猪内源性逆转录病毒体外感染人胚肾细胞系HEK-293的初步研究

目的对猪内源性逆转录病毒(PERV)体外感染人胚肾细胞系HEK-293的能力进行检测。方法建立体外感染人胚肾细胞系HEK-293的方法,用免疫电镜的方法观察PERV粒子,聚合酶链反应(PCR)、逆转录聚合酶链反应(RT—PCR)检测与PERV共培养24h后HEK-293细胞基因组中PERV前病毒基因的整合、表达并对PERV亚型进行鉴定,激光共聚焦显微镜检测PERV—gag蛋白的表达并进行定量分析。结果电镜下可观察到PERV的圆形病毒粒子;与PERV共培养24h后HEK-293细胞基因组中检测到PERV前病毒DNA,且mRNA也有效表达,PERV亚型为PERV—A,B型;激光共聚焦显微镜观察到PERV—gag蛋白在感染细胞HEK-293的胞质表达,但表达量下降。结论PERV具有体外感染人胚肾细胞系HEK-293的能力,病毒蛋白可以有效表达,因此在猪到人的异种器官移植研究中对PERV可能引起的人兽共患病进行安全性评价是极有必要的。     

Endogenous modification of macronutrient selection pattern in sea bass (Dicentrarchus labrax, L.)

The use of a single diet with a well defined composition to feed fish throughout their life cycle is an oversimplification that probably does not respond to their metabolic requirements. For example, the seasonal reproduction that characterizes most fish species demands changes in nutritional requirements. Bearing this in mind, the macronutrient selection pattern was studied from January to August in twelve individually housed sea bass exposed to a constant photoperiod (12L:12D h) and temperature (23+/-0.5 degrees C). The endogenous "seasonal" effect on food and energy intake regulation and macronutrient selection was determined, using protein (P), carbohydrate (CH), and fat (F) packaged separately into gelatine capsules, a method that prevents the diet chemosensory properties at oropharyngeal level from interfering with macronutrient selection. Energy intake changed monthly, the highest values being recorded in May and June and the lowest values in March and April. The preliminary results illustrated "seasonal" changes in the sea bass macronutrient selection pattern with, which showed a predominantly proteinic selection during April (53% P, 21% CH, 25% F) and lipidic in July (35% P, 19% CH, 42% F); the increase in fat selection from May to July being statistically significant. This is the first evidence supporting the existence of an endogenous rhythm in the "seasonal" energy regulation and macronutrient selection in fish through post-ingestive mechanisms and probably involving chemosensory detection in the gut and/or post-absorptive mechanisms, although the exact mechanisms involved have yet to be clarified.     

Digoxin immune fab protects endothelial cells from ouabain-induced barrier injury

PROBLEM Endogenous digitalis-like factors (EDLF) inhibit sodium pump Na(+) /K(+) ATPase activity, and maternal EDLF levels are elevated in preeclampsia (PE). This study determined whether digoxin immune Fab (DIF) could protect endothelial cells (ECs) from EDLF-induced endothelial barrier dysfunction. METHOD OF STUDY ECs were treated with escalating doses of ouabain (a known EDLF) in the presence or absence of DIF. EC barrier integrity was examined by junction protein VE-cadherin and occludin expressions. EC permeability was determined by horseradish-peroxidase (HRP) leakage and transendothelial electrical resistance (TEER). RESULTS EC junction protein VE-cadherin distribution was disrupted in cells treated with ouabain. DIF, but not control IgG Fab fragment, blocked ouabain-induced decreases in VE-cadherin and occludin expressions and prevented ouabain-induced HRP leakage and TEER changes. CONCLUSION DIF protects ECs from ouabain-induced barrier injury, providing evidence of beneficial effects of DIF on EC function and supporting that Na(+) /K(+) ATPase might be a therapeutic target to ameliorate endothelial dysfunction.     

督脉电针对早期受损伤的脊髓神经营养素-3表达的影响

目的 观察督脉电针对早期受损伤的脊髓神经营养素-3(NT-3)表达的影响及其细胞定位.方法 成年雌性大鼠分为损伤组和电针加损伤组.全横断损伤两组大鼠的脊髓1d后,开始对电针+损伤组大鼠进行督脉电针,损伤组大鼠不做督脉电针.电针加损伤组在电针后1、3和7d时间点取出脊髓损伤区组织,损伤组也在相应时间点取出损伤区组织,用ELISA方法检测损伤区组织NT-3水平.再取电针后7d大鼠脊髓损伤区及其邻近组织切片做NT-3的免疫荧光组织化学双标染色.结果 电针加损伤组和损伤组的脊髓损伤区组织NT-3表达在时间上基本一致;在前3 d,NT-3水平是下降的,在后3 d,NT-3水平是增高的.但是,与损伤组相比,电针加损伤组的NT-3水平是明显增高(P＜0.05).免疫荧光双标染色结果显示,神经元、星形胶质细胞、少突胶质细胞和巨噬细胞都有NT-3的表达.结论 督脉电针可以促进受损伤早期的脊髓组织细胞合成和分泌内源性NT-3,这可能是督脉电针促进急性脊髓损伤修复的适宜微环境因素之一.     

Multiplex high resolution melting assay for estimation of Porcine Endogenous Retrovirus (PERV) relative gene dosage in pigs and detection of PERV infection in xenograft recipients

Porcine Endogenous Retrovirus (PERV) poses an infectious risk in the field of xenotransplantation. This risk may be mitigated by breeding selectively animals bearing favorable PERV genetic characteristics including pigs with low levels of PERV integrated in the genome. A real-time quantitative polymerase chain reaction (PCR) assay employing the Roche High Resolution Melting (HRM) Master was used to estimate the relative gene dosage of PERV pol integrated within the pig genome. When assessed across 99 pigs of the Auckland Island breed numerous animals bearing low gene dosage were identified. The assay was adapted further to perform multiplex PCR for the detection of PERV infection within xenograft recipients. Besides PERV, amplification targets for the multiplex PCR include a pig cell marker for the determination of microchimerism and an internal amplification control (IAC) to assess the efficiency of nucleic acid isolation and effects of PCR inhibition. When 12 patients who had received porcine islet transplants were tested no evidence of PERV infection was found. The assay was shown to be specific, highly reproducible with superior performance over conventional nested PCR. This assay can be used as both a screening tool for PERV proviral levels within donor pigs and as a diagnostic tool to examine PERV transmission in human patients treated with porcine xenotransplantation material.     

Blood coagulation and fibrinolysis after long-duration treadmill exercise controlled by individual anaerobic threshold

For rehabilitation training it is recommended that the intensity of exercise should be clearly below the individual anaerobic threshold (IAT). We investigated blood coagulation, particularly endogenous thrombin potential (ETP) and fibrinolysis following a standardized treadmill (TR) ergometer test at 90% IAT for 60–120 min. Sixteen healthy male non-smokers underwent the TR test. Blood samples were taken after a 30-min rest, immediately after exercise, and 2 h after exercise completion. Extrinsic and intrinsic total (TTP_ex+in) and endogenous (ETP_ex+in) thrombin potential, prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), plasmin-2-antiplasmin complex (PAP), D-dimer, tissue plasminogen activator antigen and activity (tPA-AG and tPA-ACT) and plasminogen activator inhibitor type 1 antigen and activity (PAI-1-AG and PAI-1-ACT) were measured. Immediately after TR, F1+2, TAT and TTP_ex+in were increased (P<0.05) while ETP_ex+in remained unchanged. In contrast, PAP, D-dimer, tPA-AG, tPA-ACT (P<0.05) were distinctly enhanced while PAI-1-ACT was decreased (P<0.05) immediately after exercise. The changes in tPA-AG, tPA-ACT, and PAI-1-ACT were reversed to nearly baseline while the enhancement in PAP and D-dimer was prolonged by more than 2 h after exercise. Long-duration exercise between 60 and 120 min controlled by IAT (90%) on a TR ergometer only implicates a small increase in thrombin generation markers and total (free and ₂-macroglubulin-bound thrombin), but not in endogenous (free) thrombin potential alone. In contrast, fibrinolysis is distinctly increased after this type of exercise. Endurance exercise with an intensity below 90% IAT and a duration below 2 h generates a more favourable condition for fibrinolysis than for blood coagulation in healthy young subjects. Data are given as mean (SD).     

Human endogenous retrovirus-K (HML-2): a comprehensive review

Abstract

The human genome contains a large number of retroviral elements acquired over the process of evolution, some of which are specific to primates. However, as many of these are defective or silenced through epigenetic changes, they were historically considered “junk DNA” and their potential role in human physiology or pathological circumstances have been poorly studied. The most recently acquired, human endogenous retrovirus-K (HERV-K), has multiple copies in the human genome and some of them have complete open reading frames that are transcribed and translated, especially in early embryogenesis. Phylogenetically, HERV-K is considered a supergroup of viruses. One of the subtypes, termed HML-2, seems to be the most active and hence, it is the best studied. Aberrant expression of HML-2 in adult tissues has been associated with certain types of cancer and with neurodegenerative diseases. This review discusses the discovery of these viruses, their classification, structure, regulation and potential for replication, physiological roles, and their involvement in disease pathogenesis. Finally, it presents different therapeutic approaches being considered to target these viruses.     

过滤和离心对热原实验影响的初步研究

目的了解医疗器械和生物材料的浸提液能否过滤或离心后再进行热原实验。方法采用《中国药典》2010年版(二部)附录检测细菌内毒素的动态浊度法,进行定量测定浓度为0.2 EU/mL的原液中的细菌内毒素含量,并在过滤和离心后分别测定溶液中细菌内毒素的含量。结果溶液中的细菌内毒素含量过滤后大大减少,甚至小于0.040 EU/mL。离心则不会影响溶液中的细菌内毒素含量,与原液中含量相近,为0.260 EU/mL。结论为除去医疗器械和生物材料的浸提液中的悬浮颗粒,可以离心后再进行热原实验。     

Voluntary and involuntary attention affect face discrimination differently

Do voluntary (endogenous) and involuntary (exogenous) attention have the same perceptual consequences? Here we used fMRI to examine activity in the fusiform face area (FFA--a region in ventral visual cortex responsive to faces) and frontal-parietal areas (dorsal regions involved in spatial attention) under voluntary and involuntary spatial cueing conditions. The trial and stimulus parameters were identical for both cueing conditions. However, the cue predicted the location of an upcoming target face in the voluntary condition but was nonpredictive in the involuntary condition. The predictable cue condition led to increased activity in the FFA compared to the nonpredictable cue condition. These results show that voluntary attention leads to more activity in areas of the brain associated with face processing than involuntary attention, and they are consistent with differential behavioral effects of attention on recognition-related processes.     

The effects of graded exercise on plasma proenkephalin peptide F and catecholamine responses at sea level

Summary The purpose of this study was to evaluate the effects of graded treadmill exercise on plasma preproenkephalin peptide F immunoreactivity and concomitant catecholamine responses at sea level (elevation, 50 m). Few data exist regarding the sea-level responses of plasma peptide F immunoreactivity to exercise. Thirty-five healthy men performed a graded exercise test on a motor-driven treadmill at the relative exercise intensities of 25, 50, 75, and 100% of maximum oxygen consumption (VO_2max). Significant (P<0.05) increases above rest were observed for plasma peptide F immunoreactivity and norepinephrine at 75 and 100% of the VO_2max and at 5 min into recovery. Significant increases in plasma epinephrine were observed at 75 and 100% of VO_2max. Whole blood lactate significantly increased above resting values at 50, 75, and 100% of the VO_2max and at 5 min into recovery. These data demonstrate that exercise stress increases plasma peptide F immunoreactivity levels at sea level. While the exercise response patterns of peptide F immunoreactivity are similar to catecholamines and blood lactate responses, no bivariate relationships were observed. These data show that sea-level response patterns to graded exercise are similar to those previously observed at moderate altitude (elevation, 2200 m).Human subjects participated in these studies after giving their free and informed voluntary consent. Investigators adhered to AR 70-25 and USAMRDC regulation 70-25 on Use of Volunteers in Research. The views, opinion, and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy, or decision, unless to designated by other official documentation.