Siderosomal ferritin. The missing link between ferritin and haemosiderin?

A minor electrophoretically fast component was found in ferritin from iron-loaded rat liver in addition to a major electrophoretically slow ferritin similar to that observed in control rats. The electrophoretically fast ferritin showed immunological identity with the slow component, but on electrophoresis in SDS it gave a peptide of 17.3 kDa, in contrast with the electrophoretically slow ferritin, which gave a major band corresponding to the L-subunit (20.7 kDa). Thus the electrophoretically fast ferritin resembles that reported by Massover [(1985) Biochim. Biophys. Acta 829, 377-386] in livers of mice with short-term parenteral iron overload. The electrophoretically fast ferritin had a lower iron content (2000 Fe atoms/molecule) than the electrophoretically slow ferritin (3000 Fe atoms/molecule). Removal and re-incorporation of iron was possible without effect on the electrophoretic mobility of either ferritin species. On subcellular fractionation the electrophoretically fast ferritin was enriched in pellet fractions and was the sole soluble ferritin isolated from iron-laden secondary lysosomes (siderosomes). The amount and relative proportion of the electrophoretically fast species increased with iron loading. Haemosiderin isolated from siderosomes was found to contain a peptide reactive to anti-ferritin serum and corresponding to the 17.3 kDa peptide of the electrophoretically fast ferritin species. Unlike the electrophoretically slow ferritin, the electrophoretically fast ferritin did not become significantly radioactive in a 1 h biosynthetic labelling experiment. We conclude that the minor ferritin is not, as has been suggested for mouse liver ferritin, 'a completely new species of smaller holoferritin that represents a shift in the ferritin phenotype' in response to siderosis, but a precursor of haemosiderin, in agreement with the proposal by Richter [(1984) Lab. Invest. 50, 26-35] concerning siderosomal ferritin.     

Mössbauer spectroscopy of the iron cores in human liver ferritin, ferritin in normal human spleen and ferritin in spleen from patient with primary myelofibrosis: preliminary results of comparative analysis

Comparative study of human liver ferritin and spleen tissues from healthy human and patient with primary myelofibrosis was carried out using Mössbauer spectroscopy with a high velocity resolution at 295 and 90 K and with a low velocity resolution at 20 K. The results obtained demonstrated that the iron content in patient’s spleen in the form of iron storage proteins was about ten times larger than that in normal tissue. However, in the case of patient with primary myelofibrosis the magnetic anisotropy energy barrier differed from that in normal case and, probably, the iron core size was supposed to be slightly larger than that in both normal spleen tissue and normal human liver ferritin in contrast to well-known data for iron overload in patients with thalassemia accompanied by the iron-core size increase. Therefore, the iron overload in the case of patient with primary myelofibrosis may be related to increase in the ferritin content mainly. It was also found that Mössbauer hyperfine parameters for normal and patient’s spleen and normal human liver ferritin demonstrated some small differences related, probably, to some small structural variations in the ferritin iron cores of patient’s spleen.     

Synthesis of (±)-Homosarkomycin and (±)-Rosaprostol

(±)-Homosarkomycin (2) and (±)-rosaprostol (3) were synthesized from (±)-methyl 2-oxo-bicyclo[3.1.0]hexane-1-carboxylate (1) by using the nucleophilic ring opening reaction on the double-activated cyclopropane ring as the key step.     

Synthesis of (±)-Lamprolobine and (±)-Epilamprolobine

(±)-Lamprolobine, the (+)-enatiomer of which was isolated from the leaves of Lamprolobium fruticosum, and (±)-epilamprolobine were synthesized from δ-valerolactam.     

Biomimetic synthesis and characterization of cobalt nanoparticles using apoferritin, and investigation of direct electron transfer of Co(NPs)–ferritin at modified glassy carbon electrode to design a novel nanobiosensor

Oxyhydroxy cobalt CoO(OH) nanoparticles (Co-NPs) were prepared in horse spleen apoferritin (HsAFr) cavity. Transmission electron microscopy revealed the particle size was 5.5-6 nm. Mineralization effect on HsAFr was investigated by fluorescence and far-UV circular dichroism (far-UV CD) spectroscopies. The far-UV CD experiments indicated an increase in the α-helical content after mineralization. Intrinsic fluorescence data showed that mineralization acts as a quencher of HsAFr. For the first time, direct electron transfer between Co(NPs)-HsAFr and a glassy carbon electrode in the thin film of dihexadecylphosphate (DHP) was investigated by cyclic voltammetry (CV) to design a biosensor. The anionic surfactant DHP was used to achieve direct electron-transfer between Co(NPs)-HsAFr molecules and the GC electrode surface. CV result showed clearly a pair of well-defined and quasi-reversible redox peaks arise from Co(NPs)-HsAFr embedded in DHP film. This novel biosensor can be used in medical and industrial fields to detect different analytes.     

Synthesis and characterization of tris(β-diketonato)technetium(III) and -(IV) complexes

The preparations and properties of tris(dipivaloylmethanato)technetium(III), tris(trifluoroacetylacetonato)technetium(III), and tris(hexafluoroacetonato)technetium(III) are described. The oxidation of the dipivaloyl derivative to tris(dipivaloyl)technetium(IV) hexafluorophosphate was shown to take place readily. Voltammetric studies and magnetic resonance results on the new complexes are reported. The large shifts observed for the complexes seem to be due to a contact interaction.     

Syntheses of (±)-Epoformin (Desoxyepoxydon) and (±)-Epiepoformin (Desoxyepiepoxydon)

Boron is an essential nutrient for plants, but it is toxic in excess. Transgenic rice plants expressing an Arabidopsis thaliana borate efflux transporter gene, AtBOR4, at a low level exhibited increased tolerance to excess boron. Those lines with high levels of expression exhibited reduced growth. These findings suggest a potential of the borate transporter BOR4 for the generation of high-boron tolerant rice.     

Synthesis and characterization of β-diketiminate germanium(II) and tin(II) bromides

The equimolar reaction of a β-diketiminate lithium salt LLi(OEt₂) [L = HC(CMeNAr)₂; Ar = 2,6-iPr₂C₆H₃] with either GeBr₂ or SnBr₂ in diethyl ether affords the synthetically useful monomeric β-diketiminate-element halides LGeBr (1) and LSnBr (2), respectively. Both are soluble in hydrocarbon solvents, stable in inert atmosphere, and have been characterized by elemental analysis, NMR spectroscopy, and single-crystal X-ray diffraction analysis.     

Assignment of human ferritin genes to chromosomes 11 and 19q13.3→19qter

Summary Extracts of hamster-human and mouse-human hybrids, some with translocations involving chromosome 19, have been assayed for both human spleen ferritin (rich in L subunits) and human heart ferritin (rich in H subunits). Hybrid lines retaining part of the long arm of chromosome 19 including the region 19q13.319qter produced human L type ferritin. This confirms the previous assignment of the ferritin gene to chromosome 19 (Caskey et al. 1983). However, lines retaining chromosome 11 were found to contain human H type ferritin suggesting that the gene for the H subunit is on this chromosome. The presence of chromosome 6 was not necessary for the expression of either H or L type human ferritin. It thus seems unlikely that the gene for idiopathic haemochromatosis is a ferritin gene.     

Synthesis of ( ± )-Methyl Epijasmonate and ( ± )-Methyl Cucurbate

A novel synthesis of ( ± )-methyl epijasomonate (2) and the first synthesis of ( ± )-methyl cucurbate (4) were achieved starting from 2-allylcyclohexane-1,3-dione (8). The synthetic epimer 2 had a stronger jasmin flavor than the trans-isomer 1 with 95% purity.     

Biological function of modified nucleotides T_(54) and ψ_(55) of yeast tRNA~(Ala)

The 3' half molecule of yeast tRNAAla (nucleotides 36-75) was hybridized with a DNA fragment (5'GGAATCGAACC 3') and the hybrid was then digested with E. coli RNase H (from Boehringer). The enzyme can specifically cleave the 3' half molecule at the 3' side of nucleotide ψ55, thus a fragment C36-ψ55 was prepared. The 3'-terminal T or Tψ of this fragment was removed by one or two cycles of periodate oxidation and $-elimination. The products were fragments C36-T54 and C36-G53. Three yeast tRNAAla fragments C56-A76, U55-A76 (with ψ55 replaced by U), U54-A76 (with T54ψ55 replaced by UU) were synthesized and ligated with three prepared fragments (C36-ψ55 C36-T54 and C36-G53) respectively by T4 RNA ligase. The products were further ligated with the 5' half molecule (nu-cleotides 1-35). Using this method, one reconstituted yeast tRNAAla (tRNAr) and two yeast tRNAAla analogs: (i) tRNAa with U55 instead of ψ55; (ii) tRNAb with U54U55 instead of T54ψ55 were synthesized. The charging and incorporation activi     

Mössbauer spectroscopic studies of deproteinised,sub-fractionated and reconstituted ferritins: the relationship between haemosiderin and ferritin

In a previous study of human haemosiderin and ferritin by a combination of Mössbauer spectroscopy and electron microscopy, it was observed that the Mössbauer spectra of haemosiderin showed a very different temperature dependence to those of ferritin. These differences were related to the superparamagnetic behaviour of small particles of a magnetic material and suggested that the magnetic anisotropy constant of the haemosiderin was considerably larger than that of the ferritin. In the present work, samples of ferritin have been examined by Mössbauer spectroscopy following partial deproteinisation, subfractionation, and reconstitution with and without phosphate, in order to investigate whether these procedures lead to changes in the magnetic anisotropy constant of the iron-containing cores. There is no evidence from the present data that changes in the protein shell, in the size of the iron-containing cores of ferritin, or in the phosphate content lead to any significant changes in the magnetic anisotropy constant, as obtained from the temperature dependence of the Mössbauer spectra. These results indicate that the different magnetic anisotropy constant observed in the case of human haemosiderin resulting from transfusional iron overload must arise from other significant differences in the composition or structure of the iron-containing cores.     

Antioxidant and Cytoprotective Activity of Oxydiacetate Complexes of Cobalt(II) and Nickel(II) with 1,10-Phenantroline and 2,2′-Bipyridine

Biological Trace Element Research - The antioxidant properties of oxydiacetate complexes of cobalt(II) and nickel(II) with 1,10-phenantroline and 2,2′-bipyridine have been investigated...     

Characterization of Nuclear Localization Signals (NLSs) and Function of NLSs and Phosphorylation of Serine Residues in Subcellular and Subnuclear Localization of Transformer-2β (Tra2β)

The serine/arginine-rich (SR) proteins are one type of major actors in regulation of pre-mRNA splicing. Their functions are closely related to the intracellular spatial organization. The RS domain and phosphorylation status of SR proteins are two critical factors in determining the subcellular distribution. Mammalian Transformer-2β (Tra2β) protein, a member of SR proteins, is known to play multiple important roles in development and diseases. In the present study, we characterized the subcellular and subnuclear localization of Tra2β protein and its related mechanisms. The results demonstrated that in the brain the nuclear and cytoplasmic localization of Tra2β were correlated with its phosphorylation status. Using deletional mutation analysis, we showed that the nuclear localization of Tra2β was determined by multiple nuclear localization signals (NLSs) in the RS domains. The point-mutation analysis disclosed that phosphorylation of serine residues in the NLSs inhibited the function of NLS in directing Tra2β to the nucleus. In addition, we identified at least two nuclear speckle localization signals within the RS1 domain, but not in the RS2 domain. The nuclear speckle localization signals determined the localization of RS1 domain-contained proteins to the nuclear speckle. The function of the signals did not depend on the presence of serine residues. The results provide new insight into the mechanisms by which the subcellular and subnuclear localization of Tra2β proteins are regulated.     

Structure and ultrastructure of the silk glands and spinneret of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

This study provides comprehensive documentation of silk production in the pest moth Helicoverpa armigera from gland secretion to extrusion of silk thread. The structure of the silk glands, accessory structures and extrusion apparatus are reported. The general schema of the paired silk glands follows that found for Lepidoptera. Morphology of the duct, silk press, muscle attachments and spigot are presented as a three-dimensional reconstruction and the cuticular crescent-shaped profile of the silk press is demonstrated in both open and closed forms with attendant muscle blocks, allowing advances in our knowledge of how the silk press functions to regulate the extrusion of silk. Growth of the spigot across instars is documented showing a distinctive developmental pattern for this extrusion device. Its shape and structure are related to use and load-bearing activity.     

Crystal structure of bullfrog M ferritin at 2.8 Å resolution: analysis of subunit interactions and the binuclear metal center

Ferritins concentrate and store iron as a mineral in all bacterial, plant, and animal cells. The two ferritin subunit types, H or M (fast) and L (slow), differ in rates of iron uptake and mineralization and assemble in vivo to form heteropolymeric protein shells made up of 24 subunits; H/L subunit ratios reflect cell specificity of H and L subunit gene expression. A diferric peroxo species that is the initial reaction product of Fe(II) in H-type ferritins, as well as in ribonucleotide reductase (R2) and methane monooxygenase hydroxylase (MMOH), has recently been characterized, exploiting the relatively high accumulation of the peroxo intermediate in frog H-subunit type recombinant ferritin with the M sequence. The stability of the diferric reaction centers in R2 and MMOH contrasts with the instability of diferric centers in ferritin, which are precursors of the ferric mineral. We have determined the crystal structure of the homopolymer of recombinant frog M ferritin in two crystal forms: P4(1)2(1)2, a = b = 170.0 A and c = 481.5 A; and P3(1)21, a = b = 210.8 A and c = 328.1 A. The structural model for the trigonal form was refined to a crystallographic R value of 19.0% (Rfree = 19.4%); the two structures have an r.m.s.d. of approximately 0.22 A for all C alpha atoms. Comparison with the previously determined crystal structure of frog L ferritin indicates that the subunit interface at the molecular twofold axes is most variable, which may relate to the presence of the ferroxidase site in H-type ferritin subunits. Two metal ions (Mg) from the crystallization buffer were found in the ferroxidase site of the M ferritin crystals and interact with Glu23, Glu58, His61, Glu103, Gln137 and, unique to the M subunit, Asp140. The data suggest that Gln137 and Asp140 are a vestige of the second GluxxHis site, resulting from single nucleotide mutations of Glu and His codons and giving rise to Ala140 or Ser140 present in other eukaryotic H-type ferritins, by additional single nucleotide mutations. The observation of the Gln137xxAsp140 site in the frog M ferritin accounts for both the instability of the diferric oxy complexes in ferritin compared to MMOH and R2 and the observed kinetic variability of the diferric peroxo species in different H-type ferritin sequences.     

Comparative studies of α-lactalbumin and lysozyme: The proteins of kangaroo (Megaleia rufa and Macropus giganteus) and horse (Equus caballus)

Summary As part of a study of the whey proteins of various mammals, a comparison is made of the -lactalbumins and lysozymes of the kangaroo and horse. In the milk of the red kangaroo (Megaleia rufa) there is only one -lactalbumin and it occurs throughout lactation, but no lysozyme has been detected. There are two -lactalbumins in the milk of the grey kangaroo (Macropus giganteus), one, designated -lactalbumin Zone B, is present throughout lactation; the second, designated -lactalbumin Zone A, is present only in late lactation. One lysozyme is also present. The milk of the horse (Equus caballus) contains one -lactalbumin and at least one lysozyme. Partial amino acid sequences are proposed from sequence determination and from analyses of tryptic peptides compared with the known sequences of other -lactalbumins and lysozymes.     

Binding of α-synuclein with Fe(III) and with Fe(II) and biological implications of the resultant complexes

Parkinson’s disease (PD) is hallmarked by the abnormal intracellular inclusions (Lewy bodies or LBs) in dopaminergic cells. Amyloidogenic protein α-synuclein (α-syn) and iron (including both Fe(III) and Fe(II)) are both found to be present in LBs. The interaction between iron and α-syn might have important biological relevance to PD etiology. Previously, a moderate binding affinity between α-syn and Fe(II) (5.8 × 10³ M⁻¹) has been measured, but studies on the binding between α-syn and Fe(III) have not been reported. In this work, electrospray mass spectrometry (ES-MS), cyclic voltammetry (CV), and fluorescence spectroscopy were used to study the binding between α-syn and Fe(II) and the redox property of the resultant α-syn-Fe(II) complex. The complex is of a 1:1 stoichiometry and can be readily oxidized electrochemically and chemically (by O₂) to the putative α-syn-Fe(III) complex, with H₂O₂ as a co-product. The reduction potential was estimated to be 0.025 V vs. Ag/AgCl, which represents a shift by −0.550 V vs. the standard reduction potential of the free Fe(III)/Fe(II) couple. Such a shift allows a binding constant between α-syn and Fe(III), 1.2 × 10¹³ M⁻¹, to be deduced. Despite the relatively high binding affinity, α-syn-Fe(III) generated from the oxidation of α-syn-Fe(II) still dissociates due to the stronger tendency of Fe(III) to hydrolyze to Fe(OH)₃ and/or ferrihydrite gel. The roles of α-syn and its interaction with Fe(III) and/or Fe(II) are discussed in the context of oxidative stress, metal-catalyzed α-syn aggregation, and iron transfer processes.     

Reactivity of platina-β-diketones towards chelating nitrogen and sulfur donors: formation of acyl(hydrido)platinum(IV) and acyl(chloro)platinum(II) complexes

The platina-β-diketone [Pt₂{(COMe)₂H}₂(μ-Cl)₂] (1) was found to react with chelating N,N-ligands 2(R^′NCR)C₅H₄N (R/R^′=Ph/OH, H/Ph, Me/Ph) to form acyl(hydrido)platinum(IV) complexes [Pt(COMe)₂Cl(H){2-(R^′NCR)C₅H₄N}] (R/R^′=Ph/OH 2a; H/Ph 2b; Me/Ph (2c)). Reactions of complex 1 with chelating S,S- and N,S-donors (RS-CH₂-CH₂-SR, 2-(RSCH₂)C₅H₄N, R=Et, Ph, t-Bu) afforded acyl(chloro)platinum(II) complexes [Pt(COMe)Cl(RSCH₂CH₂SR)] (R=Et, 3a; Ph, 3b; t-Bu, 3c) and [Pt(COMe)Cl{2-(RSCH₂)C₅H₄N}] (R=Et, 4a; Ph, 4b; t-Bu, 4c), respectively. All complexes were fully characterized by microanalysis, IR and NMR (¹H, ¹³C) spectroscopy. Furthermore, molecular structures of complexes 3b and 4b were determined by single-crystal X-ray diffraction analyses revealing close to square-planar configuration. In complex 4b the acetyl ligand is trans to pyridine N atom (configuration index SP-4-2). The reactions are discussed in terms of consecutive oxidative addition and reductive elimination reactions.