Roger L Miesfeld

Roger L Miesfeld

Distinguished Professor, Chemistry and Biochemistry
Professor, BIO5 Institute
Professor, Chemistry and Biochemistry
Professor, Entomology / Insect Science - GIDP
Professor, Molecular and Cellular Biology
Primary Department
Contact
(520) 626-2343

Research Interest

Research Interest
Roger L. Miesfeld, Ph.D., Professor and Co-Chair, Dept. of Chemistry & Biochemistry, College of Science, University of Arizona. Mosquitoes are human disease vectors that transmit pathogens through blood feeding. One of these disease vectors is the Aedes aegypti mosquito, which have rapidly expanded their habitat and are contributing annually to 500,000 cases of Dengue hemorrhagic fever. On an even greater scale, Anopheline mosquitoes account for 250 million cases of malaria/yr, with up to 1 million deaths annually. The most common adult insecticides used for mosquito control are pyrethroids, which inhibit evolutionarily conserved sodium channels in the mosquito nervous system. Although these compounds have proven to be effective, mosquito resistance is an increasing problem and there is a pressing need to develop the next generation of safe and effective agents. Since blood meal feeding creates a unique metabolic challenge as a result of the extremely high protein and iron content of blood, it is possible that interfering with blood meal metabolism could provide a novel control strategy for mosquito born diseases. Our long term goal is to identify small molecule inhibitors that block blood meal metabolism in vector mosquitoes, resulting in feeding-induced death of the adult female, or a significant reduction in egg viability, as a strategy to control vector mosquito populations in areas of high disease transmission.

Publications

Rundlett, S. E., & Miesfeld, R. L. (1995). Quantitative differences in androgen and glucocorticoid receptor DNA binding properties contribute to receptor-selective transcriptional regulation. Molecular and Cellular Endocrinology, 109(1), 1-10.

PMID: 7789609;Abstract:

Androgen receptor (AR) and glucocorticoid receptor (GR) belong to the same subfamily of steroid/nuclear receptors and have been shown to bind qualitatively to the same hormone response element (HRE) DNA sequences. Despite this similarity in target gene recognition, AR and GR have differential affects on the transcriptional regulation of genes containing both simple and complex HRE control regions. Using HREs from the mouse mammary tumor virus (MMTV), tyrosine aminotransferase (TAT), prostatein (C3) or sexlimited protein (SLP) genes, linked to the thymidine kinase promoter, we found receptor-selective differences in the ability of rat AR and rat GR to induce transcription of these various reporter genes. Since AR and GR have a 20% amino acid sequence difference in their DNA binding domains (DBDs), which could result in altered DNA binding affinities, we measured the ability of purified AR and GR DBDs to bind selectively and with high affinity to these HRE sequences in vitro. Gel shift mobility assays showed that the GR DBD had a higher affinity for a consensus HRE than did the AR DBD, and quantitative DNase I footprinting revealed that AR and GR DBDs bound to the MMTV, TAT, C3 and SLP HREs with different affinities. It was found that AR had a dissociation constant (Kd) that was 2-3 times higher than GR on the TAT, C3 and SLP HREs and that the Kd of AR for the C3 and SLP HREs differed by an order of magnitude (43 nM and 460 nM, respectively). Taken together, these data suggest that amino acid differences in the AR and GR DBDs contribute to altered receptor-DNA interactions, however it is likely that non-receptor factors are involved in further modulating receptor-selective DNA binding and transactivation functions. © 1995.

Isoe, J., Rascón Jr., A. A., Kunz, S., & Miesfeld, R. L. (2009). Molecular genetic analysis of midgut serine proteases in Aedes aegypti mosquitoes. Insect Biochemistry and Molecular Biology, 39(12), 903-912.

PMID: 19883761;PMCID: PMC2818436;Abstract:

Digestion of blood meal proteins by midgut proteases provides anautogenous mosquitoes with the nutrients required to complete the gonotrophic cycle. Inhibition of protein digestion in the midgut of blood feeding mosquitoes could therefore provide a strategy for population control. Based on recent reports indicating that the mechanism and regulation of protein digestion in blood fed female Aedes aegypti mosquitoes is more complex than previously thought, we used a robust RNAi knockdown method to investigate the role of four highly expressed midgut serine proteases in blood meal metabolism. We show by Western blotting that the early phase trypsin protein (AaET) is maximally expressed at 3 h post-blood meal (PBM), and that AaET is not required for the protein expression of three late phase serine proteases, AaLT (late trypsin), AaSPVI (5G1), and AaSPVII. Using the trypsin substrate analog BApNA to analyze in vitro enzyme activity in midgut extracts from single mosquitoes, we found that knockdown of AaSPVI expression caused a 77.6% decrease in late phase trypsin-like activity, whereas, knockdown of AaLT and AaSPVII expression had no significant effect on BApNA activity. In contrast, injection of AaLT, AaSPVI, and AaSPVII dsRNA inhibited degradation of endogenous serum albumin protein using an in vivo protease assay, as well as, significantly decreased egg production in both the first and second gonotrophic cycles (P 

Miesfeld, R., Rusconi, S., Godowski, P. J., Maler, B. A., Okret, S., Wikström, A., Gustafsson, J., & Yamamoto, K. R. (1986). Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA. Cell, 46(3), 389-399.

PMID: 3755378;Abstract:

We isolated and sequenced 6.3 kb of cDNA encoding the rat glucocorticoid receptor, a protein that binds and activates a class of hormone-dependent transcriptional enhancers. Receptor-containing cells produce receptor mRNAs of ≅6.5 kb and ≅4.8 kb that differ only in their 3′ nontranslated regions; an open reading frame of 795 amino acids resides within the 5′ portion of the transcripts. The coding region was expressed in vitro, in transient transfections, and in stable transfectants of a receptor-deficient cell line. The protein products are indistinguishable from bona fide receptor with respect to sedimentation and electrophoretic mobility, antibody reactivity, and hormone and DNA binding. Moreover, the cloned receptor protein activates its corresponding enhancers, restoring to the receptor-deficient cells the full capacity for regulated enhancement. © 1986.

Distelhorst, C. W., & Miesfeld, R. (1987). Characterization of glucocorticoid receptors and glucocorticoid receptor mRNA in human leukemia cells: Stabilization of the receptor by diisopropylfluorophosphate. Blood, 69(3), 750-756.

PMID: 3545320;Abstract:

We have shown that cytosol samples from human leukemia cells frequently contain glucocorticoid receptor fragments that have a mol wt (M(r)) of ~52,000. In the present study we demonstrate that the M(r) ~52,000-receptor fragments are derived from intact glucocorticoid receptors (M(r) ~97,000) by the action of a serine protease. M(r) ~52,000-receptor fragments were present in cytosol from 24 of 52 leukemia cell samples. Only normal size glucocorticoid receptors were present in cytosol samples if diisopropylfluorophosphate (DFP), a potent inhibitor of serine proteases, was added to the hypotonic buffer used for cytosol preparation. Receptor proteolysis was not inhibited by hydrolyzed DFP, benzamidine, phenylmethylsulfonylfluoride, aprotinin, iodoacetamide, or mercuric chloride. The leukemia cell protease digests the receptor at a different site than chymotrypsin, which digests the intact receptor to produce a M(r) ~40,000 receptor fragment. Receptor messenger RNA (mRNA) in S49 mouse lymphoma cells and in human leukemia cells was analyzed by Northern hybridization with a cDNA for the normal glucocorticoid receptor. Mutant S49 mouse lymphoma cells that have abnormally small glucocorticoid receptors (M(r) ~ 48,000) make a 5.0-kilobase receptor transcript in addition to the normal size 6.5-kilobase receptor transcript. A normal size receptor transcript of 6.5 kilobases was present in all of the human leukemia cells whether or not M(r) ~ 52,000-receptor fragments were present. Therefore, abnormalities of glucocorticoid receptor mRNA, which may give rise to the synthesis of foreshortened receptors in certain mutant mouse lymphoma cells, are apparently absent from human leukemia cells.

Miesfeld, R., Scaraffia, P. Y., Tan, G., Isoe, J., Wysocki, V. H., Wells, M. A., & Miesfeld, R. L. (2008). Discovery of an alternate metabolic pathway for urea synthesis in adult Aedes aegypti mosquitoes. Proceedings of the National Academy of Sciences of the United States of America, 105(2).

We demonstrate the presence of an alternate metabolic pathway for urea synthesis in Aedes aegypti mosquitoes that converts uric acid to urea via an amphibian-like uricolytic pathway. For these studies, female mosquitoes were fed a sucrose solution containing (15)NH4Cl, [5-(15)N]-glutamine, [(15)N]-proline, allantoin, or allantoic acid. At 24 h after feeding, the feces were collected and analyzed in a mass spectrometer. Specific enzyme inhibitors confirmed that mosquitoes incorporate (15)N from (15)NH4Cl into [5-(15)N]-glutamine and use the (15)N of the amide group of glutamine to produce labeled uric acid. More importantly, we found that [(15)N2]-uric acid can be metabolized to [(15)N]-urea and be excreted as nitrogenous waste through an uricolytic pathway. Ae. aegypti express all three genes in this pathway, namely, urate oxidase, allantoinase, and allantoicase. The functional relevance of these genes in mosquitoes was shown by feeding allantoin or allantoic acid, which significantly increased unlabeled urea levels in the feces. Moreover, knockdown of urate oxidase expression by RNA interference demonstrated that this pathway is active in females fed blood or (15)NH4Cl based on a significant increase in uric acid levels in whole-body extracts and a reduction in [(15)N]-urea excretion, respectively. These unexpected findings could lead to the development of metabolism-based strategies for mosquito control.