Erythropoietin non-viral gene therapy does not affect motility,viability, morphology or concentration of rabbit sperm

Erythropoietin (EPO) gene therapy can be used for several purposes; however, its effects on reproductive performance are unknown. The aim of this study was to evaluate the toxicological effects of non-viral (EPO) gene transfer on sperm motility, viability, morphology and concentration. Rabbit EPO cDNA was cloned into a pTarget mammalian expression vector. Rabbits were administered with: (1) pTarget/EPO vector, (2) recombinant human EPO (rHuEpo) and (3) saline (control). Both pTarget/EPO and rHuEpo significantly increased (P < 0.05) hematocrit levels 1 week after injection and they remained significantly higher than the control for up to 5 weeks (P < 0.05), showing that both EPO treatments were effective in stimulating the production of red blood cells in rabbits. The EPO gene transfer or rHuEPO administration had no significant effect (P > 0.05) on sperm motility, vigor, viability, concentration or morphology in the testis.     

缺氧培养对大鼠神经干细胞体外增殖影响及其机制的研究

目的：研究应用缺氧对体外培养的大鼠神经干细胞增殖的影响。方法：将细胞分为4小时缺氧组、12小时缺氧组、促红细胞生成素（EPO）中和抗体组、IgG组以及对照组．测定大鼠神经干细胞经缺氧培养后的各组细胞克隆形成率以及EPO的表达变化。结果：单细胞培养条件下,与对照组相比,4小时缺氧组和IgG组克隆形成率明显增高;中和抗体组无明显变化;12小时组克隆形成率降低。但无统计学意义。缺氧4小时后,EPO蛋白在预处理后即刻出现表迭,4h达高峰,8h仍有部分表达。结论：短时间缺氧可以促进神经干细胞增殖．长时间缺氧则作用相反。缺氧对NSCs增殖作用的影响可能是由EPO介导产生。     

Enhancement of recombinant erythropoietin production in CHO cells in an incubator without CO2 addition

The effect of low levels of carbon dioxide (CO₂) in the gas phase on the production of recombinant human erythropoietin (EPO)in CHO cells was explored. A T-flask culture in an incubator without CO₂ addition showed a slow cell growth initially followed by the cessation of growth, while other cultures incubated under 0.5–5% CO₂ concentrations grew normally at the same rate during the entire period of cultivation. Interestingly, the production of EPO in the culture incubated under no CO₂ supply was highest among the tested cultures. The cell specific secretion rate of EPO (q_EPO) of the culture under no CO₂ supply was about 3 times higher than that of the culture under 5% CO₂ supply. Western blot analysis and in vivo bioassay of EPO showed no apparent changes in EPO quality between the two cases of different CO₂ environments (air vs. 5% CO₂), suggesting robust glycosylation of EPO by CHO cells even under very reduced CO₂ environment. Various combinations of the two extreme cases, with 5% CO₂ supply (suitable for cell growth) and no CO₂ addition (better for EPO production), were made in order to maximize the volumetric productivity of EPO secretion (P_V) in CHO cells. The P_V of the cultures programmed with initial incubation under 5% CO₂ followed by no CO₂ supply was about 2 times superior to that of the culture incubated only under no CO₂ supply. The P_V of the culture under no CO₂ supply was slightly lower than that of culture grown under 5% CO₂. However, the q_EPO of the no CO₂ supply case was more than 5 times higher than that of the culture under 5% CO₂ supply. In conclusion, we have demonstrated that a simple programming of CO₂ supply to an incubator can enhance the production of EPO in CHO cells remarkably, without any apparent change of the EPO quality. This revised version was published online in August 2006 with corrections to the Cover Date.     

Macromolecular docking of a three-body system: the recognition of human growth hormone by its receptor.

Human growth hormone (hGH) binds to its receptor (hGHr) in a three-body interaction: one molecule of the hormone and two identical monomers of the receptor form a trimer. Curiously, the hormone-receptor interactions in the trimer are not equivalent and the formation of the complex occurs in a specific kinetic order (Cunningham BC, Ultsch M, De Vos AM, Mulkerrin MG, Clauser KR, Wells JA, 1991, Science 254:821-825). In this paper, we model the recognition of hGH to the hGHr using shape complementarity of the three-dimensional structures and macromolecular docking to explore possible binding modes between the receptor and hormone. The method, reported previously (Hendrix DK, Kuntz ID, 1998, Pacific symposium on biocomputing 1998, pp 1234-1244), is based upon matching complementary-shaped strategic sites on the molecular surface. We modify the procedure to examine three-body systems. We find that the order of binding seen experimentally is also essential to our model. We explore the use of mutational data available for hGH to guide our model. In addition to docking hGH to the hGHr, we further test our methodology by successfully reproducing 16 macromolecular complexes from X-ray crystal structures, including enzyme-inhibitor, antibody-antigen, protein dimer, and protein-DNA complexes.     

Impact of host cell line choice on glycan profile

Protein glycosylation is post-translational modification (PTM) which is important for pharmacokinetics and immunogenicity of recombinant glycoprotein therapeutics. As a result of variations in monosaccharide composition, glycosidic linkages and glycan branching, glycosylation introduces considerable complexity and heterogeneity to therapeutics. The host cell line used to produce the glycoprotein has a strong influence on the glycosylation because different host systems may express varying repertoire of glycosylation enzymes and transporters that contributes to specificity and heterogeneity in glycosylation profiles. In this review, we discuss the types of host cell lines currently used for recombinant therapeutic production, their glycosylation potential and the resultant impact on glycoprotein properties. In addition, we compare the reported glycosylation profiles of four recombinant glycoproteins: immunoglobulin G (IgG), coagulation factor VII (FVII), erythropoietin (EPO) and alpha-1 antitrypsin (A1AT) produced in different mammalian cells to establish the influence of mammalian host cell lines on glycosylation.     

Glycan analysis of glycoprotein pharmaceuticals: Evaluation of analytical approaches to Z number determination in pharmaceutical erythropoietin products

N-Glycosylation of many glycoprotein drugs is important for biological activity and should therefore be the target of specific and quantitative analytical methods. In this study, we focus on the two N-glycan mapping approaches that are used in pharmacopoeial monograph to analyse N-glycans released from fifteen preparations of recombinant human erythropoietin supplied by ten Chinese manufacturers. Underivatised N-glycans were analysed by high performance anion-exchange chromatography with pulsed amperometric detection and fluorophore-labelled N-glycans were analysed by weak anion-exchange and normal-phase high performance liquid chromatography. N-glycans were also analysed by matrix assisted laser desorption ionisation mass spectrometry. The release of N-glycans by PNGase F was shown to be consistent. Z number, a mathematical expression of the total negatively charged N-glycans composition has provided a convenient way to summarise the complex dataset and it might be suitable for product consistency monitoring. However, this Z number reduces the information of individual acidic N-glycan structure and is also found to be method dependent. Therefore, its use requires clear specification and validation. In this study, we only found weak but positive correlation between the Z number and its bioactivity. Wide range of N-glycans yields were obtained from the fifteen preparations but the significance of their differences is unclear.     

Erythropoietin prevents PC12 cells from 1-methyl-4-phenylpyridinium ion-induced apoptosis via the Akt/GSK-3β/caspase-3 mediated signaling pathway

Apoptosis is a contributing cause of dopaminergic neuron loss in Parkinson disease. Recent work has shown that erythropoietin (EPO) offers protection against apoptosis in a wide variety of tissues. We demonstrate that exposure of PC12 cells to 1-methyl-4-phenylpyridinium ion (MPP⁺) with recombinant human EPO, significantly decreased apoptosis as measured by TUNEL and caspase-3 activity when compared to MPP⁺ treatment alone. EPO induced sustained phosphorylation of Akt and its substrate, GSK-3β, reduced caspase-3 activities in PC12 cells. The anti-apoptotic effect of EPO was abrogated by co-treatment with LY294002, the specific blocker of phosphatidylinositol 3-kinase (PI3K). The effects of EPO on GSK-3β and caspase-3 activities were also blocked by LY294002. LiCl, the inhibitor of GSK-3β, downregulated the caspase-3 activity and blocked the apoptosis induced by MPP⁺. Finally, we determined that EPO transiently activated the ERK signaling pathway, but PD98059, a specific inhibitor of ERK, does not alter the survival effect of EPO in this model system. Thus, these findings indicate that EPO protects against apoptosis in PC12 cells exposed to MPP⁺, through the Akt/GSK-3β/caspase-3 signaling pathway, but the ERK pathway is not involved in the EPO-dependent survival enhancing effect in this model system. The authors Yan Wu and You Shang are equally contributed to this work.     

Moss-based production of asialo-erythropoietin devoid of Lewis A and other plant-typical carbohydrate determinants

Protein therapeutics represent one of the most increasing areas in the pharmaceutical industry. Plants gain acceptance as attractive alternatives for high-quality and economical protein production. However, as the majority of biopharmaceuticals are glycoproteins, plant-specific N-glycosylation has to be taken into consideration. In Physcomitrella patens (moss), glyco-engineering is an applicable tool, and the removal of immunogenic core xylose and fucose residues was realized before. Here, we present the identification of the enzymes that are responsible for terminal glycosylation (α1,4 fucosylation and β1,3 galactosylation) on complex-type N-glycans in moss. The terminal trisaccharide consisting of α1,4 fucose and β1,3 galactose linked to N-acetylglucosamine forms the so-called Lewis A epitope. This epitope is rare on moss wild-type proteins, but was shown to be enriched on complex-type N-glycans of moss-produced recombinant human erythropoietin, while unknown from the native human protein. Via gene targeting of moss galactosyltransferase and fucosyltransferase genes, we identified the gene responsible for terminal glycosylation and were able to completely abolish the formation of Lewis A residues on the recombinant biopharmaceutical.     

悬浮培养CHO 细胞生产重组人促红细胞生成素条件的优化*

目的:通过对贴壁培养CHO细胞筛选驯化,得到高表达的细胞后进行悬浮培养生产重组人促红细胞生成素(rHuEPO)。方法:利用96孔板和24孔板对CHO细胞进行筛选,得到高表达细胞株后进行驯化,使其适合悬浮培养,经过摇瓶扩增后接种到生物反应器中无血清培养,每天监测葡萄糖含量,测rHuEPO表达量。结果:悬浮培养CHO细胞生产rHuEPO,生产周期短,表达量比贴壁培养高出很多,操作方便,减少污染,易于放大,并建立了适合悬浮培养的CHO细胞株,为工业化悬浮培养CHO细胞生产rHuEPO提供了技术基础。结论:经过工艺优化后利用无血清悬浮培养生产促红细胞生成素平均表达量较贴壁培养高,生产周期短,有利于降低生产成本。     

Erythropoietin attenuates the sequels of ischaemic spinal cord injury with enhanced recruitment of CD34+ cells in mice

Erythropoietin has been shown to promote tissue regeneration after ischaemic injury in various organs. Here, we investigated whether Erythropoietin could ameliorate ischaemic spinal cord injury in the mouse and sought an underlying mechanism. Spinal cord ischaemia was developed by cross-clamping the descending thoracic aorta for 7 or 9 min. in mice. Erythropoietin (5000 IU/kg) or saline was administrated 30 min. before aortic cross-clamping. Neurological function was assessed using the paralysis score for 7 days after the operation. Spinal cords were histologically evaluated 2 and 7 days after the operation. Immunohistochemistry was used to detect CD34(+) cells and the expression of brain-derived neurotrophic factor and vascular endothelial growth factor. Each mouse exhibited either mildly impaired function or complete paralysis at day 2. Erythropoietin-treated mice with complete paralysis demonstrated significant improvement of neurological function between day 2 and 7, compared to saline-treated mice with complete paralysis. Motor neurons in erythropoietin-treated mice were more preserved at day 7 than those in saline-treated mice with complete paralysis. CD34(+) cells in the lumbar spinal cord of erythropoietin-treated mice were more abundant at day 2 than those of saline-treated mice. Brain-derived neurotrophic factor and vascular endothelial growth factor were markedly expressed in lumbar spinal cords in erythropoietin-treated mice at day 7. Erythropoietin demonstrated neuroprotective effects in the ischaemic spinal cord, improving neurological function and attenuating motor neuron loss. These effects may have been mediated by recruited CD34(+) cells, and enhanced expression of brain-derived neurotrophic factor and vascular endothelial growth factor.