Three ferritin subunits involved in immune defense from bay scallop Argopecten irradians

Ferritin is a ubiquitous protein that plays an important role in iron storage and iron-withholding strategy of innate immunity. In this study, three genes encoding different ferritin subunits were cloned from bay scallop Argopecten irradians (AiFer1, AiFer2 and AiFer3) by rapid amplification of cDNA ends (RACE) approaches based on the known ESTs. The open reading frames of the three ferritins are of 516 bp, 522 bp and 519 bp, encoding 171,173 and 172 amino acids, respectively. All the AiFers contain a putative Iron Regulatory Element (IRE) in their 5′-untranslated regions. The deduced amino acid sequences of AiFers possess both the ferroxidase center of mammalian H ferritin and the iron nucleation site of mammalian L ferritin. Gene structure study revealed two distinct structured genes encoding a ferritin subunit (AiFer3). Quantitative real-time PCR analysis indicated the significant up-regulation of AiFers in hemocytes after challenged with Listonella anguillarum, though the magnitudes of AiFer1 and AiFer2 were much higher than that of AiFer3. Taken together, these results suggest that AiFers are likely to play roles in both iron storage and innate immune defense against microbial infections.     

Increased whole blood manganese concentrations observed in children with iron deficiency anaemia

A prospective observational study was carried out at Alder Hey Children's Hospital, Liverpool, England, UK on children aged 1–6 years attending the pathology department for routine blood tests (n = 225). Whole blood manganese concentrations were measured plus the following markers of iron status; haemoglobin, MCV, MCH, RBC count, ferritin, transferrin saturation and soluble transferrin receptors. Multiple regression analysis was performed, with blood manganese as the dependent variable and factors of iron status, age and gender as independent variables. A strong relationship between blood manganese and iron deficiency was demonstrated (adjusted R² = 34.3%, p < 0.001) and the primary contributing factors to this relationship were haematological indices and soluble transferrin receptors. Subjects were categorised according to iron status using serum ferritin, transferrin saturation and haemoglobin indices. Children with iron deficiency anaemia had higher median blood manganese concentrations (16.4 μg/L, range 11.7–42.4, n = 20) than children with iron sufficiency (11 μg/L, range 5.9–20.9, n = 59, p < 0.001). This suggests that children with iron deficiency anaemia may be at risk from manganese toxicity (whole blood manganese >20 μg/L), and that this may lead to neurological problems. Treatment of iron deficiency in children is important both to improve iron status and to reduce the risk of manganese toxicity.     

High iron level in early pregnancy increased glucose intolerance

High iron stores in pregnancy are essential in preventing negative outcomes for both infants and mothers; however the risk of gestational diabetes mellitus (GDM) might also be increased. We intend to study the relationship between increased iron stores in early pregnancy and the risk of glucose intolerance and GDM. This prospective, observational, single-hospital study involved 104 non-anemic pregnant women, divided into 4 groups based on the quartile values for ferritin at the first trimester of pregnancy. All participants were screened for GDM with 75-g oral glucose tolerance test (OGTT) at 24–28 weeks’ gestation. We observed that ferritin levels at early pregnancy were significantly correlated to glucose level after OGTT at 1-h and 2-h (rho = 0.21, p < 0.05; rho = 0.43, p < 0.001 respectively). Furthermore, in the higher quartile for ferritin (>38.8 μg/L) glycemia at 2-h OGTT was significantly higher than in the others quartiles (p = 0.002). In multivariate regression analysis, serum ferritin was a significant determinant of glycemia at 2-h OGTT. Although we did not find a significant association in the incidence of GDM in women with higher serum ferritin levels, probably in reason to the limit power of our study, our data demonstrated that the role of iron excess is tightly involved in the pathogenesis of glucose intolerance. We report for the first time that high ferritin values in early pregnancy are predictors of impaired glucose tolerance in non-anemic women. Individual iron supplementation should be evaluated in order to minimize glucose impairment risk in women with high risk of diabetes.     

Spatial variability of domoic acid concentration in king scallops Pecten maximus off the southeast coast of Ireland

Amnesic shellfish poisoning (ASP) has proved problematic in Irish king scallop, Pecten maximus fisheries necessitating restrictions on the sale of fresh scallops (in-shell), which achieve a higher market price than frozen processed product. An investigation of variability in domoic acid (DA) concentration in king scallop from 69 sampling sites within a limited area off the southeast coast of Ireland was performed. Variation in DA concentration was examined in the whole area, within smaller sub-areas, with size, age, and water depth. Mean DA concentrations in whole scallop ranged from 6.5 to 154.3 μg g⁻¹, with an overall mean of 40.6 ± 30.8 μg g⁻¹. The concentration in gonad exceeded 20 μg g⁻¹ in 17 sites and in adductor muscle in 3 sites. Significant differences in DA concentration were detected between scallops from different sampling stations. Whole scallop tissue and individual scallop tissues with the exception of gonad, exhibited significant negative correlations with water depth. Highest DA concentrations were recorded in inshore, shallow sites and lowest DA concentrations in deeper offshore waters. Significant positive correlations between DA concentration in hepatopancreas and scallop size were exhibited at inshore sites but not at offshore sites. High inter-animal and spatial variability in toxin concentration demonstrated the importance of a reliable sampling protocol for the management of ASP outbreaks to ensure public safety and to avoid unnecessary fishery closures.     

Genotoxicity assessment in iron deficiency anemia patients using sister chromatid exchanges and chromosomal aberrations assays

Previous studies have shown that iron deficiency anemia is associated with oxidative stress produced by a decrease in antioxidant enzyme activities and/or a high level of oxidants. Because oxidative stress induces DNA damage, we investigated genotoxicity in lymphocytes from patients with iron deficiency anemia (IDA) using chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) assays. Eighteen IDA subjects and a similar number of age-matched healthy controls were included in the study. The results demonstrated that IDA was associated with a slight increase in the frequency of spontaneous CAs and a decrease in the frequency of SCEs (P < 0.05). In addition, the level of SCEs was positively correlated with both the ferritin concentration (r = 0.485, P < 0.05) and hemoglobin content (r = 0.514, P < 0.05) in subjects. Moreover, vitamin E treatment reduced the frequency of SCEs in IDA patients and control subjects by the same percentage (～30%) without affecting the magnitude of the difference in the levels of SCEs between the two groups. In conclusion, our results demonstrated that IDA has a differential effect on the frequency of spontaneous CAs and SCEs.     

Iron overload,measured as serum ferritin,increases brain damage induced by focal ischemia and early reperfusion

High levels of iron, measured as serum ferritin, are associated to a worse outcome after stroke. However, it is not known whether ischemic damage might increase ferritin levels as an acute phase protein or whether iron overload affects stroke outcome. The objectives are to study the effect of stroke on serum ferritin and the contribution of iron overload to ischemic damage.Swiss mice were fed with a standard diet or with a diet supplemented with 2.5% carbonyl iron to produce iron overload. Mice were submitted to permanent (by ligature and by in situ thromboembolic models) or transient focal ischemia (by ligature for 1 or 3 h).Treatment with iron diet produced an increase in the basal levels of ferritin in all the groups. However, serum ferritin did not change after ischemia. Animals submitted to permanent ischemia had the same infarct volume in the groups studied. However, in mice submitted to transient ischemia followed by early (1 h) but not late reperfusion (3 h), iron overload increased ischemic damage and haemorrhagic transformation.Iron worsens ischemic damage induced by transient ischemia and early reperfusion. In addition, ferritin is a good indicator of body iron levels but not an acute phase protein after ischemia.     

Generation and analysis of expressed sequence tags from adductor muscle of Japanese scallop Mizuhopecten yessoensis

A normalized cDNA library was constructed from the adductor muscle of M. yessoensis and acquired 4595 high quality expressed sequence tags (ESTs). After clustering and assembly of the ESTs, 3061 unigenes containing 654 contigs and 2407 singletons were identified. The contig length ranged from 266 bp to 2364 bp and the average length of these contigs was 544 bp. Blastx nonredundant protein database analysis showed that 1522 unigenes had significant homology to known genes (E value ≤ 10^? 5). By comparing to Clusters of Orthologous Groups (COG) categories, 460 unigenes were annotated (E value ≤ 10^? 10). Using Kyoto Encyclopedia of Genes and Genomes (KEGG), 345 of 3061 unigenes were assigned into 103 pathways (E value ≤ 10^? 5). For InterProScan searches, 1237 unigenes were annotated containing 727 different types of protein domains. 941 of the 1237 unigenes were annotated for Gene Ontology (GO) classification using Uniprot2GO associations in any category (biological, cellular, and molecular). By sequences comparability and analysis of Blastx NCBI nonredundant protein database and KEGG, 66 unigenes were identified that may be involved in genetic information processing based on the known knowledge. The study provides a material basis as useful information for the genomic analysis of shellfish.     

Electrochemical analysis of autodisplayed adrenodoxin (Adx) on the outer membrane of E. coli

In this work, adrenodoxin (Adx) was expressed on the outer membrane of E. coli by autodisplay and then the iron–sulfur cluster was incorporated into apo-Adx by an anaerobic reconstitution process. For the determination of the redox potentials of the iron–sulfur clusters of the autodisplayed Adx, E. coli cells with autodisplayed Adx were immobilized on a gold electrode modified with a self-assembled monolayer of mercaptoundecanoic acid (MUA). From the repeated cyclic voltammetry (CV) analysis, the E. coli (10 mM HEPES buffer, pH 7.0) with autodisplayed Adx showed significant changes in shape with an oxidation peak at + 0.4 V (vs. Ag/AgCl) and a reduction peak at − 0.3 V (vs. Ag/AgCl) after the reconstitution process for the incorporation of the iron–sulfur cluster. From the repeated CV analysis in the reduction and oxidation potential ranges, the iron–sulfur clusters of the autodisplayed Adx were observed to undergo reversible redox reactions via direct electron transfer to the MUA-modified gold electrode.     

Structural and thermodynamic characterization of metal ion binding in Streptococcus suis Dpr

The use of protein cages for the creation of novel inorganic nanomaterials has attracted considerable attention in recent years. Ferritins are among the most commonly used protein cages in nanoscience. Accordingly, the binding of various metals to ferritins has been studied extensively. Dps (DNA-binding protein from starved cells)-like proteins belong to the ferritin superfamily. In contrast to ferritins, Dps-like proteins form 12-mers instead of 24-mers, have a different ferroxidase center, and are able to store a smaller amount of iron atoms in a hollow cavity (up to ∼ 500, instead of the ∼ 4500 iron atoms found in ferritins). With the exception of iron, the binding of other metal cations to Dps proteins has not been studied in detail. Here, the binding of six divalent metal ions (Zn²⁺, Mn²⁺, Ni²⁺, Co²⁺, Cu²⁺, and Mg²⁺) to Streptococcus suisDps-like peroxide resistance protein (SsDpr) was characterized by X-ray crystallography and isothermal titration calorimetry (ITC). All metal cations, except for Mg²⁺, were found to bind to the ferroxidase center similarly to Fe²⁺, with moderate affinity (binding constants between 0.1 × 10⁵ M^− 1 and 5 × 10⁵ M^− 1). The stoichiometry of binding, as deduced by ITC data, suggested the presence of a dication ferroxidase site. No other metal binding sites were identified in the protein. The results presented here demonstrate the ability of SsDpr to bind various metals as substitutes for iron and will help in better understanding protein-metal interactions in the Dps family of proteins as potential metal nanocontainers.     

Crystal structure of the ferritin from the hyperthermophilic archaeal anaerobe <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis>

The crystal structure of the ferritin from the archaeon, hyperthermophile and anaerobe Pyrococcus furiosus (PfFtn) is presented. While many ferritin structures from bacteria to mammals have been reported, until now only one was available from archaea, the ferritin from Archaeoglobus fulgidus (AfFtn). The PfFtn 24-mer exhibits the 432 point-group symmetry that is characteristic of most ferritins, which suggests that the 23 symmetry found in the previously reported AfFtn is not a common feature of archaeal ferritins. Consequently, the four large pores that were found in AfFtn are not present in PfFtn. The structure has been solved by molecular replacement and refined at 2.75-Å resolution to R = 0.195 and R _free = 0.247. The ferroxidase center of the aerobically crystallized ferritin contains one iron at site A and shows sites B and C only upon iron or zinc soaking. Electron paramagnetic resonance studies suggest this iron depletion of the native ferroxidase center to be a result of a complexation of iron by the crystallization salt. The extreme thermostability of PfFtn is compared with that of eight structurally similar ferritins and is proposed to originate mostly from the observed high number of intrasubunit hydrogen bonds. A preservation of the monomer fold, rather than the 24-mer assembly, appears to be the most important factor that protects the ferritin from inactivation by heat.     

Development of Neoseiulus womersleyi (Schicha) and Euseius ovalis (Evans) feeding on four tetranychid mites (Acari: Phytoseiidae,Tetranychidae) and pollen

The development of the predatory mites, Neoseiulus womersleyi (Schicha) and Euseius ovalis (Evans), feeding on four tetranychid mites (Tetranychus urticae, Tetranychus kanzawai, Oligonychus mangiferus, Panonychus citri), maize pollen or Chinese loofah pollen was studied at 25 °C. Immature stages of N. womersleyi feeding on T. urticae and T. kanzawai had shorter developmental duration (4.71 and 5.02 days for females, 4.77 and 5.19 days for males, respectively) than those feeding on other food sources. Immature stages of E. ovalis females feeding on O. mangiferus and T. urticae developed in 4.99 and 5.13 days, respectively, the shortest developmental duration measured. Immature stages of E. ovalis males feeding on O. mangiferus and T. urticae developed in 5.12 and 5.37 days, respectively. The longevity of N. womersleyi males (13.31 to 14.51 days) and females (17.67 to 21.81 days) feeding on T. urticae, T. kanzawai or maize pollen was longer than the longevity of N. womersleyi feeding on O. mangiferus, P. citri or loofah pollen. E. ovalis males (12.91 to 16.74 days) and females (16.24 to 23.77 days) feeding on O. mangiferus, T. urticae or maize pollen lived longer than E. ovalis males and females feeding on T. kanzawai, P. citri or loofah pollen.     

Biological and molecular responses of Chironomus riparius (Diptera,Chironomidae) to herbicide 2,4-D (2,4-dichlorophenoxyacetic acid)

2,4-Dichlorophenoxyacetic acid (2,4-D) is an agricultural contaminant found in rural ground water. It remains to be determined whether neither 2,4-D poses environmental risks, nor is the mechanism of toxicity known at the molecular level. To evaluate the potential ecological risk of 2,4-D, we assessed the biological parameters including the survival rate, adult sex ratio of emerged adults, and mouthpart deformities in Chironomus riparius after long-term exposure to 2,4-D. The larvae were treated with 0.1, 1 or, 10 μg L^? 1 of 2,4-D for short- and long-term exposure periods. The sex ratio was changed in C. riparius exposed to only 10 μg L^? 1 of 2,4-D, whereas mouthpart deformities were observed as significantly higher in C. riparius exposed to 0.1 μg L^? 1 of 2,4-D. Survival rates were not significantly affected by 2,4-D. Furthermore, we evaluated the molecular and biochemical responses of biomarker genes such as gene expression of heat shock proteins (HSPs), ferritins and glutathione S-transferases (GSTs) in C. riparius exposed to 2,4-D for 24 h. The expressions of HSP70, HSP40, HSP90 and GST levels in C. riparius were significantly increased after exposure to a 10 μg L^? 1 concentration of 2,4-D, whereas ferritin heavy and light chain gene expressions were significantly increased at all concentrations of 2,4-D exposure. Finally, these results may provide an important contribution to our understanding of the toxicology of 2,4-D herbicide in C. riparius. Moreover, the 2,4-D-mediated gene expressions may be generated by 2,4-D is the causative effects on most probable cause of the observed alterations. These biological, molecular and morphological parameters and the measured parameters can be used to monitor 2,4-D toxicity in an aquatic environment.     

Rumen ciliates of domestic cattle (Bos taurus taurus) in Kastamonu,Turkey, with the description of a new species

Species composition and distribution of ciliates were investigated in the rumen contents of 25 domestic cattle (Bos taurus taurus L.) living in Kastamonu, Turkey. Forty-seven species and 37 morphotypes representing 15 genera were identified. Of them, a new species of Ostracodinium was recognized and described as Ostracodinium anatolicum n. sp. This new species has two caudal lobes. The dorsal lobe is small and rounded and the ventral lobe is triangular shaped and bent toward the dorsal side like a thick hook. Furthermore, the anterior end of the macronucleus (1/5 of the length) is bent toward the left like a hook. The density of rumen ciliates in cattle was 96.8 ± 43.3 × 10⁴ cells mL⁻¹ and the mean number of ciliate species per host was 14.2 ± 4.4. Entodinium longinucleatum, E. nanellum, E. simulans and Isotricha prostoma were the most abundant species, each with a prevalence of 88%. Entodinium chatterjeei, E. bifidum m. monospinosum, Hsiungia triciliata, Oligoisotricha bubali, Ostracodinium dogieli, O. mammosum and O. munham are new host records for cattle from Turkey.     

Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology

The high stability and strong self-assembly properties made ferritins the most used proteins for nanotechnological applications. Human ferritins are made of 24 subunits of the H- and L-type that coassemble in an almost spherical nanocage 12 nm across, delimiting a large cavity. The mechanism and kinetics of ferritin self-assembly and why H/L heteropolymers formation is favored over the homopolymers remain unclarified. In order to study this, we used the Fluorescence Resonance Energy Transfer (FRET) tool by binding multiple donor or acceptor Alexa Fluor fluorophores on the outer surface of human H and L ferritins and then denaturing and reassembling them in different proportions and conditions. The FRET efficiency increase from < 0.3 of the disassembled to > 0.7 in the assembled allowed to study the assembly kinetics. We found that their assembly was complete in about one hour, and that the initial rate of self-assembly of H/L heteropolymers was slightly faster than that of the H/H homopolymers. Then, by adding various proportions of unlabeled H or L-chains to the FRET system we found that the presence of the L-chains displaced the formation of H-H dimers more efficiently than that of the H-chains. This favored formation of H/L heterodimers, which is the initial step in ferritin self-assembly, contributes to explain the preferred formation of H/L heteropolymers over the H or L homopolymers. Moreover, we found that the H-chains arrange at distant positions on the heteropolymeric shell until they reach a number above eight, when they start to co-localize.     

Purification,kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccharum officinarum L.)

We report for the first time kinetic and thermodynamic properties of soluble acid invertase (SAI) of sugarcane (Saccharum officinarum L.) salt sensitive local cultivar CP 77-400 (CP-77). The SAI was purified to apparent homogeneity on FPLC system. The crude enzyme was about 13 fold purified and recovery of SAI was 35%. The invertase was monomeric in nature and its native molecular mass on gel filtration and subunit mass on SDS-PAGE was 28 kDa. SAI was highly acidic having an optimum pH lower than 2. The acidic limb was missing. Proton transfer (donation and receiving) during catalysis was controlled by the basic limb having a pKa of 2.4. Carboxyl groups were involved in proton transfer during catalysis. The kinetic constants for sucrose hydrolysis by SAI were determined to be: k_m = 55 mg ml^?1, k_cat = 21 s^?1, k_cat/k_m = 0.38, while the thermodynamic parameters were: ΔH* = 52.6 kJ mol^?1, ΔG* = 71.2 kJ mol^?1, ΔS* = ?57 J mol^?1 K^?1, ΔG*_E–S = 10.8 kJ mol^?1 and ΔG*_E–T = 2.6 kJ mol^?1. The kinetics and thermodynamics of irreversible thermal denaturation at various temperatures 53–63 °C were also determined. The half -life of SAI at 53 and 63 °C was 112 and 10 min, respectively. At 55 °C, surprisingly the half -life increased to twice that at 53 °C. ΔG*, ΔH* and ΔS* of irreversible thermal stability of SAI at 55 °C were 107.7 kJ mol^?1, 276.04 kJ mol^?1 and 513 J mol^?1K^?1, respectively.     

Inhibitory effect of terpene nerolidol on the growth of Babesia parasites

Nerolidol is a sesquiterpene present in the essential oils of many plants, approved by the U.S. FDA as a food flavoring agent. Nerolidol interferes with the isoprenoid biosynthetic pathway in the apicoplast of P. falciparum. In the present study, the in vitro growth of four Babesia species was significantly (P < 0.05) inhibited in the presence of nerolidol (IC₅₀s values = 21 ± 1, 29.6 ± 3, 26.9 ± 2, and 23.1 ± 1 µM for B. bovis, B. bigemina, B. ovata, and B. caballi, respectively). Parasites from treated cultures failed to grow in the subsequent viability test at a concentration of 50 µM. Nerolidol significantly (P < 0.05) inhibited the growth of B. microti at the dosage of 10 and 100 mg/kg BW, while the inhibition was low compared with the high doses used. Therefore, nerolidol could not be used as a chemotherapeutic drug for babesiosis.     

Doubly linked,A-type proanthocyanidin trimer and other constituents of Ixora coccinea leaves and their antioxidant and antibacterial properties

Phytochemical investigation of the ethyl acetate fraction of the methanol extract of the leaves of Ixora coccinea led to the isolation and identification of an A-type trimeric proanthocyanidin epicatechin-(2β → O → 7, 4β → 8)–epicatechin-(5 → O → 2β, 6 → 4β)–epicatechin named ixoratannin A-2 along with seven known compounds, epicatechin, procyanidin A2, cinnamtannin B-1, and four flavon-3-ol rhamnosides viz: kaempferol-7-O-α-l-rhamnnoside, kaempferol-3-O-α-l-rhamnoside, quercetin-3-O-α-l-rhamnopyranoside, and kaempferol-3,7-O-α-l-dirhamnoside. The structures were elucidated by the application of IR, UV, MS, 1D-, and 2D-NMR spectroscopic analyses and by comparison with literature data. Antioxidant evaluation of isolated compounds revealed that ixoratannin A-2 and cinnamtannin B-1 were the most active compounds in DPPH, inhibition of lipid peroxidation and nitric oxide radical scavenging assays. Antibacterial activities were assessed by means of agar-diffusion assays using Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtilis. All tested compounds inhibited the growth of B. subtilis, while only epicatechin and quercetin-3-O-α-l-rhamnopyranoside inhibited the growth of E. coli.     

Rational design,synthesis and biological evaluation of 1,3,4-oxadiazole pyrimidine derivatives as novel pyruvate dehydrogenase complex E1 inhibitors

On the basis of previous study on 2-methylpyrimidine-4-ylamine derivatives I, further synthetic optimization was done to find potent PDHc-E1 inhibitors with antibacterial activity. Three series of novel pyrimidine derivatives 6, 11 and 14 were designed and synthesized as potential Escherichia coli PDHc-E1 inhibitors by introducing 1,3,4-oxadiazole-thioether, 2,4-disubstituted-1,3-thiazole or 1,2,4-triazol-4-amine-thioether moiety into lead structure I, respectively. Most of 6, 11 and 14 exhibited good inhibitory activity against E. coli PHDc-E1 (IC₅₀ 0.97–19.21 μM) and obvious inhibitory activity against cyanobacteria (EC₅₀ 0.83–9.86 μM). Their inhibitory activities were much higher than that of lead structure I. 11 showed more potent inhibitory activity against both E. coli PDHc-E1 (IC₅₀ < 6.62 μM) and cyanobacteria (EC₅₀ < 1.63 μM) than that of 6, 14 or lead compound I. The most effective compound 11d with good enzyme-selectivity exhibited most powerful inhibitory potency against E. coli PDHc-E1 (IC₅₀ = 0.97 μM) and cyanobacteria (EC₅₀ = 0.83 μM). The possible interactions of the important residues of PDHc-E1 with title compounds were studied by molecular docking, site-directed mutagenesis, and enzymatic assays. The results indicated that 11d had more potent inhibitory activity than that of 14d or I due to its 1,3,4-oxadiazole moiety with more binding position and stronger interaction with Lsy392 and His106 at active site of E. coli PDHc-E1.