脂多糖通过补体途径诱导小鼠产生白三烯B4

目的:研究脂多糖(LPS)对人血清中补体系统的激活及在小鼠模型中诱导产生白三烯B4(LTB4)。方法:LPS包被ELISA板,利用血清中补体C4、C3沉积实验检测补体成分被LPS活化的情况,通过尾静脉注射小鼠LPS后不同时间点ELISA定量检测LTB4,评价补体系统的活化和炎症因子的产生。结果与结论:血清系统ELISA检测发现LPS可以激活补体系统,且以凝集素途径为主;动物实验中LTB4被LPS诱导后1～3 h达到峰值,之后回落。C1INH对血清补体活化和动物模型中LTB4的产生均有显著抑制。     

The role of exo-(1-->4)-beta-galactanase in the mobilization of polysaccharides from the cotyledon cell walls of Lupinus angustifolius following germination

BACKGROUND AND AIMS: The cotyledons of Lupinus angustifolius contain large amounts of cell wall storage polysaccharide (CWSP) composed mainly of (1-->4)-beta-linked D-galactose residues in the form of branches attached to a rhamnogalacturonan core molecule. An exo-(1-->4)-beta-galactanase with a very high specificity towards (1-->4)-beta-linked D-galactan has been isolated from L. angustifolius cotyledons, and shown to vary (activity and specific protein) in step with CWSP mobilization. This work aimed to confirm the hypothesis that galactan is the main polymer retrieved from the wall during mobilization at the ultrastructural level, using the purified exo-galactanase as a probe. METHODS: Storage mesophyll cell walls ('ghosts') were isolated from the cotyledons of imbibed but ungerminated lupin seeds, and also from cotyledons of seedlings after the mobilization of the CWSP. The pure exo-(1-->4)-beta-galactanase was coupled to colloidal gold particles and shown to be a specific probe for (1-->4)-beta-D-galactan. They were used to localize galactan in ultrathin sections of L. angustifolius cotyledonary mesophyll tissue during CWSP mobilization. KEY RESULTS: On comparing the morphologies of isolated cell walls, the post-mobilization 'ghosts' did not have the massive wall-thickenings of pre-mobilization walls. Compositional analysis showed that the post-mobilization walls were depleted in galactose and, to a lesser extent, in arabinose. When pre-mobilization ghosts were treated with the pure exo-galactanase, they became morphologically similar to the post-mobilization ghosts. They were depleted of approximately 70% of the galactose residues that would have been mobilized in vivo, and retained all the other sugar residues originally present. Sharply defined electron-transparent wall zones or pockets are associated with CWSP mobilization, being totally free of galactan, whereas wall areas immediately adjacent to them were apparently undepleted. CONCLUSIONS: The exo-(1-->4)-beta-galactanase is the principal enzyme involved in CWSP mobilization in lupin cotyledons in vivo. The storage walls dramatically change their texture during mobilization as most of the galactan is hydrolysed during seedling development.     

A Risk Prediction Model for the Assessment and Triage of Women with Hypertensive Disorders of Pregnancy in Low-Resourced Settings: The miniPIERS (Pre-eclampsia Integrated Estimate of RiSk) Multi-country Prospective Cohort Study

Background

Pre-eclampsia/eclampsia are leading causes of maternal mortality and morbidity, particularly in low- and middle- income countries (LMICs). We developed the miniPIERS risk prediction model to provide a simple, evidence-based tool to identify pregnant women in LMICs at increased risk of death or major hypertensive-related complications.

Methods and Findings

From 1 July 2008 to 31 March 2012, in five LMICs, data were collected prospectively on 2,081 women with any hypertensive disorder of pregnancy admitted to a participating centre. Candidate predictors collected within 24 hours of admission were entered into a step-wise backward elimination logistic regression model to predict a composite adverse maternal outcome within 48 hours of admission. Model internal validation was accomplished by bootstrapping and external validation was completed using data from 1,300 women in the Pre-eclampsia Integrated Estimate of RiSk (fullPIERS) dataset. Predictive performance was assessed for calibration, discrimination, and stratification capacity. The final miniPIERS model included: parity (nulliparous versus multiparous); gestational age on admission; headache/visual disturbances; chest pain/dyspnoea; vaginal bleeding with abdominal pain; systolic blood pressure; and dipstick proteinuria. The miniPIERS model was well-calibrated and had an area under the receiver operating characteristic curve (AUC ROC) of 0.768 (95% CI 0.735–0.801) with an average optimism of 0.037. External validation AUC ROC was 0.713 (95% CI 0.658–0.768). A predicted probability ≥25% to define a positive test classified women with 85.5% accuracy. Limitations of this study include the composite outcome and the broad inclusion criteria of any hypertensive disorder of pregnancy. This broad approach was used to optimize model generalizability.

Conclusions

The miniPIERS model shows reasonable ability to identify women at increased risk of adverse maternal outcomes associated with the hypertensive disorders of pregnancy. It could be used in LMICs to identify women who would benefit most from interventions such as magnesium sulphate, antihypertensives, or transportation to a higher level of care. Please see later in the article for the Editors'' Summary     

A Role for Topographic Cues in the Organization of Collagenous Matrix by Corneal Fibroblasts and Stem Cells

Human corneal fibroblasts (HCF) and corneal stromal stem cells (CSSC) each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7). Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-ß3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200–300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes.     

2’-5’寡聚腺苷酸合成酶（OAS）及其临床意义的研究进展

干扰素作用于靶细胞膜表面的受体后,通过信号转导系统诱导一系列抗病毒蛋白产生,干扰病毒复制以达到抗病毒目的。2’-5’寡聚腺苷酸合成酶（2’．5’oligoadenylatesynthetase,OAS）是干扰素作用于细胞后产生的一种重要的抗病毒蛋白,几十年来,国内外学者对OAS家族及其抗病毒机制进行了大量研究并取得了一定的进展,OAS被dsRNA激活后,催化生成2-5A,2-5A激活核酸内切酶RNaseL,降解病毒RNA,阻断病毒蛋白合成,从而发挥抗病毒作用。体内外研究表明,OAS的表达量或活性的变化可用于评价机体对干扰素的反应,反映干扰素抗病毒效果,另外,它还可作为系统性红斑狼疮的病情活动度的一种检测指标。因此,OAS具有重要的临床应用价值。本文就OAS家族及其抗病毒机制,其测定方法与对于病毒性肝炎和系统性红斑狼疮疾病的临床意义展开综述,以期对OAS的研究和应用提供参考。OAS是典型的干扰素诱导产物,可反映机体内干扰素的抗病毒水平,具有广阔的应用前景。     

氨氯地平联合依那普利治疗原发性高血压的临床疗效及对患者左心室肥厚的影响

摘要目的：探讨氨氯地平联合依那普利治疗原发性高血压的临床效果,观察联合用药对左心室肥厚的影响。方法：选择本院收治的原发性高血压患者92例,随机分为观察组和对照组,各46例,对照组给予苯磺酸左旋氨氯地平5mg,1次／d,口服;观察组在对照组基础上加用马来酸依那普利10mg,2次／d,口服,疗程均为24周。观察两组治疗前后血压变化,应用超声心动图测量两组左心室厚度变化。结果：治疗后,观察组总有效率为91_3％;对照组总有效率为73．9％,观察组总有效率高于对照组（P〈0．05）。治疗前两组心率、血压比较无统计学差异（P〉0．05）,治疗后两组血压均明显降低,观察组收缩压、舒张压明显低于对照组（P〈O．05）;观察组心率明显低于对照组（P〈0．01）。治疗前两组左心室舒张末期室间隔厚度（Leaventricularend—diastolicventricularseptalthickness,IVST）、左心室后壁厚度（1eftventricularposteriorwallthickness,U,PwT）和左室射血分数（Leftventricularejectionfxaction,LVEF）比较无统计学差异（P〉0．05）;治疗后观察组IVST、L、,PwT明显低于对照组,LVEF明显高于对照组（P〈0．05）。结论：氨氯地平联合依那普利治疗原发性高血压能有效扭转左心室肥厚,降压效果较单独应用氨氯地平更佳。     

利用伴刀豆球蛋白检测O-甘露糖基化

目的:基于伴刀豆球蛋白A(ConA)特异性识别并结合甘露糖的特性,建立一种检测O-甘露糖基化的方法,为酵母等宿主表达蛋白的O-糖基化提供一种高效筛选和分析的方法。方法:利用糖苷酶F(PNGF)切除检测蛋白的N-糖链,排除N-糖基化的干扰;通过Q阴离子交换柱和ConA Sepharose 4B柱纯化Western印迹膜封闭蛋白牛血清白蛋白(BSA),除去BSA中甘露糖修饰的蛋白的干扰,优化膜封闭条件;利用辣根过氧化物酶标记的ConA检测具有低甘露糖型N-糖基化修饰能力的毕赤酵母GJK01-HL(Δoch1)表达的抗Her-2抗体是否存在O-甘露糖基化现象。结果:通过PNGF酶切处理,可以完全去除糖蛋白的N-糖链的干扰;BSA经过Q阴离子交换柱和ConASepharose 4B柱纯化后,除去了大部分甘露糖蛋白,可作为封闭蛋白;用建立的方法检测,发现毕赤酵母工程菌GJK01-HL(Δoch1)表达的抗Her-2抗体存在O-甘露糖基化现象。结论:本方法是研究糖蛋白是否发生O-甘露糖基化的有效检测手段,可用于酵母等表达蛋白的O-糖基化的高效筛选和分析。     

重组人MASP2蛋白在大肠杆菌中的表达和纯化

目的:获得酶原形式的重组人甘露聚糖结合凝集素相关丝氨酸蛋白酶2(MASP2)。方法:在大肠杆菌中诱导表达重组人MASP2全长蛋白,包涵体裂解后,经复性、透析、浓缩、考马斯亮蓝染色、SDS-PAGE及Western印迹,鉴定纯化结果及酶活性。结果:复性后的MASP2蛋白经考马斯亮蓝染色未见杂带。自激活实验表明,当MASP2浓度在1μmool/L以下时,无论在4℃还是37℃,都能较稳定地保持酶原形式;蛋白浓度为3.5μmool/L时只能在4℃保持稳定,37℃发生自激活;蛋白浓度达到12μmool/L后,在4℃时已不能稳定存在。结论:获得了较纯的重组人MASP2蛋白,且具有自激活活性。     

Sphingolipid Domains in the Plasma Membranes of Fibroblasts Are Not Enriched with Cholesterol

The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. Thus, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.     

Within‐plant distribution of 1,4‐benzoxazin‐3‐ones contributes to herbivore niche differentiation in maize

Plant defences vary in space and time, which may translate into specific herbivore‐foraging patterns and feeding niche differentiation. To date, little is known about the effect of secondary metabolite patterning on within‐plant herbivore foraging. We investigated how variation in the major maize secondary metabolites, 1,4‐benzoxazin‐3‐one derivatives (BXDs), affects the foraging behaviour of two leaf‐chewing herbivores. BXD levels varied substantially within plants. Older leaves had higher levels of constitutive BXDs while younger leaves were consistently more inducible. These differences were observed independently of plant age, even though the concentrations of most BXDs declined markedly in older plants. Larvae of the well‐adapted maize pest Spodoptera frugiperda preferred and grew better on young inducible leaves irrespective of plant age, while larvae of the generalist Spodoptera littoralis preferred and tended to grow better on old leaves. In BXD‐free mutants, the differences in herbivore weight gain between old and young leaves were absent for both species, and leaf preferences of S. frugiperda were attenuated. In contrast, S. littoralis foraging patterns were not affected. In summary, our study shows that plant secondary metabolites differentially affect performance and foraging of adapted and non‐adapted herbivores and thereby likely contribute to feeding niche differentiation.