Can uranium follow the iron-acquisition pathway? Interaction of uranyl-loaded transferrin with receptor 1

Transferrin receptor 1 (R_D) binds iron-loaded transferrin and allows its internalization in the cytoplasm. Human serum transferrin also forms complexes with metals other than iron, including uranium in the uranyl form (UO₂ ²⁺). Can the uranyl-saturated transferrin (TUr₂) follow the receptor-mediated iron-acquisition pathway? In cell-free assays, TUr₂ interacts with R_D in two different steps. The first is fast, direct rate constant, k ₁ = (5.2 ± 0.8) × 10⁶ M^?1 s^?1; reverse rate constant, k _?1 = 95 ± 5 s^?1; and dissociation constant K ₁ = 18 ± 6 μM. The second occurs in the 100-s range and leads to an increase in the stability of the protein–protein adduct, with an average overall dissociation constant K _d = 6 ± 2 μM. This kinetic analysis implies in the proposed in vitro model possible but weak competition between TUr₂ and the C-lobe of iron-loaded transferrin toward the interaction with R _D.     

Potential therapeutic radiotracers: preparation, biodistribution and metabolic characteristics of 177Lu-labeled cyclic RGDfK dimer

In this study, we reported the preparation and evaluation of ¹⁷⁷Lu-DOTA-RGD2, ¹⁷⁷Lu-DOTA-Bz-RGD2 and ¹⁷⁷Lu-DTPA-Bz-RGD2 (RGD2 = E[c(RGDfK)]₂) as a potential therapeutic radiotracers for the treatment of integrin α_vβ₃-positive tumors. The BALB/c nude mice bearing the U87MG human glioma xenografts were used to evaluate the biodistribution characteristics and excretion kinetics of ¹⁷⁷Lu-DOTA-RGD2, ¹⁷⁷Lu-DOTA-Bz-RGD2 and ¹⁷⁷Lu-DTPA-Bz-RGD2. It was found that there were no major differences in their lipophilicity and biodistribution characteristics, particularly at latter time points. A major advantage of using DTPA-Bz as the bifunctional chelator (BFC) was its high radiolabeling efficiency (fast and high yield radiolabeling) at room temperature. Using DOTA and DOTA-Bz as BFCs, the radiolabeling kinetics was slow, and heating at 100°C and higher DOTA-conjugate concentration were needed for successful ¹⁷⁷Lu-labeling. Therefore, DTPA-Bz is an optimal BFC for routine preparation of ¹⁷⁷Lu-labeled cyclic RGDfK peptides, and ¹⁷⁷Lu-DTPA-Bz-RGD2 is worthy of further investigation for targeted radiotherapy of integrin α_vβ₃-positive tumors.     

The induction of apoptosis in SGC-7901 cells through the ROS-mediated mitochondrial dysfunction pathway by a Ir(III) complex

A new ligand BTCP and its iridium(III) complex [Ir(ppy)₂(BTCP)]PF₆ (Ir-1) were synthesized and characterized by elemental analysis, ESI–MS, IR, ¹H NMR and ¹³C NMR. The cytotoxic activity in vitro of the ligand and its complex against SGC-7901, HeLa, HOS, PC-12, BEL-7402, MG-63, SiHa, A549, HepG2 and normal cell LO2 were evaluated by MTT method [MTT = (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)]. The apoptosis was assayed with AO/EB and Hoechst 33258 staining methods. The reactive oxygen species (ROS), mitochondrial membrane potential, autophagy and cell invasion were studied under fluorescent microscope. The expression of caspases and Bcl-2 family proteins were investigated by western blot. The IC₅₀ values of complex toward SGC-7901, BEL-7402 and MG-63 cells are 3.9 ± 0.5, 5.4 ± 1.2 and 4.2 ± 0.6 µM. The complex can increase the levels of ROS, and induce a decrease in the mitochondrial membrane potential. Ir-1 inhibits the cell growth at G0/G1 phase in SGC-7901 cells, and the complex can induce both autophagy and apoptosis and inhibit the cell invasion. And the complex induces apoptosis through a ROS-mediated mitochondrial dysfunction pathway.     

Subcellular Localization and Kinetic Characterization of a Gill (Na+, K+)-ATPase from the Giant Freshwater Prawn Macrobrachium rosenbergii

The stimulation by Mg²⁺, Na⁺, K⁺, NH₄ ⁺, and ATP of (Na⁺, K⁺)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na⁺, K⁺)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M _r. Under saturating Mg²⁺, Na⁺, and K⁺ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V _M = 115.0 ± 2.3 U mg^?1, K _0.5 = 0.10 ± 0.01 mmol L^?1. Stimulation by Na⁺ (V _M = 110.0 ± 3.3 U mg^?1, K _0.5 = 1.30 ± 0.03 mmol L^?1), Mg²⁺ (V _M = 115.0 ± 4.6 U mg^?1, K _0.5 = 0.96 ± 0.03 mmol L^?1), NH₄ ⁺ (V _M = 141.0 ± 5.6 U mg^?1, K _0.5 = 1.90 ± 0.04 mmol L^?1), and K⁺ (V _M = 120.0 ± 2.4 U mg^?1, K _M = 2.74 ± 0.08 mmol L^?1) followed single saturation curves and, except for K⁺, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K _I = 12.4 ± 1.3 mol L^?1. Complementary inhibition studies suggest the presence of F₀F₁–, Na⁺-, or K⁺-ATPases, but not V(H⁺)- or Ca²⁺-ATPases, in the gill microsomal preparation. K⁺ and NH₄ ⁺ synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH₄ ⁺, and K⁺ of the gill enzyme.     

Desferrioxamine-caffeine (DFCAF) as a cell permeant moderator of the oxidative stress caused by iron overload

Desferrioxamine (DFO) is a potent iron chelator used in the treatment of iron overload (IO) disorders. However, due to its low cell permeability and fast clearance, DFO administration is usually prolonged and of limited use for the treatment of IO in tissues such as the brain. Caffeine is a safe, rapidly absorbable molecule that can be linked to other compounds to improve their cell permeability. In this work, we successfully prepared and described DFO-caffeine, a conjugate with iron scavenging ability, antioxidant properties and enhanced permeation in the HeLa cell model.     

Cloning,expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP⁺ (k _cat/K _m = 2618 mM^?1 s^?1, k _cat = 249 s^?1, K _m = 0.10 ± 0.01 mM) as cofactor, although NAD⁺ (k _cat/K _m = 138 mM^?1 s^?1, k _cat = 604 s^?1, K _m = 4.37 ± 0.56 mM) could also be accepted. The K _m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP⁺ and NAD⁺ as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8–9.0 for NADP⁺ and at pH 7.0–8.6 for NAD⁺ while the optimal temperature was 80 °C for NADP⁺ and 70 °C for NAD⁺. This was the first observation that the NADP⁺-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD⁺-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.     

Rational Improvement of Peptide Affinity to Human Pregnancy-Related Serine Protease HtrA3 PDZ Domain by Introducing a Halogen Bond to the Domain–Peptide Complex Interface

The pregnancy-related serine protease HtrA3 plays an important role in human placental development and has recently been recognized as a potential therapeutic target in the treatment of cancer. Previously, a C-terminal pentapeptide FGRWV^–COOH was identified to bind at the PDZ domain of HtrA3 with a moderate affinity. Here, based on the high-resolution complex crystal structure of HtrA3 PDZ domain with the pentapeptide ligand we successfully introduced a rationally designed halogen bond to the complex interface by substituting R₄-hydrogen atom of the indole moiety of peptide Trp_-1 residue with a halogen atom. High-level theoretical calculations suggested that bromine is the best choice that can render strong interaction energy for the halogen bond and can confer high affinity to the PDZ–peptide complex. Fluorescence spectroscopy characterizations revealed that the resulting R₄-brominated peptide (K _d = 0.15 ± 0.03 μM) exhibited 12-fold affinity improvement relative to its nonhalogenated counterpart (K _d = 1.8 ± 0.4 μM). In contrast, the PDZ-binding affinity of R₆-brominated peptide (K _d = 1.2 ± 0.1 μM), a negative control that was unable to form the halogen bond according to theoretical investigations, did not change substantially as compared to the nonhalogenated peptide.     

Interaction of dimeric horse cytochrome c with cyanide ion

We have previously shown that methionine–heme iron coordination is perturbed in domain-swapped dimeric horse cytochrome c. To gain insight into the effect of methionine dissociation in dimeric cytochrome c, we investigated its interaction with cyanide ion. We found that the Soret and Q bands of oxidized dimeric cytochrome c at 406.5 and 529 nm redshift to 413 and 536 nm, respectively, on addition of 1 mM cyanide ion. The binding constant of dimeric cytochrome c and cyanide ion was obtained as 2.5 × 10⁴ M^?1. The Fe–CN and C–N stretching (ν _Fe–CN and ν _CN) resonance Raman bands of CN^?-bound dimeric cytochrome c were observed at 443 and 2,126 cm^?1, respectively. The ν _Fe–CN frequency of dimeric cytochrome c was relatively low compared with that of other CN^?-bound heme proteins, and a relatively strong coupling between the Fe–C–N bending and porphyrin vibrations was observed in the 350–450-cm^?1 region. The low ν _Fe–CN frequency suggests weaker binding of the cyanide ion to dimeric cytochrome c compared with other heme proteins possessing a distal heme cavity. Although the secondary structure of dimeric cytochrome c did not change on addition of cyanide ion according to circular dichroism measurements, the dimer dissociation rate at 45 °C increased from (8.9 ± 0.7) × 10^?6 to (3.8 ± 0.2) × 10^?5 s^?1, with a decrease of about 2 °C in its dissociation temperature obtained with differential scanning calorimetry. The results show that diatomic ligands may bind to the heme iron of dimeric cytochrome c and affect its stability.     

Biochemical and spectroscopic characterization of the catalytic domain of MMP16 (cdMMP16)

Membrane-bound matrix metalloproteinase 16 (MMP16/MT3-MMP) is considered a drug target due to its role(s) in disease processes such as cancer and inflammation. Biochemical characterization of MMP16 is critical for developing new generation MMP inhibitors (MMPi), which exhibit high efficacies and selectivities. Herein, a modified over-expression and purification protocol was used to prepare the catalytic domain of MMP16 (cdMMP16). The resulting recombinant enzyme exhibited steady-state kinetic constants of K _m = 10.6 ± 0.7 μM and k _cat = 1.14 ± 0.02 s^?1, when using FS-6 as substrate, and the enzyme bound 1.8 ± 0.1 eq of Zn(II). The enzymatic activity of cdMMP16 is salt concentration-dependent, and cdMMP16 exhibits autoproteolytic activity under certain conditions, which may be related to an in vivo regulatory mechanism of MMP16 and of other membrane-type MMPs (MT-MMPs). Co(II)-substituted analogs (Co₂- and ZnCo) of cdMMP16 were prepared and characterized using several spectroscopic techniques, such as UV–Vis, ¹H NMR, and EXAFS spectroscopies. A well-characterized cdMMP16 is now available for future inhibitor screening efforts.     

Spin concentration measurements of high-spin (g′ = 4.3) rhombic iron(III) ions in biological samples: theory and application

Electron paramagnetic resonance (EPR) signals at g′ = 4.3 are commonly encountered in biological samples owing to mononuclear high-spin (S = 5/2) Fe³⁺ ions in sites of low symmetry. The present study was undertaken to develop the experimental method and a suitable g′ = 4.3 intensity standard and for accurately quantifying the amount of Fe³⁺ responsible for such signals. By following the work of Aasa and Vänngård (J. Magn. Reson. 19:308–315, 1975), we present equations relating the EPR intensity of S = 5/2 ions to the intensities of S = 1/2 standards more commonly employed in EPR spectrometry. Of the chelates tested, Fe³⁺–EDTA (1:3 ratio) in 1:3 glycerol/water (v/v), pH 2, was found to be an excellent standard for frozen-solution S = 5/2 samples at 77 K. The spin concentrations of Cu²⁺–EDTA and aqua VO²⁺, both S = 1/2 ions, and of Fe³⁺–transferrin, an S = 5/2 ion, were measured against this standard and found to agree within 2.2% of their known metal ion concentrations. Relative standard deviations of ±3.6, ±5.3 and ±2.9% in spin concentration were obtained for the three samples, respectively. The spin concentration determined for Fe³⁺–desferrioxamine of known Fe³⁺ concentration was anomalously low suggesting the presence of EPR-silent multimeric iron species in solution.     

Effects of chelators on iron uptake and release by the brain in the rat

The iron chelators desferrioxamine (DFO), pyridoxal isonicotinoyl hydrazone (PIH), 2,2-bipyridine, diethylenetriamine penta-acetic acid (DTPA) and 1,2 dimethyl-3-hydroxy pyrid-4-one (CP20) were analysed for their ability to change⁵⁹Fe uptake and release from the brain of 15- and 63-day rats either during or after intravenous injection of⁵⁹Fe-¹²⁵I-transferrin. DTPA was the only chelator unable to significantly reduce iron uptake into the brain of 15-day rats. This indicates that iron is not released from transferrin at the luminal surface of brain capillary endothelial cells. CP20 was able to reduce iron uptake in the brain by 85% compared to 28% with DFO. Only CP20 was able to significantly reduce brain iron uptake in 63 day rats. Once⁵⁹Fe had entered the brain no chelator used was able to mediate its release. All of the chelators except CP20 had similar effects on femur iron uptake as they did on brain uptake, suggesting similar iron uptake mechanisms. It is concluded that during the passage of transferrin-bound iron into the brain the iron is released from transferrin within endothelial cells after endocytosis of transferrin.     

Diurnal variation in repeated sprint performance cannot be offset when rectal and muscle temperatures are at optimal levels (38.5°C)

The present study investigated whether increasing morning rectal temperatures (T_rec) to evening levels, or increasing morning and evening T_rec to an “optimal” level (38.5°C), resulting in increased muscle temperatures (T_m), would offset diurnal variation in repeated sprint (RS) performance in a causal manner. Twelve trained males underwent five sessions [age (mean ± SD) 21.0 ± 2.3 years, maximal oxygen consumption (V?O₂max) 60.0 ± 4.4 mL.kg^–1 min^–1, height 1.79 ± 0.06 m, body mass 78.2 ± 11.8 kg]. These included control morning (M, 07:30 h) and evening (E, 17:30 h) sessions (5-min warm-up), and three further sessions consisting of a warm-up morning trial (M_E, in 39–40°C water) until T_rec reached evening levels; two “optimal” trials in the morning and evening (M_38.5 and E_38.5, in 39–40°C water) respectively, until T_rec reached 38.5°C. All sessions included 3 × 3-s task-specific warm-up sprints, thereafter 10 × 3-s RS with 30-s recoveries were performed a non-motorised treadmill. T_rec and T_m measurements were taken at the start of the protocol and following the warm-up periods. Values for T_rec and T_m at rest were higher in the evening compared to morning values (0.48°C and 0.69°C, p < 0.0005). RS performance was lower (7.8–8.3%) in the M for distance covered (DC; p = 0.002), average power (AP; p = 0.029) and average velocity (AV; p = 0.002). Increasing T_rec in the morning to evening values or optimal values (38.5°C) did not increase RS performance to evening levels (p = 1.000). However, increasing T_rec in the evening to “optimal” level through a passive warm-up significantly reduced DC (p = 0.008), AP (p < 0.0005) and AV (p = 0.007) to values found in the M condition (6.0–6.9%). Diurnal variation in T_rec and T_m is not wholly accountable for time-of-day oscillations in RS performance on a non-motorised treadmill; the exact mechanism(s) for a causal link between central temperature and human performance are still unclear and require more research.     

Association between the rs6950982 polymorphism near the SERPINE1 gene and blood pressure and lipid parameters in a high-cardiovascular-risk population: interaction with Mediterranean diet

The SERPINE1 (serpin peptidase inhibitor, clade E, member 1) gene, better known by its previous symbol PAI-1 (plasminogen activator inhibitor 1), has been associated with cardiovascular phenotypes with differing results. Our aim was to examine the association between the rs6950982 (G > A) near the SERPINE1 gene, blood pressure (BP) and plasma lipid concentrations as well as the modulation of the polymorphism effects by adherence to Mediterranean diet (AMD). We studied 945 high-cardiovascular-risk subjects. Biochemical, clinical, dietary and genetic data (rs6950982) were obtained. We also determined the common rs1799768 (4G/5G), for checking independent effects. AMD was measured by a validated questionnaire, and four groups were considered. rs6950982 (A > G) and rs1799768 (4G/5G) were only in moderate–low linkage disequilibrium (D′ = 0.719; r ² = 0.167). The most significant associations we obtained were with rs6950982 (A > G). In males, the G allele was nominally associated with higher diastolic BP (AA: 81.5 ± 10.9, AG: 82.1 ± 11.4, GG: 85.7 ± 10.5 mmHg; P _additive = 0.030) and systolic BP (AA + AG: 141.4 ± 6.9 mmHg vs. GG: 149.8 ± 8.0 mmHg; P _recessive = 0.036). In the whole population, the rs6950982 was also associated with plasma lipids. Subject with the G allele presented higher total cholesterol (P _additive = 0.016, P _recessive = 0.011), low-density lipoprotein cholesterol (P _additive = 0.032, P _recessive = 0.031) and triglycerides (P _additive = 0.040, P _recessive = 0.029). AMD modulated the effect of rs6950982 on triglyceride concentrations (P for interaction = 0.036). Greater AMD reduced the higher triglyceride concentrations in GG subjects. No significant interactions were found for the other parameters. The rs6950982 was associated with higher BP in men and higher triglycerides in the whole population, this association being modulated by AMD.     

Sedimentation and belowground carbon accumulation rates in mangrove forests that differ in diversity and land use: a tale of two mangroves

Increased sea level is the climate change effect expected to have the greatest impact on mangrove forest survival. Mangroves have survived extreme fluctuations in sea level in the past through sedimentation and belowground carbon (C) accumulation, yet it is unclear what factors may influence these two parameters. We measured sedimentation, vertical accretion, and belowground C accumulation rates in mangrove forests from the Republic of Palau and Vietnam to examine how diversity (high-Vietnam vs. low-Palau), land use, and location (fringe vs. interior) might influence these parameters. Land use in this study was identified as disturbance and restoration for all mangrove forests sampled in Palau and Vietnam, respectively. Vertical accretion rates were significantly greater in Vietnam (2.44 ± 1.38 cm/year) than Palau mangrove forests (0.47 ± 0.08 cm/year; p < 0.001, F_1,17 = 24.96). Vertical accretion rates were positively correlated to diversity (R = 0.43, p < 0.05). However, stronger correlations of accretion to bulk density (R = 0.64, p < 0.01) and significantly higher bulk densities in Vietnamese (0.67 ± 0.04 g/cm³) than Palau mangroves (0.30 ± 0.03 g/cm³; p < 0.001, F_1,17 = 54.4) suggests that suspended sediments played a greater role in mangrove forest floor maintenance relative to sea level rise. Average vertical accretion rates were similar between naturally colonized (1.01 ± 0.10 cm/year) and outplanted sites (1.06 ± 0.05 cm/year) and between fringe (1.06 ± 0.12 cm/year) and interior mangrove (0.99 ± 0.09 cm/year) in Vietnam. In Palau, vertical accretion rates did not differ between disturbed (0.42 ± 0.11 cm/year) and undisturbed (0.51 ± 0.13 cm/year) mangrove forests and were higher in fringe (0.61 ± 0.15 cm/year) than interior sites (0.33 ± 0.09 cm/year; p = 0.1, F_1,7 = 3.45). Belowground C accumulation rates did not differ between any factors examined. C accumulation rates (69–602 gC/m²/year) were similar to those reported elsewhere in the literature and suggest that intact coastal ecosystems play an important role in the global C cycle, sequestering C at rates that are 10–20× greater than upland forests. Assuming vertical accretion rates measured using ²¹⁰Pb are an effective proxy for surface elevation, the Vietnamese and Palauan mangroves appear to be keeping up with current rates of sea level rise.     

Investigation of binding mechanism of novel 8-substituted coumarin derivatives with human serum albumin and α-1-glycoprotein

Coumarin molecules have biological activities possessing lipid-controlling activity, anti-hepatitis C activity, anti-diabetic, anti-Parkinson activity, and anti-cancer activity. Here, we have presented an inclusive study on the interaction of 8-substituted-7-hydroxy coumarin derivatives (Umb-1/Umb-2) with α-1-glycoprotein (AGP) and human serum albumin (HSA) which are the major carrier proteins in the human blood plasma. Binding constants obtained from fluorescence emission data were found to be K_Umb-1=3.1 ± .01 × 10⁴ M^?1, K_Umb-2 = 7 ± .01 × 10⁴ M^?1, which corresponds to ?6.1 and ?6.5 kcal/mol of free energy for Umb-1 and Umb-2, respectively, suggesting that these derivatives bind strongly to HSA. Also these molecules bind to AGP with binding constants of K_Umb-1-AGP=3.1 ± .01 × 10³ M^?1 and K_Umb-2-AGP = 4.6 ± .01 × 10³ M^?1. Further, the distance, r between the donor (HSA) and acceptor (Umb-1/Umb-2) was calculated based on the Forster’s theory of non-radiation energy transfer and the values were observed to be 1.14 and 1.29 nm in Umb-1–HSA and Umb-2–HSA system, respectively. The protein secondary structure of HSA was partially unfolded upon binding of Umb-1 and Umb-2. Furthermore, site displacement experiments with lidocaine, phenylbutazone (IIA), and ibuprofen (IIIA) proves that Umb derivatives significantly bind to subdomain IIIA of HSA which is further supported by docking studies. Furthermore, Umb-1 binds to LYS402 with one hydrogen bond distance of 2.8 Å and Umb-2 binds to GLU354 with one hydrogen bond at a distance of 2.0 Å. Moreover, these molecules are stabilized by hydrophobic interactions and hydrogen bond between the hydroxyl groups of carbon-3 of coumarin derivatives.     

Performance evaluation of an integrated constructed wetland used to treat a contaminated aquatic environment

The treatment performance of an integrated constructed wetland (ICW) that was in operation for 3 years was evaluated. Artificial neural network modeling was used to predict contaminant treatment efficiencies based on easily measured field parameters. The estimates for average yearly removals of total phosphorus (TP), total nitrogen (TN), chemical oxygen demand (COD), and total suspended solids (TSS) were 0.81 ± 0.18, 7.17 ± 1.62, 63.80 ± 17.41, and 126.12 ± 48.61 g m^?2 d^?1, respectively. Removal velocities of contaminants were determined from analyses of inlet–outlet datasets. The areal removal rate constants were 0.46, 0.73, 0.44, and 0.82 m d^?1 for TP, TN, COD, and TSS, respectively. The presence of high background concentrations of contaminants (TP: 0.01 mg L^?1, TN: 0.13 mg L^?1, COD: 6.43 mg L^?1, TSS: 14.83 mg L^?1) indicated that the water in the ICW was mesotrophic. Statistical methods (i.e., principal component analysis (PCA), forward selection, and correlation analysis) were used to select optimal input subsets for different contaminants. These data subsets were subsequently used for model development. To find the optimal network architectures, a genetic algorithm was introduced to the learning processes. The models were competent at providing reasonable matches between the measured and the predicted effluent concentrations of TP (R² = 0.9711), TN (R² = 0.8875), COD (R² = 0.9359), and TSS (R² = 0.9164). The results of the models provided information that will be useful for the design and modification of constructed wetlands.     

Inhibitory effects of deferasirox on the structure and function of bovine liver catalase: a spectroscopic and theoretical study

Deferasirox (DFX), as an oral chelator, is used for treatment of transfusional iron overload. In this study, we have investigated the effects of DFX as an iron chelator, on the function and structure of bovine liver catalase (BLC) by different spectroscopic methods of UV–visible, fluorescence, and circular dichroism (CD) at two temperatures of 25 and 37 °C. In vitro kinetic studies showed that DFX can inhibit the enzymatic activity in a competitive manner. K _I value was calculated 39 nM according to the Lineweaver–Burk plot indicating a high rate of inhibition of the enzyme. Intrinsic fluorescence data showed that increasing the drug concentrations leads to a significant decrease in the intrinsic emission of the enzyme indicating a significant change in the three-dimensional environment around the chromophores of the enzyme structure. By analyzing the fluorescence quenching data, it was found that the BLC has two binding sites for DFX and the values of binding constant at 25 and 37 °C were calculated 1.7 × 10⁷ and 3 × 10⁷ M^?1, respectively. The static type of quenching mechanism is involved in the quenching of intrinsic emission of enzyme. The thermodynamic data suggest that hydrophobic interactions play a major role in the binding reaction. UV–vis spectroscopy results represented the changes in tryptophan (Trp) absorption and Soret band spectra, which indicated changes in Trp and heme group position caused by the drug binding. Also, CD data represented that high concentrations of DFX lead to a significant decreasing in the content of β-sheet and random coil accompanied an increasing in α-helical content of the protein. The molecular docking results indicate that docking may be an appropriate method for prediction and confirmation of experimental results and also useful for determining the binding mechanism of proteins and drugs. According to above results, it can be concluded that the DFX can chelate the Fe(III) on the enzyme active site leading to changes in the function and structure of catalase which can be considered as a side effect of this drug and consequently has an important role in hepatic complications and fibrosis.     

RBE of thermal neutrons for induction of chromosome aberrations in human lymphocytes

The induction of chromosome aberrations in human lymphocytes irradiated in vitro with slow neutrons was examined to assess the maximum low-dose RBE (RBE_M) relative to ⁶⁰Co γ-rays. For the blood irradiations, cold neutron beam available at the prompt gamma activation analysis facility at the Munich research reactor FRM II was used. The given flux of cold neutrons can be converted into a thermally equivalent one. Since blood was taken from the same donor whose blood had been used for previous irradiation experiments using widely varying neutron energies, the greatest possible accuracy was available for such an estimation of the RBE_M avoiding the inter-individual variations or differences in methodology usually associated with inter-laboratory comparisons. The magnitude of the coefficient α of the linear dose–response relationship (α = 0.400 ± 0.018 Gy^?1) and the derived RBE_M of 36.4 ± 13.3 obtained for the production of dicentrics by thermal neutrons confirm our earlier observations of a strong decrease in α and RBE_M with decreasing neutron energy lower than 0.385 MeV (RBE_M = 94.4 ± 38.9). The magnitude of the presently estimated RBE_M of thermal neutrons is—with some restrictions—not significantly different to previously reported RBE_M values of two laboratories.     

Pectin Esterification Degree in the Bioavailability of Non-heme Iron in Women

Pectins are a type of soluble fiber present in natural and processed foods. Evidence regarding the effect of esterification degree of pectins on iron absorption in humans is scarce. In the present study, the effect of pectins with different degrees of esterification on non-heme iron absorption in women was evaluated. A controlled experimental study was conducted with block design, involving 13 apparently healthy, adult women. Each subject received 5 mg Fe (FeSO₄) without pectin (control) or accompanied by 5 g citrus pectin, two with a low degree of esterification (27 and 36%), and one with a high degree of esterification (67 to 73%), each on different days. Each day, the 5 mg Fe doses were marked with radioactive ⁵⁹Fe or ⁵⁵Fe. Radioactivity incorporated into erythrocytes was determined in blood samples 14 days after the marked Fe doses were consumed. On days 18 and 36 of study, 30 and 20 mL blood samples were obtained, respectively, and blood sample radioactivity incorporated into erythrocytes was determined. Body iron status was determined from blood taken on day 18. Whole body blood volume was estimated for calculate iron bioavailability; it was assumed that 80% of absorbed radioactivity was incorporated into the Hb. All women participants signed an informed consent of participation at baseline. Iron bioavailability (mean geometric ±1 SD) alone (control) was 18.2% (12.3–27.1%), iron + pectin27 was 17.2% (10.2–29.2%), iron + pectin36 was 15.3% (9.5–24.6%), and iron + pectin67 was 19.5% (10.0–38.0%). No statistically significant differences between iron bioavailability (repeated measures ANOVA, p = 0.22) were observed. Pectin esterification degree does not influence the bioavailability of non-heme iron in women.     

The carbohydrate-binding module of Fragaria × ananassa expansin 2 (CBM-FaExp2) binds to cell wall polysaccharides and decreases cell wall enzyme activities “in vitro”

A putative carbohydrate binding module (CBM) from strawberry (Fragaria × ananassa Duch.) expansin 2 (CBM-FaExp2) was cloned and the encoding protein was over-expressed in Escherichia coli and purified in order to evaluate its capacity to bind different cell wall polysaccharides “in vitro”. The protein CBM-FaExp2 bound to microcrystalline cellulose, xylan and pectin with different affinities (K_ad = 33.6 ± 0.44 mL g^?1, K_ad = 11.37 ± 0.87 mL g^?1, K_ad = 10.4 ± 0.19 mL g^?1, respectively). According to “in vitro” enzyme assays, this CBM is able to decrease the activity of cell wall degrading enzymes such as polygalacturonase, endo-glucanase, pectinase and xylanase, probably because the binding of CBM-FaExp2 to the different substrates interferes with enzyme activity. The results suggest that expansins would bind not only cellulose but also a wide range of cell wall polymers.