一种基于EGFP的、安全的抗HIV药物评价系统

目的：建立基于EGFP的、安全的抗人免疫缺陷病毒（HIV）药物评价系统。方法：用增强型绿色荧光蛋白（EGFP）基因替代HIV感染性克隆质粒pUC18-HIV-NL4-3中的部分包膜基因（env）,构建重组假病毒质粒pUC18-NL4-3-EGFP,将其与水疱性口炎病毒糖蛋白（VSV-G）真核表达载体共转染人胚肾293FT细胞,观察绿色荧光蛋白的表达,同时用该细胞培养上清进一步感染其他293FT细胞培养物。为了检验该假病毒系统能否用于抗病毒药物的评价,在假病毒复制和感染过程中加入不同浓度的抗HIV药物AZT（Zidovudine）,采用荧光显微镜检测和流式细胞仪定量检测,分析AZT对假病毒的抑制作用。结果：假病毒质粒pUC18-NL4-3-EGFP能够在转染细胞和再感染细胞中有效地表达绿色荧光蛋白基因,不同浓度的AZT能以剂量依赖方式抑制假病毒的感染和报告基因的表达。结论：建立了一种基于EGFP表达和检测的、安全的HIV假病毒复制和感染系统,该系统可以用于抗HIV药物的筛选和评价。     

Transport of L-lysine in the fission yeast Schizosaccharomyces pombe

Systems of L-lysine transport in Schizosaccharomyces pombe are not constitutive, as at no phase of growth in a rich medium is lysine taken up. Transport activity appears only after preincubation of harvested cells with glucose or another suitable source of energy. If cycloheximide is added during this preincubation no transport systems are synthesized. After removal of glucose, the activity of the transport system decays with a half-time of 13 min. The transport of L-lysine into S. pombe cells from the stationary phase of growth preincubated for 60 min with 1% D-glucose is mediated by at least two systems, the high-affinity one with a Kt of 26 mumol/l and Jmax of 4.95 nmol/min per mg dry wt., the low-affinity one with a KT of 1.1 mmol/l and Jmax of 11.8 nmol/min per mg dry wt. The transport of lysine mediated by these two systems proceeds uphill. The high-affinity system has a pH optimum at 4.0-4.2, the accumulation ratio is highest at a cell density 2-5 mg dry wt. per ml and decreases with increasing lysine concentrations. Lysine accumulated by this system does not exit from cells. The only potent competitive inhibitors are L-arginine, L-histidine and D-lysine. The other amino acids tested do not behave as competitive inhibitors. Of the various metabolic inhibitors tested, the most potent were proton conductors and antimycin A.     

Mutation analysis of 4 candidate genes for hereditary neuralgic amyotrophy (HNA)

Hereditary neuralgic amyotrophy (HNA) is a rare autosomal dominant disorder. It is characterised by recurrent episodes of focal neuropathy involving the brachial plexus. Genetic linkage analysis has mapped HNA to chromosome 17q25 within a 3.5-cM interval flanked by the short tandem repeat markers D17S785 and D17S802. Here, we report the mutation analysis of four candidate genes. Mutation analysis was performed on the complete coding regions of these genes. Several exonic and intronic single nucleotide polymorphisms were detected. However, no disease-causing mutations were found, indicating that these genes are most probably not involved in the pathogenesis of HNA. In addition, we have characterised and localised a putative pseudogene of the SEC14-like 1 gene.     

Assessment of the proliferation of human mesenchymal stromal cells in the presence of human demineralised bone matrix

Tissue engineering integrates discoveries from biochemistry, cell and molecular biology, material science and biomedical engineering to produce innovative three-dimensional composites that can be used to replace or correct damaged tissues and organs. Precise classification of osteoinductive properties of human demineralised bone is often the problem, because it varies from batch to batch. An in vitro assay using bone marrow derived human mesenchymal stromal cells (hMSCs) was developed to improve the classification of the osteoinductive quality of demineralised bone matrix. In this study, three-dimensional, partially demineralised bone scaffolds were investigated for their ability to induce osteogenic differentiation of hMSCs in vitro. Proliferation of the hMSCs was measured by the CelTiter 96? AQueous One Solution Cell assay. Chemical structure was evaluated using quantitative and qualitative X-ray analysis. Scanning electron microscopy revealed primary proliferation of the cells cultivated 14 days and showed elevations in the content of Ca²⁺. These results demonstrate that partially demineralised human bone material supports osteogenic differentiation of hMSCs in vitro. This study documents that in vitro test using hMSCs can be used for classification of the osteoinductive quality of human demineralised bone matrix.     

Physical fitness of Czechoslovak population between the ages of 12 and 55 years. Pulse rate and blood pressure.

In loading test of a representative sample of the Czechoslovak population aged 12--55 years, the authors, examined the reaction of the pulse rate and blood pressure (in watts--W) to loading on a bicycle ergometer. On increasing the submaximal load, the pulse rate rose linearly with age--more gradually in the phase of growth and along an approximately the same trend from the age of 18. The course in adult women corresponded to the course in boys aged about 13. The maximum pulse rate fell linearly in correlation to age, by about 4--5 beats for every 10 years, from 195/min in 12-year-old boys and 198/min in girls. The working capacity at pulse rate 170 (W 170) attained the maximum at 25 years (men 198, women 112 W). The systolic pressure rose non-linearly with loading. It increased more rapidly at lower loads. Children had the smallest pressure reaction and the oldest subjects the greatest. Diastolic pressure fell gradually and non-linearly with loading. The maximum blood pressure values, according to age, rose from 138/56 torr in the oldest males and from 139/57 to 182/84 torr in females.