV-1与慢性牙周炎活动性的关系

目的　建立临床检测龈下菌斑标本中人巨细胞病毒 (HCMV)、Epstein Barr病毒 (EBV)、单纯疱疹病毒 1型 (HSV 1 )巢式PCR方法 ,研究这 3种病毒与慢性牙周炎活动性的关系。方法　收集6 2例慢性牙周炎患者 (男性 2 7例、女性 35例 ,平均年龄 5 3岁 )的牙周炎活动部位、牙周炎静止部位的龈下菌斑 ,提取DNA后使用巢式PCR检测HCMV、EBV、HSV 1 ,比较分析其在同一病人不同部位的检出率。结果　牙周炎活动部位的HCMV检出率为 38.7%,EBV的检出率为 5 8%,HSV 1的检出率为30 .6 %,两种以上病毒合并感染的检出率为 4 0 .3%;牙周炎静止部位的HCMV检出率为 1 4 .5 %,EBV为 2 2 .6 %,HSV 1为 1 1 .3%,两种以上病毒合并感染的检出率为 1 1 .3%。这 3种病毒及其合并感染在牙周炎活动部位的检出率均高于牙周炎静止部位 ,差异有统计学意义 (P <0 .0 5 )。结论　提示HC MV、EBV、HSV 1与慢性牙周炎的活动性相关。     

一种快速分离纯化外周血细胞线粒体DNA方法的建立

目的探索快速分离纯化人外周血细胞线粒体DNA(mtDNA)的方法. 方法收集抗凝外周血,首先破红细胞,然后裂解白细胞,去除细胞膜和核DNA后获得mtDNA;再经过去除蛋白和RNA,获得纯mtDNA.用PCR扩增人线粒体ND1基因片段,PCR产物经纯化后测序和核苷酸同源性分析.结果用本研究中建立的方法制备的mtDNA纯度高,每毫升抗凝血可获得100ng左右的mtDNA.经过PCR扩增ND1基因433bp片段,较用细胞总DNA为模板,模板用量少,扩增产物多.测序后经核苷酸同源性分析证实扩增片段为ND1基因.结论本研究中建立的快速分离外周血细胞mtDNA的方法,可制备高纯度的mtDNA用于线粒体相关研究.     

人染色体11p15.5 BWS致病位点新基因C11orf21的初步功能分析

目的研究人染色体11p15.5Beckwith-Wiedemann综合征致病位点新基因C11orf21的生物学功能。方法用RT-PCR和Northern印记方法分析C11orf21基因的表达;研究C11orf21蛋白功能用Western印记和细胞培养方法。结果检测到C11orf21基因在成人和胎儿心脏和肝脏等组织转录,在心脏有较强的表达并有0.9和3.1kb两个转录物。C11orf21蛋白在COS-1和Hela细胞细胞质表达,蛋白相对分子质量为14kDa。结论初步分析显示C11orf21是11p15.5Beckwith-Wiedemann综合征致病位点的一个非印记基因,在成人心脏有较强表达的局限细胞质蛋白。     

Analysis of polyadenylated RNA from human heart mitochondria

Summary The content of the mitochondrial poly(A)-RNA fraction of human heart has been analyzed by electrophoresis through agarose slab gels in the presence of methyl mercury hydroxide and staining with ethidium bromide. It is possible to identify all the heavy strand coded mRNAs in the electrophoretic pattern. This pattern is qualitatively similar to that obtained from the mitochondria of cells cultured in vitro (HeLa cells), although differences in the relative amount of some components have been detected.     

β-Adrenergic receptor coupled-adenylate cyclase of human fat cell ghosts

Summary The effects of beta-adrenergic agonists such as isoproterenol, norepinephrine and epinephrine upon the adenylate cyclase activity of human fat cell ghosts were tested, each alone and in combination with the beta-blocking agent propranolol. Saturating concentrations of these agents showed a 2–6.5-fold increase of enzyme activity without addition of any artificial cofactors. Isoproterenol was more potent in stimulating the enzyme system than epinephrine and nor-epinephrine. Propranolol caused a dose-dependent rightward shift of the log-dose response curve of these beta-adrenergic agonists. The assay of human fat cell adenylate cyclase in vitro may provide a simple and convenient assay system for the screening of beta-adrenergic drugs of potential therapeutic importance.Herrn Prof. Dr. Dr. h.c. G. Schettler zum 60. Geburtstag gewidmet     

Local thermal unpleasantness and discomfort prediction in the vicinity of thermoneutrality

This work emphasizes a better understanding of the origin of human thermal discomfort under heterogeneous but steady environments, in subjects in the vicinity of physiological and sensory thermoneutrality. The knowledge of skin temperatures allows a psychophysiological study aiming at linking the body thermal state (local and global) to thermal sensation (perceptive and affective judgements). By using two driving simulators, 345 subjects were exposed to different thermal environments, modulated by factors such as the air distribution in the automotive cockpit or the clothing insulation (winter or summer). This work shows that consideration of the local thermal state is essential for the evaluation of thermal comfort in the case of non-uniform environments. Our experimental conditions point out that the overall sensation of discomfort is quantitative, with local unpleasantness needing to be felt for a certain number of body surfaces. A local origin is suggested for cold discomfort, in opposition to the global characteristics of warm discomfort.     

腺病毒伴随病毒表达载体表达人乳头瘤病毒18型E6E7基因构建及其转化作用的鉴定

目的：探讨人乳头瘤病毒（HPV）的E6E7基因在细胞恶性转化中所起的作用。方法：将人乳头瘤病毒（HPV）的E6E7基因克隆至腺病毒伴随病毒表达载体中，通过包装的重组病毒感染，将E6E7基因导入并整合到永生293细胞的基因组中。结果：本研究成功地构建了HPV18 E6E7 AAV病毒并感染了永生293细胞，PCR／Southern杂交分析表明E6E7基因在转化细胞293TL中确有表达，转化细胞293TC和293TL具有明显的转化表型，和亲本293细胞相比，生长速度快，接触抑制消失，集落形成率提高20倍，且集落明显增大，形成时间短。结论：成功地构建了HPV18 E6E7 AAV病毒，HPV18 E6E7基因引起永生化人上皮细胞293的恶性转化。此病毒可用于感染正常上皮细胞，研究其致癌机制。     

Galanin receptors in the post-mortem human brain. Regional distribution of I-galanin binding sites using the method of in vitro receptor autoradiography

The distribution of putative receptors for the peptide galanin was studied in the normal post-mortem human brain by using ¹²⁵I-galanin (0.5 nM) in combination with in vitro receptor autoradiography. Specific binding of ¹²⁵I-galanin was found in a large number of brain areas throughout the neuraxis. Highest binding densities occurred in the basal forebrain and hypothalamus, while the basal ganglia, major parts of the thalamus and the tectum were found to be poor in binding sites. All cortical areas harboured ¹²⁵I-galanin binding, and in the visual cortex a laminated pattern was present. In the hippocampus, ¹²⁵I-galanin binding occurred in layer 2 of the entorhinal cortex, in the uncus and in the hippocampal-amygdala area. In the brain-stem, ¹²⁵I-galanin binding was found in serotoninergic and noradrenergic cellgroups as well as in the reticular formation and in the parabrachial nuclei. Galanin receptors may, thus, mediate the response of galanin in numerous structures in the human brain.