V3环缺失的HIV-1 ADA株包膜表达载体构建及鉴定

目的构建V3环缺失的HIV-1 ADA株包膜糖蛋白表达体系。方法分别设计包膜糖蛋白两端及V3环两端的两对引物，采用重叠延伸剪接法，进行HIV-1 ADA株包膜糖蛋白V3环缺失体的构建。得到的PCR产物经EcoRⅠ和XhoⅠ双酶切，与pSM载体连接，构建HIV-1 ADA株包膜糖蛋白V3环缺失的表达载体(pSM—ADA△V3)，转化大肠杆菌后筛选阳性克隆，经PCR及基因序列测定进行鉴定。结果获得HIV-1 ADA株包膜糖蛋白V3环缺失体PCR产物，构建了其表达载体pSM—ADA△V3，经转化和筛选获得了重组菌，经PCR及基因测序结果显示重组质粒序列正确，为预期目的片段。结论成功构建了V3环缺失的HIV-1 ADA株包膜糖蛋白的表达载体。此结果为进一步构建伪病毒，观察V3环完全或部分缺失对病毒侵入靶细胞过程的影响，为开发阻止HIV-1进入靶细胞的药物或疫苗打下基础。     

A Nationwide Questionnaire Survey on Awake Craniotomy in Japan

The number of awake craniotomies is increasing because of its beneficial features. However, not enough information is available regarding the current status of awake craniotomy in Japan. To evaluate the current status of awake craniotomy in institutes, a nationwide questionnaire survey was conducted. From June to August 2019, we conducted a questionnaire survey on awake craniotomy in the neurosurgery department of 45 institutes that perform awake craniotomies in Japan. Responses were obtained from 39 institutes (response rate, 86.7%). The main methods of awake craniotomy were almost the same in all institutes. Twenty-six institutes (66.7%) had fewer than 10 awake craniotomies (low-volume institutes) per year, and 13 high-volume institutes (33.3%) performed more than 10 awake craniotomies annually. Some institutes experienced a relatively high frequency of adverse events. In 11 institutes (28.2%), the frequency of intraoperative seizures was more than 10%. An intraoperative seizure frequency of 1%-9%, 10%-29%, and over 30% was identified in 12 (92%), 0 (0%), and 1 (8%) of the high-volume institutes, which was significantly less than in 16 (62%), 10 (38%), and 0 (0%) of the low-volume institutes (p = 0.0059). The routine usage of preoperative antiepileptic drugs was not different between them, but the old type was used more often in the low-volume institutes (p = 0.0022). Taken together, the annual number of awake craniotomies was less than 10 in over two-thirds of the institutes. Fewer intraoperative seizures were reported in the high-volume institutes, which tend not to preoperatively use the old type of antiepileptic drugs.     

Application of Peroral Cholecystoscopy in a Case of Cholecystocolic Fistula

A patient with a cholecystocolic fistula, complicating cholecysto-choledocholithiasis, was treated endoscopically with gallbladder observation by peroral cholecystoscopy (POCCS). The patient was a 71-year-old female admitted to our hospital for investigation and treatment of biliary stones and a cholecystocolic fistula. Endoscopic lithotripsy and extracorporeal shock wave lithotripsy (ESWL) were performed after endoscopic sphincterotomy, and the gallbladder was subsequently investigated using a duodenoscope/ cholangioscope of the mother and baby type. Full distension of the gallbladder with saline solution allowed POCCS observation of the entire surface. The gallbladder mucosa was smooth, and neither stones nor tumors were observed. Though the cholecystocolic fistula itself could not be observed, we concluded that it had resulted from cholecystitis. Peroral cholecystoscopy is thus a useful method of investigating the gallbladder in such cases.     

The comparative potency of cholesterol crystallization-effector proteins in supersaturated model bile systems: Association with vesicle transformation

Various proteins which affect cholesterol crystallization are known to be present in bile, although the relative potency of their action is yet to be established. In this study, we evaluated the comparative potency of nucleating-effector proteins using a recently developed method for quantitative assessment of vesicle transformation in supersaturated model bile systems, to partially characterize mechanisms of their action. Concanavalin A-bound glycoproteins isolated from human gall-bladder bile shortened cholesterol crystallization time by 40% and increased cholesterol growth rate and final crystal mass by 161 and 19%, respectively, when compared to the control. In addition, immunoglobulins isolated from human gall-bladder bile increased cholesterol growth rate by 9%, but showed no significant effect on cholesterol crystallization time and final crystal mass. In contrast, human serum apolipoproteins A-I and B reduced cholesterol growth rate by 26 and 31% and reduced final crystal mass by 12 and 21%, but did not affect cholesterol crystallization time. Gel permeation chromatography revealed that proteins were distributed to both vesicles and bile salt micelles, but that no marked redistribution of lipids was caused by addition of these proteins. Furthermore, no significant difference in crystal structure was observed by video-enhanced contrast microscopy. These results indicate that nucleatingeffector substances tested in this study may modulate vesicular cholesterol-holding capacity, thus affecting cholesterol crystallization. Such modulation is based upon the protein-vesicle association which defines the physico-chemical metastability of vesicular cholesterol.     

Heterogeneity in F-actin polymerization of cord blood polymorphonuclear leukocytes stimulated by N-formyl- methionyl-leucyl-phenylalanine

BACKGROUND: To elucidate the mechanism responsible for defects of polymorphonuclear leukocyte (PMNL) chemotaxis of neonates, we determined actin polymerization of NBD (7-nitrobenz-2-oxa-diazol)-phallacidin-stained PMNL following stimulation with either N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA) in cord blood and adult controls. METHODS: We measured F-actin content in PMNL stained with NBD-phallacidin using flow cytometry. RESULTS: Relative F-actin polymerization, that is, a ratio of stimulated F-actin to basal F-actin, was significantly decreased in cord blood PMNL when compared with that of adult PMNL. Distribution of fMLP-stimulated F-actin showed a bimodal pattern, while adult PMNL disclosed a single pattern. Following stimulation with PMA, however, F-actin levels were equal in both cord and adult PMNL. A fluorescein isothiocyanate-conjugated fMLP receptor assay showed no significant difference in binding capacity of fMLP receptors between adult and cord PMNL. CONCLUSION: These results indicate that a deficiency of PMNL chemotaxis in neonates may be due, in part, to decreased relative F-actin polymerization, which may be caused by functional heterogeneity in cord blood PMNL.