The precursor to B-type natriuretic peptide is an O-linked glycoprotein

Human pro-B-type natriuretic peptide (proBNP), the precursor for B-type natriuretic peptide (BNP), was expressed in Chinese hamster ovary cells (CHO) and compared by Western blot analysis to BNP cross-reacting material immunoprecipitated from the plasma of heart failure patients. Both recombinant and native forms co-migrated as a diffuse band centered around 25 kDa and were reduced to a 12 kDa species by treatment with a mixture of O-link deglycosylation enzymes. The 108-amino acid CHO-expressed protein was examined by tryptic mapping and LC-MS and found to be an O-linked glycoprotein. Determination of the sites of O-glycosyl addition by blank cycle sequencing of tryptic and Glu-C (Staphylococcus aureus V8 protease) peptides showed that there are seven sites of glycosylation confined to a 36-amino acid residue stretch within the center of the propeptide region. This data is consistent with previous observations of higher molecular weight isoforms of BNP.     

Value of proBNP1–108 testing for the risk stratification of patients with systolic heart failure

The study objectives were to determine the circulating levels of proBNP_1–108, the precursor of B-type natriuretic peptide (BNP) and amino-terminal pro-BNP (NT-proBNP), in patients with systolic heart failure (HF) and to assess their prognosis value for cardiovascular (CV) death over a long-term follow-up. Seventy-three patients with systolic HF and 68 healthy volunteers were included. ProBNP_1–108, BNP and NT-proBNP levels were measured with automated immunoassays and their predictive value for long-term survival was assessed through an 8 years follow-up. ProBNP_1–108 levels were markedly increased in patients with systolic HF in comparison to healthy volunteers. In univariate proportional hazard model, survival was related to proBNP_1–108, BNP, NT-proBNP, age, EF and glomerular filtration rate (eGFR). Kaplan–Meier survival curves according to proBNP tertiles diverged significantly, and the highest proBNP levels were related to patients with the highest risk of CV death. In a multivariate analysis including age, EF, proBNP_1–108, BNP, NT-proBNP, and eGFR levels, NT-proBNP was the strongest predictor of long term CV death. Our study therefore demonstrated that high levels of proBNP_1–108, measured with an assay with enhanced analytical specificity, are related to the long-term risk of cardiovascular death in systolic heart failure.     

The human cardiac hormone fragment N-terminal pro B-type natriuretic peptide is an intrinsically unstructured protein

The cardiac hormone B-type natriuretic peptide (BNP) is synthesized as a prepro 134 residue molecule which is further proteolytically processed into a 76 residue fragment termed N-terminal proBNP (NT-proBNP) and the active portion of this hormone, a 32-residue disulfide-linked peptide (BNP-32). The active hormone regulates cardiac hemodynamic output while as yet no biological function has been attributed to NT-proBNP. Some solution properties of synthetically generated NT-proBNP in benign media are known. The protein is monomeric, elutes aberrantly on size-exclusion chromatography as an apparent larger molecular species, and possesses little global secondary structure as assessed by circular dichroism. To explore the solution structure of NT-proBNP in greater detail, we use 2D-NOESY and 2D-TOCSY NMR on recombinant NT-proBNP to obtain a high resolution solution conformation at the alpha-carbon level. Importantly, NH(i)-NH(i+1) coupling is virtually absent at room temperature implying that large stretches of primary sequence are unordered. Together, the results of these physicochemical measurements classify NT-proBNP as a naturally unfolded protein referred to as an Intrinsically Unstructured Protein (IUP). The calculations of FoldIndex, a computer program which predicts disorder, were compared to the experimental results described here for NT-proBNP in addition to proBNP. NT-proBNP thus appears to be an ideal candidate for the study of native, unfolded proteins.     

Direct Immunochemiluminescent Assay for proBNP and Total BNP in Human Plasma proBNP and Total BNP Levels in Normal and Heart Failure

Background

Recent studies have shown that in addition to brain (or B-type) natriuretic peptide (BNP) and the N-terminal proBNP fragment, levels of intact proBNP are also increased in heart failure. Moreover, present BNP immunoassays also measure proBNP, as the anti-BNP antibody cross-reacts with proBNP. It is important to know the exact levels of proBNP in heart failure, because elevation of the low-activity proBNP may be associated with the development of heart failure.

Methodology/Principal Findings

We therefore established a two-step immunochemiluminescent assay for total BNP (BNP+proBNP) and proBNP using monoclonal antibodies and glycosylated proBNP as a standard. The assay enables measurement of plasma total BNP and proBNP within only 7 h, without prior extraction of the plasma. The detection limit was 0.4 pmol/L for a 50-µl plasma sample. Within-run CVs ranged from 5.2%–8.0% in proBNP assay and from 7.0%–8.4% in total BNP assay, and between-run CVs ranged from 5.3–7.4% in proBNP assay and from 2.9%–9.5% in total BNP assay, respectively. The dilution curves for plasma samples showed good linearity (correlation coefficients = 0.998–1.00), and analytical recovery was 90–101%. The mean total BNP and proBNP in plasma from 116 healthy subjects were 1.4±1.2 pM and 1.0±0.7 pM, respectively, and were 80±129 pM and 42±70 pM in 32 heart failure patients. Plasma proBNP levels significantly correlate with age in normal subjects.

Conclusions/Significance

Our immunochemiluminescent assay is sufficiently rapid and precise for routine determination of total BNP and proBNP in human plasma.     

BNP及NT-proBNP与2型糖尿病关系的研究进展

BNP及NT-proBNP是诊断心衰的重要指标。近年来BNP及NT-proBNP与2型糖尿病关系的研究有了新的进展。我们收集近年来国内外关于2型糖尿病中BNP及NT-proBNP的相关文献并进行研究。结果显示2型糖尿病合并冠心病、高血压、糖尿病肾病患者BNP或NT-proBNP有升高趋势。单纯2型糖尿病及糖尿病视网膜病变患者以及低血糖患者BNP或NT-proBNP差异无统计学意义。高血压、年龄、性别、体重指数、肾功能及心脏结构功能改变是2型糖尿病患者BNP及NT-proBNP的影响因素。降糖药物对2型糖尿病患者BNP及NT-proBNP水平的研究尚少,糖尿病病程、FPG以及HbA1c对BNP及NT-proBNP的影响尚存在争议。BNP及NT-proBNP升高对2型糖尿病合并冠心病、高血压、糖尿病肾病患者病情评估,预后判断及诊治具有非常重要的意义。降糖药物、糖尿病病程、FPG以及HbA1c对BNP及NT-proBNP的影响需要进一步研究。     

基质细胞衍生因子-1 与围生期心肌病患者心力衰竭的相关性研究

目的:探讨基质细胞衍生因子-1与围生期心肌病患者心力衰竭的相关性。方法:采用前瞻性研究纳入59例围生期心肌病并发心力衰竭患者,33例单纯围生期心肌病患者作为对照组。患者均接受体检、实验室检查、心电图、心脏彩超评估。选取基质细胞衍生因子-1(SDF-1)、超敏C反应蛋白(hs-CRP)、氨基末端脑钠肽前体(NT-pro BNP)、血清肌钙蛋白(TNI)及心脏彩超相关参数为评价指标。结果:(l)围生期心肌病患者循环中基质细胞衍生因子-1水平明显高于对照组;(2)循环中基质细胞衍生因子-1与超敏C反应蛋白(CRP)、氨基末端脑钠肽前体(NT-pro BNP)呈正相关,与超声心动图左心室射血分数(LVEF)呈负相关。结论:基质细胞衍生因子-1与围生期心肌病患者心力衰竭具有显著相关性。     

Head-to-head comparison of BNP and IL-6 as markers of clinical and experimental heart failure: Superiority of BNP

Activation of BNP and IL-6 are hallmarks of left ventricular (LV) dysfunction and congestive heart failure (CHF). To assess the relative activation of BNP and IL-6 in clinical and experimental heart failure, we performed a human study in which plasma N-terminal proBNP (NT-proBNP) and IL-6 were measured in a large group of patients in the chronic phase after myocardial infarction (MI) and an animal study in which LV gene expression of BNP and IL-6 was assessed in rapid ventricular pacing-induced heart failure. In the human study, NT-proBNP and IL-6 were measured by non-extracted, enzyme-linked immunoassay in 845 subjects (n=468 outpatients after MI, MONICA MI register Augsburg; and 377 siblings without MI, control). NT-proBNP (295+/-23pg/mL vs. CTRL 84+/-8, P<0.05) and IL-6 (2.7+/-0.1pg/mL vs. CTRL 2.1+/-0.1, P<0.05) were both elevated in subjects with MI. These increases were particularly pronounced in the presence of concomitant CHF (both P<0.01 vs. CTRL) and LV dysfunction (EF<45%, both P<0.05 vs. CTRL). However, NT-proBNP was significantly correlated with several cardiac structural and functional parameters (EF, LVMI, history of MI, CHF symptoms; all P<0.05) upon regression analysis whereas IL-6 was only correlated with history of MI (P<0.001). Accordingly, MI subjects with symptomatic LV dysfunction were detected by NT-proBNP with a greater sensitivity, specificity, and ROC-area (85%, 88%, and 0.87, respectively) as compared to IL-6 (69%, 53%, and 0.67, respectively). In the animal study, IL-6 and BNP expression were both significantly elevated in CHF (both P<0.05) but with a much greater absolute activation of BNP. In addition, BNP mRNA expression displayed a stronger inverse correlation with LV function (r=-0.74; P<0.001) than IL-6 (r=-0.53; P=0.001) and was a markedly more sensitive and specific molecular marker of LV dysfunction (sensitivity 91%, specificity 100%, ROC-area 0.94) than IL-6 (sensitivity 74%, specificity 83%, ROC-area 0.87). Our animal study provides evidence that IL-6 expression is activated in heart failure but to a significantly lesser degree than that of BNP. Both the stronger expression of BNP and the better correlation with LV function provide the molecular basis for a diagnostic superiority of NT-proBNP in clinical LV dysfunction and heart failure.     

N末端脑钠肽前体在围冠状动脉搭桥术期的变化及意义

N-末端脑钠肽前体(N-terminal pro brain natriuretic peptide,NT-proBNP)是体内脑钠肽前体(proBNP)裂解成脑钠肽(brain natriuretic peptide,BNP)时的产物,NT-proBNP的血浆浓度及稳定性比BNP更高,半衰期更长,属于钠尿肽系统的重要一员,本身无生物学活性。NT-proBNP主要由正常的心肌细胞合成和分泌,在心肌损伤或坏死后迅速升高,可反映机体代偿病理改变和恢复循环的能力,是心功能障碍性疾病,如心力衰竭、左室肥厚等诊断、疗效监测和预后评估等最佳的心肌标志物,临床通过测定血浆NT-proBNP水平用于急慢性充血性心力衰竭(congestive heart failure,CHF)的诊治及预后,本文主要就NT-proBNP在围冠状动脉搭桥术(Coronary Artery Bypass Grafting,CABG)期的变化及临床意义的新进展进行综述。     

Recent advances in natriuretic peptide research

The natriuretic peptides are a family of related hormones that play a crucial role in cardiovascular and renal homeostasis. They have recently emerged as potentially important clinical biomarkers in heart failure. Natriuretic peptides, particularly brain natriuretic peptide (BNP) and the inactive N-terminal fragment of BNP, NT-proBNP, that has an even greater half-life than BNP, are elevated in heart failure and therefore considered to be excellent predictors of disease outcome. Nesiritide, a recombinant human BNP, has been shown to provide symptomatic and haemodynamic improvement in acute decompensated heart failure, although recent reports have suggested an increased short-term risk of death with nesiritide use. This review article describes: the current use of BNP and its inactive precursor NT-proBNP in diagnosis, screening, prognosis and monitoring of therapy for congestive heart failure, the renoprotective actions of natriuretic peptides after renal failure and the controversy around the therapeutic use of the recombinant human BNP nesiritide.     

Dissecting Antibodies with Regards to Linear and Conformational Epitopes

An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets.     

The Role of the Circulation in Processing pro-Brain Natriuretic Peptide (proBNP) to Amino-Terminal BNP and BNP-32

Hunt, P. J., E. A. Espiner, G. M. Nicholls, A. M. Richards and T. G. Yandle. The role of the circulation in processing pro-brain natriuretic peptide (proBNP) to amino-terminal BNP and BNP-32. Peptides 18(10) 1475–1481, 1997.—Human proBNP (purified from cardiac tissue) was incubated at 37°C in whole blood, serum and plasma and the products analyzed by size exclusion high pressure liquid chromatography and radioimmunoassay (RIA). In addition to RIAs for BNP-32 and NT-proBNP(1-13), a newly developed RIA for proBNP(62-76) was also used to identify the peptides. Incubation with serum resulted in the formation of a 9 kDa and a 3 kDa peptide, consistent with the N-terminal and the C-terminal peptides of the propeptide. Minimal processing of proBNP was seen in blood or plasma, suggesting that the circulation does not play a major role in the activation of proBNP. Analysis of degradation products of human proBNP using site directed specific antisera indicates that removal of N-terminal amino acids from proBNP occurs in serum. These findings support the view that the “high molecular weight BNP-32” previously identified in human plasma comprises amino-terminal deleted forms, and is unlikely to be intact proBNP(1-108).     

Immunoreactive forms of natriuretic peptides in ovine brain: response to heart failure

In order to elucidate how brain natriuretic peptides (NPs) are affected by experimentally induced heart failure, we have measured the immunoreactive (IR) levels of the NP in extracts from 10 regions of ovine brain, including pituitary, and clarified their molecular forms using high performance liquid chromatography (HPLC). Using species-specific radioimmunoassay (RIA), atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were all detected in extracts taken from control animals and sheep that had undergone rapid ventricular pacing for 7 days to induce heart failure. CNP was the most abundant NP as assessed by specific RIA, and the pituitary contained the highest IR levels for all three NP. Compared with control animals, the pituitary content of BNP in animals with heart failure was reduced by 40% (control, 0.26±0.02 pmol/g wet weight versus heart failure 0.16±0.01; P<0.01, n=7). No other significant changes were observed. The molecular forms of ANP and CNP in whole brain extracts as assessed by HPLC were proANP and CNP22, CNP53 and proCNP, respectively. BNP in pituitary extracts was assessed to be primarily proBNP with a minor component of mature BNP26.     

应用NT-proBNP评价常规药物加心区交感神经阻滞对心衰治疗的有效性

目的:利用常规药物加心区交感神经阻滞（CSNB）技术治疗（以下简称治疗）慢性心力衰竭，观察治疗前后患者血浆N- 端脑 利钠肽前体（NT-proBNP）水平变化情况及利用NT-proBNP评价常规药物加CSNB对心衰治疗的有效性。方法：对54 例慢性心衰 患者给予常规药物加CSNB治疗，比较治疗前后血浆NT-proBNP 的水平、左室射血分数（LVEF）、左室舒张末内径（LVEDD）变化 情况，随访6 个月内不良事件（以下简称事件）发生率（包括全因病死率和再入院率之和）。结果：治疗后患者3-8 分钟即感觉呼吸 困难等临床症状体征明显减轻，住院期间患者尿量明显增多，无一例死亡；治疗14日及出院时血浆NT-proBNP 明显下降（出院时 下降比例为61.4%），与治疗前比较其差异有显著统计学意义（P＜0.01）；LVEF升高，与治疗前比较其差异有显著统计学意义（P＜ 0.01）；住院14日及出院时LVEDD较入院时缩小。随访6 个月，发生事件12 例（22.2%）。结论：NT-proBNP是评价心衰治疗有效 性的可信指标，常规药物加CSNB治疗心衰的临床疗效明显优于单纯常规药物治疗。     

Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells

Background

Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and post-translational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for large-scale expression of cytokines typically results in much lower volumetric productivity.

Results

We have engineered a HEK293 cell clone for high level production of human recombinant glycosylated IFNα2b and developed a rapid and efficient method for its purification. This clone steadily produces more than 200 mg (up to 333 mg) of human recombinant IFNα2b per liter of serum-free culture, which can be purified by a single-step cation-exchange chromatography following media acidification and clarification. This rapid procedure yields 98% pure IFNα2b with a recovery greater than 70%. Purified IFNα2b migrates on SDS-PAGE as two species, a major 21 kDa band and a minor 19 kDa band. N-terminal sequences of both forms are identical and correspond to the expected mature protein. Purified IFNα2b elutes at neutral pH as a single peak with an apparent molecular weight of 44,000 Da as determined by size-exclusion chromatography. The presence of intramolecular and absence of intermolecular disulfide bridges is evidenced by the fact that non-reduced IFNα2b has a greater electrophoretic mobility than the reduced form. Treatment of purified IFNα2b with neuraminidase followed by O-glycosidase both increases electrophoretic mobility, indicating the presence of sialylated O-linked glycan. A detailed analysis of glycosylation by mass spectroscopy identifies disialylated and monosialylated forms as the major constituents of purified IFNα2b. Electron transfer dissociation (ETD) shows that the glycans are linked to the expected threonine at position 106. Other minor glycosylated forms and non-sialylated species are also detected, similar to IFNα2b produced naturally by lymphocytes. Further, the HEK293-produced IFNα2b is biologically active as shown with reporter gene and antiviral assays.

Conclusion

These results show that the HEK293 cell line is an efficient and valuable host for the production of biologically active and glycosylated human IFNα2b.     

左西孟旦联合脑心通胶囊治疗急性心力衰竭的疗效及对血清NT-proBNP，Galectin-3，ET-1及CystatinC水平的影响

目的:分析左西孟旦联合脑心通胶囊治疗急性心力衰竭的临床疗效及对血清氨基末端B型钠尿肽前体(NT-proBNP)、半乳糖凝集素(Galectin-3)、内皮素-1(ET-1)、半胱氨酸蛋白酶抑制剂C(CystatinC)水平的影响。方法:选取2015年3月至2016年6月我院收治的90例急性心力衰竭患者,按抽签法将患者随机分为观察组(n=45)和对照组(n=45),对照组患者给予单纯左西孟旦治疗,观察者患者在此基础上联合应用脑心通胶囊治疗。比较两组患者的临床疗效、心功能指标、血压、心率、不良反应的发生情况及治疗前后血清NT-proBNP、Galectin-3、ET-1和CystatinC水平的变化。结果:治疗后,观察组总有效率为93.33%,比对照组显著升高(77.78%)(P0.05)。两组治疗后患者的左室部分缩短(LVFS)、左心射血分数(LVEF)、每搏输出量(SV)水平均较治疗前增高,血压、心率及血清NT-proBNP、Galectin-3、ET-1、CystatinC水平均较治疗前明显下降,且观察组患者的LVFS、LVEF、SV水平明显高于对照组(P0.05),血压、心率及血清NT-proBNP、Galectin-3、ET-1、CystatinC水平明显低于对照组(P0.05)。结论:左西孟旦联合脑心通胶囊治疗能提高急性心力衰竭的治疗效果,显著降低血清NT-proBNP、Galectin-3、ET-1和CystatinC水平,安全性较高。     

Utility of plasma apelin and other indices of cardiac dysfunction in the clinical assessment of patients with dilated cardiomyopathy

Apelin is a recently discovered peptide ligand reported to be involved in the regulation of cardiovascular homeostasis. The exact role of apelin in the pathophysiology of congestive heart failure has remained obscure, and the reported circulating levels of apelin in patients with heart failure have been contradictory. To establish the role of apelin in the assessment of cardiac dysfunction we measured plasma apelin levels in 65 patients with congestive heart failure caused by idiopathic dilated cardiomyopathy (IDC) and 14 healthy volunteers by specific radioimmunoassay. IDC patients were carefully examined including echocardiography, both-sided cardiac catheterization and cardiopulmonary exercise test. In addition, plasma levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), N-terminal pro-atrial natriuretic peptide (NT-proANP), interleukin (IL)-6, tumor necrosis factor alpha (TNF-), epinephrine and norepinephrine were determined. Plasma apelin levels were similar in IDC patients (median 26.5 pg/ml, range < 3.40–97.6 pg/ml) and in control subjects (median 24.1 pg/ml, range 19.0–28.7 pg/ml; p = NS). Unlike the levels of NT-proBNP, IL-6, TNF-, and norepinephrine, plasma apelin levels did not reflect the severity of heart failure. Our study demonstrates that although disturbed apelin–APJ signalling in heart may play a role in the pathophysiology of heart failure, circulating apelin levels cannot be applied in the clinical assessment of patients with chronic left ventricular dysfunction.     

HDR imaging evaluation of a NT-proBNP test with a mobile phone

The determination of NT-proBNP levels is key for the monitoring of patients with diagnosed heart failure and it is a routine measurement typically performed at health care centers, which would benefit from decentralized alternatives. Here we investigate the quantitative evaluation of a well-established NT-proBNP test using a standard mobile phone (Nokia 6720) as measuring platform rather than a dedicated instrument. A Java ME software developed for this application controls the illumination and imaging of the proBNP test under defined time intervals, which enables the composition of multi-exposure sets that are processed as high dynamic range (HDR) images for contrast enhancement. The results show that HDR processing significantly increases the sensitivity and resolution of the technique achieving a performance within the diagnostics range. These results demonstrate the feasibility to exploit a ubiquitous device to decentralize the evaluation of a routine test and identify key processing alternatives to bring the performance of such systems within the diagnostics range.     

Glycoproteomics of milk: differences in sugar epitopes on human and bovine milk fat globule membranes

Oligosaccharides from human and bovine milk fat globule membranes were analyzed by LC-MS and LC-MS/MS. Global release of N-linked and O-linked oligosaccharides showed both to be highly sialylated, with bovine peak-lactating milk O-linked oligosaccharides presenting as mono- and disialylated core 1 oligosaccharides (Galbeta1-3GalNAcol), while human milk had core type 2 oligosaccharides (Galbeta1-3(GlcNAcbeta1-6)GalNAcol) with sialylation on the C-3 branch. The C-6 branch of these structures was extended with branched and unbranched N-acetyllactosamine units terminating in blood group H and Lewis type epitopes. These epitopes were also presented on the reducing terminus of the human, but not the bovine, N-linked oligosaccharides. The O-linked structures were found to be attached to the high molecular mass mucins isolated by agarose-polyacrylamide composite gel electrophoresis, where MUC1 and MUC4 were present. Analysis of bovine colostrum showed that O-linked core 2 oligosaccharides are present at the early stage (3 days after birth) but are down-regulated as lactation develops. This data indicates that human milk may provide different innate immune protection against pathogens compared to bovine milk, as evidenced by the presence of Lewis b epitope, a target for the Helicobacter pylori bacteria, on human, but not bovine, milk fat globule membrane mucins. In addition, non-mucin-type O-linked fucosylated oligosaccharides were found (NeuAc-Gal-GlcNAc1-3Fuc-ol in bovine milk and Gal-GlcNAc1-3Fuc-ol in human milk). The O-linked fucose structure in human milk is the first to our knowledge to be found on high molecular mass mucin-type molecules.     

The Biological Variation of N-Terminal Pro-Brain Natriuretic Peptide in Postmenopausal Women with Type 2 Diabetes: A Case Control Study

Background

The incidence of heart failure in type 2 diabetes is high and it has poorer prognosis when compared with patients without diabetes. Access to echocardiography is limited and alternative methods to identify early heart failure such as the measurement of natriuretic peptides levels have been proposed. However, their wide biological variation could limit their clinical utility. Our aim was to determine if the intrinsic biological variation of one of these peptides, N-terminal proBNP, is as wide in type 2 diabetes as it is in health and to calculate the critical difference values that could be utilised in clinical practice to ensure changes observed between two samples are due to intervention rather than to its biological variability.

Methodology/Principal Findings

12 postmenopausal women with diet controlled type 2 diabetes and without heart failure were compared with 11 control postmenopausal women without diabetes. N-terminal proBNP levels were measured on 10 occasions. The biological variation was calculated according to Fraser''s methods. The mean NT-proBNP level was similar in both groups (mean ± standard deviation; type 2 diabetes, 10.7 pmol/L± 8.5 versus 8.49±6.0 pmol/L, p = 0.42). The biological variation was also similarly wide. The critical difference in patients with type 2 diabetes was between −70% and ±236%.

Conclusions

Type 2 diabetes does not appear to significantly influence the marked biological variation of N-terminal proBNP in postmenopausal women. The critical difference values reported in this study could be used to titrate therapy or monitor response to interventions although the change required in between samples is wide and this might limit its utility.     

Mammalian Notch1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor-like modules

Notch is a large cell-surface receptor known to be an essential player in a wide variety of developmental cascades. Here we show that Notch1 endogenously expressed in Chinese hamster ovary cells is modified with O-linked fucose and O-linked glucose saccharides, two unusual forms of O-linked glycosylation found on epidermal growth factor-like (EGF) modules. Interestingly, both modifications occur as monosaccharide and oligosaccharide species. Through exoglycosidase digestions we determined that the O-linked fucose oligosaccharide is a tetrasaccharide with a structure identical to that found on human clotting factor IX: Sia-alpha2,3-Gal-beta1, 4-GlcNAc-beta1,3-Fuc-alpha1-O-Ser/Thr. The elongated form of O-linked glucose appears to be a trisaccharide. Notch1 is the first membrane-associated protein identified with either O-linked fucose or O-linked glucose modifications. It also represents the second protein discovered with an elongated form of O-linked fucose. The sites of glycosylation, which fall within the multiple EGF modules of Notch, are highly conserved across species and within Notch homologs. Since Notch is known to interact with its ligands through subsets of EGF modules, these results suggest that the O-linked carbohydrate modifications of these modules may influence receptor-ligand interactions.