Metabolic fate of [14C]-labeled meal protein amino acids in Aedes aegypti mosquitoes

We developed a method to follow the metabolic fate of [(14)C]-labeled Euglena gracilis protein amino acids in Aedes aegypti mosquitoes under three different adult nutritional regimes. Quantitative analysis of blood meal protein amino acid metabolism showed that most of the carbon of the amino acids was either oxidized to CO(2) or excreted as waste. Under the three different adult nutritional regimes, no significant differences in the metabolism of amino acids were found, which indicated that the female A. aegypti mosquitoes possess a substantial capacity of maintaining metabolic homeostasis during a gonotrophic cycle. The amount of maternal glycogen and lipid after egg laying were significantly lower in the mosquitoes that underwent a partial starvation before a blood meal and/or starvation after the blood meal. The content of egg lipid or protein or the number of eggs laid did not show a significant difference among the three different regimes, which indicates that stable fecundity of A. aegypti under the partial starvation before a blood meal and/or starvation after the blood meal seemed to result from a trade-off between current fecundity and future survival after the eggs laid. The methods described in this paper can be applied to a wide range of questions about the effects of environmental conditions on the utilization of blood meal amino acids.     

Utilization of host iron sources by Corynebacterium diphtheriae: identification of a gene whose product is homologous to eukaryotic heme oxygenases and is required for acquisition of iron from heme and hemoglobin.

Corynebacterium diphtheriae was examined for the ability to utilize various host compounds as iron sources. C. diphtheriae C7(-) acquired iron from heme, hemoglobin, and transferrin. A siderophore uptake mutant of strain C7 was unable to utilize transferrin but was unaffected in acquisition of iron from heme and hemoglobin, which suggests that C. diphtheriae possesses a novel mechanism for utilizing heme and hemoglobin as iron sources. Mutants of C. diphtheriae and Corynebacterium ulcerans that are defective in acquiring iron from heme and hemoglobin were isolated following chemical mutagenesis and streptonigrin enrichment. A recombinant clone, pCD293, obtained from a C7(-) genomic plasmid library complemented several of the C. ulcerans mutants and three of the C. diphtheriae mutants. The nucleotide sequence of the gene (hmuO) required for complementation was determined and shown to encode a protein with a predicted mass of 24,123 Da. Sequence analysis revealed that HmuO has 33% identity and 70% similarity with the human heme oxygenase enzyme HO-1. Heme oxygenases, which have been well characterized in eukaryotes but have not been identified in prokaryotes, are involved in the oxidation of heme and subsequent release of iron from the heme moiety. It is proposed that the HmuO protein is essential for the utilization of heme as an iron source by C. diphtheriae and that the heme oxygenase activity of HmuO is involved in the release of iron from heme. This is the first report of a bacterial gene whose product has homology to heme oxygenases.     

Kinetics of the removal of ferric ion from transferrin by aminoalkylphosphonic acids

The kinetics of ion removal at 25 degrees C in 0.1 M Tris, pH 7.4 by a series of phosphonic acids have been evaluated. The initial rate of iron removal is first order in ferric-transferrin, but shows a hyperbolic dependence on the concentration of the phosphonate ligand. At high ligand concentrations the reaction is clearly biphasic, and the data are interpreted in terms of nonequivalent rate constants for iron removal from the two transferrin iron-binding sites. Rate constants for three phosphonic acid ligands are approximately 0.025 min-1 and approximately 0.007 min-1 for the faster and slower binding sites. The results are discussed in relation to the conformational change mechanism for iron removal from transferrin proposed by Coward et al. [21].     

Transferrin and iron uptake by human lymphoblastoid and K-562 cells

Two human lymphoblastoid cell lines and K-562 cells were found to take up radioiodinated transferrin and transferrin-bound iron in amounts comparable to reticulocytes. These cell lines were also shown to possess transferrin receptors whose numbers and affinity for transferrin were similar to those of reticulocytes. However, unlike reticulocytes, in which at least 90% of the iron taken up is incorporated into heme, in the lymphoblastoid and K-562 cells only around 10% of the incorporated iron is found in heme. In addition, in contrast to the hemoglobin synthesizing cells, excess heme does not inhibit the removal of iron from transferrin by the lymphoblastoid and K-562 cells, suggesting that only during erythroid differentiation do cells acquire a specific mechanism for removing iron from transferrin which is subject to feedback inhibition by heme.     

Non-transferrin donors of iron for heme synthesis in immature erythroid cells

The mechanism of iron uptake from several iron-containing compounds by transferrin-depleted rabbit reticulocytes and mouse spleen erythroid cells was investigated. Iron complexes of DL-penicillamine, citrate and six different aroyl hydrazones may be utilized by immature erythroid cells for hemoglobin synthesis, although less efficiently than iron from transferrin. HTF-14, a monoclonal antibody against human transferrin, reacts with rabbit transferrin and inhibits iron uptake and heme synthesis by rabbit reticulocytes. HTF-14 had no significant effect on iron uptake and heme synthesis when non-transferrin donors of iron were examined. Ammonium chloride (NH4Cl) increases intracellular pH and blocks the release or utilization of iron from the internalized transferrin. NH4Cl only slightly affected iron incorporation and heme synthesis from non-transferrin donors of iron. Hemin inhibited transferrin iron uptake and heme synthesis, but had a much lesser effect on iron incorporation and heme synthesis from non-transferrin donors of iron. These results allow us to conclude that transferrin-depleted reticulocytes take up iron from all of the examined non-transferrin iron donors without the involvement of the transferrin/transferrin receptor pathway.     

The effects of resistance exercise and post-exercise meal timing on the iron status in iron-deficient rats

Resistance exercise increases heme synthesis in the bone marrow and the hemoglobin in iron-deficient rats. Post-exercise early nutrient provision facilitates skeletal muscle protein synthesis compared to late provision. However, the effects of post-exercise nutrition timing on hemoglobin synthesis are unclear. The current study investigated the effect of post-exercise meal timing on the activity of the key enzyme involved in hemoglobin synthesis, δ-aminolevulinic acid dehydratase (ALAD), in the bone marrow and examined the hemoglobin concentration in iron-deficient rats. Male 4-week-old Sprague-Dawley rats were fed an iron-deficient diet containing 12 mg iron/kg and performed climbing exercise (5 min × 6 sets/day, 3 days/week) for 3 weeks. The rats were divided into a group fed a post-exercise meal early after exercise (E) or a group fed the meal 4 h after exercise (L). A single bout of exercise performed after the 3-week training period increased the bone marrow ALAD activity, plasma iron concentration, and transferrin saturation. Although the plasma iron concentration and transferrin saturation were lower in the E group than the L group after a single bout of exercise, the basal hematocrit, hemoglobin, and TIBC after 3 weeks did not differ between the groups. Therefore, resistance exercise increases the bone marrow ALAD activity, while the post-exercise meal timing has no effect on the hemoglobin concentration in iron-deficient rats.     

Aedes aegypti peritrophic matrix and its interaction with heme during blood digestion

A large amount of heme is produced upon digestion of red cell hemoglobin in the midgut of mosquitoes. The interaction between heme and the peritrophic matrix (PM) was studied in Aedes aegypti. By light microscopy, the PM appeared as a light brownish layer between the intestinal epithelium and the alimentary bolus. This natural color can be attributed to the presence of heme bound to the matrix. In histochemical studies, a diffuse peroxidase activity of the heme molecules was clearly observed between the erythrocytes and the PM at 14 h after the blood meal. This activity tends to increase and concentrate in the PM reaching its maximum thickness at 24 h after feeding. Most of the heme of the PM was found associated to with enormous number of small electron-dense granules. The amount of heme bound to the PM increased in parallel with the progression of digestion, reaching a maximum at 48 h after feeding, when 18 nmol of heme were found in an individual matrix. The association of heme with PM from insects fed with plasma is saturable, suggesting the existence of specific binding sites for hemin in the PM. Taken all together, our data indicate that the PM performs a central role in heme detoxification in this insect.     

Macrophages function as a ferritin iron source for cultured human erythroid precursors

Iron is essential for the survival as well as the proliferation and maturation of developing erythroid precursors (EP) into hemoglobin-containing red blood cells. The transferrin-transferrin receptor pathway is the main route for erythroid iron uptake. Using a two-phase culture system, we have previously shown that placental ferritin as well as macrophages derived from peripheral blood monocytes could partially replace transferrin and support EP growth in a transferrin-free medium. We now demonstrate that in the absence of transferrin, ferritin synthesized and secreted by macrophages can serve as an iron source for EP. Macrophages trigger an increase in both the cytosolic and the mitochondrial labile iron pools, in heme and in hemoglobin synthesis, along with a decrease in surface transferrin receptors. Inhibiting macrophage exocytosis, binding extracellular ferritin with specific antibodies, inhibiting EP receptor-mediated endocytosis or acidification of EP lysosomes, all resulted in a decreased EP growth when co-cultured with macrophages under transferrin-free conditions. The results suggest that iron taken up by macrophages is incorporated mainly into their ferritin, which is subsequently secreted by exocytosis. Nearby EP are able to take up this ferritin probably through clathrin-dependent, receptor-mediated endocytosis into endosomes, which following acidification and proteolysis release the iron from the ferritin, making it available for regulatory and synthetic purposes. Thus, macrophages support EP development under transferrin-free conditions by delivering essential iron in the form of metabolizable ferritin.     

Control of heme synthesis during Friend cell differentiation: role of iron and transferrin

In many types of cells the synthesis of delta-aminolevulinic acid (ALA) limits the rate of heme formation. However, results from our laboratory with reticulocytes suggest that the rate of iron uptake from transferrin (Tf), rather than ALA synthase activity, limits the rate of heme synthesis in erythroid cells. To determine whether changes occur in iron metabolism and the control of heme synthesis during erythroid cell development Friend erythroleukemia cells induced to erythroid differentiation by dimethylsulfoxide (DMSO) were studied. While added ALA stimulated heme synthesis in uninduced Friend cells (suggesting ALA synthase is limiting) it did not do so in induced cells. Therefore the possibility was investigated that, in induced cells, iron uptake from Tf limits and controls heme synthesis. Several aspects of iron metabolism were investigated using the synthetic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH). Both induced and uninduced Friend cells take up and utilize Fe for heme synthesis directly from Fe-SIH without the involvement of transferrin and transferrin receptors and to a much greater extent than from saturating levels of Fe-Tf (20 microM). Furthermore, in induced Friend cells 100 microM Fe-SIH stimulated 2-14C-glycine incorporation into heme up to 3.6-fold as compared to the incorporation observed with saturating concentrations of Fe-Tf. In contrast, Fe-SIH, even when added in high concentrations, did not stimulate heme synthesis in uninduced Friend cells but was able to do so as early as 24 to 48 h following induction. In addition, contrary to previous results with rabbit reticulocytes, Fe-SIH also stimulated globin synthesis in induced Friend cells above the level seen with saturating concentrations of transferrin. These results indicate that some step(s) in the pathway of iron from extracellular Tf to protoporphyrin, rather than the activity of ALA synthase, limits and controls the overall rate of heme and possibly hemoglobin synthesis in differentiating Friend erythroleukemia cells.     

Utilization of pre-existing energy stores of female Aedes aegypti mosquitoes during the first gonotrophic cycle

Pre-existing energy reserves may play an important role in regulating the utilization of blood meal proteins in female anautogenous mosquitoes. Determining the fate of reserves derived from the sugar meal and larval food during the first gonotrophic cycle would help to elucidate the relative contributions of larval and adult nutrition to survival and reproduction. We measured the allocation of pre-blood-meal reserves to egg production or energy production during the first gonotrophic cycle by using [14C]-labeled female Aedes aegypti mosquitoes. Feeding adults [3,4-14C]-glucose labeled the glycogen and sugar stores (approximately 50%), lipid stores (approximately 25%), and protein and amino acid stores (approximately 25%). During the first gonotrophic cycle, about 60% of the glycogen and sugar stores were metabolized and all were used for energy production. About 33% of the labeled protein and 72% of the labeled amino acid stores were metabolized, with about 9% being transferred to the eggs and the rest oxidized. About 30% of the lipid was metabolized, with about 65% being transferred to the eggs and the rest oxidized. Feeding [1-14C]-oleic acid to larvae effectively labeled adult lipid stores with about 75% of the label in lipid stores and 16% in proteins and 6% in glycogen. During the first gonotrophic cycle, about 35% of the labeled lipid stores were metabolized, with equal amounts being oxidized and transferred to the eggs. None of the other maternal stores labeled by fatty acid were metabolized during the first gonotrophic cycle. These results show that carbohydrate reserves are a critical source of energy during the first gonotrophic cycle, while lipid reserves are used equally for energy production and provisioning the eggs.     

Impaired iron status in rats as induced by copper deficiency

The hypothesis was tested that marginal copper deficiency affects iron status. Copper restriction (1 vs 5 mg Cu/kg diet) significantly lowered iron concentrations and transferrin saturation in plasma and reduced blood hemoglobin, hematocrit, and iron concentrations in tibia and femur, but raised iron concentrations in liver. Marginal copper deficiency did not affect feed intake and body-weight gain.     

In vitro heme and non-heme iron capture from hemoglobin,myoglobin and ferritin by bovine lactoferrin and implications for suppression of reactive oxygen species in vivo

Lactoferrin (Lf), present in colostrum and milk is a member of the transferrin family of iron-binding glyco-proteins, with stronger binding capacity to ferric iron than hemoglobin, myoglobin or transferrin. Unlike hemoglobin and myoglobin, iron-bound Lf is reasonably stable to gastric and duodenal digestive conditions. Unlike ferrous iron, ferric iron is not directly reactive with oxygen supporting the capacity of Lf capture of heme iron to suppress reactive oxygen species (ROS) production. We therefore hypothesized that bovine Lf could capture and thereby terminate the cycle of ROS production by heme iron. The transfer of heme iron from either intact or digested forms of hemoglobin and myoglobin and from intact ferritin was demonstrated by in vitro methods, monitoring Fe-saturation status of Lf by changes in absorptivity at 465 nm. The results are discussed in the context of new proposed opportunities for orally administered Lf to regulate oxidative damage associated with heme iron. In addition to potentially suppressing oxidative heme–iron-mediated tissue damage in the lumen, Lf is expected to also reverse the overload of ferritin-bound iron, that accompanies chronic inflammation and aging. These new proposed uses of Lf are additional to known host defense functions that include anti-microbial, anti-viral properties, immune and cancer cell growth regulation. The findings and interpretations presented require clinical substantiation and may support important additional protective and therapeutic uses for Lf in the future.     

Effects of variations in a formulated protein meal on the fecundity and fertility of female mosquitoes

Abstract. A formulated protein meal developed by Kogan (1990) for adult female Aedes aegypti mosquitoes was evaluated and modified to increase egg and pupal yield. A vigorous laboratory colony was maintained with the females fed exclusively on this dietary formula for about twenty-five generations over more than 2 years. Extra modifications were made to produce a diet suitable for Anopheles arabiensis and Anopheles stephensi females to produce eggs. Both formulations contain bovine albumin, haemoglobin and globulin in a ringer based solution, plus ATP as a phagostimulant for Ae.aegypti. Compared to Kogan's original, our Aedes formula doubled the production of pupae per female after a single meal, although the yield was still significantly lower than from mosquitoes fed on animal hosts or defibrinated pig blood. In varying the proportions of different constituents during attempts to optimize the formula, no relationship was found between total protein content (within the range 80–220 mg/ml) and fecundity, percentage hatch or pupal yield of Ae.aegypti. Equivocal results were found when an isoleucine supplement was added to the formula.     

Heme inhibits transferrin endocytosis in immature erythroid cells

The inhibitory effect of heme on iron uptake from transferrin by rat and rabbit reticulocytes and erythroid cells from the fetal rat liver was studied in vitro. Addition of hemin was shown to cause a decrease in the rate of transferrin endocytosis, the degree of inhibition being proportional to the reduction in iron uptake. The heme synthesis inhibitors, isoniazid and succinylacetone, stimulated the rate of transferrin endocytosis by 15-30% and caused a proportional increase in the rate of iron uptake, possibly by reducing the intracellular free heme concentration. It is concluded from these results that heme affects iron uptake by influencing the rate of transferrin endocytosis and recycling.     

Heme regulation of HeLa cell transferrin receptor number

The number of diferic transferrin receptors on HeLa cells decreases when cells are grown in iron-supplemented media. The experiments reported here suggest that heme is the iron-containing compound which serves as the signal for receptor number regulation. When HeLa cells were grown in the presence of hemin, transferrin receptor number decreased to a greater degree than when cells were grown in equivalent amounts of iron supplied as ferric ammonium citrate. Incubation of cells in conditions which increased cellular heme content resulted in a decrease in cellular transferrin receptors. Incubating cells with 5-aminolevulinic acid (thus bypassing the rate-limiting step in heme biosynthesis, 5-aminolevulinic acid synthase) led to a decrease in transferrin receptor number. Incubation of cells with an inhibitor of heme oxygenase, Sn-protoporphyrin IX, also led to a decrease in transferrin receptor number. When cellular heme content was decreased by inhibiting heme synthesis with succinylacetone (an inhibitor of 5-aminolevulinic acid dehydratase), or by depriving cells of iron with deferoxamine, an increase in HeLa cell transferrin receptor number was seen. When HeLa cells were incubated with inducers of heme oxygenase (CoCl2, SnCl2, Co-protoporphyrin IX), transferrin receptor number also increased. The effects of all compounds which alter transferrin receptor number were dependent on the concentration of the supplement, as well as the duration of the supplementation. These experiments suggest that intracellular heme content may be an important signal controlling transferrin receptor number.     

The antioxidant role of xanthurenic acid in the Aedes aegypti midgut during digestion of a blood meal

In the midgut of the mosquito Aedes aegypti, a vector of dengue and yellow fever, an intense release of heme and iron takes place during the digestion of a blood meal. Here, we demonstrated via chromatography, light absorption and mass spectrometry that xanthurenic acid (XA), a product of the oxidative metabolism of tryptophan, is produced in the digestive apparatus after the ingestion of a blood meal and reaches milimolar levels after 24 h, the period of maximal digestive activity. XA formation does not occur in the White Eye (WE) strain, which lacks kynurenine hydroxylase and accumulates kynurenic acid. The formation of XA can be diminished by feeding the insect with 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl] benzenesulfonamide (Ro-61-8048), an inhibitor of XA biosynthesis. Moreover, XA inhibits the phospholipid oxidation induced by heme or iron. A major fraction of this antioxidant activity is due to the capacity of XA to bind both heme and iron, which occurs at a slightly alkaline pH (7.5-8.0), a condition found in the insect midgut. The midgut epithelial cells of the WE mosquito has a marked increase in occurrence of cell death, which is reversed to levels similar to the wild type mosquitoes by feeding the insects with blood supplemented with XA, confirming the protective role of this molecule. Collectively, these results suggest a new role for XA as a heme and iron chelator that provides protection as an antioxidant and may help these animals adapt to a blood feeding habit.     

Abnormal iron delivery to the bone marrow in neonatal hypotransferrinemic mice

Hypotransferrinemic (HP) mice have a splicing defect inthe transferrin gene, resulting in <1% of the normal plasma levels of transferrin. They have severe anemia, suggesting that transferrin is essential for iron uptake by erythroid cells in the bone barrow. To clarify the significance of transferrin on iron delivery to the bone marrow, iron concentration and ⁵⁹Fe distribution were determined in 7-day-old HP mice. Iron concentration in the femur, bone containing the bone marrow, of HP mice was approximately twice higher than in wild type mice. Twenty-four h after injection of ⁵⁹FeCl₃, ⁵⁹Fe concentration in the bone and bone marrow of HP mice was also twice higher than in wild type mice. The present findings indicate that iron is abnormally delivered to the bone marrow of HP mice. However, the iron seems to be unavailable for the production of hemoglobin. These results suggest that transferrin-dependent iron uptake by erythroid cells in the bone marrow is essential for the development of erythrocytes.     

Iron metabolism in K562 erythroleukemic cells

Iron delivery to K562 cells is enhanced by desferrioxamine through induction of transferrin receptors. Experiments were performed to further characterize this event with respect to iron metabolism and heme synthesis. In control cells, up to 85% of the iron taken up from iron-transferrin was incorporated into ferritin, 7% into heme, and the remainder into compartments not yet identified. In cells grown with desferrioxamine, net accumulation of intracellular desferrioxamine (14-fold) was observed and iron incorporation into ferritin and heme was inhibited by 86% and 75%, respectively. In contrast, complete inhibition of heme synthesis in cells grown with succinylacetone had no effect on transferrin binding or iron uptake. Exogenous hemin (30 microM) inhibited transferrin binding and iron uptake by 70% and heme synthesis by 90%. These effects were already evident after 2 h. Thus, although heme production could be reduced by desferrioxamine, succinylacetone, and hemin, cell iron uptake was enhanced only by the intracellular iron chelator. The effects of exogenous heme are probably unphysiologic and the greater inhibition of iron flow into heme can be explained by effects on early steps of heme synthesis. We conclude that in this cell model a chelatable intracellular iron pool rather than heme synthesis mediates regulation of iron uptake.     

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system¹. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm^2,3. This pathway has been dissected through numerous in vitro studies^4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models^8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.     

Arrest, resorption, or maturation of oöcytes in Aedes aegypti: dependence on the quantity of blood and the interval between blood meals

ABSTRACT. After emergence, the follicles of A. aegypti double in length and the oöcytes may deposit a small amount of yolk, but within 2 days growth is arrested. Renewed growth and vitellogenesis, as well as the number of eggs finally produced, depends on the quantity of blood ingested. All females, given either a small (1 μl) or large (4 μl) meal of rat blood by enema, began yolk deposition in a nearly equal number of oöcytes, and each oöcyte had about the same amount of yolk 8 h later. Within 48 h, females fed 4 μl had each produced more than 100 eggs, whereas females fed 1 μl either had continued yolk deposition in some oöcytes, while most degenerated, or had all re-entered oögenic arrest. Consequently, 48 h after the 1 μl meal, a female had either c. 50 or 0 eggs. Even by 14 h after a 1-μl meal, females were either committed to re-enter oögenic arrest or to complete maturation of some oöcytes and resorb the yolk of others. This was shown by removing and examining one ovary 14 h after a blood meal and then giving a second blood meal. The second meal stimulated meal maturation in the remaining ovary, but only in those females whose oöcytes had been in oögenic arrest when the first ovary was examined; the second meal had no effect on females whose first ovary had contained both vitellogenic and degenerating oöcytes. Oösorption was not reversed by a second blood meal. Our results do not support the hypothesis that the female 'evaluates' the ingested meal and begins vitellogenesis in an 'appropriate' number of oöcytes. The results demonstrate that the ovary is an unreliable indicator of the frequency of blood-feeding, when females take a small meal.