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J J Winzerling

Publications and source records attributed to J J Winzerling.

At least 19 recordsLinked to original sources
PubMed2002

Cloning and molecular characterization of two mosquito iron regulatory proteins.

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.

Aedes↗
PubMed2002

Ribonucleotide reductase subunits from the yellow fever mosquito, Aedes aegypti: cloning and expression.

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.

Aedes↗
PubMed2002

Biliary excretion of [(GS)(2)AsSe](-) after intravenous injection of rabbits with arsenite and selenate.

It has been shown that the seleno-bis (S-glutathionyl) arsinium ion, [(GS)(2)AsSe](-), 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)(2)AsSe](-) 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)(2)AsSe](-) 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.

Animals↗
PubMed2001

Manduca sexta IRP1: molecular characterization and in vivo response to iron.

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 beta-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.

Animals↗
PubMed2001

Repression of Manduca sexta ferritin synthesis by IRP1/IRE interaction.

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.

Amino Acid Sequence↗
PubMed2000

Structure and location of a ferritin gene of the yellow fever mosquito Aedes aegypti.

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.

5' Untranslated Regions↗
PubMed1999

Transcriptional control is relevant in the modulation of mosquito ferritin synthesis by iron.

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.

Aedes↗
PubMed1998

Human peritoneal monocytic cells: lipoprotein uptake and foam cell formation.

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.

Foam Cells↗
PubMed1997

Comparative nutrition of iron and copper.

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.

Animals↗
PubMed1996

Manduca sexta hemolymph ferritin: cDNA sequence and mRNA expression.

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.

Amino Acid Sequence↗
PubMed1996

Purification of recombinant insect transferrin from large volumes of cell culture medium using high capacity Ni(2+)-dipicolylamine gel.

We report the purification of secreted recombinant Manduca sexta transferrin from Spodoptera frugiperda (Sf9) cell culture medium in a single step using high capacity Ni(2+)-dipicolylamine (DPA)-Novarose gel. Although the original sample was highly diluted (approximately 10 micrograms transferrin/ml medium) and the cell culture medium contained 10% surfactant (Pluronic F68) and a lipid emulsion, we were able to recover the recombinant transferrin (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.

Animals↗
PubMed1996

Lipoprotein metabolism in human peritoneal cells.

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 < 0.05) and acetyl-LDL (r = 0.931; p < 0.01) 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.

Acetylation↗
PubMed1995

Rapid one-step purification of goat immunoglobulins by immobilized metal ion affinity chromatography.

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.

Animals↗
PubMed1995

Isolation and characterization of mosquito ferritin and cloning of a cDNA that encodes one subunit.

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.

Aedes↗
PubMed1995

1,25-Dihydroxyvitamin D3-induced HL-60 macrophages: regulation of cholesterol and LDL metabolism.

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 x 10(3) LDL receptors/cell vs 79 +/- 7 x 10(3) 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.

Acyl Coenzyme A↗
PubMed1995

Rapid and efficient isolation of transferrin and ferritin from Manduca sexta.

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.

Animals↗