Spectroscopic studies on the binding of iron, terbium, and zinc by apoferritin

Ultraviolet difference spectroscopy has been used to study Fe (III)-apoferritin complexes formed after addition of Fe (II) to apoferritin in air. At constant iron, the recorded spectra varied with time after Fe (II) addition and with the number of iron atoms/molecule (protein concentration). The results indicate that after production of an initial complex, rearrangement or migration of Fe (III) atoms occurs, with polynuclear species forming as end-product, probably by hydrolytic polymerization. The presence of Tb3+ or Zn2+ ions affected the Fe (III) spectra and their development in different ways. The combined data suggest that more than one site, or processes, are involved in ferritin iron-core formation and that some of the metal sites are clustered.     

M?ssbauer spectroscopic investigation of structure-function relations in ferritins.

Ferritin plays an important role in iron metabolism and our aim is to understand the mechanisms by which iron is sequestered within its protein shell as the mineral ferrihydrite. We present M?ssbauer spectroscopic data on recombinant human and horse spleen ferritin from which we draw the following conclusions: (1) that apoferritin catalyses Fe(II) oxidation as a first step in ferrihydrite deposition, (2) that the catalysis of Fe(II) oxidation is associated with residues situated within H chains, at the postulated 'ferroxidase centre' and not in the 3-fold inter-subunit channels previously suggested as the initial Fe(II) binding and oxidation site; (3) that both isolated Fe(III) and Fe(III) mu-oxo-bridged dimers found previously by M?ssbauer spectroscopy to be intermediates in iron-core formation in horse spleen ferritin, are located on H chains; and (4) that these dimers form at ferroxidase centres. The importance of the ferroxidase centre is suggested by the conservation of its ligands in many ferritins from vertebrates, invertebrates and plants. Nevertheless iron-core formation does occur in those ferritins that lack ferroxidase centres even though the initial Fe(II) oxidation is relatively slow. We compare the early stages of core formation in such variants and in horse spleen ferritin in which only 10-15% of its chains are of the H type. We discuss our findings in relation to the physiological role of isoferritins in iron storage processes.     

The European Renal Genome Project: An Integrated Approach Towards Understanding the Genetics of Kidney Development and Disease

Rapid progress in genome research creates a wealth of information on the functional annotation of mammalian genome sequences. However, as we accumulate large amounts of scientific information we are facing problems of how to integrate and relate the data produced by various genomic approaches. Here, we propose the novel concept of an organ atlas where diverse data from expression maps to histological findings to mutant phenotypes can be queried, compared and visualized in the context of a three-dimensional reconstruction of the organ. We will seek proof of concept for the organ atlas by elucidating genetic pathways involved in development and pathophysiology of the kidney. Such a kidney atlas may provide a paradigm for a new systems-biology approach in functional genome research aimed at understanding the genetic bases of organ development, physiology and disease.Key Words: EuReGene, kidney, genome, development, pathophysiology, genetics     

A new form of ferritin heterogeneity explained. Isolation and identification of a nineteen-amino-acid-residue fragment from siderosomal ferritin of rat liver.

Ferritin present within siderosomes of iron-loaded rats has a faster anodal mobility than that of cytosolic ferritin from the same rats. A 19-amino-acid-residue peptide was isolated from this fast ferritin and shown to be derived from the C-terminal end of its L-subunit. A 17.3 kDa peptide seen on electrophoresis in denaturing gels of this ferritin accounts for the major portion of the original 182-residue subunit. The two peptides arise from cleavage within the 'insertion region' of the L-subunit sequence that occurs between the D and E helices and lies on the outside of the assembled molecule. This cleavage is present in about 80% of the L-subunits of siderosomal ferritin but nevertheless leaves the molecular structure otherwise intact. It gives rise to an apparent decrease in molecular size, accounting for the faster anodal mobility on native gels. Hence a new form of heterogeneity in ferritin preparations has been explained.     

Iron-induced changes in rat liver isoferritins.

The effects of single and of multiple iron injection on the distribution of isoferritins was studied in rat liver with the aid of 14C-labelling either after or before iron treatment. Several effects of iron can be seen. Analysis of protein and labelling patterns show that it not only produces a disproportionate increase in the more-basic isoferritins, but may, in sufficient dose, actually lead to a decrease in the more-acidic isoferritins. Use of iron injection after radioactivity shows that it must give rise to post-assembly changes causing acidic isoferritins to become more basic. With a moderate iron dose this change is relatively slow, taking several hours, and seems to occur in addition to a differential stimulation of the synthesis of the more-basic isoferritins. With higher iron dosage the post-assembly changes may be so rapid that it is difficult to distinguish them from a switch in the pattern of synthesis.     

Rejecting strictly allopatric speciation on a continental island: prolonged postdivergence gene flow between Taiwan (Leucodioptron taewanus,Passeriformes Timaliidae) and Chinese (L. canorum canorum) hwameis

Allopatry is conventionally considered the geographical mode of speciation for continental island organisms. However, strictly allopatric speciation models that assume the lack of postdivergence gene flow seem oversimplified given the recurrence of land bridges during glacial periods since the late Pliocene. Here, to evaluate whether a continental island endemic, the Taiwan hwamei (Leucodioptron taewanus, Passeriformes Timaliidae) speciated in strict allopatry, we used weighted‐regression‐based approximate Bayesian computation (ABC) to analyse the genetic polymorphism of 18 neutral nuclear loci (total length: 8500 bp) in Taiwan hwamei and its continental sister species, the Chinese hwamei (L. canorum canorum). The nonallopatry model was found to fit better with observed genetic polymorphism of the two hwamei species (posterior possibility = 0.82). We also recovered unambiguous signals of nontrivial bidirectional postdivergence gene flow (N_em » 1) between Chinese hwamei and Taiwan hwamei until 0.5 Ma. Divergence time was estimated to be 3.5 to 2 million years earlier than that estimated from mitochondrial cytochrome b sequences. Finally, using the inferred nonallopatry model to simulate genetic variation at 24 nuclear genes examined showed that the adiponectin receptor 1 gene may be under divergent adaptation. Our findings imply that the role of geographical barrier may be less prominent for the speciation of continental island endemics, and suggest a shift in speciation studies from simply correlating geographical barrier and genetic divergence to examining factors that facilitate and maintain divergence, e.g. differential selection and sexual selection, especially in the face of interpopulation gene flow.     

Improved gold chloride staining method for anatomical analysis of sensory nerve endings in the shoulder capsule and labrum as examples of loose and dense fibrous tissues

Consistency in gold chloride staining is essential for anatomical analysis of sensory nerve endings. The gold chloride stain for this purpose has been modified by many investigators, but often yields inconsistent staining, which makes it difficult to differentiate structures and to determine nerve ending distribution in large tissue samples. We introduce additional steps and major changes to the modified Gairns’ protocol. We controlled the temperature and mixing rate during tissue staining to achieve consistent staining and complete solution penetration. We subjected samples to sucrose dehydration to improve cutting efficiency. We then exposed samples to a solution containing lemon juice, formic acid and paraformaldehyde to produce optimal tissue transparency with minimal tissue deformity. We extended the time for gold chloride impregnation 1.5 fold. Gold chloride was reduced in the labrum using 25% formic acid in water for 18 h and in the capsule using 25% formic acid in citrate phosphate buffer for 2 h. Citrate binds gold nanoparticles, which minimizes aggregation in the tissue. We stored samples in fresh ultrapure water at 4° C to slow reduction and to maintain color contrast in the tissue. Tissue samples were embedded in Tissue Tek and sectioned at 80 and 100 μm instead of using glycerin and teasing the tissue apart as in Gairns’ modified gold chloride method. We attached sections directly to gelatin subbed slides after sectioning with a cryostat. The slides then were processed and coverslipped with Permount. Staining consistency was demonstrated throughout the tissue sections and neural structures were clearly identifiable.     

Effect of ferritin-containing fractions with different iron loading on lipid peroxidation.

Ferritin-containing fractions with different degrees of iron loading were prepared. All ferritin fractions stimulated the peroxidation of bovine brain phospholipid liposomes, as measured by the formation of thiobarbituric acid-reactive material. This stimulation was increased in the presence of ascorbate. Iron salts of equivalent concentration to those of the ferritin fractions were more stimulatory to lipid peroxidation at the higher iron concentrations. None of the fractions inhibited ascorbate-dependent peroxidation in the presence of added iron salts.