Biomedical subjects

Michael R Miller

Publications and source records attributed to Michael R Miller.

11 recordsLinked to original sources

PubMed2006

Rapid and cost-effective polymorphism identification and genotyping using restriction site associated DNA (RAD) markers.

Restriction site associated DNA (RAD) tags are a genome-wide representation of every site of a particular restriction enzyme by short DNA tags. Most organisms segregate large numbers of DNA sequence polymorphisms that disrupt restriction sites, which allows RAD tags to serve as genetic markers spread at a high density throughout the genome. Here, we demonstrate the applicability of RAD markers for both individual and bulk-segregant genotyping. First, we show that these markers can be identified and typed on pre-existing microarray formats. Second, we present a method that uses RAD marker DNA to rapidly produce a low-cost microarray genotyping resource that can be used to efficiently identify and type thousands of RAD markers. We demonstrate the utility of the former approach by using a tiling path array for the fruit fly to map a recombination breakpoint, and the latter approach by creating and using an enriched RAD marker array for the threespine stickleback. The high number of RAD markers enabled localization of a previously identified region, as well as a second region also associated with the lateral plate phenotype. Taken together, our results demonstrate that RAD markers, and the method to develop a RAD marker microarray resource, allow high-throughput, high-resolution genotyping in both model and nonmodel systems.

Animals↗

PubMed2006

Biochemical and structural domain analysis of xeroderma pigmentosum complementation group C protein.

XPC is a 940-residue multidomain protein critical for the sensing of aberrant DNA and initiation of global genome nucleotide excision repair. The C-terminal portion of XPC (residues 492-940; XPC-C) has critical interactions with DNA, RAD23B, CETN2, and TFIIH, whereas functional roles have not yet been assigned to the N-terminal portion (residues 1-491; XPC-N). In order to analyze the molecular basis for XPC function and mutational defects associated with xeroderma pigmentosum (XP) disease, a series of stable bacterially expressed N- and C-terminal fragments were designed on the basis of sequence analysis and produced for biochemical characterization. Limited proteolysis experiments combined with mass spectrometry revealed that the full XPC-C is stable but XPC-N is not. However, a previously unrecognized folded helical structural domain was found within XPC-N, XPC(156-325). Pull-down and protease protection assays demonstrated that XPC(156-325) physically interacts with the DNA repair factor XPA, establishing the first functional role for XPC-N. XPC-C exhibits binding characteristics of the full-length protein, including stimulation of DNA binding by physical interaction with RAD23B and CETN2. Analysis of an XPC missense mutation (Trp690Ser) found in certain patients with XP disease revealed that this mutation is associated with a diminished ability to bind DNA. Evidence of contributions to protein interactions from regions in both XPC-N and XPC-C along with recently recognized homologies to yeast PNGase prompted construction of a structural model of a folded XPC core. This model offers key insights into how domains from the two portions of the protein may cooperate in generating specific XPC functions.

Binding Sites↗

PubMed2006

Identification of specific chemoattractants and genetic complementation of a Borrelia burgdorferi chemotaxis mutant: flow cytometry-based capillary tube chemotaxis assay.

Measuring the chemotactic response of Borrelia burgdorferi, the bacterial species that causes Lyme disease, is relatively more difficult than measuring that of other bacteria. Because these spirochetes have long generation times, enumerating cells that swim up a capillary tube containing an attractant by using colony counts is impractical. Furthermore, direct counts with a Petroff-Hausser chamber is problematic, as this method has a low throughput and necessitates a high cell density; the latter can lead to misinterpretation of results when assaying for specific attractants. Only rabbit serum and tick saliva have been reported to be chemoattractants for B. burgdorferi. These complex biological mixtures are limited in their utility for studying chemotaxis on a molecular level. Here we present a modified capillary tube chemotaxis assay for B. burgdorferi that enumerates cells by flow cytometry. Initial studies identified N-acetylglucosamine as a chemoattractant. The assay was then optimized with respect to cell concentration, incubation time, motility buffer composition, and growth phase. Besides N-acetylglucosamine, glucosamine, glucosamine dimers (chitosan), glutamate, and glucose also elicited significant chemoattractant responses, although the response obtained with glucose was weak and variable. Serine and glycine were nonchemotactic. To further validate and to exploit the use of this assay, a previously described nonchemotactic cheA2 mutant was shown to be nonchemotactic by this assay; it also regained the wild-type phenotype when complemented in trans. This is the first report that identifies specific chemical attractants for B. burgdorferi and the use of flow cytometry for spirochete enumeration. The method should also be useful for assaying chemotaxis for other slow-growing prokaryotic species and in specific environments in nature.

Animals↗

PubMed2006

Evidence for a novel endocrine disruptor: the pesticide propanil requires the ovaries and steroid synthesis to enhance humoral immunity.

Steroid hormones are known to affect the humoral immune response to a variety of antigens. However, the mechanisms regulating these effects are poorly understood. The immunotoxic chemical propanil and estrogen have similar effects on the immune system including augmentation of humoral immune responses. Propanil enhances the number of phosphorylcholine (PC)-specific IgG2b, IgG3, and IgM antibody-secreting cells (ASCs) in the spleen four- to sixfold 7 days after vaccination of female C57BL/6 mice with heat-killed Streptococcus pneumoniae. Several experiments were performed to test the hypothesis that propanil increases the response via an estrogenic pathway. Ovariectomy abrogated the effect of propanil on the PC-specific ASC response. Both in vitro and in vivo assays indicate that propanil does not bind either estrogen receptor (ER) alpha or beta. Exogenous estradiol administration in ovariectomized mice failed to restore the effect of propanil on the PC response. Treatment of female mice with a pure ER antagonist, ICI 182,780, or the progesterone antagonist RU486 did not inhibit the increase in ASCs. These data suggest that estrogen and progesterone do not regulate the effect of propanil. However, complete inhibition of steroid synthesis with the gonadotropin-releasing hormone (GnRH) antagonist antide abrogated the increased response in propanil-treated mice, indicating a necessary role for steroid synthesis. Experiments in male mice demonstrated that propanil increased the number of ASCs comparable to female mice. However, orchiectomy did not inhibit this effect, suggesting that androgens do not regulate the amplification of the humoral response. These data suggest a novel role for the ovarian hormones in the regulation of the PC-specific antibody response.

Animals↗

PubMed2005

Difficulty demonstrating estradiol-mediated Erk1/2 phosphorylation in MCF-7 cells.

While some studies report that estradiol (E2) activates extracellular-signal regulated kinase (Erk1/2) in MCF-7 breast cancer cells, others report E2 does not activate this signaling pathway. This study attempted to resolve the conflicting reports by investigating experimental variables that could impact Erk1/2 activation using a high through-put assay that quantitatively assessed Erk1/2 phosphorylation. Variables tested included: cell staging and dosing regimes with and without charcoal-stripped serum, different MCF-7 cell sublines and culture densities and several E2 formulations and solvents. Levels of phosphorylated Erk1/2 were normalized to cellular protein rather than to total Erk1/2 protein because an antibody purported to recognize total Erk1/2 preferentially reacted with non-phosphorylated Erk1/2, potentially exaggerating the apparent level of Erk1/2 activation. Dosing MCF-7 cells with E2 containing small amounts of stripped serum induced Erk1/2 phosphorylation; however, this induction was largely attributed to serum factors. E2 administered in serum-free medium did not significantly alter Erk1/2 phosphorylation under any condition tested; immunocytochemistry corroborated this conclusion. While phosphatase inhibitors generally increased Erk1/2 phosphorylation, they did not impact E2-altered Erk1/2 phosphorylation. It remains important to resolve the basis of conflicting reports regarding E2-induced Erk1/2 activation due to the potential importance of this pathway on breast cancer and other processes.

Animals↗

PubMed2005

CheX is a phosphorylated CheY phosphatase essential for Borrelia burgdorferi chemotaxis.

Motility and chemotaxis are believed to be important in the pathogenesis of Lyme disease caused by the spirochete Borrelia burgdorferi. Controlling the phosphorylation state of CheY, a response regulator protein, is essential for regulating bacterial chemotaxis and motility. Rapid dephosphorylation of phosphorylated CheY (CheY-P) is crucial for cells to respond to environmental changes. CheY-P dephosphorylation is accomplished by one or more phosphatases in different species, including CheZ, CheC, CheX, FliY, and/or FliY/N. Only a cheX phosphatase homolog has been identified in the B. burgdorferi genome. However, a role for cheX in chemotaxis has not been established in any bacterial species. Inactivating B. burgdorferi cheX by inserting a flgB-kan cassette resulted in cells (cheX mutant cells) with a distinct motility phenotype. While wild-type cells ran, paused (stopped or flexed), and reversed, the cheX mutant cells continuously flexed and were not able to run or reverse. Furthermore, swarm plate and capillary tube chemotaxis assays demonstrated that cheX mutant cells were deficient in chemotaxis. Wild-type chemotaxis and motility were restored when cheX mutant cells were complemented with a shuttle vector expressing CheX. Furthermore, CheX dephosphorylated CheY3-P in vitro and eluted as a homodimer in gel filtration chromatography. These findings demonstrated that B. burgdorferi CheX is a CheY-P phosphatase that is essential for chemotaxis and motility, which is consistent with CheX being the only CheY-P phosphatase in the B. burgdorferi chemotaxis signal transduction pathway.

Bacterial Proteins↗

PubMed2004

C/EBPbeta activity and HPV-16 E6/E7 mRNA expression are not altered by imiquimod (ALDARA) in human cervical cancer cells in vitro.

OBJECTIVE: The purpose of this study was to determine the potential relationship between imiquimod and C/EBPbeta by investigating the extent to which imiquimod could alter C/EBPbeta binding activity to known sequences of the HPV-16 NCR, which could lead to the repression of HPV-16 E6/E7 oncogene expression, possibly impacting a major mechanism by which HPV causes cellular transformation. METHODS: The effect of imiquimod treatment on C/EBPbeta binding activity to its consensus sequence as well as to two specific regions of the HPV-16 NCR was determined by electromobility shift assay (EMSA) in CaSki cervical cancer cells. HPV-16 E6/E7 gene expression was evaluated by RNase protection assay (RPA) in CaSki and in W12-E cells treated with imiquimod. In addition, C/EBPbeta mRNA expression and protein production in response to imiquimod were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively, in the cervical cancer cell lines, CaSki, HeLa, and C33A. RESULTS: C/EBPbeta binding activity, mRNA expression, and protein production remained unchanged with imiquimod treatment. Initially, HPV-16 E6/E7 expression appeared to be increased with imiquimod treatment in CaSki cells, but this effect was not reproducible. HPV-16 E6/E7 expression was not reproducibly altered with imiquimod treatment in W12-E cells. CONCLUSION: While these results indicate that imiquimod does not alter C/EBPbeta binding activity, nor does it appear to decrease HPV-16 E6/E7 oncogene expression in vitro, it remains possible that imiquimod may have utility in treating cervical dysplasia or cervical cancer via another mechanism.

Aminoquinolines↗

PubMed2004

Combinatorial treatment of ovarian cancer cells with harringtonine and cisplatin results in increased cisplatin-DNA adducts.

The current studies represent the first step in assessing the utility of harringtonine in combination with cisplatin as an improved approach for treating ovarian cancer. Three ovarian cancer cell lines, platinum-sensitive A2780, and platinum-resistant A2780/CP70 and OvCar-3, were exposed to their respective IC(50) dose of cisplatin for 1 h with or without a 24-h pretreatment with harringtonine. The level of platinum-DNA adducts was determined by atomic absorption spectrometry (AAS). These studies show for the first time that harringtonine pretreatment significantly increased the amount of platinum-DNA adducts in all ovarian cancer cell lines by 2-4 fold, immediately following 1-h exposure to cisplatin. Moreover, the level of cisplatin-DNA adducts in harringtonine-pretreated cells remained elevated by 3-4.7-fold for at least 6 h after cisplatin was removed, relative to cells only exposed to cisplatin. In all three cell lines the removal (repair) of platinum-DNA adducts was not significantly altered by harringtonine. In addition, the extent to which harringtonine altered the expression of select DNA damage response genes (p53, P16, ERCC1 and XPB) was determined using RT-PCR and Southern hybridization in A2780 and A2780/CP70 cells. The expression of ERCC1 and XPB RNAs were only modestly altered by harringtonine, consistent with a lack of effect of harringtonine on repair of cisplatin-DNA damage. However, harringtonine altered expression of p53 and P16 RNAs in both cell lines, although the down-regulation of p53 and P16 RNAs by harringtonine were more pronounced in A2780 cells. The novel observation that harringtonine augments platinum-DNA adducts in both platinum-sensitive and -resistant ovarian cancer cells indicates this combination of drugs may have utility in treating ovarian cancer and may be especially useful in managing platinum-resistant cancers. Additional studies are required to determine which sequence of these drugs is most beneficial, as well as the mechanism by which harringtonine increases cisplatin-DNA damage in ovarian cancer cells.

Actins↗

PubMed2003

Octamethylcyclotetrasiloxane exhibits estrogenic activity in mice via ERalpha.

Octamethylcyclotetrasiloxane (D4) is a low molecular weight cyclic silicone used in the synthesis of larger silicone polymers and in the formulation of a variety of personal care products. The effects of oral D4 exposure in mice on serum estradiol levels, uterine wet weight, and uterine peroxidase activity were investigated. Additionally, in vitro estrogen receptor binding activity was evaluated. Serum estradiol levels decreased in a dose-dependent manner after exposure to 100 mg/kg to 1000 mg/kg D4. Studies with adrenalectomized animals demonstrated that the decreased serum estradiol levels were not due to elevated serum corticosterone levels. Uterine wet weights in ovariectomized mice were significantly increased in a dose-dependent manner by exposure to 250-1000 mg of D4/kg, but not by exposure to other silicone compounds tested (hexamethylcyclotrisiloxane, decamethylcyclopentasiloxane, decamethyltetrasiloxane, and octaphenylcyclotetrasiloxane). Uterine peroxidase activity, a marker for estrogenic activity, was also significantly increased in D4-exposed mice, but not in mice exposed to the other siloxanes. Pretreating mice with the estrogen receptor antagonist ICI 182,780 completely blocked the D4-induced increase in uterine weight, and ovariectomized estrogen receptor-alpha knockout mice showed no increases in uterine weights when orally exposed to D4 or estradiol. In an in vitro estrogen receptor binding assay, D4 showed significant competition with (3)H-estradiol for binding to estrogen receptor-alpha, but not estrogen receptor-beta. The data presented here indicate that D4 has weak estrogenic activity, and that these effects are mediated through estrogen receptor-alpha.

Adjuvants, Immunologic↗

PubMed2003

Acetaminophen exhibits weak antiestrogenic activity in human endometrial adenocarcinoma (Ishikawa) cells.

The purpose of this study was to test the hypothesis that acetaminophen would alter an estrogen-regulated process in human cells that express endogenous estrogen receptor alpha and beta (ERalpha and ERbeta). Specifically, the extent to which acetaminophen altered the expression of estrogen-inducible alkaline phosphatase in endometrial adenocarcinoma (Ishikawa) cells and directly interacted with ERbeta and ERalpha was determined. Ishikawa cells were exposed to estradiol and/or to a range of concentrations of acetaminophen for four days, and alkaline phosphatase activity was measured spectrophotometrically. Acetaminophen inhibited both basal and estradiol-induced alkaline phosphatase activity in Ishikawa cells in a concentration-dependent manner. The reduction of Ishikawa cell alkaline phosphatase was not due to direct inhibition of enzyme activity by acetaminophen. Toxic effects of acetaminophen on Ishikawa cells were determined by measuring loss of cellular lactate dehydrogenase to culture medium. High concentrations of acetaminophen (>/=0.5 mM) induced lactate dehydrogenase release from cells and reduced the amount of cellular protein in culture dishes, indicating some acetaminophen-induced reduction of alkaline phosphatase activity might be attributed to toxic effects. However, lower concentrations of acetaminophen significantly reduced alkaline phosphatase activity in the absence of detectable toxicity. Acetaminophen also augmented 4-hydroxy-tamoxifen reduction of alkaline phosphatase activity. Competition binding assays with human ERalpha and ERbeta demonstrated 10(6)-fold molar excess acetaminophen did not directly interact significantly with the ligand-binding domain of either receptor. These studies indicate acetaminophen exerts weak antiestrogenic activity in Ishikawa cells without directly binding ERalpha or ERbeta.

Acetaminophen↗

PubMed2002

Acetaminophen-induced proliferation of estrogen-responsive breast cancer cells is associated with increases in c-myc RNA expression and NF-kappaB activity.

Studies reported here tested the hypothesis that acetaminophen stimulates proliferation of E2-responsive cells by inducing expression of E2-regulated genes. Ribonuclease protection assays compared the effects of acetaminophen and E2 on expression of selected genes (c-myc, c-fos, cyclin D1, bcl-2, bax, gadd45, mcl-1, p53, p21(CIP1/WAF1), and bcl-xL) in E2-responsive breast cancer (MCF-7) and endometrial adenocarcinoma (Ishikawa) cells as well as in E2-nonresponsive (MDA-MB-231) breast cancer cells. Acetaminophen and E2 increased c-myc RNA levels in MCF-7 cells, consistent with a mitogenic activity of these compounds in MCF-7 cells. However, the magnitude and time course of acetaminophen and E2 induction of c-myc differed. Neither acetaminophen nor E2 induced c-myc in MDA-MB-231 cells, whereas E2, but not acetaminophen, weakly induced c-myc expression in Ishikawa cells. Furthermore, in these 3 cell types, the expression patterns of the other genes differed dramatically in response to acetaminophen and to E2, indicating that acetaminophen does not activate ER as a transcription factor in the same manner as does E2. Additionally, gel shift assays demonstrated that in MCF-7 cells, acetaminophen increased NF-kappaB activity approximately 40% and did not alter AP-1 activity, whereas E2 increased AP-1 activity approximately 50% and did not increase NF-B activity. These studies indicate that acetaminophen effects on gene expression and cell proliferation depend more on cell type/context than on the presence of ER.

Acetaminophen↗