Sterol Composition in the Pollens of Citrus unshiu Marcov. and Citrus sinensis Osbeck

Bacillus brevis secretes a large amount of cell wall proteins into the culture medium. For construction of Bacillus brevis expression-secretion vectors of human erythropoietin (EPO) and the extracellular domain of mouse erythropoietin receptor (sEPOR), cDNA for each mature form was inserted into a plasmid containing the promoter region and the signal-peptide encoding region of a cell wall protein. Culture supernatants of transformants were affinity purified using a monoclonal antibody-fixed gel for EPO and an EPO-fixed gel for sEPOR. The affinity purification efficiently removed unwanted proteins, giving samples with sufficiently high purity to analyze amino acid sequences of N-terminal regions and biological activities. Combination of this secretory production and affinity purification may facilitate isolation of a large amount of pure EPO and sEPOR, and is useful for further understanding the molecular mechanism of interaction between EPO and EPOR.     

左卡尼汀联合促红细胞生成素治疗肾性贫血的临床疗效观察

目的观察不同给药方案对治疗肾性贫血的疗效及安全性。方法将我院收治的198例肾性贫血患者依据治疗方案差异进行分组,于每次血透后给予rHuEPO治疗的98例患者为对照组,于每次血透后应用左卡尼汀联合rHuEPO治疗的100例患者为观察组,比较两组疗效及不良反应情况。结果治疗后,两组患者的Hb、Hct水平变化与治疗前比较,差异均有统计学意义(P0.05)。两组的rHuEPO用量比较,差异无统计学意义(P0.05)。观察组治疗总有效率为93.00%明显高于对照组的82.65%,不良反应发生率为10.00%明显低于对照组的21.43%,组间比较差异均有统计学意义(均P0.05)。结论肾性贫血患者给予左卡尼汀联合rHuEPO治疗的疗效确切,且不良反应低,具有临床推广价值。     

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

Human recombinant erythropoietin (rHuEPO) was produced from Chinese hamster ovary (CHO) cells transfected with the human EPO gene. The cells were grown in batch cultures in controlled bioreactors in which the set-points for dissolved oxygen varied between 3% and 200%. The cell-specific growth rate and final cell yield was significantly lower under hyperoxic conditions (200% DO). However, there was no significant difference in growth rates at other oxygen levels compared to control cultures run under a normoxic condition (50% DO). The specific productivity of EPO was significantly lower at a DO set-point of 3% and 200% but maintained a consistently high value between 10% to 100% DO. The EPO produced under all conditions as analyzed by two-dimensional electrophoresis showed a molecular weight range of 33 to 37 kDa and a low isoelectric point range of 3.5 to 5.0. This corresponds to a highly glycosylated and sialylated protein with a profile showing at least seven distinct isoforms. The glycan pattern of isolated samples of EPO was analyzed by weak anion exchange (WAX) HPLC and by normal-phase HPLC incorporating sequential digestion with exoglycosidase arrays. Assigned structures were confirmed by mass spectrometry (MALDI-MS). The most prominent glycan structures were core fucosylated tetranntenary with variable sialylation. However, significant biantennary, triantennary, and non-fucosylated glycans were also identified. Detailed analysis of these glycan structures produced under variable dissolved oxygen levels did not show consistently significant variations except for the ratio of fucosylated to non-fucosylated isoforms. Maximum core fucosylation (80%) was observed at 50% and 100% DO, whereas higher or lower DO levels resulted in reduced fucosylation. This observation of lower fucosylation at high or low DO levels is consistent with previous data reported for glycoprotein production in insect cells.     

人促红细胞生成素受体胞外区基因的克隆及其在大肠肝菌中的表达

以人胎肝为材料,通过ＰＴ－ＰＣＲ的方法扩增出人促红细胞生成素受体（ｈＰｅｏＲ）的胞外区基因。将获得的或熟体胞外区基因起始密码子改构后克隆原核表达载体ｐＢＶ２２０中,进行原核温控诱导表达。表达产物经蛋白Ｎ端测序及Ｗｅｓｔｅｒｎ－ｂｌｏｔ实验证实表达产物是ｈＥｐｏＲ胞外区蛋白。利用上罐发酵培养获得的包涵体蛋白经复性纯化后,体外生物学活性检测表明表达产物可特 抑制ＴＦ－１细胞在Ｅｐｏ刺激下的生长,证实了     

Effect of Erythropoietin on the interaction of Concanavalin A with rat erythrocytes

Effect of Erythropoietin (Ep) on the interaction of Concanavalin A (Con A) with rat erythrocytes was studied using ¹²⁵I-labelled Con A. Binding of Con A to erythrocytes was dependent on time and cell concentration. Starvation caused an elevation of the lectin binding capacity of red cells which again came down towards the normal level on Ep administration to starved rats. Binding of Con A to erythrocytes decreased linearly with increasing concentration of Ep. Specificity of binding was confirmed by inhibition studies with -methyl-D-mannopyranoside (Me Man) Cells from the starved rats compared to those from normal and Ep treated animals were less prone to inhibition by this sugar analog. Positive cooperative binding of Con A to rat erythrocyte was observed at low concentration of Con A but was absent at higher lectin concentrations. Starvation caused an increase in the number of binding sites per cell which returned to normal level after Ep treatment. Under identical conditions, binding affinities were not much changed in these cells. Cells from the starved animals were more susceptible to agglutination compared to those from normal and Ep-treated rats. Microviscosity and cholesterol/phospholipid ratio of red cell membrane decreased in the starved animals which retraced its way back towards the normal level after Ep treatment.     

Erythroid cell growth and differentiation in vitro in the simulated microgravity environment of the nasa rotating wall vessel bioreactor

Prolonged exposure of humans and experimental animals to the altered gravitational conditions of space flight has adverse effects on the lymphoid and erythroid hematopoietic systems. Although some information is available regarding the cellular and molecular changes in lymphocytes exposed to microgravity, little is known about the erythroid cellular changes that may underlie the reduction in erythropoiesis and resultant anemia. We now report a reduction in erythroid growth and a profound inhibition of erythropoietin (Epo)-induced differentiation in a ground-based simulated microgravity model system. Rauscher murine erythroleukemia cells were grown either in tissue culture vessels at 1 x g or in the simulated microgravity environment of the NASA-designed rotating wall vessel (RWV) bioreactor. Logarithmic growth was observed under both conditions; however, the doubling time in simulated microgravity was only one-half of that seen at 1 x g. No difference in apoptosis was detected. Induction with Epo at the initiation of the culture resulted in differentiation of approximately 25% of the cells at 1 x g, consistent with our previous observations. In contrast, induction with Epo at the initiation of simulated microgravity resulted in only one-half of this degree of differentiation. Significantly, the growth of cells in simulated microgravity for 24 h prior to Epo induction inhibited the differentiation almost completely. The results suggest that the NASA RWV bioreactor may serve as a suitable ground-based microgravity simulator to model the cellular and molecular changes in erythroid cells observed in true microgravity.     

Erythropoietin and the skin: a role for epidermal oxygen sensing?

Erythropoietin (EPO), long appreciated as the chief endocrine regulator of red blood cell formation, is now recognized to exert many additional functions outside the bone marrow. Thus, the quest is on to define the full range of EPO functions in the physiology and pathology of non‐hematopoietic tissues. Two recent studies in man and mice have highlighted the importance of the mammalian skin as one peripheral tissue with a previously unsuspected role in EPO biology; both, as a target and as a source of EPO. In addition, the skin has been proposed to function as an oxygen sensor. The present hypothesis essay critically reviews the currently available evidence for this and provides a unifying theoretical scenario for intracutaneous EPO functions and for a potential role of the skin in the control of EPO production. Mainly, we propose that the skin itself directly contributes to the up‐regulation of EPO plasma levels in response to hypoxia.     

人红细胞生成素工程细胞株的特性鉴定

目的：为了对人红细胞生成素工程细胞株(F10—6)进行特性鉴定。方法：将人红细胞生成素基因构建的重组表达质粒pCDB与标志质粒pSV—dhfr共转染CHO-dhfr^-细胞中,经氨甲喋呤加压筛选和亚克隆挑选,获得高效稳定表达EPO的工程细胞株并对其进行鉴定。结果：实验结果表明,工程细胞株无细菌、支原体、真菌和病毒污染;并无致瘤性;染色体数目不变;连续传代50次和三次冻存复苏后,细胞表达稳定。结论：该工程细胞株可用于工业化生产。     

Neurorestorative effect of erythropoietin and environmental enrichment in the early stage of stroke recovery

The purpose of this study was to investigate whether erythropoietin (EPO) and environmental enrichment (EE) after hypoxic ischemic insult can prevent brain damage, improve neuronal plasticity, and reduce sensorimotor deficits and whether the combination of EPO and EE has a synergistic effect. Sixty Sprague-Dawley rats were used for this study. The rats were divided into three groups: middle cerebral artery occlusion (MCAO) without treatment, MACO?+?EPO, and MCAO?+?EPO?+?EE. For the behavior test, the foot-fault test and measurement of the crossing time in the parallel bars were used. Infarct volume measurement, Neuronal nuclei (NeuN) immunohistochemistry, western blot of the synaptophysin (SYP), and Growth-Associated protein 43 (GAP-43) were used. On postoperative days (POD) at 1 week and 4 weeks, group of EPO and EE increased the recovery as seen in the behavior test. The infarct volume of the EPO group was 32.4%, the EPO?+?EE group was 28.5%, and the control group was 44.0% on POD 4 weeks. The SYP and GAP-43 expression showed an increase in the EPO and EPO?+?EE group compared with the MCAO group. EPO and EPO?+?EE treatment creates a neurorestorative effect, improved neural plasticity, and reduced sensorimotor deficits. But the synergistic effect of the combination of EPO and EE was significantly not proved. In the results of this study, we suggest that EPO and EE treatment may offer a neruorestoration, which can improve the functional brain disturbance of stroke patients.     

Different Expression Patterns of Ngb and EPOR in the Cerebral Cortex and Hippocampus Revealed Distinctive Therapeutic Effects of Intranasal Delivery of Neuro-EPO for Ischemic Insults to the Gerbil Brain

The purpose of this study was to evaluate the neuroprotective effects of intranasally delivered recombinant human neuronal erythropoietin (Neuro-EPO) on brain injury induced by unilateral permanent ischemia in the Mongolian gerbil. Expression of EPO receptor (EPOR) and neuroglobin (Ngb) over 5 weeks after intranasal treatment with Neuro-EPO was determined using immunohistochemistry. Mortality of Neuro-EPO-treated gerbils decreased after surgery, and the sensory and motor function was significantly improved. Histopathological mapping showed that Neuro-EPO significantly reduced delayed neuronal death in the brain. Expression of Ngb was upregulated in the cerebral cortex at most time points (expect for 10 min and 48 hr) and in the hippocampus at 10 min and from 48 hr to 5 weeks, whereas EPOR was almost downregulated or unchanged in the brain (expect for 48 hr). The 10 min and 48 hr seemed to be two time points for the brain to switch the expression of both Ngb and EPOR to early and late recovery phase, respectively. In addition, there were two phases, 10 min to 1 hr and 24 hr to 72 hr, respectively, closing to the “golden hour” of about 60 min and the “silver day” of 1 to 3 days, for the brain to recover from stroke onset with intranasal Neuro-EPO treatment. Therefore, the results suggest that the intranasal administration of Neuro-EPO is effective in the treatment of acute brain ischemia. The different expression patterns of Ngb and EPOR is probably due to ischemic tolerance in the cerebral cortex and ischemic sensitivity in the hippocampus.     

Recombinant human erythropoietin (rHuEPO): cross-linking with disuccinimidyl esters and identification of the interfacing domains in EPO.

Several amino groups of recombinant human erythropoietin are selectively cross-linked by specific cross-linkers including disuccinimidyl suberate or dithiobis(succinimidyl propionate). Intramolecular cross-linkings are obtained without significant change of the protein conformation using appropriate concentrations (0.2 mM) of the cross-linkers, which possess an 11-12-A length of a spacer between two reacting groups. Intramolecularly cross-linked peptides obtained suggest that several amino groups in erythropoietin (EPO) are positioned at a distance of near 12 A in the solution state. These interfacing amino groups include Lys 20-Lys 154, Lys 45-Lys 140, Lys 52-Lys 154, Lys 52-Lys 140, and Ala 1-Lys 116. A comparison of the cross-linking results between nonglycosylated EPO and glycosylated EPO suggests that both proteins retain high similarity regarding protein conformation. These results fit a structural model similar to that of human growth hormone, in which four alpha-helical bundles and a long stretch of beta-sheet structure are involved in the active protein.     

Isolation and characterization of a subset of erythropoietin glycoforms with cytoprotective but minimal erythropoietic activity

Although historically used for the treatment of anemia, erythropoietin (EPO) has emerged as a neurotrophic and neuroprotective agent in different conditions of neuronal damage (traumatic brain injury, ischemia, spinal cord compression, peripheral neuropathy, retinal damage, epilepsy, Parkinson's Disease, among others). Nonetheless, EPO's therapeutic application is limited due to its hematological side‐effects. With the aim of obtaining EPO derivatives resembling the hormone isolated from cells and tissues of neural origin, a novel combination of less acidic EPO glycoforms ‐designated as neuroepoetin (rhNEPO)‐ was purified to homogeneity from the supernatant of a CHO‐producing cell line by a four‐step chromatographic procedure. This simple and single process allowed us to prepare two EPO derivatives with distinct therapeutic expectations: the hematopoietic version and a minimally hematopoietic, but mainly in vitro cytoprotective, alternative. Further biological characterization showed that the in vivo erythropoietic activity of rhNEPO was 25‐times lower than that of rhEPO. Interestingly, using different in vitro cytoprotective assays we found that this molecule exerts cytoprotection equivalent to, or better than, that of rhEPO in cells of neural phenotype. Furthermore, despite its shorter plasma half‐life, rhNEPO was rapidly absorbed and promptly detected in the cerebrospinal fluid after intravenous administration in rats (5 min postinjection, in comparison with 30 min for rhEPO). Therefore, our results support the study of neuroepoetin as a potential drug for the treatment of neurological diseases, combining high cytoprotective activity with reduced hematological side‐effects. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

氨甲酰促红细胞生成素的神经保护作用及机制的研究进展

氨甲酰促红细胞生成素(Carbamylated erythropoietin,CEPO)是促红细胞生成素(Erythropoietin,EPO)的一种衍生物,与EPO一样具有神经保护作用,但是没有其造血功能。已有大量实验研究证实了CEPO的神经保护作用,并有最新研究阐述了EPO和CEPO具有不同的信号转导通路。本文主要介绍了CEPO的减轻神经细胞水肿、促进神经细胞分化、改善神经功能恢复等神经保护作用及CEPO可能通过激活CEPO受体、CD131、神经胶质细胞源性的神经营养因子(Glial cell line derived neurotrophic factor,GDNF)、蛋白激酶B(Protein kinase B,Akt)等机制发挥其神经保护作用。     

Production of erythropoietin by BHK cells growing on the microcarriers trapped in alginate gel beads

Baby hamster kidney (BHK) cells engineered to produce recombinant human erythropoietin (EPO) were cultured at high density on microcarriers entrapped by calcium alginate gel particles. In this system, the BHK cells proliferated not only on the microcarriers but also in vacant spaces in the alginate gel particles. These spaces contributed greatly to high-density cultivation of the cells and a high productivity of EPO.Abbreviations BHK Baby Hamster Kidney - EPO Erythropoietin     

Differential effects of haloperidol and olanzapine on the expression of erythropoietin and its receptor in rat hippocampus and striatum

Compared with first-generation antipsychotics (FGAs), second-generation antipsychotics (SGAs) seem to be neuroprotective and trigger neuroplasticity. Because neuroplasticity is regulated by a variety of neurotrophic factors we studied differential effects of haloperidol (HAL, a FGA) and olanzapine (OLZ, a SGA) on temporal expression of erythropoietin (EPO), a potent neuroprotective factor and its receptor (EPOr) in rat brain. Rats (8-10/group) were treated with HAL or OLZ for 14 days (HAL-14 or OLZ-14) or 45 days (HAL-45 or OLZ-45). Animals were killed by decapitation or by perfusion to collect brains for immunoblotting and immunohistochemical analysis respectively. In hippocampus, the levels of both EPO and EPOr were significantly increased in HAL-14 (p < 0.001) and OLZ-14 (p < 0.001) groups. Their levels decreased in HAL-45 compared with levels in HAL-14 (EPO, p < 0.001; EPOr, p < 0.05), whereas the levels were further increased (EPO, p < 0.05) in OLZ-45 compared with OLZ-14. In striatum, the levels of both EPO and EPOr were unchanged in HAL-14 and EPO levels significantly decreased in HAL-45 (p < 0.05), whereas their levels were significantly increased in OLZ-14 and OLZ-45 compared with the vehicle-treated control (p < 0.001). Both EPO and EPOr were primarily expressed by neurons and endothelial cells. These data suggest that SGAs such as OLZ may have neuroprotective effects through expression of EPO that may be clinically relevant for long-term safe and beneficial management of psychotic patients.     

用三步法纯化重组人促红细胞生成素

在生物反应器中培养中国仓鼠卵巢细胞—促红细胞生成素(CHO-EPO)C2细胞株,培养上清液中重组人促红细胞生成素(rHuEPO)表达水平达2000～3000U/ml。培养上清经过三步纯化:第一步为反相柱色谱,可将样品浓缩约96.7％,其收集液经充分透析后进行第二步的DEAE-离子交换色谱,最后进行分子筛色谱,总回收率为30％以上,经纯化的rHuEPO比活性为1.5×10~8U/g蛋白,SDSPAGE一条带,扫描测试纯度达98％以上。