Further studies of the role of Ser-16 in the regulation of the activity of phenylalanine hydroxylase.

It was previously proposed that the activation of rat liver phenylalanine hydroxylase (EC 1.14.16.1) by cAMP-dependent protein kinase-mediated phosphorylation of Ser-16 is due to the introduction of the negatively charged phosphate group. To explore the validity of this proposal, we have applied site-directed mutagenesis to specifically replace Ser-16 with negatively charged amino acids, glutamic and aspartic; with polar uncharged amino acids, asparagine and glutamine; with the positively charged amino acid lysine; and with the nonpolar hydrophobic amino acid alanine. The wild-type and mutant enzymes were purified to homogeneity, and the importance of Ser-16 in the activation of phenylalanine hydroxylase was examined by comparing the state of activation of the phosphorylated form of the wild-type hydroxylase with that of the mutants. The kinetic studies carried out on the wild-type phosphorylated hydroxylase showed that all the activation could be accounted for by an increase in Vmax with no change in Km for either phenylalanine or the pterin cofactor. Replacement of Ser-16 with a negatively charged residue, glutamate of aspartate, resulted in the activation of the hydroxylase by 2- to 4-fold, whereas replacement with glutamine, asparagine, lysine, or alanine resulted in a much more modest increase. Further, lysolecithin was found to stimulate the phosphorylated hydroxylase and the mutant enzymes S16E and S16D by a factor of 6-7. In contrast, the mutants S16Q, S16N, and S16A all showed the same magnitude of activation as the wild-type with lysolecithin. Therefore, this study demonstrates that activation of the enzyme by phosphorylation of Ser-16 by cAMP-dependent protein kinase is due to the introduction of negative charge(s) and strongly suggests the involvement of electrostatic interaction between the regulatory and catalytic domains of the hydroxylase.     

低温保存同种心脏瓣膜活性及临床应用的研究

取正常成人心脏瓣膜３６个，置于低浓度广谱抗生素的营养液（ＲＰ－ＭＩ１６４０细胞培养液）中灭菌４８ｈ，－８０℃冰箱初冻，置于液氮中长期保存。灭菌前后瓣膜细菌培养阴性率分别为９４．３％和１００％，解冻后经光镜、电镜检查证实瓣组织细胞结构正常，组织培养葡萄糖消耗率测定示瓣组织存活良好。临床同种心脏瓣膜原位移植３例，随访６～３０个月，瓣功能良好     

有效控制与控制不佳的MRI阴性的GTCS病人发作间期SPECT对照研究

目的:利用单光子发射计算机断层摄影(SPECT)半定量分析有效控制与控制不良的MRI阴性的全面性强直阵挛发作癫(GTCS)病人的局部脑血流差异,探讨脑血流灌注与其预后的关系.材料和方法:对29例有效控制的和12例控制不佳的MRI阴性的GTCS病人进行发作间期99mTc-ECD-SPECT脑血灌流显像,10例年龄匹配的健康人作对照,用感兴趣区(ROI)的不对称指数(%AI)进行半定量分析.将SPECT分析结果与病人的临床表现与EEG相比较.结果:①控制不佳组与有效控制组在丘脑和基底节区的%AI存在显著性差异(P<0.05);②控制不佳组SPECT脑显像的异常率(83.3%,10/12)明显高于有效控制组的异常率(17.2%,5/29),两组具有显著性差异(P<0.01);而两组病人的EEG异常率分别为58.3%、44.8%(7/12、13/29),无显著性差异(P>0.05).结论:控制不佳的MRI阴性的GTCS病人往往存在发作间期的低血流灌注脑区,提示癫的难治性;而控制良好的病人多无明显异常发现,可能预后较好.     

新型胫骨髓内钉的研制与生物力学研究

目的　研制新型胫骨串孔型髓内钉并对其进行生物力学研究。方法　用TAMZ合金制造远端锁孔由一个三个同样直径的串孔所替代的 9根新型胫骨髓内钉。将髓内钉安装在自行设计的夹具中 ,先将 3根新钉进行静态垂直压缩实验 ,根据静态实验结果 ,分别测试 6根新钉的动态疲劳强度。结果　新钉的静态压缩破坏载荷为 2 1 33N ,是人平均体重的 3倍多 ;通过 5 0万次压缩循环的条件疲劳强度为 1 2 0 0N ,约是人体重的 2倍。结论　新钉不仅能够方便临床使用 ,而且拥有较好的力学特性