Biomedical subjects

Billy W Day

Publications and source records attributed to Billy W Day.

At least 19 recordsLinked to original sources

PubMed2007

Cyclostreptin binds covalently to microtubule pores and lumenal taxoid binding sites.

Cyclostreptin (1), a natural product from Streptomyces sp. 9885, irreversibly stabilizes cellular microtubules, causes cell cycle arrest, evades drug resistance mediated by P-glycoprotein in a tumor cell line and potently inhibits paclitaxel binding to microtubules, yet it only weakly induces tubulin assembly. In trying to understand this paradox, we observed irreversible binding of synthetic cyclostreptin to tubulin. This results from formation of covalent crosslinks to beta-tubulin in cellular microtubules and microtubules formed from purified tubulin in a 1:1 total stoichiometry distributed between Thr220 (at the outer surface of a pore in the microtubule wall) and Asn228 (at the lumenal paclitaxel site). Unpolymerized tubulin was only labeled at Thr220. Thus, the pore region of beta-tubulin is an undescribed binding site that (i) elucidates the mechanism by which taxoid-site compounds reach the kinetically unfavorable lumenal site and (ii) explains how taxoid-site drugs induce microtubule formation from dimeric and oligomeric tubulin.

Alkanes↗

PubMed2006

Production and characterization of monoclonal antibodies against ochratoxin B.

Monoclonal antibodies against ochratoxin B (OTB) were generated by immunizing Balb/c mice with OTB conjugated to keyhole limpet hemocyanin (KLH) via carbodiimide reactions with CHMC and EDAC. A stable hybridoma cell line 2F1.E10 was produced by fusion of murine splenocytes and myeloma cells. The obtained antibodies were characterized using an indirect competitive ELISA. The detection limit was calculated (27+/-2 nM OTB) and 50% binding inhibition was reached at 500 nM free OTB. A low cross-reactivity to ochratoxin A (OTA) of 3.3% and no cross-reactivities to either coumarin or DL-phenylalanine were observed, suggesting a highly specific OTB antibody. The antibody type was identified as IgG class 1 with the light chain being of the kappa configuration. These antibodies can be used in an indirect competitive ELISA to detect OTB in the nanomolar to micromolar concentration range and may be useful for the analysis of contaminated food items.

Animals↗

PubMed2006

Albumin marks pseudopodia of astrocytoma cells responding to hepatocyte growth factor or serum.

It is well accepted that dysfunction in the blood brain barrier (BBB) allows permeation of albumin from the bloodstream into astrocytic brain tumors, especially glioblastomas, the most aggressive astrocytomas. In vitro, bovine serum albumin (BSA) aids functional cell assays by maintaining cytokines and growth factors in solution and delivering its cargo of fatty acids. Earlier, we showed that BSA was prominent in lysates prepared from pseudopodia formed by U87 astrocytoma cells. The present studies investigated the association of albumin with pseudopodia formed by U87 and LN229 astrocytoma cells. With hepatocyte growth factor (HGF) stimulation, cell migration was enhanced and BSA, especially its dimerized form, was prominent in pseudopodia compared to unmigrated cells on one-dimensional gels and immunoblots. When lysates were equalized for levels of glyceraldehyde-3-phosphate dehydrogenase, the rise for BSA levels in pseudopodia vs migrated cells was comparable or greater than levels noted for established pseudopodial proteins, beta-actin and ezrin. The increase for dimerized BSA in pseudopodia compared to unmigrated cells was greater than the rise in levels of beta-actin, ezrin, HGF, and phosphorylated Met when pseudopodia were harvested from filters with 1 mum pores using either cell line. Fluorescein (F)-labeled BSA co-localized with HGF on actin-rich cellular protrusions and with CM-DiI labeled pseudopodial plasma membranes. The F-BSA highlighted small, individual pseudopodial profiles more so than complex pseudopodial networks (reticulopodia) or unmigrated cells. Labeled human serum albumin also decorated pseudopodia preferentially. Albumin's association with pseudopodia may help to explain its selective accumulation in astrocytomas in vivo. The leaky BBB permits serum albumin to enter the microenvironment of astrocytomas thus allowing their invasive cells contact with serum albumin as a source of fatty acids that would be useful for remodeling cell membranes in pseudopodia. Thus, albumin potentially aids and marks invasion as it accumulates in these tumors.

Actins↗

PubMed2006

Synergistic effects of peloruside A and laulimalide with taxoid site drugs, but not with each other, on tubulin assembly.

Previous studies on the drug content of pelleted tubulin polymers suggest that peloruside A binds in the laulimalide site, which is distinct from the taxoid site. In a tubulin assembly system containing microtubule-associated proteins and GTP, however, peloruside A was significantly less active than laulimalide, inducing assembly in a manner that was most similar to sarcodictyins A and B. Because peloruside A thus far seems to be the only compound that mimics the action of laulimalide, we examined combinations of microtubule-stabilizing agents for synergistic effects on tubulin assembly. We found that peloruside A and laulimalide showed no synergism but that both compounds could act synergistically with a number of taxoid site agents [paclitaxel, epothilones A/B, discodermolide, dictyostatin, eleutherobin, the steroid derivative 17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol, and cyclostreptin]. None of the taxoid site compounds showed any synergism with each other. From an initial study with peloruside A and cyclostreptin, we conclude that the synergism phenomenon derives, at least in part, from an apparent lowering of the tubulin critical concentration with drug combinations compared with single drugs. The apparent binding of peloruside A in the laulimalide site led us to attempt construction of a pharmacophore model based on superposition of an energy-minimized structure of peloruside A on the crystal structure of laulimalide. Although the different sizes of the macrocycles limited our ability to superimpose the two molecules, atom correspondences that were observed were consistent with the difficulty so far experienced in creation of fully active analogs of laulimalide.

Bridged Bicyclo Compounds, Heterocyclic↗

PubMed2006

Synthesis and biological evaluation of purealin and analogues as cytoplasmic dynein heavy chain inhibitors.

Cytoplasmic dynein plays important roles in membrane transport, mitosis, and other cellular processes. A few small-molecule inhibitors of cytoplasmic dynein have been identified. We report here the first synthesis of purealin, a natural product isolated from the sea sponge Psammaplysilla purea, which is known to inhibit axonemal dynein. Also described are the first syntheses, by modular amide coupling reactions, of the natural product purealidin A (a component of purealin) and a small library of analogues. The library was examined for inhibition of cytoplasmic dynein heavy chain and cell growth. The compounds showed effective antiproliferative activity against a mouse leukemia cell line but selective activities against human carcinoma cell lines. Purealin and some of the analogues inhibited the microtubule-stimulated ATPase activity of recombinant cytoplasmic dynein heavy chain motor domain. The inhibitory effect of purealin was concentration dependent and uncompetitive, supporting the hypothesis that it does not compete with the binding of ATP.

Adenosine Triphosphatases↗

PubMed2006

Fluorous mixture synthesis of (-)-dictyostatin and three stereoisomers.

[reaction: see text] A mixture of four stereoisomers whose configurations are encoded by fluorous silyl protecting groups has been prepared and converted over 22 steps to a mixture of protected dictyostatins. Demixing by fluorous HPLC followed by removal of the fluorous protecting groups (detagging) provides dictyostatin and three C6,C7 stereoisomers. Biological evaluation showed that the monoepimers of the natural product retained highly potent activity.

Fluorine↗

PubMed2006

Altered expression and localization of creatine kinase B, heterogeneous nuclear ribonucleoprotein F, and high mobility group box 1 protein in the nuclear matrix associated with colon cancer.

Identification of biomarkers could lead to the development of effective screening tests for colorectal cancer. A previous study from our laboratory showed specific alterations of nuclear structure in colon cancer. In an effort to characterize these biomarkers, protein spots were selected from separations made by two-dimensional gel electrophoresis, which were analyzed by mass spectrometry. The sequences obtained from the isolated spots revealed that they have close similarity to creatine kinase B (CKB) isoforms, heterogeneous nuclear ribonucleoprotein F (hnRNP F) and high mobility group box 1 protein (HMGB1) isoforms. To determine the expression of these proteins in colon cancer, expression was studied in 9 tumor and matched adjacent normal pairs, 5 donor colons, 16 polyps, 4 metastatic liver lesions and matched adjacent normal pairs, and 3 liver donors. CKB and hnRNP F were expressed in 78% and 89% of colon tumors, respectively. hnRNP F had a higher frequency of expression than CKB in premalignant polyps. With the establishment of differential expression of the proteins in colon cancer, their subcellular localization was analyzed. The subcellular fractions studied both showed high protein levels of hnRNP F in colon tumors compared with normal colon tissues. Surprisingly, subcellular levels of CKB were decreased in colon tumors, suggesting that the observed high CKB levels in nuclear matrix extracts are caused by the enhanced localization of CKB to the nuclear matrix during colon tumorigenesis. These results suggest an involvement of hnRNP F and CKB in colorectal cancer. Additionally, they suggest that hnRNP F is a potential marker for colorectal cancer progression.

Adenocarcinoma↗

PubMed2006

Myeloperoxidase-catalyzed metabolism of etoposide to its quinone and glutathione adduct forms in HL60 cells.

Etoposide is a widely used antineoplastic agent that has provided great success in the treatment of childhood leukemias and other malignancies. Unfortunately, its use is associated with the increased risk of development of secondary acute myelogenous leukemias involving translocations at the MLL gene in chromosome band 11q23. Previous studies showed that the phenoxyl radical of etoposide can be generated by myeloperoxidase (MPO), an enzyme prevalent in myeloid progenitor cells that can derive myelogenous leukemias. Disproportionation of this radical leads to formation of the redox active etoposide ortho-quinone metabolite. We hypothesized that etoposide ortho-quinone could therefore form in myeloid progenitor cells and might be a contributor to the development of treatment-related secondary leukemias. Etoposide ortho-quinone is an inherently unstable compound and readily reacts with glutathione in aqueous media without any requirement for catalytic assistance from glutathione S-transferase. We looked for the presence of its glutathione adduct as an indicator of etoposide ortho-quinone in cells. MPO-expressing human myeloid leukemia HL60 cells were treated with etoposide for 0.5 h in the presence and absence of the cosubstrate of MPO, hydrogen peroxide. Cell lysates and medium were analyzed by LC-ESI-ion trap-MS and MS/MS, which yielded clear evidence of the intracellular formation of the etoposide ortho-quinone-glutathione adduct. A stable isotope-labeled form of the GSH adduct was synthesized and employed as an isotope dilution internal standard in LC-ESI-quadrupole-MS analyses. The glutathione adduct level was dependent on the concentration of etoposide added to the cells. More importantly, the formation of the glutathione adduct was significantly suppressed by the pretreatment of HL60 cells with the heme synthesis inhibitor succinylacetone (p < 0.001), which resulted in a decreased level and activity of MPO. These results are consistent with the idea that MPO is responsible for the conversion of etoposide to its ortho-quinone in these cells.

Benzoquinones↗

PubMed2005

Brisk production of nitric oxide and associated formation of S-nitrosothiols in early hemorrhage.

The results of previous inhibitor studies suggest that there is some increase in nitric oxide (NO) production from constitutive NO synthase in early hemorrhage (H), but the magnitude of NO production early after H has not been previously assessed. It is generally believed that only modest production rates are possible from the constitutively expressed NO synthases. To study this, anesthetized male Sprague-Dawley rats were subjected to 90 min of isobaric (40 mmHg) H. During this period of time, the dynamics of accumulation of NO intermediates in the arterial blood was assessed using electron paramagnetic resonance spectroscopy, chemiluminescence, fluorescence imaging, and mass spectrometry. Electron paramagnetic resonance-detectable NO adducts were also measured with spin traps in blood plasma and red blood cells. H led to an increase in the concentration of hemoglobin-NO from 0.9 +/- 0.2 to 4.8 +/- 0.7 microM. This accumulation was attenuated by a nonselective inhibitor of NO synthase, NG-nitro-L-argininemethyl ester (L-NAME), but not by NG-nitro-D-argininemethyl ester (D-NAME) or 1400W. Administration of L-NAME (but not 1400W or D-NAME) during H produced a short-term increase in mean arterial pressure ( approximately 90%). In H, the level of N oxides in red blood cells increased sevenfold. S-nitrosylation of plasma proteins was revealed with "biotin switch" techniques. The results provide compelling evidence that there is brisk production of NO in early H. The results indicate that the initial compensatory response to H is more complicated than previously realized, and it involves an orchestrated balance between intense vasoconstrictor and vasodilatory components.

Animals↗

PubMed2005

Tubulin assembly, taxoid site binding, and cellular effects of the microtubule-stabilizing agent dictyostatin.

(-)-Dictyostatin is a sponge-derived, 22-member macrolactone natural product shown to cause cells to accumulate in the G2/M phase of the cell cycle, with changes in intracellular microtubules analogous to those observed with paclitaxel treatment. Dictyostatin also induces assembly of purified tubulin more rapidly than does paclitaxel, and nearly as vigorously as does dictyostatin's close structural congener, (+)-discodermolide (Isbrucker et al. (2003), Biochem. Pharmacol. 65, 75-82). We used synthetic (-)-dictyostatin to study its biochemical and cytological activities in greater detail. The antiproliferative activity of dictyostatin did not differ greatly from that of paclitaxel or discodermolide. Like discodermolide, dictyostatin retained antiproliferative activity against human ovarian carcinoma cells resistant to paclitaxel due to beta-tubulin mutations and caused conversion of cellular soluble tubulin pools to microtubules. Detailed comparison of the abilities of dictyostatin and discodermolide to induce tubulin assembly demonstrated that the compounds had similar potencies. Dictyostatin inhibited the binding of radiolabeled discodermolide to microtubules more potently than any other compound examined, and dictyostatin and discodermolide had equivalent activity as inhibitors of the binding of both radiolabeled epothilone B and paclitaxel to microtubules. These results are consistent with the idea that the macrocyclic structure of dictyostatin represents the template for the bioactive conformation of discodermolide.

Alkanes↗

PubMed2005

Rac1 leads to phosphorylation-dependent increase in stability of the p66shc adaptor protein: role in Rac1-induced oxidative stress.

The rac1 GTPase and the p66shc adaptor protein regulate intracellular levels of reactive oxygen species (ROS). We examined the relationship between rac1 and p66shc. Expression of constitutively active rac1 (rac1V12) increased phosphorylation, reduced ubiquitination, and increased stability of p66shc protein. Rac1V12-induced phosphorylation and up-regulation of p66shc was suppressed by inhibiting p38MAPK and was dependent on serine 54 and threonine 386 in p66shc. Phosphorylation of recombinant p66shc by p38MAPK in vitro was also partly dependent on serine 54 and threonine 386. Reconstitution of p66shc in p66shc-null fibroblasts increased intracellular ROS generated by rac1V12, which was significantly dependent on the integrity of residues 54 and 386. Overexpression of p66shc increased rac1V12-induced apoptosis, an effect that was also partly dependent on serine 54 and threonine 386. Finally, RNA interference-mediated down-regulation of endogenous p66shc suppressed rac1V12-induced cell death. These findings identify p66shc as a mediator of rac1-induced oxidative stress. In addition, they suggest that serine 54 and threonine 386 are novel phosphorylatable residues in p66shc that govern rac1-induced increase in its expression, through a decrease in its ubiquitination and degradation, and thereby mediate rac1-stimulated cellular oxidative stress and death.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Orphan nuclear receptor pregnane X receptor sensitizes oxidative stress responses in transgenic mice and cancerous cells.

Efficient handling of oxidative stress is critical for the survival of organisms. The orphan nuclear receptor pregnane X receptor (PXR) is important in xenobiotic detoxification through its regulation of phase I and phase II drug-metabolizing/detoxifying enzymes and transporters. In this study we unexpectedly found that the expression of an activated human PXR in transgenic female mice resulted in a heightened sensitivity to paraquat, an oxidative xenobiotic toxicant. Heightened paraquat sensitivity was also seen in wild-type mice treated with the mouse PXR agonist pregnenolone-16alpha-carbonitrile. The PXR-induced paraquat sensitivity was associated with decreased activities of superoxide dismutase and catalase, enzymes that scavenge superoxide and hydrogen peroxide, respectively. Paradoxically, the general expression and activity of glutathione S-transferases, a family of phase II enzymes that detoxify electrophilic and cytotoxic substrates, was also induced in the transgenic mice. PXR regulates glutathione S-transferase expression in an isozyme-, tissue-, and sex-specific manner, and this regulation is independent of the nuclear factor-erythroid 2 p45-related factor 2/Kelch-like Ech-associated protein 1 pathway. In cell cultures, expression of activated human PXR sensitizes the cancerous colon and liver cells to the cytotoxic effect of paraquat, which is associated with an increased production of the reactive oxygen species. The current study reveals a novel function of PXR in the mammalian oxidative stress response, and this regulatory pathway may be implicated in carcinogenesis by sensitizing normal and cancerous tissues to oxidative cellular damage.

Adaptor Proteins, Signal Transducing↗

PubMed2005

A common pharmacophore for a diverse set of colchicine site inhibitors using a structure-based approach.

Modulating the structure and function of tubulin and microtubules is an important route to anticancer therapeutics, and therefore, small molecules that bind to tubulin and cause mitotic arrest are of immense interest. A large number of synthetic and natural compounds with diverse structures have been shown to bind at the colchicine site, one of the major binding sites on tubulin, and inhibit tubulin assembly. Using the recently determined X-ray structure of the tubulin:colchicinoid complex as the template, we employed docking studies to determine the binding modes of a set of structurally diverse colchicine site inhibitors. These binding models were subsequently used to construct a comprehensive, structure-based pharmacophore that in combination with molecular dynamics simulations confirms and extends our understanding of binding interactions at the colchicine site.

2-Methoxyestradiol↗

PubMed2005

Redox regulation of Cdc25B by cell-active quinolinediones.

Intracellular reduction and oxidation pathways regulate protein functionality through both reversible and irreversible mechanisms. The Cdc25 phosphatases, which control cell cycle progression, are potential subjects of oxidative regulation. Many of the more potent Cdc25 phosphatase inhibitors reported to date are quinones, which are capable of redox cycling. Therefore, we used the previously characterized quinolinedione Cdc25 inhibitor DA3003-1 [NSC 663284 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione] and a newly synthesized congener JUN1111 [7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione] to test the hypothesis that quinone inhibitors of Cdc25 regulate phosphatase activity through redox mechanisms. Like DA3003-1, JUN1111 selectively inhibited Cdc25 phosphatases in vitro in an irreversible, time-dependent manner and arrested cells in the G1 and G2/M phases of the cell cycle. It is noteworthy that both DA3003-1 and JUN1111 directly inhibited Cdc25B activity in cells. Depletion of glutathione increased cellular sensitivity to DA3003-1 and JUN1111, and in vitro Cdc25B inhibition by these compounds was sensitive to pH, catalase, and reductants (dithiothreitol and glutathione), consistent with oxidative inactivation. In addition, both DA3003-1 and JUN1111 rapidly generated intracellular reactive oxygen species. Analysis of Cdc25B by mass spectrometry revealed sulfonic acid formation on the catalytic cysteine of Cdc25B after in vitro treatment with DA3003-1. These results indicate that irreversible oxidation of the catalytic cysteine of Cdc25B is indeed a mechanism by which these quinolinediones inactivate this protein phosphatase.

Cell Cycle Proteins↗

PubMed2005

Synthesis and biological evaluation of (-)-16-normethyldictyostatin: a potent analogue of (-)-dictyostatin.

[structure: see text] (-)-16-Normethyldictyostatin has been made by total synthesis and is a potent antitumor agent in cells expressing wild-type tubulin and in one mutant cell line that is resistant to paclitaxel, but it is much less active than dictyostatin in another paclitaxel-resistant cell line where Val is substituted for Phe270. This provides strong evidence that the C16 methyl group of the dictyostatins is oriented toward Phe270 in the paclitaxel-binding site on beta-tubulin.

Animals↗

PubMed2005

A pregnane X receptor agonist with unique species-dependent stereoselectivity and its implications in drug development.

Pregnane X receptor (PXR) is an orphan nuclear receptor that regulates the expression of genes encoding drug-metabolizing enzymes and transporters. In addition to affecting drug metabolism, potent and selective PXR agonists may also have therapeutic potential by removing endogenous and exogenous toxins. In this article, we report the synthesis and identification of novel PXR agonists from a library of peptide isosteres. Compound S20, a C-cyclopropylalkylamide, was found to be a PXR agonist with both enantiomer- and species-specific selectivity. S20 has three chiral carbons and was resolved into its two enantiomers. The individual S20 enantiomers exhibited striking mouse/human-specific PXR activation, whereby enantiomer (+)-S20 preferentially activated hPXR, and enantiomer (-)-S20 was a better activator for mPXR. As a human PXR (hPXR) agonist, (+)-S20 was more potent and efficacious than rifampicin. Mutagenesis studies revealed that the ligand binding domain residue Phe305 is critical for the preference for the (-)-S20 enantiomer by the rodent PXR. Treatment of S20 induced the expression of drug-metabolizing enzymes and transporters in reporter gene assays, in primary human hepatocytes, and in "humanized" hPXR transgenic mice. To our knowledge, S20 represents the first compound whose enantiomers have opposite species preference in activating a xenobiotic receptor. The stereoselectivity may be used to guide the development of safer drugs to avoid drug-drug interactions or to achieve human-specific therapeutic effects when a xenobiotic receptor is being used as a drug target.

Amides↗

PubMed2005

HPRT gene alterations in umbilical cord blood T-lymphocytes in newborns of mothers exposed to tobacco smoke during pregnancy.

Prenatal exposure to tobacco smoke has been associated with an increased risk of pediatric malignancies, yet the transplacental induction of genetic alterations by tobacco smoke carcinogens and their implication to childhood diseases remain poorly understood. We characterized mutations in the HPRT gene in umbilical cord blood T-lymphocytes of self-reported 103 never-smoking mothers and 104 smoking mothers (54 mothers smoked throughout and 50 mothers quit smoking during pregnancy). The results showed the illegitimate V(D)J recombinase-mediated deletion of HPRT exons 2-3 was the most prominent alteration occurring in 48.2% (26/54) of mutants from neonates of the smoking mothers who smoked during pregnancy, compared with 28.0% (14/50) from those of smoking mothers who quit smoking during pregnancy (p=0.035, Fisher's exact test), 34.9% (36/103) from never-smoking mothers (p=0.08), or 32.7% (50/153) of those of neonates born from the latter two groups of mothers combined (p=0.043). There was no significant difference in the frequency of this deletion between neonates of the never-smoking mothers and the smoking mothers who quit smoking during pregnancy (34.9% versus 28.0%, respectively, p=0.39). The results show an increase in illegitimate V(D)J recombinase-mediated deletion of HPRT exons 2-3 in cord blood T-lymphocytes of newborns of mothers who smoked during pregnancy, compared with the group of mothers who did not smoke during pregnancy, implying an increase in illegitimate V(D)J recombinase-mediated alteration, a genetic recombination event associated with childhood malignancies, may be induced in utero during pregnancy by maternal exposure to tobacco smoke-derived genotoxicants.

Base Sequence↗

PubMed2005

New antiestrogens from a library screen of homoallylic amides, allylic amides, and C-cyclopropylalkylamides.

A new structural scaffold for antiestrogens was identified from the cell-based screening of transcriptional regulation properties of a 67-member library of homoallylic amides, allylic amides, and C-cyclopropylalkylamides. C-Cyclopropylalkylamide 3a (O-ethyl-N-{2-[(1S*,2R*)-2-{(R*)-[(diphenylphosphinoyl)amino](phenyl)methyl}cyclopropyl]ethyl}-N-[(4-methylphenyl)sulfonyl]carbamate) had antagonistic activity similar to that of tamoxifen and was further evaluated. Compound 3a inhibited estradiol-induced proliferation of the ER-positive MCF-7 cells but had no effect on ER-negative MDA-MB231 human breast cancer cells. Furthermore, high micromolar concentrations of 3a exhibited minimal cytotoxicity to the ER-negative line. The biological activities of the enantiomers of 3a did not differ from one another nor from that of racemic 3a.

Amides↗