Kinetic and mechanistic considerations to assess the biological fate of peroxynitrite

Background

Peroxynitrite, the product of the reaction between superoxide radicals and nitric oxide, is an elusive oxidant with a short half-life and a low steady-state concentration in biological systems; it promotes nitroxidative damage.

Scope of review

We will consider kinetic and mechanistic aspects that allow rationalizing the biological fate of peroxynitrite from data obtained by a combination of methods that include fast kinetic techniques, electron paramagnetic resonance and kinetic simulations. In addition, we provide a quantitative analysis of peroxynitrite production rates and conceivable steady–state levels in living systems.

Major conclusions

The preferential reactions of peroxynitrite in vivo include those with carbon dioxide, thiols and metalloproteins; its homolysis represents only < 1% of its fate. To note, carbon dioxide accounts for a significant fraction of peroxynitrite consumption leading to the formation of strong one-electron oxidants, carbonate radicals and nitrogen dioxide. On the other hand, peroxynitrite is rapidly reduced by peroxiredoxins, which represent efficient thiol-based peroxynitrite detoxification systems. Glutathione, present at mM concentration in cells and frequently considered a direct scavenger of peroxynitrite, does not react sufficiently fast with it in vivo; glutathione mainly inhibits peroxynitrite-dependent processes by reactions with secondary radicals. The detection of protein 3-nitrotyrosine, a molecular footprint, can demonstrate peroxynitrite formation in vivo. Basal peroxynitrite formation rates in cells can be estimated in the order of 0.1 to 0.5 μM s^− 1 and its steady-state concentration at ~ 1 nM.

General significance

The analysis provides a handle to predict the preferential fate and steady-state levels of peroxynitrite in living systems. This is useful to understand pathophysiological aspects and pharmacological prospects connected to peroxynitrite. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Hypoxia mediated expression of stem cell markers in VHL-associated hemangioblastomas

Hemangioblastomas of the retina, central nervous system, and kidney are observed in patients with mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. Mutations in the VHL lead to constitutive activation of hypoxia-inducible-factor (HIF) pathway. HIF-mediated expression of pro-angiogenic genes causes extensive pathological neovascularization in hemangioblastomas. A number of studies have shown coexistence of pro-angiogenic and stem cell markers in ‘tumorlet-like stromal cells’ in the retinal and optic nerve hemangioblastomas, leading to suggestions that hemangioblastomas originate from developmentally arrested stem cells or embryonic progenitors. Since recent studies have shown that the HIF pathway also plays a role in the maintenance/de-differentiation of normal and cancerous stem cells, we evaluated the role of the HIF pathway in the expression of stem cell markers in VHL−/− renal cell carcinoma cells under normoxia or VHL+/+ retinal pigment epithelial cells under hypoxia. Here we show that the expression of stem cell markers in hemangioblastomas is due to activation of the HIF pathway. Further, we show that honokiol, digoxin, and doxorubicin, three recently identified HIF inhibitors from natural sources, blocks the expression of stem cell markers. Our results show the mechanism for the cytological origin of neoplastic stromal cells in hemangioblastomas, and suggest that inhibition of the HIF pathway is an attractive strategy for the treatment of hemangioblastomas.     

ERK-mediated production of neurotrophic factors by astrocytes promotes neuronal stem cell differentiation by erythropoietin

Erythropoietin (EPO), a hematopoietic factor, is also required for normal brain development, and its receptor is localized in brain. Our previous study showed that EPO promotes differentiation of neuronal stem cells into astrocytes. Since astrocytes have influence on the neuronal function, we investigated whether EPO-activated astrocytes could stimulate differentiation of neuronal stem cells into neurons. EPO did not promote neuronal differentiation of neuronal stem cells isolated from 17 day embryos, however, neuronal differentiation was promoted when the neuronal stem cells were co-cultured with astrocyte isolated from post neonatal (Day 1) rat brain. Moreover, neuronal differentiation was further promoted when the neuronal stem cells were cultured with astrocyte culture medium treated by EPO (10U/ml) showing increase of morphological differentiation, and expression of neuronal differentiation marker proteins, neurofilament, and tyrosine hydroxylase. The promoting effect of EPO-treated astrocyte medium was also found in the differentiation of PC12 cells. EPO-promoted morphological differentiation of neuronal stem cells as well as astrocytes was dose dependently reduced by treatment with anti-EPO receptor antibodies in culture with astrocyte culture medium. To clarify whether EPO itself or via production of well-known neurotropic factor could promote neuronal cell differentiation, we determined the level of neurotropic factors in the EPO-treated astrocytes. Compared to untreated astrocytes, EPO-treated astrocytes increased about 2-fold in beta-NGF and 3-4-fold in BMP2, but did not increase BNDF and NT-3 levels. Since the previous study showed that extracellular signal-regulated kinase (ERK) is involved in activation of astrocytes by EPO, we determined whether generation of neurotrophic factor may also be involved with the ERK pathway. In the presence of ERK inhibitor, PD98059, the generation of beta-NGF was diminished in a dose dependent manner consistent with the inhibiting effect on neuronal differentiation. These data demonstrate that EPO promotes neuronal cell differentiation through increased release of beta-NGF and BMP2 from astrocytes, and this effect may be associated with ERK pathway signals.     

Purification of erythropoietin from human plasma samples using an immunoaffinity well plate

A method is described to isolate human erythropoietin (hEPO) from plasma using an EPO-specific immunoaffinity micro well plate (IAP). The operating conditions of the method (binding, blocking and elution) were optimised to avoid isoform discrimination and cross-contamination with other glycoproteins. The overall hEPO recovery was ca. 56% and significant clean-up for plasmatic hEPO was achieved. Polyvinylpyrrolidone (PVP) was used as a blocking reagent and elution took place at pH 11.0. Under these conditions all isoforms from recombinant human EPOs (rhEPOs) and analogues were uniformly recovered guaranteeing lack of discrimination. The resulting procedure allowed isolating erythropoietin from plasma in conditions amenable to hEPO analysis by other techniques such as SDS-PAGE or IEF. Moreover, avoiding contamination with other glycosylated material allowed the identification in human plasma samples of the non-human N-glycolyl-neuraminic acid (Neu5Gc) using HPLC-FLD. Neu5Gc is present as 1–2% of the sialic acid content in rhEPO so this approach could be used to unequivocally detect abuse of rhEPOs or analogues as part of the doping control.     

促红细胞生成素对神经干细胞缺氧性损伤保护作用的实验研究

目的观察外源性EPO对神经干细胞缺氧性损伤的保护作用,为缺氧缺血性脑损伤的治疗提供新思路。方法从孕11.5d（E11.5d）大鼠获得神经干细胞,经无血清培养基悬浮培养并传代,对所获细胞的自我增殖、自我更新及其多分化潜能进行检测。取传3代神经干细胞中添加不同剂量的EPO,在5%O2培养箱中培养120h。通过计数干细胞克隆形成率和MTT法检测神经干细胞的增殖情况。于含血清分化培养基中加入不同剂量的EPO,用NSE和GFAP免疫细胞化学染色观察神经干细胞的分化情况。采用AnnexinⅤ-FITC/PI染色,激光扫描共聚焦显微镜观察、检测细胞凋亡率。结果加入EPO后神经干细胞的克隆形成率和MTT检测的OD值显著增高,细胞凋亡率显著下降,NSE阳性细胞的比例明显升高,其作用随剂量增加而增大,50U/ml时作用最大。结论EPO对神经干细胞缺氧性损伤具有明显的保护作用,并可促进神经干细胞向神经元方向分化。EPO的这种作用随剂量增加而增大,50U/ml时达高峰。     

人EPO高效表达细胞株的筛选与鉴定

用氨甲喋呤 (MTX)对稳定转染人促红细胞生成素 (EPO)基因的CHOˉ(DHFR缺陷型 )细胞株进行梯度加压 ,并使用α -MEM培养基进行培养 ,使DHFR基因在CHOˉ细胞中大量扩增 ,从而使EPO的表达量提高。经检测分析EPO的表达量 ,筛选出EPO高效表达的细胞株。结果表明 ,用MTX加压到 16× 10 - 7mol l和 6 4× 10 - 7mol l浓度时 ,收集的细胞培养液经 (NH4 ) 2 SO4 盐析、透析等处理得到EPO抽提液 ,经SDS -聚丙烯酰胺凝胶电泳 (SDS -PAGE)检测出与标准品相对应的一条带 ,WesternBlot证明该带确为EPO。     

MafG controls the hypoxic response of cells by accumulating HIF-1alpha in the nuclei

We identified MafG as a protein that interacts with HIF-1alpha, a key factor in hypoxic response, using the yeast two-hybrid system. Interaction between MafG and HIF-1alpha was confirmed by surface plasmon resonance and by translocation to the nucleolus with the NoLS signal. A knockdown of MafG reduced erythropoietin (EPO) mRNA levels as well as luciferase reporter activity with the hypoxia response element. The knockdown of MafG did not change total HIF-1alpha protein, but reduced the accumulation of HIF-1alpha in the nuclei. These results suggest that MafG regulates the hypoxic response of cells by detaining HIF-1alpha in the nuclei.     

Application of a Reversible Immortalization System for the Generation of Proliferation-Controlled Cell Lines

To employ physiological mechanisms to control cell growth primary cells were reversibly immortalized using the SV40 TAg. The cells showed a fibroblast-like morphology. When the expression of the TAg was turned off, the cells arrested in the G0/G1 cell cycle phase. The cell culture could be kept for over 1 week in the proliferation-controlled state while the growth arrest remained fully reversible. The regulation was highly efficacious in that the arrested cell population did not spontaneously resume growth, suggesting that in the absence of the immortalizing gene expression endogenous growth-control mechanisms can keep these cells in a viable state for a prolonged time. Recombinant protein expression increased in growth-controlled cells when compared to conventionally cultured cells. Analysis of a secreted pharmaceutical protein revealed high product integrity without any signs of degradation. Therefore, it is feasible to apply genetic regulation of cell immortalization to obtain proliferation-controlled cell lines and this technique may be of interest to generate novel biotechnological producer cells.     

An assessment of emerging molecular farming activities based on patent analysis (2002∼2006)

The products of Plant Molecular Farming are recombinant proteins or their metabolic products. In this study, patent data was employed to assess industrial trend in the research and innovation process of Plant Molecular Farming within national and international context. The US Patent and Trade Organization (USPTO), the European Patent Office (EPO) issued a total of 585 patents covering Plant Molecular Farming from 2002 through 2006. By nationality, US inventors predominated as recipients of PMF patents, followed by Germany, Denmark, and Japan. The PMF patents were catagorized in five major areas of research namely pharmaceutical and nutraceuticals with 170 patents (31%) and plant expression tools and methods for alternative production systems with 169 patents (29%) were the dominating patent applications, followed by 102 patent claims associated with antibodies (17%), 71 patents of industrial molecules (12%), 48 patents of vaccines (8%), and finally 18 patents related to post-translational protein glycosylation (3%). The greatest proportion of patentees was of US origin (52%), and PMF associated patenting activities at the USPTO and EPO were dominated with 67% by private organizations. Disclaimer: The views expressed in this study do not necessarily reflect those of the European Commission.