Roger L Miesfeld
Publications
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.
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.
PMID: 20447372;PMCID: PMC2900518;Abstract:
The fragmentation patterns of various 13C-labeled glucose molecules were analyzed by electrospray ionization tandem mass spectrometry. Derivatization of glucose to yield methylglucosamine makes the C-C bond between C1 and C2 a favored cleavage site. This is in contrast to underivatized glucose, which favorably undergoes loss of a fragment containing both C1 and C2. Based on the fragmentation pattern of methylglucoasmine, we developed a method to distinguish and quantify C1 and C2 13C-labeled glucose by derivatization with methylamine followed by multiple reaction monitoring scans in a Q-trap mass spectrometer. Fragment ion ratios in the tandem mass spectra showed an isotope effect with 13C or deuterium labeling, so a " correction factor" was introduced to make the quantification more accurate. The current approach can be applied to individually monitor the metabolic origin and fate of C1 and C2 atoms in 13C-labeled glucose. This method provides a new means of quantifying glucose isotopomers in metabolic studies. © 2010 Elsevier Inc.
PMID: 18977440;PMCID: PMC2673731;Abstract:
To gain a better understanding of coordinate regulation of protease gene expression in the mosquito midgut, we undertook a comprehensive molecular study of digestive carboxypeptidases in Aedes aegypti. Through a combination of cDNA cloning using degenerate PCR primers, and database mining of the recently completed A. aegypti genome, we cloned and characterized 18 A. aegypti carboxypeptidase genes. Bioinformatic analysis revealed that 11 of these genes belong to the carboxypeptidase A family (AaCPA-I through AaCPA-XI), and seven to the carboxypeptidase B gene family (AaCPB-I through AaCPB-VII). Phylogenetic analysis of 32 mosquito carboxypeptidases from five different species indicated that most of the sequence divergence in the carboxypeptidase gene family occurred prior to the separation of Aedes and Anopheles mosquito lineages. Unlike the CPA genes that are scattered throughout the A. aegypti genome, six of seven CPB genes were found to be located within a single 120 kb genome contig, suggesting that they most likely arose from multiple gene duplication events. Quantitative expression analysis revealed that 11 of the A. aegypti carboxypeptidase genes were induced up to 40-fold in the midgut in response to blood meal feeding, with peak expression times ranging from 3 to 36 h post-feeding depending on the gene. © 2008 Elsevier Ltd. All rights reserved.
PMID: 6287426;PMCID: PMC320769;Abstract:
A Hela cell S-100 extract primed with a purified human rDNA containing clone, has been shown to be capable of initiating specific α-amanitin-resistent RNA transcripts. By using a number of truncated templates, the site of RNA polymerase I initiation in vitro has been identified. The origin of transcription in vitro and in vivo was further defined by S1-mapping studies with total Hela cell RNA or RNA isolated from the in vitro transcription reaction. The initiation site was found to be the same. The nucleotide sequence of an 848 bp region around the initiation site, has also been determined. A perfect 15 bp homology has been found to exist between human and mouse rDNA very close to the origin of transcription, although little homology exists elsewhere. Sequences homolgous to the origin of transcription region were not found repeated within a 12 kb non-transcribed spacer segment upstream from it. © 1982 IRL Press Limited.