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Aedes aegypti [Linnaeus in Hasselquist; yellow fever mosquito] transmits several viruses that infect millions of people each year, including Zika, dengue, yellow fever, chikungunya, and West Nile. Pathogen transmission occurs during blood feeding. Only the females blood feed as they require a bloodmeal for oogenesis; in the bloodmeal, holo-transferrin and hemoglobin provide the females with a high iron load. We are interested in the effects of the bloodmeal on the expression of iron-associated proteins in oogenesis. Previous data showed that following digestion of a bloodmeal, ovarian iron concentrations doubles by 72 hr. We have used shotgun proteomics to identify proteins expressed in Ae. aegypti ovaries at two oogenesis developmental stages following blood feeding, and tandem mass tag-labeling proteomics to quantify proteins expressed at one stage following feeding of a controlled iron diet. Our findings provide the first report of mosquito ovarian protein expression in early and late oogenesis. We identify proteins differentially expressed in the two oogenesis development stages. We establish that metal-associated proteins play an important role in Ae. aegypti oogenesis and we identify new candidate proteins that might be involved in mosquito iron metabolism. Finally, this work identified a unique second ferritin light chain subunit, the first reported in any species. The shotgun proteomic data are available via ProteomeXchange with identifier PXD005893, while the tandem mass tag-labeled proteomic data are available with identifier PXD028242.

Erythropoietin (Epo) drives iron (Fe) utilization for erythropoiesis, but the potentially resultant tissue iron deficiency (ID) can also impede brain development. Conversely, Epo binds to Epo receptors (EpoR) on immature brain oligodendrocytes and neurons, promoting growth and differentiation. The objective of the study was to examine the interaction between Epo and Fe on myelination in brain development during daily Epo treatment. Male and female Sprague-Dawley rats from postnatal day (P) P4-P12 modeled premature newborns. Dam-fed Fe-sufficient (IS) or postnatal ID groups were given daily subcutaneous sham or erythropoietic Epo injections (425 U. kg-1. d-1 ), ± oral Fe (6 mg. kg-1. d-1 ). Tissues and blood were collected and studied at P12. Epo in the ID groups, in the absence of oral Fe, stimulated microcytic ID anemia along with raising inflammatory markers. Both the microcytic anemia and inflammation improved in the ID + Epo + Fe group. Fe treatment positively impacted erythropoiesis and body Fe (µg/g) in all groups. Relative brain Fe (µg/g rat) was improved in the IS + Epo + Fe group. Brain Fe was not worsened in +Epo groups. Brain weight and brain Fe were related to plasma Epo levels. Amount of myelination was impacted by feeding type, but was not inhibited by Epo. Expression of a protein in myelin, mylein basic protein, was greater in all +Fe groups than -Fe groups. With therapeutic Epo, available body Fe was prioritized for erythropoiesis instead of brain, but Epo did not worsen brain Fe and potentially Epo improved myelination and maturation in the brain.

Feedback from employers, internship supervisors, and graduate schools tells us that we are doing a good job of teaching students academically/technically, but our graduates require greater training in communications, critical thinking/problem solving, and leadership/management. Because of their long-term importance to our graduates, we call these “career skills.” To address this issue a task force was established, and over a 2-year period, this group defined the College of Agriculture and Life Sciences [CALS (University of Arizona, Tucson)] Students Career Skills Competencies and vetted them through college faculty and more than 50 companies. We divided the CALS Student Career Skills Competencies into three categories: 1) communication skills, 2) critical thinking/problem solving skills, and 3) leadership/management skills. We are currently asking each degree-granting unit in the college how they do, or will, incorporate these competencies into their curricula, with the idea of incorporating the competencies into existing courses first, to avoid increasing the number of required credit hours for graduation and time to degree. Our goal is that by graduation, each student should have had multiple opportunities to hone each of these competencies.

Dengue, yellow fever, and Zika are viruses transmitted by yellow fever mosquito, Aedes aegypti [Linnaeus (Diptera: Culicidae)], to thousands of people each year. Mosquitoes transmit these viruses while consuming a blood meal that is required for oogenesis. Iron, an essential nutrient from the blood meal, is required for egg development. Mosquitoes receive a high iron load in the meal; although iron can be toxic, these animals have developed mechanisms for dealing with this load. Our previous research has shown iron from the blood meal is absorbed in the gut and transported by ferritin, the main iron transport and storage protein, to the ovaries. We now report the distribution of iron and ferritin in ovarian tissues before blood feeding and 24 and 72 h post-blood meal. Ovarian iron is observed in specific locations. Timing post-blood feeding influences the location and distribution of the ferritin heavy-chain homolog, light-chain homolog 1, and light-chain homolog 2 in ovaries. Understanding iron deposition in ovarian tissues is important to the potential use of interference in iron metabolism as a vector control strategy for reducing mosquito fecundity, decreasing mosquito populations, and thereby reducing transmission rates of vector-borne diseases.

The available genomic and proteomic information of non-model organisms is often underrepresented in public databases hindering their study at molecular, cellular, and physiological levels. Information on Zabrotes subfasciatus (Mexican bean weevil) is poorly represented in databases, yet it is a major pest of common beans. We report the transcriptome of Z. subfasciatus larvae; transcripts were sequenced using an Illumina RNA-Seq technology and assembled de novo identifying 29,029 unigenes with an average size of 1168 bp and an N50 value of 2196 bp. About 15,124 unigenes (52%) were functionally annotated and categorized. Further analysis revealed 30 unigene sequences encoding putative targets of the insecticidal PF2 lectin. The complete deduced amino acid sequences of eight selected proteins potentially related to insecticidal mechanism of Palo Fierro 2 (PF2) were used for predicting probable N-glycosylation sites and analyzing phylogenetic relationships with insect sequences. This work provides a dramatic increase in the genetic resources available for Coleopterans and set the basis for developing future studies on biological aspects and potential control strategies for Z. subfasciatus.

Ferritin is required for iron storage in vertebrates and for iron transport and storage in invertebrates, specifically insects. Classical ferritins consist of 24 subunits configured as a polyhedron wherein iron is held. The 24 subunits include light and heavy chains, each with specific functions. Several homologues of the light and heavy chains have been sequenced and studied in insects. In addition to iron transport and storage, ferritin has a role in dietary iron absorption, and functions as a protective agent preventing iron overload, decreasing oxidative stress, and reducing infection in these animals. The expression profile and regulation of a second ferritin light chain homologue (LCH2) in Aedes aegypti [Linnaeus (Diptera: Culicidae), yellow fever mosquito] was characterized in cells, animal developmental stages, and tissues post bloodmeal (PBM) by real-time PCR and immunoblot. Two previously studied ferritin subunits from Ae. aegypti, HCH and LCH1, along with LCH2 were immunoprecipitated and analyzed by mass spectrometry. The three Ae. aegypti ferritin subunits, HCH, LCH1, and LCH2, have different expression profiles and regulation with iron exposure, developmental stage, and tissue response PBM. Ae. aegypti expresses multiple and unique ferritin light chain subunits. Ae. aegypti, the vector for Zika, Dengue, and yellow fever, requires iron for oogenesis that is transported and stored in ferritin; this vector expresses a second light chain ferritin subunit homologue unlike any other species in which ferritin has been studied to date.

Secreted ferritin is the major iron storage and transport protein in insects. Here, we characterize the message and protein expression profiles of yellow fever mosquito (Aedes aegypti) ferritin heavy chain homologue (HCH) and light chain homologue (LCH) subunits in response to iron and bacterial challenge. In vivo experiments demonstrated tissue-specific regulation of HCH and LCH expression over time post-blood meal (PBM). Transcriptional regulation of HCH and LCH was treatment specific, with differences in regulation for naive versus mosquitoes challenged with heat-killed bacteria (HKB). Translational regulation by iron regulatory protein (IRP) binding activity for the iron-responsive element (IRE) was tissue-specific and time-dependent PBM. However, mosquitoes challenged with HKB showed little change in IRP/IRE binding activity compared to naive animals. The changes in ferritin regulation and expression in vivo were confirmed with in vitro studies. We challenged mosquitoes with HKB followed by a blood meal to determine the effects on ferritin expression, and demonstrate a synergistic, time-dependent regulation of expression for HCH and LCH.

Zabrotes subfasciatus (Boheman) is the main pest of common beans ( Phaselous vulgaris ). Wild legume seeds from Olneya tesota contain a lectin, PF2, that shows insecticidal activities against this insect. The binding of PF2 to midgut glycoproteins of 20-day-old larvae was evaluated using PF2 affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the proteins retained on the gel revealed several putative glycoproteins, ranging in mass from 17 to 97 kDa. Subsequent protein digestion and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) provided amino acid fragments that identified an α-tubulin, cytochrome c oxidase subunit I, an odorant receptor, and a lysozyme from available insect sequence databases. The potential of these proteins to serve as part of the mechanisms involved in the insecticidal activity of PF2 to Z. subfasciatus is discussed.

To gain a better understanding of the proteins associated with cold acclimation and winter dormancy in apple trees (Malus domestica), we studied the expression of a dehydrin gene encoding one of the important proteins involved in these processes. A novel gene, designated mddhn, was obtained from M. domestica cv. “Golden Delicious”. The full-length complementary DNA (cDNA) consists of 993 nucleotides with an open reading frame of 687 bp. The deduced amino acid sequence of 229 residues shows similarity to members of the dehydrin (DHN) family. The putative DHN presents a structure typical of DHN proteins that are composed of two consensus Y-segments, a stretch of serine residues, and four lysine-rich repeats (Y2SK4). This apple DHN shows the greatest similarity to members of both the YnKn and YnSKn classes of DHNs from peach and almond: 57% to peach (Y2SK3) and almond (Y2SK3 and Y2K4) and a lower percentage (35%) to peach (Y2K9). When we analyzed the available expressed sequence tag databases for additional apple DHNs, we identified a DHN of the class Y2SK4 with a deduced amino acid sequence with 79–98% identity among the cultivars “Royal Gala”, “Goldrush”, and the M9 rootstock that showed high identity to our DHN from “Golden Delicious” (92–98%). The transcripts of mddhn are highly expressed in bark and bud tissues when the plant is dormant and cold hardy in midwinter, but are not expressed in early spring when cold hardiness is lost and the buds are growing.

Ferritin is a multimer of 24 subunits of heavy and light chains. In mammals, iron taken into cells is stored in ferritin or incorporated into iron-containing proteins. Very little ferritin is found circulating in mammalian serum; most is retained in the cytoplasm. Female mosquitoes, such as Aedes aegypti (yellow fever mosquito, Diptera), require a blood meal for oogenesis. Mosquitoes receive a potentially toxic level of iron in the blood meal which must be processed and stored. We demonstrate by (59)Fe pulse-chase experiments that cultured A. aegypti larval CCL-125 cells take up iron from culture media and store it in ferritin found mainly in the membrane fraction and secrete iron-loaded ferritin. We observe that in these larval cells ferritin co-localizes with ceramide-containing membranes in the absence of iron. With iron treatment, ferritin is found associated with ceramide-containing membranes as well as in cytoplasmic non-ceramide vesicles. Treatment of CCL-125 cells with iron and CI-976, an inhibitor of lysophospholipid acyl transferases, disrupts ferritin secretion with a concomitant decrease in cell viability. Interfering with ferritin secretion may limit the ability of mosquitoes to adjust to the high iron load of the blood meal and decrease iron delivery to the ovaries reducing egg numbers.

Available evidence has shown that transferrins are involved in iron metabolism, immunity and development in eukaryotic organisms including insects. Here we characterize the gene and message expression profile of Aedes aegypti transferrin 2 (AaTf2) in response to iron, bacterial challenge and life stage. We show that AaTf2 shares a low similarity with A. aegypti transferrin 1 (AaTf1), but higher similarity with mammalian transferrins and avian ovotransferrin. Iron-binding pocket analysis indicates that AaTf2 has residue substitutions of Y188F, T120S, and R124S in the N lobe, and Y517N, H585N, T452S, and R456T in the C lobe, which could alter or reduce iron-binding activity. In vivo studies of message expression reveal that AaTf2 message is expressed at higher levels in larva and pupa, as well as adult female ovaries 72h post blood meal (PBM) and support that AaTf2 could play a role in larval and pupal development and in late physiological events of the gonotrophic cycle. Bacterial challenge significantly increases AaTf1 expression in ovaries at 0 and 24h PBM, but decreases AaTf2 expression in ovaries at 72h PBM, suggesting that AaTf1 and AaTf2 play different roles in immunity of female adults during a gonotrophic cycle.

Ticks are among the most important vectors of a wide range of human and animal diseases. During blood feeding, ticks are exposed to an enormous amount of free iron that must be appropriately used and detoxified. However, the mechanism of iron metabolism in ticks is poorly understood. Here, we show that ticks possess a complex system that efficiently utilizes, stores and transports non-heme iron within the tick body. We have characterized a new secreted ferritin (FER2) and an iron regulatory protein (IRP1) from the sheep tick, Ixodes ricinus , and have demonstrated their relationship to a previously described tick intracellular ferritin (FER1). By using RNA interference-mediated gene silencing in the tick, we show that synthesis of FER1, but not of FER2, is subject to IRP1-mediated translational control. Further, we find that depletion of FER2 from the tick plasma leads to a loss of FER1 expression in the salivary glands and ovaries that normally follows blood ingestion. We therefore suggest that secreted FER2 functions as the primary transporter of non-heme iron between the tick gut and the peripheral tissues. Silencing of the fer1 , fer2 , and irp1 genes by RNAi has an adverse impact on hatching rate and decreases postbloodmeal weight in tick females. Importantly, knockdown of fer2 dramatically impairs the ability of ticks to feed, thus making FER2 a promising candidate for development of an efficient anti-tick vaccine.

Zabrotes subfasciatus (Boheman) is the main pest of common beans (Phaselous vulgaris). Some wild legume seeds may contain lectins with insecticidal activities against this insect. The larval developments of Z. subfasciatus on seeds of Olneya tesota (a desert wild legume) and on artificial seeds containing purified PF2 lectin were evaluated. PF2 susceptibility to proteolysis was assessed by incubation with midgut extract at different times. PF2 binding to midgut glycoconjugates was assessed by histochemistry. A reduced level of oviposition and a lack of emergence of adult beetles were observed in O. tesota seeds (compared to common beans), and in artificial seeds containing PF2 at 0.5 and 1%. PF2 was resistant to larval midgut proteolysis for 24 h, while PHA-E (lectin control) was fully digested after 4 h. Histochemistry analysis of midguts incubated with PF2 showed recognition for microvillae and possibly with peritrophic gel. On the other hand, PHA-E exhibited no interaction with larval midgut glycoproteins. Proteolysis resistance and glycan recognition could in part explain why PF2 is toxic to Z. subfasciatus while PHA is not.

Mosquitoes must blood feed in order to complete their life cycle. The blood meal provides a high level of iron that is required for egg development. We are interested in developing control strategies that interfere with this process. We report the temporal effects of iron exposure on iron metabolism of Aedes aegypti larval cells. These cells take up iron in linear relationship to exposure time and distribute the iron primarily to the membranes. Iron uptake increases cytoplasmic, membrane and secreted ferritin. Membrane ferritin is abundant in cells treated with iron, increases in cells in the absence of iron exposure and is associated with the secretory pathway. Our data suggest that in contrast to mammals, mosquitoes control intracellular iron levels by producing membrane ferritin in anticipation of an iron load such as that provided by a blood meal and support the hypothesis that secreted ferritin is a primary iron storage protein for these animals.

Ferritin is a 24-subunit molecule, made up of heavy chain (HC) and light chain (LC) subunits, which stores and controls the release of dietary iron in mammals, plants, and insects. In mosquitoes, dietary iron taken in a bloodmeal is stored inside ferritin. Our previous work has demonstrated the transport of dietary iron to the ovaries via ferritin during oogenesis. We evaluated the localization of ferritin subunits inside CCL-125 [Aedes aegypti Linnaeus (Diptera: Culicidae), yellow fever mosquito] and 4a3b [Anopheles gambiae Giles (Diptera: Culicidae), African malaria mosquito] cells under various iron treatment conditions to further elucidate the regulation of iron metabolism in these important disease vectors and to observe the dynamics of the intracellular ferritin subunits following iron administration. Deconvolution microscopy captured 3D fluorescent images of iron-treated mosquito cells to visualize the ferritin HC and LC homologue subunits (HCH and LCH, respectively) in multiple focal planes. Fluorescent probes were used to illuminate cell organelles (i.e., Golgi apparatus, lysosomes, and nuclei) while secondary probes for specific ferritin subunits demonstrated abundance and co-localization within organelles. These images will help to develop a model for the biochemical regulation of ferritin under conditions of iron exposure, and to advance novel hypotheses for the crucial role of iron in mosquito vectors.

Female mosquitoes require a blood meal for oogenesis and receive a substantial iron load in this meal in the form of holo-transferrin and hemoglobin. During the course of digestion and absorption i...

All animals require iron for survival. This requirement reflects the role of this mineral as a cofactor of numerous proteins. However, under physiological conditions, Fe(2+) oxidizes to Fe(3+) encouraging the formation of toxic free radicals. In mammals, the potential for oxidative damage from iron is minimized by binding iron to proteins. Mammalian iron metabolism is complex and numerous proteins are involved in iron absorption, transport, uptake and utilization. We have analyzed the Anopheles gambiae translated protein database for candidates that show identity to proteins involved in mammalian iron metabolism (Holt et al., 2002. The genome sequence of the malaria mosquito Anopheles gambiae. Science 298, 129-149). Our results indicate that proteins involved in iron absorption and intracellular iron utilization are, for the most part, conserved in A. gambiae. In contrast, proteins involved in the pathways of iron export from the gut, transport in hemolymph and uptake at peripheral tissues in mosquitos differ from those for mammals.

The yellow fever mosquito, Aedes aegypti, must blood feed in order to complete her life cycle. The blood meal provides a high level of iron that is required for egg development. We are interested in developing control strategies that interfere with this process. We show that A. aegypti larval cells synthesize and secrete ferritin in response to iron exposure. Cytoplasmic ferritin is maximal at low levels of iron, consists of both the light chain (LCH) and heavy chain (HCH) subunits and reflects cytoplasmic iron levels. Secreted ferritin increases in direct linear relationship to iron dose and consists primarily of HCH subunits. Although the messages for both subunits increase with iron treatment, our data indicate that mosquito HCH synthesis could be partially controlled at the translational level as well. Importantly, we show that exposure of mosquito cells to iron at low concentrations increases cytoplasmic iron, while higher iron levels results in a decline in cytoplasmic iron levels indicating that excess iron is removed from mosquito cells. Our work indicates that HCH synthesis and ferritin secretion are key factors in the response of mosquito cells to iron exposure and could be the primary mechanisms that allow these insects to defend against an intracellular iron overload.

Inhalation of airborne pollution particles that contain iron can result in a variety of detrimental changes to lung cells and tissues. The lung iron burden can be substantially increased by exposure to cigarette smoke, and cigarette smoke contains iron particulates, as well as several environmental toxins, that could influence intracellular iron status. We are interested in the effects of environmental contaminants on intracellular iron metabolism. We initiated our studies using lung A549 type II epithelial cells as a model, and we evaluated the effects of iron dose and smoke treatment on several parameters of intracellular iron metabolism. We show that iron at a physiological dose stimulates ferritin synthesis without altering the transferrin receptor (TfR) mRNA levels of these cells. This is mediated primarily by a reduction of iron regulatory protein 2. Higher doses of iron reduce iron regulatory protein-1 binding activity and are accompanied by a reduction in TfR mRNA. Thus, for A549 cells, different mechanisms influencing IRP-IRE interaction allow ferritin translation in the presence of TfR mRNA to provide for iron needs and yet prevent excessive iron uptake. More importantly, we report that smoke treatment diminishes ferritin levels and increases TfR mRNA of A549 cells. Ferritin serves as a cytoprotective agent against oxidative stress. These data suggest that exposure of lung cells to low levels of smoke as are present in environmental pollutants could result in reduced cytoprotection by ferritin at a time when iron uptake is sustained, thus enhancing the possibility of lung damage by iron-mediated oxidative stress.

Diseases transmitted by hematophagous (blood-feeding) insects are responsible for millions of human deaths worldwide. In hematophagous insects, the blood meal is important for regulating egg maturation. Although a high concentration of iron is toxic for most organisms, hematophagous insects seem unaffected by the iron load in a blood meal. One means by which hematophagous insects handle this iron load is, perhaps, by the expression of iron-binding proteins, specifically the iron storage protein ferritin. In vertebrates, ferritin is an oligomer composed of two types of subunits called heavy and light chains, and is part of the constitutive antioxidant response. Previously, we found that the insect midgut, a main site of iron load, is also a primary site of ferritin expression and that, in the yellow fever mosquito, Aedes aegypti, the expression of the ferritin heavy-chain homologue (HCH) is induced following blood feeding. We now show that the expression of the Aedes ferritin light-chain homologue (LCH) is also induced with blood-feeding, and that the genes of the LCH and HCH are tightly clustered. mRNA levels for both LCH- and HCH-genes increase with iron, H2O2 and hemin treatment, and the temporal expression of the genes is very similar. These results confirm that ferritin could serve as the cytotoxic protector in mosquitoes against the oxidative challenge of the bloodmeal. Finally, although the Aedes LCH has no iron responsive element (IRE) at its 5′-untranslated region (UTR), the 5′-UTR contains several introns that are alternatively spliced, and this alternative splicing event is different from any ferritin message seen to date.

Iron regulatory protein (IRP) blocks ribosomal assembly by binding to an iron responsive element (IRE) located proximal (

Ribonucleotide reductase catalyses the de novo synthesis of deoxyribonucleotides. Class I reductases use an iron center to generate a tyrosyl free radical that can initiate formation of the deoxyribonucleotide. These reductases are alpha 2 beta 2 holoenzymes, and the subunits are denoted as R1 and R2. R1 contains the allosteric binding site and the active site, whereas R2 contains a binuclear iron center that initiates formation of the tyrosyl radical. We have cloned and sequenced the cDNAs encoding the R1 and R2 subunit in the yellow fever mosquito, Aedes aegypti. The messages for these proteins are increased in response to blood-feeding.

Iron regulatory proteins (IRPs) control the synthesis of various proteins at the translational level by binding to iron responsive elements (IREs) in the mRNAs. Iron, infection, and stress can alter IRP/IRE binding activity. Insect messenger RNAs for ferritin and succinate dehydrogenase subunit b have IREs that are active translational control sites. We have cloned and sequenced cDNAs encoding proteins from the IRP1 family for the mosquitoes, Aedes aegypti and Anopheles gambiae. Both deduced amino acid sequences show substantial similarity to human IRP1 and Drosophila IRP1A and IRP1B, and all of the residues thought to be involved in aconitase activity and iron-sulfur cluster formation are conserved. Recombinant A. aegypti IRP1 binds to transcripts of the IREs of mosquito or human ferritin subunit mRNAs. No significant change in A. gambiae IRP1 messenger RNA could be detected during the various developmental stages of the life cycle, following iron loading by blood feeding, or after bacterial or parasitic infections. These data suggest that there is no change in gene transcription. Furthermore, bacterial challenge of A. gambiae cells did not change IRP1 protein levels. In contrast, IRP1 binding activity for the IRE was elevated following immune induction. These data show that changes in IRP1/IRE binding activity occur as part of the insect immune response.

Africa's Bane Tsetse are blood-feeding, fast-flying flies that transmit a range of Trypanosoma spp. protozoan pathogens, which cause sleeping sickness in humans and their nagana in their livestock. The International Glossina Genome Initiative (p. 380 ) sequenced the genome of Glossina morsitans and identified the genes for many attributes of the tsetse's remarkable biology, including viviparity and the expression of analogs of mammalian milk proteins. Tsetse are host to several specific symbionts that appear to synthesize essential nutrients for the fly and also to hitherto undiscovered parasitoid-derived viruses. Deeper exploration of this genome will reveal what makes these fly species so host- and trypanosome specific.

Insect midguts, as well as other animals, are rich in glycoconjugates that have important biological functions required for growth and development. Dietary lectins can bind to gut glycoconjugates a...

Mammalian ferritin subunit synthesis is controlled at the translational level by the iron regulatory protein 1 (IRP1)/iron responsive element (IRE) interaction. Insect haemolymph ferritin subunit messages have an IRE in the 5′-untranslated region (UTR). We have shown that recombinant M. sexta IRP1 represses the in vitro translation of both the heavy and light chain ferritin subunits from this species without altering transcription. Deletion of either the 5′-UTR or the IRE from the mRNA abolishes IRP1 repression. Our studies indicated that the translational control of ferritin synthesis by IRP/IRE interaction could occur in insects in a manner similar to that of mammals. To our knowledge, this is the first report of the control of insect ferritin synthesis by IRP1/IRE interaction. Furthermore, this is the first indication that the synthesis of a secreted ferritin subunit can also be controlled in this manner.

We have isolated and sequenced a genomic clone encoding the 24- and 26-kDa ferritin subunits in the mosquito Aedes aegypti (Rockefeller strain). The A. aegypti gene differs from other known ferritin genes in that it possesses an additional intron and an unusually large second intron. The additional intron is located within the 5' untranslated region, between the CAP site and the start codon. The second intron contains numerous putative transposable elements. In addition, unlike the human and rat ferritin genes, the A. aegypti ferritin gene is a single copy gene, located at 88.3% FLpter on the q-arm of chromosome 1. Primer extension analysis indicates that the A. aegypti ferritin gene has multiple transcriptional start sites. A differential usage of these sites is observed with varied cellular iron concentrations.

In yellow fever mosquito cells (Aag2 clone), iron treatment induces a threefold increase in ferritin message (fer mRNA) and protein (ferritin) by 16 h. These data contrast with work in mammalian hepatocytes and fibroblasts in which fer mRNA levels do not change with iron stimulation, but ferritin levels increase 50-fold. Pretreatment of the Aag2 cells with actinomycin D blocks induction of fer mRNA and reduces the ferritin subunit synthesis, suggesting that iron induction of ferritin subunit synthesis is subjected to transcriptional control. A putative iron-regulatory protein has also been identified in cytoplasmic extracts from Aag2 cells.

Human peritoneal cells isolated from dialysis effluent have in vivo maturated human macrophages that could serve as a model for studying lipoprotein metabolism and foam cell formation. We previously characterized the low density lipoprotein (LDL) and acetylated LDL (acetyl-LDL) receptor activities of human total peritoneal cells. Now, we provide evidence that both LDL and acetyl-LDL stimulate acylCoA cholesterol:acyl transferase (ACAT) activity of peritoneal cells. Prolonged incubation of cells with LDL results in suppression of ACAT activity, while incubation with acetyl-LDL results in elevated and sustained enzyme activity. When human peritoneal cells were analyzed using flow cytometry, the cell population showed reactivity for CD2, CD4, CD8, CD20, CD14 and HLA-DR antigens. Purified human peritoneal mononuclear cells degraded LDL. Human peritoneal macrophages formed foam cells when exposed to LDL or acetyl-LDL in culture, and lipid deposition increased with incubation time. Macrophages incubated in the presence of butylated hydroxy toluene and LDL did not form foam cells.

Amylases are an important family of enzymes involved in insect carbohydrate metabolism that are required for the survival of insect larvae. For this reason, enzymes from starch-dependent insects are targets for insecticidal control. PF2 ( Olneya tesota ) is a lectin that is toxic to Zabrotes subfasciatus (Coleoptera: Bruchidae) larvae. In this study, we evaluated recognition of the PF2 lectin to α-amylases from Z. subfasciatus midgut and the effect of PF2 on α-amylase activity. PF2 caused a decrease of total amylase activity in vitro. Subsequently, several α-amylase isoforms were isolated from insect midgut tissues using ion exchange chromatography. Three enzyme isoforms were verified by an in-gel assay for amylase activity; however, only one isoform was recognized by antiamylase serum and PF2. The identity of this Z. subfasciatus α-amylase was confirmed by liquid chromatography−tandem mass spectrometry. The findings strongly suggest that a glycosylated α-amylase isoform from larval Z. subfasciatus midgut interacts with PF2, which interferes with starch digestion.

▪ Abstract The suggestion from nutritional studies with mammals of a link between iron and copper metabolism has been reinforced by recent investigations with yeast cells. Iron must be in the reduced ferrous (FeII) state for uptake by yeast cells, and reoxidation to ferric (FeIII) by a copper oxidase is part of the transport process. Thus, yeast cells deficient in copper are unable to absorb iron. In an analogous way, animals deficient in copper appear to be unable to move FeII out of cells, probably because it cannot be oxidized to FeIII. Invertebrate animals use copper and iron in ways very similar to vertebrates, with some notable exceptions. In the cases where vertebrates and invertebrates are similar, the latter may be useful models for vertebrate metabolism. In cases where they differ (e.g. predominance of serum ferritin in insects, oxygen transport by a copper protein in many arthropods, central importance of phenoloxidase, a copper enzyme in arthropods), the differences may represent processes that are exaggerated in invertebrates and thus more amenable to study in these organisms. On the other hand, they may represent processes unique to invertebrates, thus providing novel information on species diversity.

The feasibility of using human cells isolated from peritoneal dialysis effluent as a model for studying lipoprotein and cholesterol metabolism was investigated. Human peritoneal cells degraded low density lipoproteins (LDL) and acetylated LDL (acetyl-LDL) by saturable, high affinity receptor-mediated processes. Positive correlations of the percentage of macrophage cells with degradation rates of LDL (r = 0.742; p0.05) and acetyl-LDL (r = 0.931; p0.01) indicated that macrophage cells significantly contributed to lipoprotein degradation. LDL receptor-mediated degradation was calcium dependent, and sensitive to pronase and chloroquine treatments. The receptor exhibited specificity for lipoproteins containing apolipoprotein B (apoB) or apolipoprotein E (apoE). Exposure of cells to LDL for 24 hrs significantly down-regulated LDL receptor-mediated degradation. Acetyl-LDL receptor-mediated degradation was calcium independent, inhibited by chloroquine, and was sensitive to pronase and fucoidin treatments. The scavenger receptor exhibited specificity for only acetyl-LDL. These results demonstrate that human peritoneal cells can provide a source of human tissue macrophages suitable for studies of cholesterol and lipoprotein metabolism and offer the opportunity for comparison of metabolic characteristics of in vivo maturated macrophages with available macrophage-like cell lines.

We report methods for the rapid purification of two iron-binding proteins from larval hemolymph of Manduca sexta. Ferritin was purified in two steps by density gradient ultracentrifugation. To accomplish this, we utilized the relatively high level of ferritin present in the hemolymph of this animal and augmented the density of the protein in vivo by injection of iron sulfate. Nitrocellulose blots analyzed by laser densitometry showed hemolymph from iron-injected insects contained about 0.4 mg of ferritin per ml (approximately 0.7% of total hemolymph protein); of this, 62% was found as pure ferritin in the pellet formed during ultracentrifugation. Following the density ultracentrifugation, we purified transferrin from the hemolymph subphase by immobilized metal ion affinity chromatography using a new gel, Novarose-SE1000/40 coupled to dipicolylamine (DPA) chelated with nickel. Higher capacity Ni 2+ DPA-gel permitted good resolution of transferrin in the first chromatography; a lower capacity of the same gel allowed purification of transferrin in a second step. Overall transferrin recovery was 52%. Larval hemolymph contained 0.770 mg transferrin/ml, representing about 1.3% of the total protein.

Ferritin, an iron storage protein, was isolated from larvae and pupae of Aedes aegypti grown in an iron-rich medium. Mosquito ferritin is a high molecular weight protein composed of several different, relatively small, subunits. Subunits of molecular mass 24, 26, and 28 kDa are equally abundant, while that of 30 kDa is present only in small amounts. The N-terminal sequence of the 24 and 26 kDa subunits are identical for the first 30 amino acids, while that of the 28 kDa subunit differs. Studies using antiserum raised against a subunit mixture showed that the ferritin subunits were present in larvae, pupae, and adult females, and were increased in animals exposed to excess iron. The antiserum also was used to screen a cDNA library from unfed adult female mosquitoes. Nine clones were obtained that differed only in a 27 bp insertion in the 3′ end. Rapid amplification of cDNA ends (RACE) was used to obtain the complete protein coding sequence. A putative iron-responsive element (IRE) is present in the 5′-untranslated region. The deduced amino acid sequence shows a typical leader sequence, consistent with the fact that most insect ferritins are secreted, rather than cytoplasmic proteins. The sequence encodes a mature polypeptide of 20,566 molecular weight, smaller than the estimated size of any of the subunits. However, the sequence exactly matches the N-terminal sequences of the 24 and 26 kDa subunits as determined by Edman degradation. Of the known ferritin sequences, that of the mosquito is most similar to that of somatic cells of a snail. © 1995 Wiley-Liss, Inc.

Background Many studies have been done evaluating transferrin in insects. Genomic analyses indicate that insects could have more than one transferrin. However, the most commonly studied insect transferrin, Tsf1, shows greatest homology to mammalian blood transferrin. Scope of review Aspects of insect transferrin structure compared to mammalian transferrin and the roles transferrin serves in insects are discussed in this review. Major conclusions Insect transferrin can have one or two lobes, and can bind iron in one or both. The iron binding ligands identified for the lobes of mammalian blood transferrin are generally conserved in the lobes of insect transferrins that have an iron binding site. Available information supports that the form of dietary iron consumed influences the regulation of insect transferrin. Although message is expressed in several tissues in many insects, fat body is the likely source of hemolymph transferrin. Insect transferrin is a vitellogenic protein that is down-regulated by Juvenile Hormone. It serves a role in transporting iron to eggs in some insects, and transferrin found in eggs appears to be endowed from the female. In addition to the roles of transferrin in iron delivery, this protein also functions to reduce oxidative stress and to enhance survival of infection. General significance Future studies in Tsf1 as well as the other insect transferrins that bind iron are warranted because of the roles of transferrin in preventing oxidative stress, enhancing survival to infections and delivering iron to eggs for development. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.

We measured red blood cell zinc protoporphyrin/heme (ZnPP/H) ratios in premature infants at hospital discharge. ZnPP/heme ratios correlated directly with red blood cell distribution width and reticulocyte number. As in other populations, ZnPP/H ratios may provide a simple measure of iron-deficient erythropoiesis in premature infants.

Insects transmit millions of cases of disease each year, and cost millions of dollars in agricultural losses. The control of insect-borne diseases is vital for numerous developing countries, and the management of agricultural insect pests is a very serious business for developed countries. Control methods should target insect-specific traits in order to avoid non-target effects, especially in mammals. Since insect cells have had a billion years of evolutionary divergence from those of vertebrates, they differ in many ways that might be promising for the insect control field—especially, in iron metabolism because current studies have indicated that significant differences exist between insect and mammalian systems. Insect iron metabolism differs from that of vertebrates in the following respects. Insect ferritins have a heavier mass than mammalian ferritins. Unlike their mammalian counterparts, the insect ferritin subunits are often glycosylated and are synthesized with a signal peptide. The crystal structure of insect ferritin also shows a tetrahedral symmetry consisting of 12 heavy chain and 12 light chain subunits in contrast to that of mammalian ferritin that exhibits an octahedral symmetry made of 24 heavy chain and 24 light chain subunits. Insect ferritins associate primarily with the vacuolar system and serve as iron transporters—quite the opposite of the mammalian ferritins, which are mainly cytoplasmic and serve as iron storage proteins. This review will discuss these differences.

Porath and co-workers first described immobilized metal ion affinity chromatography (IMAC) in 1975 (Nature268, 598–599). Since that time IMAC has been used to isolate proteins, peptides, and nucleic acids and for cell separation. In IMAC, the metal ion is immobilized to a solid support matrix via coordinate bonding with a chelating agent. Similarly, retention of a solute on the column depends upon formation of coordinate bonds between electron-donating species of the solute in the mobile phase and the immobilized metal ion. The best-studied interaction of this nature is protein or peptide adsorption by coordinate bonding of the imidazole nitrogen of histidine side chains with certain metal ions. By judicious selection of the chelating agent, the metal ion, and the chromatography conditions, IMAC can be tailored to isolate desired constituents. To this end, we list several chelating agents that have been used in IMAC and introduce a new chelating agent, tris(2-aminoethyl)amine (Tren) and we provide protocols for activation of the matrix and the coupling of Tren. We also discuss the basic principles of IMAC to provide ground rules for initiating protein separation. Finally, we share a few applications of IMAC that demonstrate the substantive potential of this rapidly developing separation technology.

Iron is an essential element of living cells and organisms as a component of numerous metabolic pathways. Hemoglobin and ferric-transferrin in vertebrate host blood are the two major iron sources for female mosquitoes. We used inductively coupled plasma mass spectrometry (ICP-MS) and radioisotope labeling to quantify the fate of iron supplied from hemoglobin or as transferrin in Aedes aegypti. At the end of the first gonotrophic cycle, approximately 87% of the ingested total meal heme iron was excreted, while 7% was distributed into the eggs and 6% was stored in different tissues. In contrast, approximately 8% of the iron provided as transferrin was excreted and of that absorbed, 77% was allocated to the eggs and 15% distributed in the tissues. Further analyses indicate that of the iron supplied in a blood meal, approximately 7% appears in the eggs and of this iron 98% is from hemoglobin and 2% from ferric-transferrin. Whereas, of iron from a blood meal retained in body of the female, approximately 97% is from heme and1% is from transferrin. Evaluation of iron-binding proteins in hemolymph and egg following intake of (59)Fe-transferrin revealed that ferritin is iron loaded in these animals, and indicate that this protein plays a critical role in meal iron transport and iron storage in eggs in A. aegypti.