Mechanism of ferritin iron uptake: activity of the H-chain and deletion mapping of the ferro-oxidase site. A study of iron uptake and ferro-oxidase activity of human liver, recombinant H-chain ferritins, and of two H-chain deletion mutants

To study the functional differences between human ferritin H- and L-chains and the role of the protein shell in the formation and growth of the ferritin iron core, we have compared the kinetics of iron oxidation and uptake of ferritin purified from human liver (90% L) and of the H-chain homopolymer overproduced in Escherichia coli (100% H). As a control for iron autocatalytic activity, we analyzed the effect of Fe(III) on the iron uptake reaction. The results show that the H-chain homopolymer has faster rates of iron uptake and iron oxidation than liver ferritin in all the conditions analyzed and that the difference is reduced in the conditions in which iron autocatalysis in high: i.e. at pH 7 and in presence of iron core. We have also analyzed the properties of two engineered H-chains, one lacking the last 22 amino acids at the carboxyl terminus and the other missing the first 13 residues at the amino terminus. These mutant proteins assemble in ferritin-like proteins and maintain the ability to catalyze iron oxidation. The deletion at the carboxyl terminus, however, prevents the formation of a stable iron core. It is concluded that the ferritin H-chain has an iron oxidation site which is separated from the sites of iron transfer and hydrolysis and that either the integrity of the molecule or the presence of the amino acid sequences forming the hydrophobic channel is necessary for iron core formation.     

The genetic diversity of two brazilian vellozia (velloziaceae) with different patterns of spatial distribution and pollination biology

BACKGROUND AND AIMS: The genetic structure and variability of two species of Vellozia (Velloziaceae) with restricted distribution in high-altitude quartzitic fields in south-eastern Brazil were studied. Vellozia epidendroides is short, grows on pebbly or sandy soil, and is pollinated by bees. Vellozia leptopetala is arborescent, grows on rock outcrops, and is pollinated by bees and hummingbirds. Both are self-incompatible and have a short, massive flowering strategy. The study aimed to associate differences in their genetic diversity and structure with their microhabitat distribution and pollination ecology. METHODS: Leaves from 106 and 139 plants of V. epidendroides and V. leptopetala, respectively, were collected from five patches of each species and prepared for electrophoretic analyses. KEY RESULTS: Five enzyme systems could be reliably scored for both species. Vellozia epidendroides showed 100 % of the loci polymorphic for almost all patches. The average number of alleles per locus ranged between 2.2 and 2.4 among patches. The Wright's fixation index (F) for this species was 0.226. A significant (p) value indicates that there is a reasonable genetic divergence among patches. Vellozia leptopetala presented 47.5 % of polymorphic loci. All levels of P, A, A(p) and of heterozygosities were lower than those of V. epidendroides. Vellozia leptopetala showed high inbreeding within patches. CONCLUSIONS: The relatively high values of genetic diversity indices found for V. epidendroides may be associated with its large and widespread populations. On the other hand, the low values of genetic diversity found for V. leptopetala may be related to physical isolation on outcrops and intensive foraging by territorial hummingbirds, which may hinder gene flow among patches, aggravated by the very restricted seed dispersal characteristic of the genus, that facilitates sibling mating. It is important to stress the need to preserve the specific habitats of these species of Vellozia, in particular those of V. leptopetala that has lower genetic diversity and is restricted to rock outcrop environments.     

The pollination biology of two species of Helicteres (Malvaceae) with different mechanisms of pollen deposition

Species of Helicteres are pollinated mainly by hummingbirds and bats. Most species pollinated by hummingbirds have a mechanism of depositing pollen on the top of the pollinator's head such as is shown in this work for Helicteres sacarolha. H. brevispira has an unusual mechanism of depositing pollen grains under the tail or on the abdomen of the hummingbirds. The top of the birds’ head may be considered an efficient place to transport pollen grains, because it is plain, easily accessible for deposition and donation of pollen and not easily accessible for grooming, while the tail is movable and inclined. Thus, H. brevispira pollinators may carry or transfer fewer amounts of pollen grains than H. sacarolha pollinators from one flower to another. If a large amount of pollen grains is lost, a higher quantity of flower or pollen has to be produced to guarantee reproductive success. Plants of H. brevispira set higher number of flowers and pollen grains per flower than H. sacarolha and have also higher rates of fruit and flower abortion. Thus, pollination efficiency of H. brevispira may be reached by high pollen and flower production. Flower change mechanism presented in this species may be also involved with the optimization of pollinator feeding and pollination efficiency.     

Mutational analysis of the channel and loop sequences of human ferritin H-chain.

Human ferritin H-chain mutants were obtained by engineering the recombinant protein expressed by Escherichia coli. The mutagenesis were directed to the C-terminal sequence forming the hydrophobic channel, to the hydrophilic channel and to the loop sequence. The mutants were analysed for extent of expression, for stability, for capacity to incorporate iron and for kinetics of iron uptake and iron oxidation. Of the 22 mutants analysed only two with deletions of single residues in the loop sequence and one with deletion of the last 28 amino acid residues did not assemble into ferritin-like proteins. The other mutants assembled correctly and showed similar chemical/physical properties to the wild-type; they included duplication of an 18-amino acid-residue stretch, deletion of the last 22 and the last seven residues and various mutations of single amino acid residues. Two mutants with extensive alteration in the C-terminal sequence had a diminished thermostability associated with incapability to incorporate iron though they still catalysed iron oxidation. The mutants with alterations of the sequence around the hydrophilic channel showed diminished iron uptake and oxidation kinetics, together with a slightly larger apparent molecular size. The results indicate (i) that two of the sequences are important for ferritin assembly/stability, (ii) that the presence of the hydrophobic channel is essential for formation of the iron core and (iii) that the sites of iron interaction and the path of iron penetration into ferritin remain unidentified.     

Influence of landscape context on the abundance of native bee pollinators in tomato crops in Central Brazil

Evidence shows that pollinator abundance has declined and, consequently, so has their services, which has possible negative impacts on ecosystem functioning. The goal of this study was to evaluate the influence of landscape context at multiple spatial scales on the abundance of bee pollinators of tomato crops in Brazil. Pollinator abundance was obtained from tomato crops grown in a conventional system in the Cerrado region. Around each tomato field circular buffers of 0.75, 1, 1.5, 2, and 3 km radius were defined. Inside each buffer the landscapes were manually classified into native and non-native cover and, the proportion of native vegetation, the relative largest patch size, and the distance of the nearest native vegetation to each field were calculated. Pollinator species were categorized into five groups: Exomalopsis, Centris, Bombus/Eulaema, Halictidae, and all buzz pollinators combined (Buzzers). The results showed that the landscape context influenced the abundance of the five groups of tomato pollinators. Bees with a smaller body size, such as Exomalopsis spp., responded at smaller scales, while bees with a larger body size, such as the Centris and Bombus/Eulaema groups, responded at larger scales. The abundance of all pollinator groups increased with native vegetation cover. Most groups showed higher abundances in landscapes with similar-size fragments. The results reinforce the recommendation for maintaining natural habitats around crop areas, even if fragmented, for the conservation of the tomato pollinator assemblage. These findings are valuable for planning landscape management in the studied area to improve bee conservation, ecosystem services, and food production.     

Expression and structural and functional properties of human ferritin L-chain from Escherichia coli

The human ferritin L-chain cDNA was cloned into a vector for overproduction in Escherichia coli, under the regulation of a lambda promoter. The plasmid obtained contains the full L-chain coding region modified at the first two codons. It is able to direct the synthesis of the L-chain which can constitute up to 15% of the total soluble protein of bacterial extract. The L-chains assemble to form a ferritin homopolymer with electrophoretic mobility, molecular weight, thermal stability, spectroscopic, and immunological properties analogous to natural ferritin from human liver (95% L-chain). This recombinant L-ferritin is able to incorporate and retain iron in solution at physiological pH values. At variance with the H-ferritin, the L form does not uptake iron at acidic pH values and does not show detectable ferroxidase activity. It is concluded that ferritin L-chain lacks the ferroxidase site present in the H-chain and that the two chains may have specialized functions in intracellular iron metabolism.     

Simaba guianensis subsp. huberi, a new Venezuelan taxon of Simaroubaceae

Simaba guianensis subsp.huberi (Simaroubaceae), restricted to the savannas of the Venezuelan State of Amazonas, is described and illustrated. The new subspecies is distinguished from the other two subspecies ofS. guianensis by its smaller size, characteristics of the leaflets (size, texture, apex shape, and margin curvature), and fruit color.     

Dynamics of a dry forest fragment after the exclusion of human disturbance in southeastern Brazil

Changes in the structure and composition of a dry forest fragment were described for a 4-yr period (1994–1998) in the southeastern Brazil (19°12′05″ S and 47°08′02″ W). This is the first dynamic study of a vanishing type of dry forest, which grows on base-rich soils originating from the basalt bedrock of western Minas Gerais State. A survey of trees ≥ 3.2 cm dbh (diameter at breast height, or 1.30 m) was conducted in 26 transects of 50 m × 6 m (0.78 ha). The species were classified into three regeneration guilds – pioneer, light-demanding and shade-tolerant – on the basis of others studies and personal observation. There was a decrease of 10.1% in the number of trees between 1994 and 1998. However, the basal area had an increase of 1.5% in the same period. Size class distributions in 1994 and 1998 were significantly different. The smallest trees (diameter < 10 cm) had the highest mortality rates. Although stand composition showed little change, a few species showed significant imbalance between recruitment and mortality. The studied fragment had mortality (2.3% yr^–1) and recruitment (1.6% yr^–1) rates similar to other tropical rain forests for trees ≥ 10 cm dbh. The present fragment of forest (turnover time = 39.5 yr, at cut level 10 cm dbh) lies among the most dynamic tropical forests. The main compositional changes were the increase of shade-tolerant trees and decline of pioneer species. The changes in composition and structure over the survey period may reflect the recovery of the forest after 4 yr of conservation. This revised version was published online in June 2006 with corrections to the Cover Date.