The FTO (fat mass and obesity associated) gene codes for a novel member of the non-heme dioxygenase superfamily.

BACKGROUND: Genetic variants in the FTO (fat mass and obesity associated) gene have been associated with an increased risk of obesity. However, the function of its protein product has not been experimentally studied and previously reported sequence similarity analyses suggested the absence of homologs in existing protein databases. Here, we present the first detailed computational analysis of the sequence and predicted structure of the protein encoded by FTO. RESULTS: We performed a sequence similarity search using the human FTO protein as query and then generated a profile using the multiple sequence alignment of the homologous sequences. Profile-to-sequence and profile-to-profile based comparisons identified remote homologs of the non-heme dioxygenase family. CONCLUSION: Our analysis suggests that human FTO is a member of the non-heme dioxygenase (Fe(II)- and 2-oxoglutarate-dependent dioxygenases) superfamily. Amino acid conservation patterns support this hypothesis and indicate that both 2-oxoglutarate and iron should be important for FTO function. This computational prediction of the function of FTO should suggest further steps for its experimental characterization and help to formulate hypothesis about the mechanisms by which it relates to obesity in humans.

Nonheme-iron histochemistry for light and electron microscopy: a historical, theoretical and technical review.

We reviewed the methods of nonheme-iron histochemistry with special focus on the underlying chemical principles. The term nonheme-iron includes heterogeneous species of iron complexes where iron is more loosely bound to low-molecular weight organic bases and proteins than that of heme (iron-protoporphyrin complex). Nonheme-iron is liberated in dilute acid solutions and available for conventional histochemistry by the Perls and Turnbull and other methods using iron chelators, which depend on the production of insoluble iron compounds. Treatment with strong oxidative agents is required for the liberation of heme-iron, which therefore is not stained by conventional histochemistry. The Perls method most commonly used in laboratory investigations largely stains ferric iron, but stains some ferrous iron as well, while the Turnbull method is specific for the latter. Although the Turnbull method performed on sections fails in staining ferrous iron or stains only such parts of the tissue where iron is heavily accumulated, an in vivo perfusion-Turnbull method demonstrated the ubiquitous distribution of ferrous iron, particularly in lysosomes. The Perls or Turnbull reaction is enhanced by DAB/silver/gold methods for electron microscopy. The iron sulfide method and the staining of redox-active iron with H(2)O(2) and DAB are also applicable for electron microscopy. Although the above histochemical methods have advantages for visualizing iron by conventional light and electron microscopy, the quantitative estimation of iron is not easy. Recent methods depending on the quenching of fluorescent divalent metal indicators by Fe(2+) and dequenching by divalent metal chelators have enabled the quantitative estimation of chelatable Fe(2+) in isolated viable cells.

Relationship between body iron stores and diquat toxicity in male Fischer-344 rats.

The effects of body iron stores on diquat (DQ)-induced toxicity were examined in male Fischer-344 rats, which are sensitive to this herbicide. The rats (5 weeks old) were fed diets containing 40 (lower iron storage [LIS] group) or 320 ppm iron (higher iron storage [HIS] group) for 5 weeks. The concentrations of nonheme iron and ferritin in the liver and kidney were significantly higher in the HIS group than in the LIS group (P<0.0001), although there was no significant differences between the HIS and LIS groups in hematological parameters, including red blood cell count, hemoglobin concentration, and mean corpuscular volume. Three hours after administration of 0.1 mmol DQ/kg, serum alanine aminotransferase and urea nitrogen were significantly higher than in controls (saline injection) for both the LIS and HIS groups (P<0.01), and, after DQ injection, these parameters were significantly higher in the HIS group than in the LIS group (P<0.01). When the rats were injected with 0.075 or 0.1 mmol DQ/kg, the survival time was significantly shorter in the HIS group than in the LIS group (P<0.05). These findings suggest that higher body iron stores result in more severe DQ toxicity in Fischer-344 rats.

Visualization of non-heme ferric and ferrous iron by highly sensitive non-heme iron histochemistry in the stress-induced acute gastric lesions in the rat.

Redox-active non-heme iron catalyzes hydroxyl radical [Formula: see text] generation through Haber-Weiss reaction. Oxidative tissue damage by OH* has been suggested in the development of stress-induced gastric lesion. Using highly sensitive non-heme iron histochemistry, the perfusion-Perls and -Turnbull methods plus DAB intensification, we studied the distribution of non-heme ferric and ferrous iron (NHF[III] and NHF[II]) in the normal stomach and its changes in the acute gastric lesions induced by restraint water immersion (RWI) stress in the rat. Both NHF[III] and NHF[II] staining increased in the oncotic parietal cells located at the erosive lesion which developed on the gastric mucosal folds after 3 h RWI. It was considered that increase in non-heme iron in these cells catalyzed OH* generation under the presence of O(2)(*-) released from abundant injured mitochondria. This was supported by the increase in H(2)O(2) staining in the erosive region and the obvious reduction of the gastric lesion following administration of deferoxamine before RWI. NHF[II] was stained in the arterial endothelium in the tela submucosa of the normal gastric wall and increase in the entire gastric mucosa after 3 h RWI suggests that the changes in the vascular non-heme iron metabolism were also involved in the response of the stomach to stressful conditions.

Quantitative analysis of iron-containing protein myoglobin in different foodstuffs by liquid chromatography coupled to high-resolution inductively coupled plasma mass spectrometry.

Quantitative determination was made of the iron-containing protein myoglobin in a range of different foods, including meat, processed meat, fish, and shellfish, by liquid chromatography coupled to a double-focusing sector field inductively coupled plasma mass spectrometry (ICP-MS). The concentration of myoglobin determined in the samples ranged from 0 to 6.5 mg/kg, and the analytical precision (coefficient of variation) for the analysis of 8 replicate raw steak extracts was 2.1%. By using a double-focusing ICP-MS instrument, direct on-line detection of the most abundant iron isotope 56Fe was possible without interference from a major polyatomic interference (40Ar16O). Separation of myoglobin from other iron-containing compounds was facilitated by use of a gel filtration column (TSK Gel G2000SW) and Tris buffer (pH 7.2). The chromatographic column was coupled directly to the nebulizer of the ICP-MS instrument by a short piece of PEEK tubing. To ensure sufficient quality control throughout the study, a raw beefsteak sample was developed as an in-house reference material. The concentration of the heme-iron-containing protein myoglobin in this sample was determined by the developed method and independently by a conventional spectrophotometric method. The agreement between the 2 analytical techniques was very good. The detection limit (3 times the signal/noise ratio for a blank) of the reported method for myoglobin was 0.85 ng Fe/L.

A density functional investigation of the extradiol cleavage mechanism in non-heme iron catechol dioxygenases.

The mechanism for extradiol cleavage in non-heme iron catechol dioxygenase was modelled theoretically via density functional theory. Based on the Fe(II)-His,His,Glu motif observed in enzymes, an active site model complex, [Fe(acetate)(imidazole)(2)(catecholate)(O(2))](-), was optimized for states with six, four and two unpaired electrons (U6, U4 and U2, respectively). The transfer of the terminal atom of the coordinated dioxygen leading to "ferryl" Fe=O intermediates spontaneously generates an extradiol epoxide. The computed barriers range from 19 kcal mol(-1) on the U6 surface to approximately 25 kcal mol(-1) on the U4 surface, with overall reaction energies of +11.6, 6.3 and 7.1 kcal mol(-1) for U6, U4 and U2, respectively. The calculations for a protonated process reveal the terminal oxygen of O(2) to be the thermodynamically favoured site but subsequent oxygen transfer to the catechol has a barrier of approximately 30-40 kcal mol(-1), depending on the spin state. Instead, protonating the acetate group gives a slightly higher energy species but a subsequent barrier on the U4 surface of only 7 kcal mol(-1) relative to the hydroperoxide complex. The overall exoergicity increases to 13 kcal mol(-1). The favoured proton-assisted pathway does not involve significant radical character and has features reminiscent of a Criegee rearrangement which involves the participation of the aromatic ring pi-orbitals in the formation of the new carbon-oxygen bond. The subsequent collapse of the epoxide, attack by the coordinated hydroxide and final product formation proceeds with an overall exoergicity of approximately 75 kcal mol(-1) on the U4 surface.

Vitamin E modulates radiation-induced oxidative damage in mice fed a high-lipid diet.

The Vitamin E (VE) effect was examined on oxidative damage to DNA, lipids, and protein in mice that were fed various levels of lipid diets after total body irradiation (TBI) with X-rays at 2 Gy. No increase of 8-hydroxydeoxyguanosine (8OHdG) by TBI was observed in the + VE group; however, in the case of the -VE group, a significantly higher 8OHdG level was observed in the high-lipid group than in the low- or basal-lipid group. In the groups with TBI, the concentration of thiobarbituric reactive substances (TBARS) only significantly increased in the high-lipid (-VE) group. These changes in TBARS, due to TBI, were not detected in other groups. The contents of protein carbonyls only increased in the (-VE) group. The contents of protein carbonyls was significantly different between the (+VE) and the (-VE) groups, regardless of the lipid levels. The concentrations of GSH, vitamins C and E in the liver were lower, and the concentration of non-heme iron in the liver was higher in the high-lipid group than in the low- and basal-lipid groups. These concentrations in the high-lipid group were significantly different between the (+VE) and the (-VE) groups. These results strongly suggest that mice that are fed a high-lipid diet are susceptible to TBI-induced oxidative damage. Also, decreases in the GSH levels and an increase in the iron level are involved in the mechanism of this susceptibility.

The concept of iron bioavailability and its assessment.

In this review a broad overview of historical and current methods for the assessment of iron bioavailability was given. These methods can be divided into iron solubility studies, iron absorption studies, endpoint measures, and arithmetic models. The pros and cons of all methods were discussed. First, studies on in vitro and in vivo iron solubility have been described. The disadvantages of iron solubility include the impossibility of measuring absorption or incorporation of iron. Furthermore, only the solubility of nonheme iron, and not heme iron, can be studied. Second, we focused on iron absorption studies (either with the use of native iron, radioiron or stable iron isotopes), in which balance techniques, whole-body counting or postabsorption plasma iron measurements can be applied. In vitro determination of iron absorption using intestinal loops or cell lines, was also discussed in this part. As far as absorption studies using animals, duodenal loops, gut sacs or Caco-2 cells were concerned, the difficulty of extrapolating the results to the human situation seemed to be the major drawback. Chemical balance in man has been a good, but laborious and expensive, way to study iron absorption. Whole-body counting has the disadvantage of causing radiation exposure and it is based on a single meal. The measurement of plasma iron response did not seem to be of great value in determining nutritional iron bioavailability. The next part dealt with endpoint measures. According to the definition of iron bioavailability, these methods gave the best figure for it. In animals, the hemoglobin-repletion bioassay was most often used, whereas most studies in humans monitored the fate of radioisotopes or stable isotopes of iron in blood. Repletion bioassays using rats or other animals were of limited use because the accuracy of extrapolation to man is unknown. The use of the rat as a model for iron bioavailability seemed to be empirically based, and there were many reasons to consider the rat as an obsolete model in this respect. The double-isotope technique was probably the best predictor of iron bioavailability in humans. Disadvantages of this method are the single meal basis and the exposure to radiation (as far as radioisotopes were used). Finally, some arithmetic models were described. These models were based on data from iron bioavailability studies and could predict the bioavailability of iron from a meal.

[Iron and nonheme iron protein metabolism in ejaculates with varying degrees of fertility]. / Obmen zheleza i negemovykh ferroproteinov v éiakuliatakh razlichnoi fertil'nosti.

Spermoplasm of 21 fertile and 96 subfertile males was examined for Fe, transferrin and scaferrin the concentrations of which were compared to 14 morphofunctional and physicochemical parameters of ejaculate fertility. Fe and non-hemic spermoplasm ferroproteins are involved in male reproduction: participate in ejaculate thinning, in formation of viscosity and sperm pH, are important for normal spermatogenesis and intercellular interactions. The findings can be applied in diagnosis of male subfertility and control of ongoing treatment, hold promise for improvement of fertilizing ability of the sperm used for artificial insemination and extracorporeal fertilization.

Electrospray mass spectrometry studies of non-heme iron-containing proteins.

The oligomeric state and the metal atom stoichiometry of a series of non-heme iron-containing, multimeric proteins have been measured using electrospray ionization (ESI) in a time-of-flight (TOF) mass spectrometer. The proteins were obtained both from natural sources and by overexpression of recombinant DNA in Escherichia coli. ESI-TOF mass spectra of the metalloproteins present in nondenaturing solutions exhibit peaks corresponding to the multimeric forms of the holoproteins containing the expected number of metal atoms. Capillary-skimmer dissociation of the holoproteins produces a series of ions, which allows an exact count of the number of metal atoms present in each subunit, and also provides an indication of the oxidation state of the metal atoms. Two recombinant proteins, Phascolopsis gouldii hemerythrin (Pg-Hr) and Desulfovibrio vulgaris rubrerythrin (Dv-Rr), have been examined as well as hemerythrin isolated from Lingula reevii (Lr-Hr). ESI-TOF measurements of the aqueous solution of Pg-Hr at pH 6 yields ions of mass 108,783 Da, in close agreement with the calculated average molecular mass of an intact octameric holoprotein. Capillary-skimmer dissociation of the ions of the holoprotein produces a mass spectrum that contains peaks corresponding to a low m/z monomer and a high m/z heptamer. The masses of the monomer ions produced in this manner are assigned to the aposubunit, [subunit + Fe - 3H]+, and [subunit + 2Fe - 6 H]+. Naturally occurring Lr-Hr is composed of two subunits with average molecular masses measured under denaturing conditions by ESI-TOF to be 13,877.0 Da for the alpha-subunit and 13,517.5 Da for the beta-subunit. Under nondenaturing conditions, a multimeric species with a molecular weight of 110,663 Da is measured by ESI-TOF, corresponding to an alpha 4 beta 4 octamer. Capillary-skimmer dissociation of the alpha 4 beta 4 oligomer produces ions corresponding to both types of monomers (alpha and beta) and the corresponding heptamers (alpha 3 beta 4 and alpha 4 beta 3). In ESI-TOF measurements of recombinant rubrerythrin Dv-Rr using nondenaturing conditions, the principal ion observed corresponds to a homotetramer with an average molecular mass of 86,844 Da. Capillary-skimmer dissociation of the rubrerythrin tetramer leads to formation of a series of peaks corresponding to the subunit of the apoprotein and to subunits containing from one to three specifically bound iron atoms.

Membranes of retinal microsomes: a new protein of the microsomal monooxigenase system.

A new component, which substitutes cytochrome P-450 as an acceptor of reducing equivalents from NADPH-cytochrome P-450 reductase, was identified in the bovine retina microsomal monooxigenase system, which does not contain cytochrome P-450. This component is a non-heme iron-containing protein with molecular mass of 66 kDa. The properties of the protein from the bovine retina are similar to those of MIP, a non-heme iron-containing protein from the heart microsomal monooxigenase system, in which cytochrome P-450 was not identified, either. Activation of the microsomal monooxigenase system (an increase in the NADPH-cytochrome P-450 reductase activity, an increased rate of microsomal NADPH oxidation) was shown in the retina upon long-term intensive illumination. It was shown also that the development of hereditary degeneration of the retina in rats was accompanied by activation of the specific microsomal monooxigenase system in the target tissues (retina, brain cortex) irrespective of its composition (cytochrome P-450 or non-heme iron-containing protein).