Transferrin receptor-1 suppresses neurite outgrowth in neuroblastoma Neuro2A cells

Transferrin receptor-1 (TfR1) is a cell membrane-associated glycoprotein responsible for incorporation of the iron bound to transferrin through an endocytotic process from the circulating blood. Iron is believed to play a dual role as an active center of the electron transfer system in mitochondria and as an endogenous cytotoxin through promoted generation of reactive oxygen species in different eukaryotic cells. In this study, we evaluated expression profiles of different genes related to iron mobilization across plasma membranes in neuronal cells. Marked mRNA expression was seen for various iron-related genes such as TfR1 in cultured mouse neocortical neurons, while TfR1 mRNA levels were more than doubled during culture from 3 to 6days. In mouse embryonal carcinoma P19 cells endowed to differentiate into neuronal and astroglial lineages, a transient increase was seen in both mRNA and corresponding protein for TfR1 in association with neuronal marker expression during culture with all-trans retinoic acid (ATRA). In neuronal Neuro2A cells cultured with ATRA, moreover, neurite was elongated together with increased expression of both mRNA and protein for TfR1. Overexpression of TfR1 significantly decreased the length of neurite elongated, however, while significant promotion was invariably seen in the neurite elongation in Neuro2A cells transfected with TfR1 siRNA as well as in Neuro2A cells cultured with an iron chelator. These results suggest that TfR1 would be highly expressed by neurons rather than astroglia to play a negative role in the neurite outgrowth after the incorporation of circulating transferrin in the brain.     

Effects of hypoxic preconditioning on the expression of iron influx and efflux proteins in primary neuron culture

The mechanisms of neuroprotection induced by hypoxic preconditioning (HP) and the effects of HP on iron metabolism proteins in the brain have not been fully elucidated. Based on the accumulated information, we hypothesized that HP would be able to affect the expression of iron metabolism proteins in the brain and that the changes in the expression of these proteins induced by HP might be partly associated with the HP-induced neuroprotection. Here, we demonstrated for the first time that HP could induce a significant increase in the expression of HIF-1alpha as well as iron uptake (TfR1 and DMT1) and release (Fpn1) proteins and thus increase transferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI) uptake and iron release, and also a progressive increase in cellular iron content in the cultured neurons. We concluded that HP has the ability to speed iron transport rate and proposed that the increase in iron transport rate and cellular iron in neurons might be one of the mechanisms involved in neuroprotection in the HP neurons. We also demonstrated that Fpn1 expression was significantly affected by HIF-1alpha, implying that the gene encoding this iron efflux protein is hypoxia-inducible.     

Chiroptical Spectra of Tetrakis (+)‐3‐Heptafluorobutylrylcamphorate Ln(III) Complexes with an Encapsulated Alkali Metal Ion: Solution Structures as Revealed by Chiroptical Spectra

The preparation of tetrakis((+)‐hfbc) lanthanide(III) complexes with an encapsulated alkali metal and ammonium ions M[Ln((+)‐hfbc)₄] (hereafter abbreviated as M‐Ln : (+)‐hfbc, (+)‐heptafluorobutyrylcamphorate; M, ammonium or benzyl ammonium ions as well as alkali metal ions) was reported and discussed. The electronic circular dichroism (CD) spectra in the intraligand π?π* transition of M–Ln were examined in view of the solvent effect. Here, the concentration, alkali metal, and ammonium ion dependences are compared with the solid CD, ⁵D₀ ← ⁷F₀(Eu(III)) excitation spectra, circularly polarized luminescence, and vibrational circular dichroism. It has been revealed that the dodecahedral eight coordinate DD‐8‐M‐Ln complexes in crystals are equilibrated between the diastereoselectively formed square antiprism eight coordinate SAPR‐8‐M‐Ln and [Ln((+)‐hfbc)₃] in EtOH and CH₃CN solutions or between the SAPR‐8‐M‐Ln and DD‐D_2d(mmmm)‐8‐M‐Ln complexes in CHCl₃ solution. The observed CD couplets are found to reflect the exciton CD couplets which are useful to determine the four‐bladed SAPR‐(llll) absolute configuration around the lanthanide(III) ion. Chirality 24:1055–1062, 2012. © 2012 Wiley Periodicals, Inc.     

A "Link-Psi" strategy using crosslinking indicates that the folding transition state of ubiquitin is not very malleable

Using a combined crosslinking-ψ analysis strategy, we examine whether the structural content of the transition state of ubiquitin can be altered. A synthetic dichloroacetone crosslink is first introduced across two β strands. Whether the structural content in the transition state ensemble has shifted towards the region containing the crosslink is probed by remeasuring the ψ value at another region (ψ identifies the degree to which an inserted bi-Histidine metal ion binding site is formed in the transition state). For sites around the periphery of the obligate transition state nucleus, we find that the resulting changes in ψ values are near or at our detection limit, thereby indicating that the structural content of the transition state has not measurably changed upon crosslinking. This work demonstrates the utility of the simultaneous application of crosslinking and ψ-analysis for examining potential transition state heterogeneity in globular proteins.     

铅胁迫对斜纹夜蛾生长发育与生殖的影响

在植食性昆虫斜纹夜蛾幼虫标准人工饲料中添加不同浓度的重金属铅(Pb),研究Pb胁迫对其生长发育与生殖的影响.结果表明:斜纹夜蛾不同发育阶段(幼虫、蛹和成虫)的存活率和体质量随着饲料中Pb浓度的增加而下降,引起存活率显著下降的最低Pb胁迫浓度是100 mg·kg-1,引起体质量显著减少的最低Pb胁迫浓度是50 mg·kg-1.在取食Pb浓度为25～ 200 mg·kg-1的人工饲料后,斜纹夜蛾成虫的产卵天数显著减少;产卵力和生育力随着饲料中Pb浓度的增加而显著下降;1000粒卵的平均质量显著低于对照;卵孵化率显著下降.重金属Pb胁迫对斜纹夜蛾生长发育和生殖具有显著的抑制作用.     

Inactivation of herpes simplex type 1 gene vector on immobilized metal affinity chromatography: oxidative damage by hydroxyl free radicals and its prevention

Metal catalyzed oxidation (MCO), which typically involves oxygen free radical generation, is an important pathway that leads to the deterioration of many biological molecules in solution. The occurrence of MCO in immobilized metal affinity chromatography (IMAC) systems and its potential for inactivating biological products has not been well recognized. In this study, we report the inactivation of herpes simplex virus type 1 (HSV-1) gene therapy vector on immobilized cobalt affinity chromatography. We observed that purification of KgBHAT, an HSV-1 mutant bearing cobalt affinity tags (HAT) on the surface, on an IDA-Co2+ column using crude supernatant as starting material resulted in signification loss in virus infectivity (<5% recovery). Electron spin resonance (ESR) revealed that the virus inactivation was caused by hydroxyl free radicals generated from the interactions between cellular impurities and the metal ions on the column. Inclusion of 20 mM ascorbate, a free radical scavenger, in the chromatography mobile phase effectively scavenged the hydroxyl radicals and dramatically augmented the infectivity recovery to 70%. This finding is the first demonstration of oxygen free radical-mediated biological inactivation in an actual IMAC purification and the way on how to effectively prevent it.     

Engineering TCE-degrading rhizobacteria for heavy metal accumulation and enhanced TCE degradation

Many superfund sites are currently co-contaminated with organic pollutants such as trichloroethene (TCE) and heavy metals. A promising strategy to address these mixed-waste situations is the use of TCE-degrading rhizobacteria that will survive and thrive in soil heavily polluted with heavy metals. In this work, a gene coding for the metal-binding peptide, EC20, was introduced into rhizobacteria engineered for TCE degradation, resulting in strains with both metal accumulation and TCE degradation capabilities. EC20 was displayed onto the cell surface of Pseudomonas strain Pb2-1 and Rhizobium strain 10320D using an ice-nucleation protein (INP) anchor. Expression of EC20 was confirmed by Western blot analysis and cells with EC20 expression showed sixfold higher cadmium accumulation than non-engineered strains in the presence of 16 microM CdCl(2). As expected, the TCE degradation rate was reduced in the presence of cadmium for cells without EC20 expression. However, expression of EC20 (higher cadmium accumulation) completely restored the level of TCE degradation. These results demonstrated that EC20 expression enhanced not only cadmium accumulation but also reduced the toxic effect of cadmium on TCE degradation. We expect that similar improvements will be observed when these engineered rhizobacteria are inoculated onto plant roots.     

Isolation of putative type 2 metallothionein encoding sequences and spatial expression pattern in the seagrass Posidonia oceanica

Metallothioneins (MTs) are a group of proteins with low molecular mass and high cysteine content that bind to heavy metals and are thought to play a role in their metabolism and detoxification. Genes encoding MT-like proteins have been isolated in a number of plants. In this work we isolated nine MT-like sequences from copper- or cadmium-exposed plants of the seagrass Posidonia oceanica, a marine Angiosperm playing a major role in maintaining infralittoral ecosystems in the Mediterranean sea. These sequences, together with two other MT genes previously isolated from this species, show high similarities with genes encoding type 2 MTs. Neighbour-joining analysis, at both deduced protein and 3′-UTR sequence level, indicates that at least two subgroups occur within Posidonia type 2 MTs, showing, however, a strong sequence uniformity. Southern analysis of two type 2 MT-encoding sequences (Pomt2b and Pomt2f) belonging to the two different subgroups showed distinct hybridisation patterns. For both type 2 MTs, we have determined, by in situ technique, the expression domain in Posidonia plants. The members of these two MT subgroups show differences in their histological expression, with Pomt2b associated with proliferative tissues whereas Pomt2f is associated with lignified or suberized cell wall.     

The nitrate/nitrite ABC transporter of Phormidium laminosum: Phosphorylation state of NrtA is not involved in its substrate binding activity

Most cyanobacteria take up nitrate or nitrite through a multisubunit ABC transporter (ATP-binding cassette) located in the cytoplasmic membrane. Nitrate and nitrite transport activity is instantaneously blocked by the presence of ammonium in the medium. Previous biochemical studies reported the existence of phosphorylation/dephosphorylation events of the nitrate transporter (NRT) related to the presence of ammonium-sensitive kinase/phosphatase activities in plasma membranes of the cyanobacterium Synechococcus elongatus PCC 6301. In this work, we have analyzed the biochemical properties of the periplasmic nitrate/nitrite-binding subunit (NrtA) of NRT from the thermophilic nondiazotrophic cyanobacterium Phormidium laminosum. Our results show that cyanobacterial NrtA is phosphorylated in vivo. However, substrate binding activity in vitro is not affected by the phosphorylation state of the protein, ruling out the possibility that phosphorylation/dephosphorylation of NrtA is involved in the regulation of the nitrate/nitrite uptake by NRT transporter. Moreover, NrtA is present as multiple isoforms showing the same molecular mass but different isoelectric points ranging from pI 5 to 6. Mass spectrometric characterization of NrtA isoforms shows that the protein is phosphorylated at residue Tyr²⁰³, and contains several methionine sulphoxide residues which account for the observed isoforms. Both phosphorylated and non-phosphorylated forms of NrtA are active in vitro, showing comparable binding affinity for nitrate and nitrite. Both substrates behave as pure competitive inhibitors with a binding stoichiometry of one molecule of anion per NrtA monomer.     

Induction of catalytic activity of plasminogen by monoclonal antibody IV-Ic in the presence of divalent metal cations and α2-antiplasmin

Investigation of the influence of divalent metal cations on the induction of plasminogen catalytic activity by monoclonal antibody IV-Ic showed that the presence of metal cations in the reaction medium changes the induction by slowing down or accelerating the process. Ions of Zn²⁺, Mn²⁺, and Cu²⁺ completely inhibit activation. Ions of Co²⁺ and Ni²⁺ decrease the rate of the first and second phases of the reaction more than 2 times. Ca²⁺ ions do not have any effect on the activation rate. Ions of Mg²⁺, Ba²⁺, and Sr²⁺ increase the rate of the first phase of the reaction by 1.5, 2.0, and 2.0 times and the rate of the second phase by 2.0, 3.8, and 4.7 times, correspondingly. Sr²⁺ ions have the strongest stimulating effect on plasminogen activation by monoclonal antibody IV-Ic. Investigation of the dose dependent effect of Sr²⁺ on the rate of plasminogen activation by monoclonal antibody IV-Ic showed stimulating effect of Sr²⁺ at concentrations from 0.1 to 1.0 mM with half maximum at 0.6 mM. However, Sr²⁺ ions do not affect amidolytic activity of plasmin and activation of plasminogen by streptokinase. Sr²⁺ ions also do not affect monoclonal antibody IV-Ic binding to plasminogen. The effect of Sr²⁺ is specific and mediated by the IV-Ic component. The presence of metal cations affects conformational changes in the process of active site formation. Metal cations also affect structure of the plasminogen molecule active site in the complex with monoclonal antibody IV-Ic and enzyme-substrate interaction. The effect of α₂-antiplasmin on the induction of plasminogen catalytic activity by monoclonal antibody IV-Ic in range of concentrations from 5 to 30 nM has been studied. α₂-Antiplasmin at concentration 30 nM almost completely inhibits induction of plasminogen catalytic activity by monoclonal antibody IV-Ic at the ratio plasminogen/α₂-antiplasmin of 3:1. This can be explained by competition of α₂-antiplasmin and monoclonal antibody IV-Ic for the lysine-binding sites of plasminogen and inhibition of the active center in activated complex plasminogen*—mAB IV-Ic. Divalent metal cations and α₂-antiplasmin are important factors in induction of plasminogen catalytic activity by monoclonal antibody IV-Ic. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 6, pp. 778–785.