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Independent StudyNSC 399 (Spring 2016)
Mineral MetabolismACBS 622A (Spring 2016)
Mineral MetabolismNSC 622A (Spring 2016)
- Ray, D. T., Winzerling, J. J., & Staten, M. E. (2017). Career Skills: Our Process and Where we are Today. HortTechnology, 27(5), 586-590. doi:10.21273/HORTTECH03671-17
- Geiser, D. L., Zhou, G., Mayo, J. J., & Winzerling, J. J. (2013). The effect of bacterial challenge on ferritin regulation in the yellow fever mosquito, Aedes aegypti. Insect Science, 20(5), 601-619.More infoPMID: 23956079;Abstract: 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 naïve 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 naïve 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. © 2012 Institute of Zoology, Chinese Academy of Sciences.
- Geiser, D. L., Zhou, G., Mayo, J. J., & Winzerling, J. J. (2013). The effect of bacterial challenge on ferritin regulation in the yellow fever mosquito, Aedes aegypti. Insect science, 20(5).More infoSecreted 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 naïve 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 naïve 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.
- Geiser, D. L., & Winzerling, J. J. (2012). Insect transferrins: Multifunctional proteins. Biochimica et Biophysica Acta - General Subjects, 1820(3), 437-451.More infoPMID: 21810453;Abstract: 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. © 2011 Elsevier B.V. All rights reserved.
- Geiser, D. L., & Winzerling, J. J. (2012). Insect transferrins: multifunctional proteins. Biochimica et biophysica acta, 1820(3).More infoMany 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.
- Lagarda-Diaz, I., Robles-Burgeño, M. R., Guzman-Partida, A. M., Geiser, D., Winzerling, J., & Vazquez-Moreno, L. (2012). Binding of PF2 lectin from Olneya tesota to gut proteins of Zabrotes subfasciatus larvae associated with the insecticidal mechanism. Journal of Agricultural and Food Chemistry, 60(9), 2398-2402.More infoPMID: 22288827;Abstract: 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. © 2012 American Chemical Society.
- Lagarda-Diaz, M., Robles-Burgeño, M., Guzman-Partida, M., Geiser, M., Winzerling, M., & Vazquez-Moreno, M. (2012). Binding of PF2 lectin from Olneya tesota to gut proteins of Zabrotes subfasciatus larvae associated with the insecticidal mechanism. Journal of agricultural and food chemistry, 60(9).More infoZabrotes 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.
- Makhoul, M., Taren, M., Duncan, M., Pandey, M., Thomson, M., Winzerling, M., Muramoto, M., & Shrestha, M. (2012). Risk factors associated with anemia, iron deficiency and iron deficiency anemia in rural Nepali pregnant women. The Southeast Asian journal of tropical medicine and public health, 43(3).More infoWe conducted a cross sectional study to investigate risk factors associated with severe anemia [hemoglobin (Hb) < 8.0 g dl(-1)] and poor iron status among Nepali pregnant women. Socio-demographic, anthropometric, health and dietary data were collected from 3,531 women living in the southeastern plains of Nepal. Stool samples were analyzed for intestinal helminthes. Dark adaptation was assessed using the Night Vision Threshold Test (NVTT). Hb levels were measured in all subjects to detect anemia and the soluble transferrin receptor (sTfR) was measured among a subsample of 479 women. The iron status categories were: 1) normal (Hb> or = 11.0 g/dl and sTfR < or = 8.5 mg/l); 2) anemia without iron deficiency (Hb or = 11.0 g/dl and sTfR>8.5 mg/l); and 4) iron deficiency anemia (IDA): (Hb8.5 mg/l). Factors associated with severe anemia and poor iron status were determined using logistic regression. Hookworm infection increased the risk for developing severe anemia [adjusted odds ratio (AOR): 4.26; 95% CI 1.67-10.89; p
- Makhoul, Z., Taren, D., Duncan, B., Pandey, P., Thomson, C., Winzerling, J., Muramoto, M., & Shrestha, A. (2012). Risk factors associated with anemia, iron deficiency and iron deficiency anemia in rural Nepali pregnant women. Southeast Asian Journal of Tropical Medicine and Public Health, 43(3), 735-745.More infoPMID: 23077854;Abstract: We conducted a cross sectional study to investigate risk factors associated with severe anemia [hemoglobin (Hb) < 8.0 g dl -1] and poor iron status among Nepali pregnant women. Socio-demographic, anthropometric, health and dietary data were collected from 3,531 women living in the southeastern plains of Nepal. Stool samples were analyzed for intestinal helminthes. Dark adaptation was assessed using the Night Vision Threshold Test (NVTT). Hb levels were measured in all subjects to detect anemia and the soluble transferrin receptor (sTfR) was measured among a subsample of 479 women. The iron status categories were: 1) normal (Hb≥11.0 g/dl and sTfR≤8.5 mg/l); 2) anemia without iron deficiency (Hb8.5 mg/l); and 4) iron deficiency anemia (IDA): (Hb8.5 mg/l). Factors associated with severe anemia and poor iron status were determined using logistic regression. Hookworm infection increased the risk for developing severe anemia [adjusted odds ratio (AOR): 4.26; 95% CI 1.67-10.89; p
- Sarabia-Sainz, A., Ramo-Clarmont, G., & Winzerling, J. (2011). Bacterial recognition of thermal glycation products derived from procine serum albumin with lactose.. Acta Biochimica Polonica, 58, 95-100.
- Sarabia-Sainz, A., Ramos-Clamont, G., Winzerling, J., & Vazquez-Moreno, L. (2011). Bacterial recognition of thermal glycation products derived from porcine serum albumin with lactose. Acta Biochimica Polonica, 58(1), 95-100.More infoPMID: 21403918;Abstract: Recently, glyco-therapy is proposed to prevent the interaction of bacterial lectins with host ligands (glycoconjugates). This interaction represents the first step in infection. Neoglycans referred to as PSA-Lac (PSA-Glu (β1-4) Gal) were obtained by conjugation of porcine serum albumin (PSA) with lactose at 80 °C, 100 °C and 120 °C. Characterization studies of the products showed that PSA could contain 1, 38 or 41 added lactoses, depending on the reaction temperature. These neoglycans were approximately 10 times more glycated than PSA-Lac obtained in previous work. Lactose conjugation occurred only at lysines and PSA-Lac contained terminal galactoses as confirmed by Ricinus communis lectin recognition. Furthermore, Escherichia coli K88+, K88ab, K88ac and K88ad adhesins showed affinity toward all PSA-Lac neoglycans, and the most effective was the PSA-Lac obtained after 100 °C treatment. In vitro, this neoglycan partially inhibited the adhesion of E. coli K88+ to piglet mucin (its natural ligand). These results provide support for the hypothesis that glycated proteins can be used as an alternative for bioactive compounds for disease prevention.
- Sarabia-Sainz, A., Ramos-Clamont, G., Winzerling, J., & Vázquez-Moreno, L. (2011). Bacterial recognition of thermal glycation products derived from porcine serum albumin with lactose. Acta biochimica Polonica, 58(1).More infoRecently, glyco-therapy is proposed to prevent the interaction of bacterial lectins with host ligands (glycoconjugates). This interaction represents the first step in infection. Neoglycans referred to as PSA-Lac (PSA-Glu (β1-4) Gal) were obtained by conjugation of porcine serum albumin (PSA) with lactose at 80 °C, 100 °C and 120 ºC. Characterization studies of the products showed that PSA could contain 1, 38 or 41 added lactoses, depending on the reaction temperature. These neoglycans were approximately 10 times more glycated than PSA-Lac obtained in previous work. Lactose conjugation occurred only at lysines and PSA-Lac contained terminal galactoses as confirmed by Ricinus communis lectin recognition. Furthermore, Escherichia coli K88+, K88ab, K88ac and K88ad adhesins showed affinity toward all PSA-Lac neoglycans, and the most effective was the PSA-Lac obtained after 100 ºC treatment. In vitro, this neoglycan partially inhibited the adhesion of E. coli K88+ to piglet mucin (its natural ligand). These results provide support for the hypothesis that glycated proteins can be used as an alternative for bioactive compounds for disease prevention.
- Pham, D. Q., & Winzerling, J. J. (2010). Insect ferritins: Typical or atypical?. Biochimica et Biophysica Acta - General Subjects, 1800(8), 824-833.More infoPMID: 20230873;PMCID: PMC2893279;Abstract: 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. © 2010 Elsevier B.V.
- Pham, D. Q., & Winzerling, J. J. (2010). Insect ferritins: Typical or atypical?. Biochimica et biophysica acta, 1800(8).More infoInsects 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.
- Garcia-Bañuelos, M. L., Gardea, A. A., Winzerling, J. J., & Vazquez-Moreno, L. (2009). Characterization of a midwinter-expressed dehydrin (DHN) gene from apple trees (Malus domestica). Plant Molecular Biology Reporter, 27(4), 476-487.More infoAbstract: 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. © Springer-Verlag 2009.
- Geiser, D. L., Shen, M., Mayo, J. J., & Winzerling, J. J. (2009). Iron loaded ferritin secretion and inhibition by CI-976 in Aedes aegypti larval cells. Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology, 152(4), 352-363.More infoPMID: 19168145;PMCID: PMC2649984;Abstract: 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 59Fe 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. © 2009 Elsevier Inc. All rights reserved.
- Hajdusek, O., Sojka, D., Kopacek, P., Buresova, V., Franta, Z., Sauman, I., Winzerling, J., & Grubhoffer, L. (2009). Knockdown of proteins involved in iron metabolism limits tick reproduction and development. Proceedings of the National Academy of Sciences of the United States of America, 106(4), 1033-1038.More infoPMID: 19171899;PMCID: PMC2633537;Abstract: 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. © 2009 by The National Academy of Sciences of the USA.
- Lagarda-Diaz, I., Guzman-Partida, A. M., Urbano-Hernandez, G., Ortega-Nieblas, M. M., Robles-Burgueño, M. R., Winzerling, J., & Vazquez-Moreno, L. (2009). Insecticidal action of PF2 lectin from Olneya tesota (palo fierro) against Zabrotes subfasciatus larvae and midgut glycoconjugate binding. Journal of Agricultural and Food Chemistry, 57(2), 689-694.More infoPMID: 19102651;Abstract: 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. © 2009 American Chemical Society.
- Zhou, G., Velasquez, L. S., Geiser, D. L., Mayo, J. J., & Winzerling, J. J. (2009). Differential regulation of transferrin 1 and 2 in Aedes aegypti. Insect Biochemistry and Molecular Biology, 39(3), 234-244.More infoPMID: 19166934;Abstract: 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 72 h 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 24 h PBM, but decreases AaTf2 expression in ovaries at 72 h PBM, suggesting that AaTf1 and AaTf2 play different roles in immunity of female adults during a gonotrophic cycle. © 2008 Elsevier Ltd. All rights reserved.
- L., M., Moreno, L. V., Winzerling, J., Orozco, J. A., & Gardea, A. A. (2008). Winter metabolism in deciduous trees: Mechanisms, genes and associated proteins. Revista Fitotecnia Mexicana, 31(4), 295-308.More infoAbstract: Plants vary greatly in their ability to survive cold temperatures; some may withstand extreme freezing conditions, while others will be irreversibly injured at temperatures above freezing. The maximum freezing tolerance of plants is induced in response to environmental signals. Temperate woody trees need to acclimate to survive the cold winter. Trees have evolved a complex dynamic process controlling the development of dormancy and cold hardiness that synchronize accurately the onset and termination of winter metabolism. Only recently has been obtained progress in elucidating the molecular mechanisms of dormancy and freezing tolerance development in woody plants, and emerging disciplines are opening a wide horizon for future studies. The purpose of this review is to highlight recent developments indicating that cold-responsive genes and proteins contribute to freezing tolerance during winter in woody plants.
- Geiser, D. L., Mayo, J. J., & Winzerling, J. J. (2007). The unique regulation of Aedes aegypti larval cell ferritin by iron. Insect Biochemistry and Molecular Biology, 37(5), 418-429.More infoPMID: 17456437;Abstract: 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. © 2007 Elsevier Ltd. All rights reserved.
- Zhou, G., Kohlhepp, P., Geiser, D., del, M., Vazquez-Moreno, L., & Winzerling, J. J. (2007). Fate of blood meal iron in mosquitoes. Journal of Insect Physiology, 53(11), 1169-1178.More infoPMID: 17689557;PMCID: PMC2329577;Abstract: 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, ∼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, ∼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, ∼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, ∼97% is from heme and
- Geiser, D. L., Zhang, D., & Winzerling, J. J. (2006). Secreted ferritin: Mosquito defense against iron overload?. Insect Biochemistry and Molecular Biology, 36(3), 177-187.More infoPMID: 16503479;Abstract: 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. © 2006 Elsevier Ltd. All rights reserved.
- Pham, D. Q., Kos, P. J., Mayo, J. J., & Winzerling, J. J. (2006). Regulation of the ribonucleotide reductase small subunit (R2) in the yellow fever mosquito, Aedes aegypti. Gene, 372(1-2), 182-190.More infoPMID: 16530987;Abstract: Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotides, a rate limiting step in DNA synthesis. Class I RNR is a tetramer that consists of two subunits, R1 and R2; enzymatic activity requires association of R1 with R2. The R2 subunit is of special interest because it dictates the interaction with R1 that is required for enzymatic activity expression, and it is expressed only during the S phase of the cell cycle. We previously sequenced an R2 cDNA clone from the yellow fever mosquito, Aedes aegypti. We found the message was upregulated by blood feeding. We now report the sequence of an R2 genomic clone. The gene consists of 4 introns and 5 exons. Both major and minor transcriptional start sites have been identified, and their use differs in sugar-fed versus blood-fed females. The gene contains putative cis-regulatory sites for E2F, Caudal (Cdx) and Dearolf (Dfd). The mosquito R2 gene contains iron-specific regulatory elements immediately upstream of the minimal promoter region. Binding of a factor to the distal putative Cdx site in the - 400 region is altered by iron treatment of cells. Further, following blood feeding, R2 message is significantly induced in mosquito ovaries (tissues that are involved in oogenesis-a process requiring DNA synthesis). © 2006 Elsevier B.V. All rights reserved.
- Winzerling, J. J., & Pham, D. Q. (2006). Iron metabolism in insect disease vectors: Mining the Anopheles gambiae translated protein database. Insect Biochemistry and Molecular Biology, 36(4 SPEC. ISS.), 310-321.More infoPMID: 16551545;Abstract: All animals require iron for survival. This requirement reflects the role of this mineral as a cofactor of numerous proteins. However, under physiological conditions, Fe2+ oxidizes to Fe3+ 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. © 2006 Elsevier Ltd. All rights reserved.
- Mayo, J. J., Kohlhepp, P., Zhang, D., & Winzerling, J. J. (2004). Effects of sham air and cigarette smoke on A549 lung cells: Implications for iron-mediated oxidative damage. American Journal of Physiology - Lung Cellular and Molecular Physiology, 286(4 30-4), L866-L876.More infoPMID: 15003939;Abstract: 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.
- Geiser, D. L., Chavez, C. A., Flores-Munguia, R., Winzerling, J. J., & Pham, D. Q. (2003). Aedes aegypti ferritin: A cytotoxic protector against iron and oxidative challenge?. European Journal of Biochemistry, 270(18), 3667-3674.More infoPMID: 12950250;Abstract: 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 heroin 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 blood-meal. 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.
- Gailer, J., George, G. N., Pickering, I. J., Prince, R. C., Younis, H. S., & Winzerling, J. J. (2002). Biliary excretion of [(GS)2AsSe]- after intravenous injection of rabbits with arsenite and selenate. Chemical Research in Toxicology, 15(11), 1466-1471.More infoPMID: 12437338;Abstract: It has been shown that the seleno-bis (S-glutathionyl) arsinium ion, [(GS)2AsSe]-, is the major arsenic and selenium excretory product in bile of rabbits treated with arsenite and selenite [Gailer, J., Madden, S., Buttigieg, G. A., Denton, M. B., and Younis, H. S. (2002) Appl. Organomet. Chem. 16, 72-75]. To investigate the in vivo interaction between the other environmentally common oxy-anions of arsenic and selenium in mammals, we have intravenously injected rabbits with different combinations of the arsenic and selenium oxo-anions (arsenite + selenate, arsenate + selenite, and arsenate + selenate) and analyzed the collected bile and whole blood samples by X-ray absorption spectroscopy. Only the injection of arsenite and selenate led to the biliary excretion of [(GS)2AsSe]- within 25 min. Whole blood collected from these animals (25 min postinjection) contained predominantly unchanged selenate, which suggests the presence of a mammalian selenate reductase in the liver. The lack of any significant biliary excretion of [(GS)2AsSe]- in the other treatment groups implies that arsenate was not reduced in the liver on the time scale of our experiments. The relevance of these results for the human toxicology of arsenic and selenium is discussed.
- Kling, P. J., & Winzerling, J. J. (2002). Iron status and the treatment of the anemia of prematurity. Clinics in Perinatology, 29(2), 283-294.More infoPMID: 12168242;Abstract: Many unanswered issues regarding rhEPO therapy in prematurity remain, including which premature infants best respond to rhEPO, what the long-term effect of decreased erythrocyte transfusions is, how nutritional supplementation optimizes the effect of rhEPO, whether or not rhEpo therapy causes iron deficiency later in life, and whether or not it is safe to supplement with parenteral iron. Further study of rhEPO therapy and iron status in prematurity is necessary.
- Nichol, H., & Winzerling, J. (2002). Structured RNA upstream of insect cap distal iron responsive elements enhances iron regulatory protein-mediated control of translation. Insect Biochemistry and Molecular Biology, 32(12), 1699-1710.More infoPMID: 12429122;Abstract: Iron regulatory protein (IRP) blocks ribosomal assembly by binding to an iron responsive element (IRE) located proximal (
- Nichol, H., Law, J. H., & Winzerling, J. J. (2002). Iron metabolism in insects. Annual Review of Entomology, 47, 535-559.More infoPMID: 11729084;Abstract: Like other organisms, insects must balance two properties of ionic iron, that of an essential nutrient and a potent toxin. Iron must be acquired to provide catalysis for oxidative metabolism, but it must be controlled to avoid destructive oxidative reactions. Insects have evolved distinctive forms of the serum iron transport protein, transferrin, and the storage protein, ferritin. These proteins may serve different functions in insects than they do in other organisms. A form of translational control of protein synthesis by iron in insects is similar to that of vertebrates. The Drosophila melanogaster genome contains many genes that may encode other proteins involved in iron metabolism.
- Pham, D. Q., Blachuta, B. J., Nichol, H., & Winzerling, J. J. (2002). Ribonucleotide reductase subunits from the yellow fever mosquito, Aedes aegypti: Cloning and expression. Insect Biochemistry and Molecular Biology, 32(9), 1037-1044.More infoPMID: 12213240;Abstract: 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 α2β2 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. © 2002 Elsevier Science Ltd. All rights reserved.
- Zhang, D., Dimopoulos, G., Wolf, A., Miñana, B., Kafatos, F. C., & Winzerling, J. J. (2002). Cloning and molecular characterization of two mosquito iron regulatory proteins. Insect Biochemistry and Molecular Biology, 32(5), 579-589.More infoPMID: 11891134;Abstract: 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. © 2002 Elsevier Science Ltd. All rights reserved.
- Gailer, J., George, G. N., Pickering, I. J., Prince, R. C., Kohlhepp, P., Zhang, D., Walker, F. A., & Winzerling, J. J. (2001). Human cytosolic iron regulatory protein 1 contains a linear iron-sulfur cluster . Journal of the American Chemical Society, 123(41), 10121-10122.More infoPMID: 11592901;
- Winzerling, J. J., & Kling, P. J. (2001). Iron-deficient erythropoiesis in premature infants measured by blood zinc protoporphyrin/heme. Journal of Pediatrics, 139(1), 134-136.More infoPMID: 11445807;Abstract: 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.
- Zhang, D., Albert, D. W., Kohlhepp, P., D.-Pham, D., & Winzerling, J. J. (2001). Repression of Manduca sexta ferritin synthesis by IRP1/ire interaction. Insect Molecular Biology, 10(6), 531-539.More infoPMID: 11903622;Abstract: 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.
- Zhang, D., Ferris, C., Gailer, J., Kohlhepp, P., & Winzerling, J. J. (2001). Manduca sexta IRP1: Molecular characterization and in vivo response to iron. Insect Biochemistry and Molecular Biology, 32(1), 85-96.More infoPMID: 11719072;Abstract: Manduca sexta IRP1 was cloned and sequenced. The deduced amino acid sequence of Manduca IRP1 shows high similarity to other IRP1 proteins. The Cys residues required as ligands for the iron sulfur cluster, as well as all residues necessary for aconitase activity are conserved in the insect protein. Purified recombinant Manduca IRP1 binds specifically to transcripts of the iron responsive element (IRE) of Manduca or human ferritin subunit mRNA. Binding activity of the recombinant protein was not influenced by the presence of β-mercaptoethanol. However, IRP/IRE binding activity of cytoplasmic extracts from fat body was decreased by reducing agents in a dose-responsive manner. Fat body IRP1/IRE binding activity was reduced for Manduca sexta larvae injected with low doses of iron, while IRP1 mRNA and protein levels remained stable. At higher iron doses, binding activity increased and stabilized. Hemolymph ferritin levels showed an inverse relationship to IRP1/IRE binding activity. These data suggest that the Manduca IRP1 is likely involved in translational control of ferritin synthesis in a manner similar to that found in vertebrates. However, factors other than iron can influence IRP/IRE interaction and hemolymph ferritin levels in insects. © 2001 Elsevier Science Ltd. All rights reserved.
- Pham, D. Q., Brown, S. E., Knudson, D. L., Winzerling, J. J., Dodson, M. S., & Shaffer, J. J. (2000). Structure and location of a ferritin gene of the yellow fever mosquito Aedes aegypti. European Journal of Biochemistry, 267(12), 3885-3890.More infoPMID: 10849008;Abstract: We have isolated and sequenced a genomic clone encodihg the 24- and 26- kDa ferritin subunits in the mosquito Aedes aegypti (Rockefeller strain). The A. aegypti gene differs from other known ferrtin 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 those sites is observed with varied cellular iron- concentrations.
- Pham, D. Q., Winzerling, J. J., Dodson, M. S., & Law, J. H. (1999). Transcriptional control is relevant in the modulation of mosquito ferritin synthesis by iron. European Journal of Biochemistry, 266(1), 236-240.More infoPMID: 10542070;Abstract: 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 offer 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.
- Winzerling, J. J., Jouni, Z. E., & McNamara, D. J. (1998). Human peritoneal monocytic cells: Lipoprotein uptake and foam cell formation. Life Sciences, 62(6), 501-513.More infoPMID: 9464462;Abstract: 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.
- Winzerling, J. J., & Law, J. H. (1997). Comparative nutrition of iron and copper. Annual Review of Nutrition, 17, 501-526.More infoPMID: 9240938;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.
- Q., D., Zhang, D., Hufnagel, D. H., & Winzerling, J. J. (1996). Manduca sexta hemolymph ferritin: CDNA sequence and mRNA expression. Gene, 172(2), 255-259.More infoPMID: 8682313;Abstract: A cDNA clone encoding a subunit of the tobacco hornworm Manduca sexta (Ms) hemolymph (serum) ferritin (Fer) has been identified and sequenced. The deduced amino acid (aa) sequence shows approx. 50% similarity to vertebrate Fer subunit sequences, and the nucleotide sequence contains a stem-loop structure in the 5' untranslated region that could serve as an iron-responsive element (IRE). The stem-loop of this putative IRE exhibits high identity to vertebrate IRE that play an essential role in the control of Fer synthesis. The Ms Fer subunit lacks one of the three Tyr residues required for the rapid biomineralization of iron shown in vertebrate heavy-chain Fer. In addition, aa residues that comprise the putative ferroxidase centers generally are not conserved, suggesting that the Ms Fer subunit more closely resembles the vertebrate light-chain subunit. Northern blot analyses indicate that the fer mRNA is expressed in the midgut, fat body and hemocytes, with the greatest expression in the midgut.
- Winzerling, J. J., Jouni, Z. E., & McNamara, D. J. (1996). Lipoprotein metabolism in human peritoneal cells. Life Sciences, 58(19), 1631-1641.More infoPMID: 8632700;Abstract: 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; p
- Winzerling, J. J., Pham, D. Q., Kunz, S., Law, J. H., & Porath, J. (1996). Purification of an Expressed Insect Transferrin from Cell Culture Media using High-capacity Ni2+-dipicolylamine Gel. Journal of Molecular Recognition, 9(5-6), 747-.More infoPMID: 9174967;Abstract: Vertebrate transferrin is a well characterized iron transport protein. In contrast, little is known concerning the role of transferrin in insects. Yet, study of iron metabolism in insects could give insights into strategies for insect control, particularly for insects that transmit disease.
- Winzerling, J. J., Pham, D. Q., Kunz, S., Samaraweera, P., Law, J. H., & Porath, J. (1996). Purification of recombinant insect transferrin from large volumes of cell culture medium using high capacity Ni2+-dipicolylamine gel. Protein Expression and Purification, 7(2), 137-142.More infoPMID: 8812846;Abstract: We report the purification of secreted recombinant Manduca sexta transferrin from Spodoptera frugiperda (Sf9) cell culture medium in a single step using high capacity Ni2+-dipicolylamine (DPA)-Novarose gel. Although the original sample was highly diluted (~10 μg transferrin/ml medium) and the cell culture medium contained 10% surfactant (Pluronic F68) and a lipid emulsion, we were able to recover the recombinant transfertin (1 mg protein/100 ml) under gentle elution conditions with 70% yield at >90% homogeneity. This work demonstrates the versatility of immobilized metal ion affinity chromatography using a high metal ion capacity gel to purify a recombinant protein and illustrates the potential of this affinity technique for protein separations from large volumes of cell culture media that contain surfactants.
- Boden, V., Winzerling, J. J., Vijayalakshmi, M., & Porath, J. (1995). Rapid one-step purification of goat immunoglobulins by immobilized metal ion affinity chromatography. Journal of Immunological Methods, 181(2), 225-232.More infoPMID: 7745251;Abstract: A rapid, single step purification of immunoglobulins from goat serum was achieved using immobilized metal ion affinity chromatography (IMAC) on a new high capacity gel, Novarose, coupled to tris(2-aminoethyl)amine (TREN) chelated with copper. When goat serum was adsorbed to this gel in buffer pH 7 at 11 cm/h (8.6 ml/h), the immunoglobulin fraction was recovered in a decreasing linear pH gradient at about pH 5.5. When the adsorption buffer was adjusted to pH 6.0 and the linear velocity increased to 110 cm/h (221 ml/h), an immunoglobulin fraction of greater than 95% homogeneity was obtained. Protein purity was assessed by silver-stained native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The capacity of the gel for immunoglobulins was 17 mg immunoglobulin/ml at the low flow rate with adsorption at pH 7 and 15 mg immunoglobulin/ml at the high flow rate with adsorption at pH 6. No problems of back pressure or gel compression were observed at the higher linear velocity. The mild elution pH, high flow rate, and synthetic nature of the ligand support make this new metal-chelating gel a powerful alternative to the use of other currently available commercial gels commonly used for immunoglobulin purification. © 1995.
- Dunkov, B. C., Zhang, D., Choumarov, K., Winzerling, J. J., & Law, J. H. (1995). Isolation and characterization of mosquito ferritin and cloning of a cDNA that encodes one subunit.. Archives of insect biochemistry and physiology, 29(3), 293-307.More infoPMID: 7655055;Abstract: 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 subunit 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.
- Jouni, Z. E., Winzerling, J. J., & McNamara, D. J. (1995). 1,25-Dihydroxyvitamin D3-induced HL-60 macrophages: regulation of cholesterol and LDL metabolism. Atherosclerosis, 117(1), 125-138.More infoPMID: 8546749;Abstract: Differentiation of human promyelocytic leukemic HL-60 cells with 1,25-dihydroxyvitamin D3 (D3) results in macrophages which exhibit specific and saturable receptor-mediated processing of both native and modified low density lipoprotein (LDL). Analysis of binding kinetics demonstrated that macrophages bind LDL and acetyl-LDL with similar affinities, yet possess significantly different numbers of receptors (55 ± 6 × 103 LDL receptors/cell vs. 79 ± 7 × 103 acetyl-LDL receptors/cell). D3-induced HL-60 macrophages challenged with LDL or acetyl-LDL exhibited suppression of HMG-CoA reductase activity as well as a significant induction in the incorporation of [14C]oleate into cholesteryl ester compared with macrophages incubated with lipoprotein depleted serum. Maximum increases in ACAT activity were obtained in macrophages incubated with 25-hydroxycholesterol plus LDL or acetyl-LDL. The increase in ACAT activity in macrophages challenged with acetyl-LDL paralleled the increase in cellular cholesterol content and the increase of oil red O lipid stainable material, imparting the macrophages with a foamy appearance. The data indicate that D3-induced HL-60 macrophages are a useful model for the study of lipoprotein -macrophage interactions as related to foam cell development and atherogenesis. © 1995.
- Winzerling, J. J., Nez, P., Porath, J., & Law, J. H. (1995). Rapid and efficient isolation of transferrin and ferritin from Manduca sexta. Insect Biochemistry and Molecular Biology, 25(2), 217-224.More infoPMID: 7711752;Abstract: 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 Ni2+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. © 1995.
- Winzerling, J. J., Berna, P., & Porath, J. (1992). How to use immobilized metal ion affinity chromatography. Methods, 4(1), 4-13.More infoAbstract: 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. © 1992.
- Kranch, R., Stern, D., Wright, A. L., Lohman, I. C., Spangenberg, A., Vercelli, D., Winzerling, J. J., Wilhelm, M. S., & Halonen, M. (2015, Spring). Temporal patterns of early life food-specific IgE and their relation to asthma, eczema, and allergic rhinitis at age 5. Frontiers in Immunobiology & Immunopathogenesis Symposium. University of Arizona.