Cardiolipin modulates allosterically peroxynitrite detoxification by horse heart cytochrome c

Upon interaction with bovine heart cardiolipin (CL), horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential out of the range required for its physiological role, binds CO and NO with high affinity, and displays peroxidase activity. Here, the effect of CL on peroxynitrite isomerization by ferric cytc (cytc-Fe(III)) is reported. In the absence of CL, hexa-coordinated cytc does not catalyze peroxynitrite isomerization. In contrast, CL facilitates cytc-Fe(III)-mediated isomerization of peroxynitrite in a dose-dependent fashion inducing the penta-coordination of the heme-Fe(III)-atom. The value of the second order rate constant for CL-cytc-Fe(III)-mediated isomerization of peroxynitrite (k_on) is (3.2 ± 0.4) × 10⁵ M⁻¹ s⁻¹. The apparent dissociation equilibrium constant for CL binding to cytc-Fe(III) is (5.1 ± 0.8) × 10⁻⁵ M. These results suggest that CL-cytc could play either pro-apoptotic or anti-apoptotic effects facilitating lipid peroxidation and scavenging of reactive nitrogen species, such as peroxynitrite, respectively.     

Metabolic response in roots of Prunus rootstocks submitted to iron chlorosis

Iron deficiency induces several responses to iron shortage in plants. Metabolic changes occur to sustain the increased iron uptake capacity of Fe-deficient plants. We evaluated the metabolic changes of three Prunus rootstocks submitted to iron chlorosis and their different responses for tolerance using measurements of metabolites and enzymatic activities. The more tolerant rootstocks Adesoto (Prunus insititia) and GF 677 (Prunus amygdalus × Prunus persica), and the more sensitive Barrier (P. persica × Prunus davidiana) were grown hydroponically in iron-sufficient and -deficient conditions over two weeks. Sugar, organic and amino acid concentrations of root tips were determined after two weeks of iron shortage by proton nuclear magnetic resonance spectroscopy of extracts. Complementary analyses of organic acids were performed by liquid chromatography coupled to mass spectrometry. The major soluble sugars found were glucose and sucrose. The major organic acids were malic and citric acids, and the major amino acid was asparagine. Iron deficiency increased root sucrose, total organic and amino acid concentrations and phosphoenolpyruvate carboxylase activity. After two weeks of iron deficiency, the malic, citric and succinic acid concentrations increased in the three rootstocks, although no significant differences were found among genotypes with different tolerance to iron chlorosis. The tolerant rootstock Adesoto showed higher total organic and amino acid concentrations. In contrast, the susceptible rootstock Barrier showed lower total amino acid concentration and phosphoenolpyruvate carboxylase activity values. These results suggest that the induction of this enzyme activity under iron deficiency, as previously shown in herbaceous plants, indicates the tolerance level of rootstocks to iron chlorosis. The analysis of other metabolic parameters, such as organic and amino acid concentrations, provides complementary information for selection of genotypes tolerant to iron chlorosis.     

Investigation and circumvention of transfection inhibition by ferric ammonium citrate in serum-free media for Chinese hamster ovary cells

While reliable transfection methods are essential for Chinese hamster ovary (CHO) cell line engineering, reduced transfection efficiencies have been observed in several commercially prepared media. In this study, we aimed to assess common media additives that impede efficiency mediated by three chemical transfection agents: liposomal-based (Lipofectamine 2000), polymer-based (TransIT-X2), and lipopolyplex-based (TransIT-PRO). An in-house GFP-expressing vector and serum-free medium (BCR-F12: developed for the purposes of this study) were used to analyze transient transfection efficiencies of three CHO cell lines (CHO-K1, DG44, DP12). Compared to a selection of commercially available media, BCR-F12 displayed challenges associated with transfection in vendor-prepared formulations, with no detection when liposomal-based methods were used, reduced (<3%) efficiency observed when polymer-based methods were used and only limited efficiency (25%) with lipopolyplexes. Following a stepwise removal protocol, ferric ammonium citrate (FAC) was identified as the critical factor impeding transfection, with transfection enabled with the liposomal- and polymer-based methods and a 1.3- to 7-fold increased lipopolyplex efficiency observed in all cell lines in FAC-depleted media (−FAC), although lower viabilities were observed. Subsequent early addition of FAC (0.5–5 hr post-transfection) revealed 0.5 hr post-transfection as the optimal time to supplement in order to achieve transfection efficiencies similar to −FAC medium while retaining optimal cellular viabilities. In conclusion, FAC was observed to interfere with DNA transfection acting at early stages in all transfection agents and all cell lines studied, and a practical strategy to circumvent this problem is suggested.     

Sorption of Cadmium and Lead by Bacteria–Ferrihydrite Composites

The sorptive behavior of bacteria—iron oxide composites was investigated in batch metal sorption assays using ferrihydrite in isolation (0.13 and 0.14 g/L ferrihydrite in cadmium and lead systems, respectively) as well as in combination with Bacillus subtilis (0.25 g/L adsorbent mixture) and Escherichia coli (0.27 g/L adsorbent mixture). A pH range from 3.0 to 6.5 was studied using total metal concentrations of 1.0 × 10 ^{? 4.0} and 3.2 × 10 ^{? 5} M with adsorbent mixtures proportioned on a 1:1 mass/volume basis. The log of the apparent surface complex formation constants (log K ^S _M ) and sorption capacity (S _max ) values were determined by fitting the experimental data to one-site Langmuir sorption isotherms. The one-site model effectively described the sorption data (r ² > 0.9), where Cd ^{2 +} exhibited somewhat lower sorption affinities (log K ^S _M = ?3 for ferrihydrite, ?1.7 for B. subtilis–ferrihydrite, and ?1.1 for E. coli–ferrihydrite) than Pb ^{2 +} (log K ^S _M = ?0.9 for ferrihydrite, ? 0.2 forB. subtilis–ferrihydrite, and –0.1 for E. coli–ferrihydrite). The corresponding S _max values for Cd ^{2 +} and Pb ^{2 +} on ferrihydrite were 0.78 mmole/g and 1.34 mmole/g, respectively. For the B. subtilis–ferrihydrite composites, Cd ^{2 +} and Pb ^{2 +} S _max values were lower at 0.29 mmole/g and 0.5 mmole/g, respectively. Similar values were determined for the E. coli–ferrihydrite composites (0.15 mmole/g and 0.68 mmole/g for Cd ^{2 +} and Pb ^{2 +} , respectively). The sorption of Cd ^{2 +} and Pb ^{2 +} by each of the sorbent systems exhibited a strong dependence on pH with sorption edges in the range of pH 4.0 to 7.3. The observed S _max of the composites were lower than values predicted upon available site additivity (Cd ^{2 +} _{B. subtilis ?ferrihydrite} : 0.29 mmole/g (observed) < 0.57 mmole/g (calculated); Cd ^{2 +} _{E. coli ?ferrihydrite} : 0.15 mmole/g (observed) < 0.44 mmole/ g (calculated); Pb ^{2 +} _{B. subtilis ?ferrihydrite} : 0.5 mmole/g (observed) < 0.805 mmole/g (calculated); Pb ^{2 +} _{E. coli –ferrihydrite} : 0.68 mmole/g (observed) < 0.775 mmole/g (calculated)), implying that a masking of reactive surface sites by attachment had occurred between the bacteria and ferrihydrite. Electrophoretic mobility analysis indicated that the ferrihydrite surface properties dominate the net surface charge for each composite system with lesser contributions from the bacteria.     

Manganese inhibition of microbial iron reduction in anaerobic sediments

Potential mechanisms for the lack of Fe(II) accumulation in Mn(IV)‐con‐taining anaerobic sediments were investigated. The addition of Mn(IV) to sediments in which Fe(III) reduction was the terminal electron‐accepting process removed all the pore‐water Fe(II), completely inhibited net Fe(III) reduction, and stimulated Mn(IV) reduction. In a solution buffered at pH 7, Mn(IV) oxidized Fe(II) to amorphic Fe(III) oxide. Mn(IV) naturally present in oxic freshwater sediments also rapidly oxidized Fe(II). A pure culture of a dissimilatory FE(III)‐ and Mn(FV)‐reducing organism isolated from the sediments reduced Fe(III) to Fe(II) in the presence of Mn(IV) when ferrozine was present to trap Fe(II) before Mn(IV) oxidized it. Depth profiles of dissolved iron and manganese reported in previous studies suggest that Fe(II) diffusing up from the zone of Fe(III) reduction is consumed within the Mn(IV)‐reducing zone. These results demonstrate that preferential reduction of Mn(IV) by Fe(III)‐reducing bacteria cannot completely explain the lack of Fe(II) accumulation in anaerobic, Mn(IV)‐containing sedments, and indicate that Mn(IV) oxidation of Fe(II) is the mechanism that ultimately prevents Fe(II) accumulation.     

Antioxidative property of T-0970, a new ureidophenol derivative

We investigated the antioxidative property of T-0970, a newly synthesized ureidophenol derivative. The inhibitory effect of T-0970 on spontaneous lipid peroxidation in rat brain was 10 times greater than those of well-known antioxidants such as butylhydroxytoluene (BHT), probucol and α-tocopherol. T-0970 also showed dose-dependent free radical scavenging activities in vitro for both superoxide anions and hydroxyl radicals. The radical-scavenging potencies of T-0970 were about 10–30 times stronger than those of BHT. We evaluated the in vivo antioxidative ability of T-0970 in the animal model of acute oxidative tissue injury in rats. Intraperitoneal injection of ferric nitrilotriacetate (Fe/NTA) caused an acute and remarkable increase in the level of thiobarbituric acid-reactive substances (TBARS) in both plasma and the liver, and also resulted in a considerable elevation of the plasma levels of GOT and GPT indicative of hepatic injury. Both oral and intravenous administration of T-0970 dose-dependently depressed these diagnostic parameters. These results indicate that T-0970 may have a therapeutic potential in various diseases associated with oxidative tissue injury.     

Effects of Schisandrin B and α-tocopherol on lipid peroxidation, in vitro and in vivo

Effects of Schisandrin B (Sch B) and -tocopherol (-TOC) on ferric chloride (Fe³⁺) induced oxidation of erythrocyte membrane lipids in vitro and carbon tetrachloride (CCl₄) induced lipid peroxidation in vivo were examined. While -TOC could produce prooxidant and antioxidant effect on Fe³⁺-induced lipid peroxidation, Sch B only inhibited the peroxidation reaction. Pretreatment with -TOC (3 mmol/kg/day × 3) did not protect against CCl₄-induced lipid peroxidation and hepatocellular damage in mice, whereas Sch B pretreatment (0.3 mmol/3.0 mmol/kg/day × 3) produced a dose-dependent protective effect on the CCl₄-induced hepatotoxicity. The ensemble of results suggests that the ability of Sch B to inhibit lipid peroxidation, while in the absence of pro-oxidant activity, may at least in part contribute to its hepatoprotective action.Abbreviations ALT alanine aminotransferase - CCl₄ carbon tetrachloride - Fe³⁺ ferric chloride - MDA malondialdehyde - Sch B Schisandrin B - TBA 2-thiobarbituric acid - TBARS thiobarbituric acid reactive substances - -TOC dl--tocopherol     

表达空肠弯曲菌CfrA蛋白重组乳酸乳球菌的构建及其免疫效果北大核心CSCD

【目的】本试验将空肠弯曲菌肠菌素受体蛋白CfrA编码基因导入食品级乳酸乳球菌表达系统,然后将重组乳酸乳球菌口服免疫鸡,降低空肠弯曲菌在鸡肠道中的定殖。【方法】利用PCR分别扩增空肠弯曲菌cfrA全基因及其N端片段,插入食品级表达载体pNZ8149多克隆位点并转化乳酸乳球菌NZ3900,通过Western blot鉴定重组菌株CfrA蛋白表达情况,同时通过筛选nisin浓度、温度、时间等诱导条件优化重组蛋白表达水平;进而将重组乳酸乳球菌经口服免疫SPF鸡,免疫后分别测定乳酸乳球菌自鸡体内的排出情况、以及诱导CfrA血清抗体和粘膜抗体水平,最后将空肠弯曲菌口服攻毒免疫后的鸡,通过测定鸡泄殖腔棉拭子中空肠弯曲菌的数目来判定口服免疫效果。【结果】Western blot检测显示CfrA全基因及其N端片段均可在重组乳酸乳球菌胞内可溶性表达,不分泌,筛选的最佳诱导表达条件为nisin浓度25 ng/mL、温度37°C、时间1 h。口服乳酸乳球菌10 d内自鸡体完全排空;鸡口服免疫后可产生CfrA蛋白特异性的血清IgG和肠粘膜sIgA抗体;重组乳酸乳球菌口服免疫后空肠弯曲菌在鸡体内的增殖速度显著低于对照组。【结论】成功构建了重组CfrA蛋白的食品级乳酸乳球菌诱导表达系统;表达CfrA蛋白的重组乳酸乳球菌口服免疫鸡对空肠弯曲菌在鸡肠道的定殖具有一定的抑制作用,为研制重组乳酸菌口服家禽免疫制剂防治空肠弯曲菌奠定了基础。     

Intraspecific responses of medicinal plants: Genotype-environment interaction may alter drug quality of aromatic plants

An open field experiment was established in 2014 in Budapest (BP) and Poznań (PZ), which are characterized by different soil and weather conditions. The purpose was to study the intraspecific responses of Melissa officinalis L. (lemon balm -Lb) and Thymus vulgaris (thyme -T) to varying locations. Five accessions of lemon balm (‘Lorelei’, ‘Lemona’, ‘Soroksár’, ‘Quedlinburger Niederliegende’, ‘Gold Leaf’) and four of thyme (‘Sloneczko’, French Summer’, ‘Varico 3′, ‘Standard Winter’) were tested for yield and chemical characteristics.Biomass of both species was significantly higher in PZ, than in BP (291 and 107 g/plant for Lb and 105 and 53 g/plant for T in PZ and BP, respectively). In Lb, the environmental circumstances of the actual vegetation period of BP location were more favourable for the accumulation of volatiles (+76%), while the level of phenolic compounds was higher in PZ (up to +53% for rosmarinic acid). In T, each chemical parameter increased (up to +27% both for volatiles and total flavonoid content) in PZ, where lower temperatures and less precipitation prevailed.In most traits a significant interaction of genotype and growing location was established, which was more characteristic in T. The results show that breeding of specific cultivars for a given location might be of primary importance.     

FurA modulates gene expression of alr3808, a DpsA homologue in Nostoc (Anabaena) sp. PCC7120

The DNA-binding protein from stationary phase (Dps) protein family plays an important role in protecting microorganisms from oxidative and nutritional stresses. In silico analysis of the promoter region of alr3808, a dpsA homologue from the cyanobacterium Nostoc sp. PCC7120 shows putative iron-boxes with high homology with those recognized by FurA (ferric uptake regulator). Evidence for the modulation of dpsA by FurA was obtained using in vitro and in vivo approaches. SELEX linked to PCR was used to identify P_dpsA as a FurA target. Concurrently, EMSA assays showed high affinity of FurA for the dpsA promoter region. DpsA expression analysis in an insertional mutant of the alr1690-αfurA message (that exhibited an increased expression of FurA) showed a reduced synthesis of DpsA. These studies suggest that FurA plays a significant role in the regulation of the DpsA.