Toward Homogeneous Erythropoietin: Application of Metal-Free Dethiylation in the Chemical Synthesis of the Ala79-Arg166 Glycopeptide Domain

We describe herein the assembly of hEPO(79–166), a key glycopeptide segment en route to erythropoietin, in minimally protected form. Key to the success of this synthetic endeavor was the application of our two-step cysteine-free native chemical ligation strategy, by which we achieved formal ligation at alanine and proline residues through the use of an N-terminal amino acid surrogate presenting a readily removable thiol functionality.     

Erythropoietin: New Directions for the Nervous System

New treatment strategies with erythropoietin (EPO) offer exciting opportunities to prevent the onset and progression of neurodegenerative disorders that currently lack effective therapy and can progress to devastating disability in patients. EPO and its receptor are present in multiple systems of the body and can impact disease progression in the nervous, vascular, and immune systems that ultimately affect disorders such as Alzheimer’s disease, Parkinson’s disease, retinal injury, stroke, and demyelinating disease. EPO relies upon wingless signaling with Wnt1 and an intimate relationship with the pathways of phosphoinositide 3-kinase (PI 3-K), protein kinase B (Akt), and mammalian target of rapamycin (mTOR). Modulation of these pathways by EPO can govern the apoptotic cascade to control β-catenin, glycogen synthase kinase-3β, mitochondrial permeability, cytochrome c release, and caspase activation. Yet, EPO and each of these downstream pathways require precise biological modulation to avert complications associated with the vascular system, tumorigenesis, and progression of nervous system disorders. Further understanding of the intimate and complex relationship of EPO and the signaling pathways of Wnt, PI 3-K, Akt, and mTOR are critical for the effective clinical translation of these cell pathways into robust treatments for neurodegenerative disorders.     

乳酸对重组CHO细胞生长代谢及EPO表达的影响

利用脉冲实验方法研究了乳酸和渗透压对重组CHO细胞生长代谢和EPO表达的影响 ,证明乳酸分子本身对重组CHO细胞的生长代谢和产物表达没有明显的影响 .乳酸浓度从 0增加到 6 9mmol·L^-1对应于渗透压从 30 0增加到 4 2 0mOsm ,细胞的比生长速率下降了 33% ,葡萄糖和谷氨酰胺的比消耗速率均增加了 2 3% ,细胞对葡萄糖和谷氨酰胺的得率系数分别下降了 4 5 %和 4 7% ,乳酸的比生成速率则保持恒定 ,表明在高渗透压下营养物的利用更倾向于能量代谢过程 ,好氧能量代谢过程所占的比例随着渗透压的增加而增加 ;另一方面 ,氨对谷氨酰胺的得率系数增加了 4 4 % ,丙氨酸对谷氨酰胺的得率系数下降了 2 4 % ,表明谷氨酰胺经谷氨酸脱氢酶途径的代谢流量随着渗透压的增加而增加 .EPO的比生成速率随着渗透压的增加而下降     

Elaborate synthesis of biological macromolecules

Egged on: Elaborate syntheses of biological macromolecules consisting of more than two different components is developing. Kajihara and co-workers have used a bio-resource to develop a new strategy for the semisynthesis of glycoproteins.     

促红细胞生成素对急性脊髓损伤患者血清降钙素基因相关肽的影响

目的:探讨促红细胞生成素(erythropoietin,EPO)对急性脊髓损伤患者血清降钙素基因相关肽(calcitonin gene-related peptide,CGRP)的影响。方法:2013年9月到2018年2月选择在本院进行急诊的急性脊髓损伤患者156例,根据治疗方法的不同分为EPO组80例与对照组76例,对照组给予甲强龙冲击治疗,EPO组给予甲强龙联合EPO治疗,两组都连续治疗4周。结果:治疗后两组患者的脊髓神经功能评分都显著高于治疗前(P<0.05),EPO组也显著高于对照组(P<0.05)。EPO组治疗后的红细胞与血红蛋白值显著高于治疗前(P<0.05),也高于对照组(P<0.05),对照组治疗前后红细胞与血红蛋白值对比差异无统计学意义(P>0.05)。EPO组治疗期间的消化道出血、感染、肝肾功能异常等并发症发生率为8.8%,对照组为9.2%(P>0.05)。治疗后两组的血清CGRP值都显著高于治疗前,EPO组也显著高于对照组(P<0.05)。结论:EPO在急性脊髓损伤患者中的应用具有很好的安全性,有利于血清CGPR的释放,改善机体营养状态,提高患者的脊髓神经功能。     

EPO对阿霉素所致心衰大鼠氧化应激能力及细胞凋亡水平的影响

目的评价红细胞生成素（EPO）对阿霉素所致心力衰竭大鼠氧化应激和细胞凋亡水平的影响,并探讨其抗心力衰竭的可能机制。方法30只SD大鼠随机分为EPO组、心衰组和对照组（予相同体积生理盐水）,每组10只。采用腹腔注射盐酸阿霉素（ADR）建立心衰大鼠模型,EPO组每日给予EPO50U·kg-1;心衰组每日给予相同体积生理盐水,观察10周。第11周行心脏超声检测并测定大鼠血清中超氧化物歧化酶（SOD）、丙二醛（MDA）水平及心肌组织中FasmRNA的表达,Tunel法测心肌细胞凋亡。结果与心衰组相比,EPO组左室射血分数显著提高、MDA及FasmRNA的水平降低、心肌细胞凋亡指数（AI）减少、SOD活力增加（均P〈0．01）;与对照组相比,心衰组大鼠SOD活力下降、MDA水平升高、FasmRNA的表达及心肌细胞AI指数增加（均P〈0．01）。结论EPO能显著减轻阿霉素诱导的血流动力学障碍、改善心功能,且此作用可能与氧化应激能力及细胞凋亡水平降低有关。     

Simvastatin downregulates the expression of hepcidin and erythropoietin in HepG2 cells

Statin therapy may improve responsiveness to erythropoietin‐stimulating agents in patients with end‐stage renal disease. Although statins increase hepatic iron uptake and storage capacity in cholestatic rats, the underlying mechanisms are unclear. Therefore, we examined the effects of a statin (simvastatin) on the expression of hepcidin, erythropoietin receptor (EPOR) and erythropoietin (EPO) in cultured HepG2 cells. HepG2 cells (6–6.5 × 10⁵ cells) were seeded in 6‐cm dishes and incubated overnight. The cells were then treated with 0, 0.5, 1, 3, 5, or 10 μM simvastatin, and the mRNA expression of hepcidin, EPOR, and EPO was determined. Data were collected from three independent experiments. The cDNA extracted from the cells was used as a template for real‐time polymerase chain reaction, and each sample was tested in duplicate. Significant differences (P < 0.05) among groups were determined using one‐way analysis of variance with Fisher's least significant difference post hoc test. Data were adjusted using Bonferroni's method. The relative mRNA expression of hepcidin in HepG2 cells treated with 0.5, 1, 3, 5, and 10 μM simvastatin, relative to the control group, was 0.7273, 0.3303, 0.2418, 0.4131, and 0.4064, respectively. The relative mRNA expression of EPOR was 0.5196, 0.2319, 0.2398, 0.4253, and 0.1245, respectively, while that of EPO was 0.9751, 0.4712, 0.4613, 0.4875, and 0.1654. There was a reverse dose‐dependent effect of simvastatin. These results suggest that statins increase erythropoiesis by targeting hepcidin and iron regulatory pathways, independent of erythropoietin.     

Hemochromatosis gene mutations and treatment of anemia in patients on hemodialysis

Hemochromatosis causes iron overload by enhanced intestinal absorption. This study examined erythropoietin and intravenous (IV) iron requirements in hemodialysis (HD) patients with HFE mutations. Patients on HD for >90 days with no cause of anemia except chronic kidney disease were tested for HFE mutations (H63D and C282Y). Intravenous iron and erythropoietin doses were adjusted to achieve recommended targets. Monthly hemoglobin (Hb), ferritin, mean corpuscular volume, mean cell hemoglobin, erythropoietin, and IV iron doses for 3 consecutive months were averaged. Of 172 patients, 71 (41.3%) had ≥1 HFE mutation: 24 (14%) C282Y heterozygotes, 40 (23.3%) H63D heterozygotes, 5 compound heterozygotes, and 2 homozygotes. Comparing patients with ≥1 HFE mutation to those without mutations showed no significant difference in Hb or serum ferritin. There was a trend toward lower median weekly erythropoietin dose in patients with ≥1 HFE mutation (94.0 vs. 135.4 U/kg body weight; P=0.13). There was no difference in median weekly IV iron dose (1.0 vs. 0.9 mg/kg body weight; P=0.56). Comparing the 30 patients with a C282Y mutation to patients without HFE mutations produced similar results. Comparing the 47 patients with an H63D mutation, with those without HFE mutations, no discernable trend was observed. In this study, patients with HFE gene mutations on HD for established renal failure do not require less iron supplementation to achieve recommended Hb targets. We observed a trend toward lower erythropoietin requirement in patients possessing C282Y mutations. Larger studies may clarify the role of HFE mutations, regulators of iron metabolism and erythropoiesis in chronic kidney disease.     

Statin use is associated with lower inflammation and erythropoietin responsiveness index in hemodialysis patients

Patients with end-stage renal disease are prone to inflammation and inflammation is related to erythropoietin-stimulating agent hyporesponsiveness and mortality in this population. Statins have been demonstrated to reduce cardiovascular mortality in selected populations of end-stage renal disease patients. These drugs have pleiotrophic effects such as anti-inflammation. In this retrospective analysis, we determined whether the use of statins improves inflammation and inflammation-related anemia in a cohort of hemodialysis patients. Data were analyzed from Fresenius Medical Care Dialysis Clinics in Turkey between 2005 and 2007. Seventy prevalent hemodialysis patients who were on statins at the start of the study and have been on statins during follow-up (statin users) and 1293 patients who were not on statin at the start of the study and had never been prescribed any lipid-modifying drugs during follow-up (statin nonusers) were included in the study. High-sensitive C-reactive protein levels were significantly decreased in statin users (1.50±1.49 vs. 1.33±1.11 mg/L, P=0.05) compared with nonusers (1.93±3.22 vs. 2.05±2.77 mg/L). Hemoglobin levels and the rate of erythropoietin-stimulating agent users were similar. However, the prescribed erythropoietin-stimulating agent dose (31.6±27.5 vs. 47.3±45.2 U/kg/week, P<0.05) and the erythropoietin response index (2.90±2.73 vs. 4.51±4.48 U/kg/week/Hb, P=0.001) were lower in statin users compared with statin nonusers. On stepwise multiple regression analysis, gender, high-sensitive C-reactive protein, duration of hemodialysis, serum ferritin, and statin use were independent determinants of the erythropoietin responsiveness index. Our results suggest that statin treatment leads to lower inflammation and improves hematopoiesis in hemodialysis patients.