Probing the role of cysteine residues in the EcoP15I DNA methyltransferase

Chemical modification using thiol-directed agents and site-directed mutagenesis has been used to investigate the role of cysteine residues of EcoP15I DNA methyltransferase. Irreversible inhibition of enzymatic activity was provoked by chemical modification of the enzyme by N-ethylmaleimide and iodoacetamide. 5, 5'-Dithiobis(2-nitrobenzoic acid) titration of the enzyme under nondenaturing and denaturing conditions confirmed the presence of six cysteine residues without any disulfides in the protein. Aware that relatively bulky reagents inactivate the methyltransferase by directly occluding the substrate-binding site or by locking the methyltransferase in an inactive conformation, we used site-directed mutagenesis to sequentially replace each of the six cysteines in the protein at positions 30, 213, 344, 434, 553, and 577. All the resultant mutant methylases except for the C344S and C344A enzymes retained significant activity as assessed by in vivo and in vitro assays. The effects of the substitutions on the function of EcoP15I DNA methyltransferase were investigated by substrate binding assays, activity measurements, and steady-state kinetic analysis of catalysis. Our results clearly indicate that the cysteines at positions other than 344 are not essential for activity. In contrast, the C344A enzyme showed a marked loss of enzymatic activity. More importantly, whereas the inactive C344A mutant enzyme bound S-adenosyl-L-methionine, it failed to bind to DNA. Furthermore, in double and triple mutants where two or three cysteine residues were replaced by serine, all such mutants in which the cysteine at position 344 was changed, were inactive. Taken together, these results convincingly demonstrate that the Cys-344 is necessary for enzyme activity and indicate an essential role for it in DNA binding.     

推进江西省水权水市场创新实践的思考

推进水权水市场改革是解决水资源供需矛盾、实现水资源节约集约利用的重要措施,也是贯彻落实节水优先方针、推动水利高质量发展的重要举措。本文通过分析江西省水权水市场改革历程和实践现状,理清当前存在的问题,从明晰用水户水权、完善水权水市场制度体系、加强水权交易市场主体培育、探索多种模式的水权交易等方面,提出了新阶段江西省水权水市场改革的对策建议,为今后江西省持续推进水权交易、健康发展水市场提供重要参考。     

仲氨改性剂制备低黄变氨基硅油的研究

以仲氨改性剂γ-环己基甲基二甲氧基硅烷（氨基硅烷偶联剂）和八甲基环四硅氧烷（D4）为原料,在氢氧化钾（KOH）催化下,制备了低黄变的氨基硅油。较佳合成条件为：氨基偶联剂用量为4.0%,反应时间为4.5 h,反应温度120℃,催化剂用量为0.10%,制得氨基硅油,将其用于纯棉布后整理,能赋予织物较好的柔软性、平滑性、蓬松性、低黄变性以及亲水性。     

介电性弹性体的研究进展

综述了介电弹性体的种类、性能、制备方法与主要发展方向,概述了介电弹性体驱动性能的表征方法,提出了制备高介电低模量介电弹性体的几点建议。     

基于形态学和像素灰度归类的背景重建算法

针对传统背景提取方法在前景运动对象密度较高,亦或前景出现过于频繁时,提取到的背景图像会掺杂很多前景成分的问题,提出了一种基于形态学和像素灰度归类的背景重建算法。该算法通过三帧差分法和形态学把每帧图像分为前景对象和背景区域;然后再用像素灰度归类法只对背景区域进行背景重建,以期在前景对象密度较高时正确重建背景。实验结果证明,新方法能够在车辆密度较高的情况下正确生成背景。     

Development of the hypothalamic vasopressin system and nephrons in Meriones shawi during ontogenesis

This study has evaluated the development of the hypothalamic vasopressin system and nephrons of the kidney in desert rodents, Meriones shawi, which effectively retain water by excretion of highly concentrated urine. The vasopressin system was studied immunocytochemically at the 18th fetal day, at the 2nd, 13th, 27th postnatal days and in adulthood. The kidneys were investigated at the 2nd, 13th postnatal days and in adulthood using microdissection technique. Occasional vasopressin-immunoreactive neurons were observed as early as the 18th fetal day, only in the paraventricular nucleus. From the 2nd postnatal day onwards, vasopressin neurons increased progressively in number, being mainly concentrated in the supraoptic and paraventricular nuclei, as well as in the ventral retrochiasmatic region. Transient neuronal populations were also observed at the 13th postnatal day in the lateral preoptic area and anterior hypothalamic nucleus. Apart from the neurons, the glandular cells of the tuberal lobe showed immunostaining from the 18th fetal day, the first age studied, until the 13th postnatal day. The fibers of differentiating vasopressin neurons grew towards the circumventricular/neurohemal organs, terminating in the organum vasculosum of the lamina terminalis and the lateral ventricles as early as the 18th fetal day, as well as the third ventricle, the posterior lobe and the external zone of the median eminence between the 2nd and 13th postnatal days. The kidney in 2-day-old Meriones comprised nephrons at different stages of development from an S-shaped body to well-differentiated nephrons. At the 13th postnatal day, as in adulthood, the nephrons were well differentiated and characterized by long, thin loops descending to different levels of papilla. Thus, according to our morphological data the hypothalamic vasopressin neurons and nephrons in the kidney of Meriones reach the definitive state by the end of the 2nd postnatal week.     

Accumulation of co-localised unesterified cholesterol and neutral lipids within vacuolised elastin fibres in athero-prone areas of the human aorta

To investigate whether there are alterations of elastin fibres in the arterial intima at the pre-atherosclerotic stage, grossly normal areas of human thoracic aorta were taken soon after death from 13 healthy trauma victims whose ages ranged from 16 to 40 years. Two areas were compared: atherosclerosis-prone (AP) areas localised to the dorsal aspect of the aorta along the rows of intercostal branch origins, and atherosclerosis-resistant (AR) areas from the ventral aorta. Electron microscopic analysis combined with cytochemical staining was applied. Unesterified cholesterol was identified using the filipin-staining technique while neutral lipids were visualised by the OTO-technique. Intimal features were studied by combining the filipin-staining and the OTO-technique. Electron microscopical examination showed that in both AR and AP areas, some elastin fibres in the intima were vacuolised. Unesterified cholesterol was found to be predominantly localised in the musculoelastic layer, in particular, inside the vacuolised elastin fibres. This localisation was seen in all 13 AP areas studied in contrast to the AR areas where it was observed in only four of 13 aortas studied (P < 0.0005, chi2-test). Accumulation of neutral lipids inside vacuolised elastin fibres was found in five out of 13 AP areas but was not observed in any of the AR areas (P=0.01, chi2). A combination of the filipin-staining and OTO-techniques showed that some deposits of neutral lipids and unesterified cholesterol within vacuolised elastin fibres were independently located from each other, but more frequently, neutral lipids were co-located with unesterified cholesterol. The present observations indicate a difference between AP and AR intimal areas which, in particular, relates to the structure of elastin fibres in the musculoelastic layer. The observations suggest that alterations of the extracellular matrix are involved in the trapping and retention of cholesterol and neutral lipids within the intima at an early stage in the development of atherosclerotic lesions.