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Find video protocols related to scientific articles indexed in Pubmed.
Preparation and long-term biodistribution studies of a PAMAM dendrimer G5-Gd-BnDOTA conjugate for lymphatic imaging.
Nanomedicine (Lond)
PUBLISHED: 11-14-2014
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Aims: To demonstrate the use of gadolinium (Gd)-labeled dendrimers as lymphatic imaging agents and establish the long-term biodistribution (90-day) of this type of agent in mice. Methods: A G5-Gd-BnDOTA dendrimer was prepared and injected into mice and monkeys for MR lymphangiography, and long-term biodistribution of the conjugate was studied. Results: Administration of G5-Gd-BnDOTA in mice demonstrated a rapid uptake in the deep lymphatic system while injection in monkeys showed enhanced internal iliac nodes, indicating its general utility for lymphatic tracking. Biodistribution studies to 90 days showed that gadolinium conjugate is slowly being eliminated from the liver and other organs. Conclusion: The use of G5-Gd-BnDOTA holds great promise for lymphatic imaging, but its slow clearance from the body might hamper its eventual clinical translation.
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Immuno-PET Imaging of Tumor Endothelial Marker 8 (TEM8).
Mol. Pharm.
PUBLISHED: 07-11-2014
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Tumor endothelial marker 8 (TEM8) is a cell surface receptor that is highly expressed in a variety of human tumors and promotes tumor angiogenesis and cell growth. Antibodies targeting TEM8 block tumor angiogenesis in a manner distinct from the VEGF receptor pathway. Development of a TEM8 imaging agent could aid in patient selection for specific antiangiogenic therapies and for response monitoring. In these studies, L2, a therapeutic anti-TEM8 monoclonal IgG antibody (L2mAb), was labeled with (89)Zr and evaluated in vitro and in vivo in TEM8 expressing cells and mouse xenografts (NCI-H460, DLD-1) as a potential TEM8 immuno-PET imaging agent. (89)Zr-df-L2mAb was synthesized using a desferioxamine-L2mAb conjugate (df-L2mAb); (125)I-L2mAb was labeled directly. In vitro binding studies were performed using human derived cell lines with high, moderate, and low/undetectable TEM8 expression. (89)Zr-df-L2mAb in vitro autoradiography studies and CD31 IHC staining were performed with cryosections from human tumor xenografts (NCI-H460, DLD-1, MKN-45, U87-MG, T-47D, and A-431). Confirmatory TEM8 Western blots were performed with the same tumor types and cells. (89)Zr-df-L2mAb biodistribution and PET imaging studies were performed in NCI-H460 and DLD-1 xenografts in nude mice. (125)I-L2mAb and (89)Zr-df-L2mAb exhibited specific and high affinity binding to TEM8 that was consistent with TEM8 expression levels. In NCI-H460 and DLD-1 mouse xenografts nontarget tissue uptake of (89)Zr-df-L2mAb was similar; the liver and spleen exhibited the highest uptake at all time points. (89)Zr-L2mAb was highly retained in NCI-H460 tumors with <10% losses from day 1 to day 3 with the highest tumor to muscle ratios (T:M) occurring at day 3. DLD-1 tumors exhibited similar pharmacokinetics, but tumor uptake and T:M ratios were reduced ?2-fold in comparison to NCI-H460 at all time points. NCI-H460 and DLD-1 tumors were easily visualized in PET imaging studies despite low in vitro TEM8 expression in DLD-1 cells indicating that in vivo expression might be higher in DLD-1 tumors. From in vitro autoradiography studies (89)Zr-df-L2mAb specific binding was found in 6 tumor types (U87-MG, NCI-H460, T-47D MKN-45, A-431, and DLD-1) which highly correlated to vessel density (CD31 IHC). Westerns blots confirmed the presence of TEM8 in the 6 tumor types but found undetectable TEM8 levels in DLD-1 and MKN-45 cells. This data would indicate that TEM8 is associated with the tumor vasculature rather than the tumor tissue, thus explaining the increased TEM8 expression in DLD-1 tumors compared to DLD-1 cell cultures. (89)Zr-df-L2mAb specifically targeted TEM8 in vitro and in vivo although the in vitro expression was not necessarily predictive of in vivo expression which seemed to be associated with the tumor vasculature. In mouse models, (89)Zr-df-L2mAb tumor uptakes and T:M ratios were sufficient for visualization during PET imaging. These results would suggest that a TEM8 targeted PET imaging agent, such as (89)Zr-df-L2mAb, may have potential clinical, diagnostic, and prognostic applications by providing a quantitative measure of tumor angiogenesis and patient selection for future TEM8 directed therapies.
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Inhibition of glutathione peroxidase mediates the collateral sensitivity of multidrug-resistant cells to tiopronin.
J. Biol. Chem.
PUBLISHED: 06-14-2014
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Multidrug resistance (MDR) is a major obstacle to the successful chemotherapy of cancer. MDR is often the result of overexpression of ATP-binding cassette transporters following chemotherapy. A common ATP-binding cassette transporter that is overexpressed in MDR cancer cells is P-glycoprotein, which actively effluxes drugs against a concentration gradient, producing an MDR phenotype. Collateral sensitivity (CS), a phenomenon of drug hypersensitivity, is defined as the ability of certain compounds to selectively target MDR cells, but not the drug-sensitive parent cells from which they were derived. The drug tiopronin has been previously shown to elicit CS. However, unlike other CS agents, the mechanism of action was not dependent on the expression of P-glycoprotein in MDR cells. We have determined that the CS activity of tiopronin is mediated by the generation of reactive oxygen species (ROS) and that CS can be reversed by a variety of ROS-scavenging compounds. Specifically, selective toxicity of tiopronin toward MDR cells is achieved by inhibition of glutathione peroxidase (GPx), and the mode of inhibition of GPx1 by tiopronin is shown in this report. Why MDR cells are particularly sensitive to ROS is discussed, as is the difficulty in exploiting this hypersensitivity to tiopronin in the clinic.
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Simultaneous ECG-gated PET imaging of multiple mice.
Nucl. Med. Biol.
PUBLISHED: 02-11-2014
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We describe and illustrate a method for creating ECG-gated PET images of the heart for each of several mice imaged at the same time. The method is intended to increase "throughput" in PET research studies of cardiac dynamics or to obtain information derived from such studies, e.g. tracer concentration in end-diastolic left ventricular blood.
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Magnetic resonance lymphography of the thoracic duct after interstitial injection of gadofosveset trisodium: a pilot dosing study in a porcine model.
Lymphat Res Biol
PUBLISHED: 02-06-2014
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BACKGROUND-RATIONALE: To investigate whether interstitial injection of gadofosveset trisodium (Ablavar®, Lantheus Medical, North Billerica, MA) would be suitable for thoracic duct (TD) imaging in a pig model.
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Tracking the luminal exposure and lymphatic drainage pathways of intravaginal and intrarectal inocula used in nonhuman primate models of HIV transmission.
PLoS ONE
PUBLISHED: 01-01-2014
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Over 80% of sexual HIV-1 transmissions originate from a single viral variant, but the underlying basis for this transmission bottleneck remains to be elucidated. Nonhuman primate models of mucosal virus transmission allow opportunities to gain insight into the basis of this mucosal bottleneck. We used simulated inocula consisting of either non-infectious vital dye or contrast dye with non-invasive magnetic resonance imaging (MRI) to visualize mucosal exposure and passive lymphatic drainage patterns following vaginal and rectal exposures in Indian origin rhesus macaques. Results revealed a limited overall distance of dye coverage from the anal verge following 1 ml (n = 8) intrarectally administered, which greatly increased with a 3 ml (n = 8) volume. Intravaginal dye exposure using 2 ml revealed complete coverage of the mucosa of the vagina and ectocervix, however dye was not detectable in the endocervix, uterus, fallopian tubes or ovaries in nuliparous sexually mature rhesus macaques (n = 9). In addition, following submucosal and intranodal injections of vital dye or MRI contrast dye in the rectum (n = 9), or distal and proximal vagina (n = 4), the lymphatic drainage pathways were identified as first the internal then common iliac chain followed by para-aortic lymph nodes. Drainage from the distal descending colon (n = 8) was via the para-colonic lymph nodes followed by the inferior mesenteric and para-aortic lymph nodes. Analysis after vaginal challenge with infectious SIVmac239 followed by euthanasia at day 3 revealed a pattern of viral dissemination consistent with the imaging results. These results provide insights into potential patterns of viral dissemination that can help guide efforts to better elucidate the earliest events of virus transmission and potential intervention strategies.
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Endosomolytic anionic polymer for the cytoplasmic delivery of siRNAs in localized in vivo applications.
Adv Funct Mater
PUBLISHED: 11-26-2013
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The use of small interfering RNAs (siRNAs) to down-regulate the expression of disease-associated proteins carries significant promise for the treatment of a variety of clinical disorders. One of the main barriers to the widespread clinical use of siRNAs, however, is their entrapment and degradation within the endolysosomal pathway of target cells. Here we report the trafficking and function of PP75, a non-toxic, biodegradable, lipid membrane disruptive anionic polymer composed of phenylalanine derivatized poly(L-lysine iso-phthalamide). PP75 is readily endocytosed by cells, safely permeabilizes endolysosomes in a pH dependent manner and facilitates the transfer of co-endocytosed materials directly into the cytoplasm. The covalent attachment of siRNAs to PP75 using disulfide linkages generates conjugates that effectively traffic siRNAs to the cytoplasm of target cells both in vitro and in vivo. In a subcutaneous malignant glioma tumor model, a locally delivered PP75-stathmin siRNA conjugate decreases stathmin expression in tumor cells and, in combination with the nitrosourea chemotherapy carmustine, is highly effective at inhibiting tumor growth. PP75 may be clinically useful for the local delivery of siRNAs, in particular for the treatment of solid tumors.
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The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2.
Glycobiology
PUBLISHED: 08-25-2011
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In recent years, sugars with a unique chemical handle have been used to detect and elucidate the function of glycoconjugates. Such chemical handles have generally been part of an N-acetyl moiety of a sugar. We have previously developed several applications using the single mutant Y289L-?1,4-galactosyltransferase I (Y289L-?4Gal-T1) and the wild-type polypeptide-?-GalNAc-T enzymes with UDP-C2-keto-Gal. Here, we describe for the first time that the GlcNAc-transferring enzymes-R228K-Y289L-?4Gal-T1 mutant enzyme, the wild-type human ?1,3-N-acetylglucosaminyltransferase-2 and human Maniac Fringe-can also transfer the GlcNAc analog C2-keto-Glc molecule from UDP-C2-keto-Glc to their respective acceptor substrates. Although the R228K-Y289L-?4Gal-T1 mutant enzyme transfers the donor sugar substrate GlcNAc or its analog C2-keto-Glc only to its natural acceptor substrate, GlcNAc, it does not transfer to its analog C2-keto-Glc. Thus, these observations suggest that the GlcNAc-transferring glycosyltransferases can generally accommodate a chemical handle in the N-acetyl-binding cavity of the donor sugar substrate, but not in the N-acetyl-binding cavity of the acceptor sugar.
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Collateral sensitivity of multidrug-resistant cells to the orphan drug tiopronin.
J. Med. Chem.
PUBLISHED: 06-24-2011
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A major challenge in the treatment of cancer is multidrug resistance (MDR) that develops during chemotherapy. Here we demonstrate that tiopronin (1), a thiol-substituted N-propanoylglycine derivative, was selectively toxic to a series of cell lines expressing the drug efflux pump P-glycoprotein (P-gp, ABCB1) and MRP1 (ABCC1). Treatment of MDR cells with 1 led to instability of the ABCB1 mRNA and consequently a reduction in P-gp protein, despite functional assays demonstrating that tiopronin does not interact with P-gp. Long-term exposure of P-gp-expressing cells to 1 sensitized them to doxorubicin and paclitaxel, both P-gp substrates. Treatment of MRP1-overexpressing cells with tiopronin led to a significant reduction in MRP1 protein. Synthesis and screening of analogues of tiopronin demonstrated that the thiol functional group was essential for collateral sensitivity while substitution of the amino acid backbone altered but did not destroy specificity, pointing to future development of targeted analogues.
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Syntheses of meta-[F]Fluorobenzaldehyde and meta-[F]Fluorobenzylbromide from Phenyl(3-Formylphenyl) Iodonium Salt Precursors.
J Labelled Comp Radiopharm
PUBLISHED: 05-03-2011
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(18)F-labeled fluorobenzaldehydes and fluorobenzylbromides are useful synthons for the preparation of PET radiopharmaceuticals. Whereas ortho- and para-[(18)F]fluorobenzaldehydes can easily be prepared with high yields, the corresponding meta- derivatives are more problematic. In order to improve the yield of meta-[(18)F]fluorobenzaldehyde we used the corresponding diaryliodonium salt precursors, since diaryliodonium salts had already been used as precursors in preparations of (18)F-labeled electron rich, as well as electron deficient, aromatic rings. Diaryliodonium salts with different counter ions [PhIPhCHO]X (X = Cl, Br, OTs, OTf) were synthesized. (18)F radiolabeling was performed using different bases at different temperatures in the presence of a radical scavenger, 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO). The best conversion (~80%) to meta-[(18)F] fluorobenzaldehyde was obtained using CsHCO(3) base at a reaction temperature of 110 °C. To study iodonium salt counter ion effects on radiofluorination, each precursor was separately treated with CsF[(18)F]/CsHCO(3) in DMF at 110 °C for 5 min in the presence of TEMPO. Our observed reactivity order was OTs
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Rh-I-UEA-1 polymerized liposomes target and image adenomatous polyps in the APC(Min/+) mouse using optical colonography.
Mol Imaging
PUBLISHED: 04-26-2011
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Mutated adenomatous polyposis coli (APC) genes predispose transformations to neoplasia, progressing to colorectal carcinoma. Early detection facilitates clinical management and therapy. Novel lectin-mediated polymerized targeted liposomes (Rh-I-UEA-1), with polyp specificity and incorporated imaging agents were fabricated to locate and image adenomatous polyps in APC(Min/+) mice. The biomarker ?-L-fucose covalently joins the liposomal conjugated lectin Ulexeuropaeus agglutinin (UEA-1), via glycosidic linkage to the polyp mucin layer. Multispectral optical imaging (MSI) corroborated a global perspective of specific binding (rhodamine B 532 nm emission, 590-620 nm excitation) of targeted Rh-I-UEA-1 polymerized liposomes to polyps with 1.4-fold labeling efficiency. High-resolution coregistered optical coherence tomography (OCT) and fluorescence molecular imaging (FMI) reveal the spatial correlation of contrast distribution and tissue morphology. Freshly excised APC(Min) bowels were incubated with targeted liposomes (UEA-1 lectin), control liposomes (no lectin), or iohexol (Omnipaque) and imaged by the three techniques. Computed tomographic quantitative analyses did not confirm that targeted liposomes more strongly bound polyps than nontargeted liposomes or iohexol (Omnipaque) alone. OCT, with anatomic depth capabilities, along with the coregistered FMI, substantiated Rh-I-UEA-1 liposome binding along the mucinous polyp surface. UEA-1 lectin denotes ?-l-fucose biomarker carbohydrate expression at the mucin glycoprotein layer; Rh-I-UEA-1 polymerized liposomes target and image adenomatous polyps in APC(Min) mice.
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Improved synthesis of UDP-2-(2-ketopropyl)galactose and a first synthesis of UDP-2-(2-ketopropyl)glucose for the site-specific linking of biomolecules via modified glycan residues using glycosyltransferases.
Tetrahedron
PUBLISHED: 03-26-2011
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The potential of wild-type and mutant glycosyltransferases to produce glycoconjugates carrying sugar moieties with chemical handles has made it possible to conjugate biomolecules with orthogonal reacting groups at specific sites. The synthesis of UDP-2-(2-ketopropyl)galactose has been previously carried out, albeit with difficulty and low efficiency. A modified approach has been developed for the synthesis of UDP-2-(2-ketopropyl)glucose and UDP-2-(2-ketopropyl)galactose, allowing better access to the desired test compounds, the UDP-2-(2-ketopropyl)glucose and UDP-2-(2-ketopropyl)galactose analogs were synthesized in 8 steps and 4.8% and 5.3% overall yield respectively, an improvement over the 1(st) generation synthesis involving 8 steps and an overall yield of 0.7%.
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68Ga-DOTA-affibody molecule for in vivo assessment of HER2/neu expression with PET.
Eur. J. Nucl. Med. Mol. Imaging
PUBLISHED: 03-18-2011
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Overexpression of HER2/neu in breast cancer is correlated with a poor prognosis. It may vary between primary tumors and metastatic lesions and change during the treatment. Therefore, there is a need for a new means to assess HER2/neu expression in vivo. In this work, we used (68)Ga-labeled DOTA-Z(HER2:2891)-Affibody to monitor HER2/neu expression in a panel of breast cancer xenografts.
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Development of a MR-visible compound for tracing neuroanatomical connections in vivo.
Neuron
PUBLISHED: 02-16-2011
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Traditional studies of neuroanatomical connections require injection of tracer compounds into living brains, then histology of the postmortem tissue. Here, we describe and validate a compound that reveals neuronal connections in vivo, using MRI. The classic anatomical tracer CTB (cholera-toxin subunit-B) was conjugated with a gadolinium-chelate to form GdDOTA-CTB. GdDOTA-CTB was injected into the primary somatosensory cortex (S1) or the olfactory pathway of rats. High-resolution MR images were collected at a range of time points at 11.7T and 7T. The transported GdDOTA-CTB was visible for at least 1 month post-injection, clearing within 2 months. Control injections of non-conjugated GdDOTA into S1 were not transported and cleared within 1-2 days. Control injections of Gd-Albumin were not transported either, clearing within 7 days. These MR results were verified by classic immunohistochemical staining for CTB, in the same animals. The GdDOTA-CTB neuronal transport was target specific, monosynaptic, stable for several weeks, and reproducible.
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Near-infrared fluorescence lifetime pH-sensitive probes.
Biophys. J.
PUBLISHED: 02-16-2011
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We report what we believe to be the first near-infrared pH-sensitive fluorescence lifetime molecular probe suitable for biological applications in physiological range. Specifically, we modified a known fluorophore skeleton, hexamethylindotricarbocyanine, with a tertiary amine functionality that was electronically coupled to the fluorophore, to generate a pH-sensitive probe. The pK(a) of the probe depended critically on the location of the amine. Peripheral substitution at the 5-position of the indole ring resulted in a compound with pK(a) ? 4.9 as determined by emission spectroscopy. In contrast, substitution at the meso-position shifted the pK(a) to 5.5. The resulting compound, LS482, demonstrated steady-state and fluorescence-lifetime pH-sensitivity. This sensitivity stemmed from distinct lifetimes for protonated (?1.16 ns in acidic DMSO) and deprotonated (?1.4 ns in basic DMSO) components. The suitability of the fluorescent dyes for biological applications was demonstrated with a fluorescence-lifetime tomography system. The ability to interrogate cellular processes and subsequently translate the findings in living organisms further augments the potential of these lifetime-based pH probes.
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Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.
J. Lipid Res.
PUBLISHED: 07-21-2010
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We previously showed that macrophages from macrophage-specific ATP-binding cassette transporter A1 (ABCA1) knockout (Abca1(-M/-M)) mice had an enhanced proinflammatory response to the Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), compared with wild-type (WT) mice. In the present study, we demonstrate a direct association between free cholesterol (FC), lipid raft content, and hyper-responsiveness of macrophages to LPS in WT mice. Abca1(-M/-M) macrophages were also hyper-responsive to specific agonists to TLR2, TLR7, and TLR9, but not TLR3, compared with WT macrophages. We hypothesized that ABCA1 regulates macrophage responsiveness to TLR agonists by modulation of lipid raft cholesterol and TLR mobilization to lipid rafts. We demonstrated that Abca1(-M/-M) vs. WT macrophages contained 23% more FC in isolated lipid rafts. Further, mass spectrometric analysis suggested raft phospholipid composition was unchanged. Although cell surface expression of TLR4 was similar between Abca1(-M/-M) and WT macrophages, significantly more TLR4 was distributed in membrane lipid rafts in Abca1(-M/-M) macrophages. Abca1(-M/-M) macrophages also exhibited increased trafficking of the predominantly intracellular TLR9 into lipid rafts in response to TLR9-specific agonist (CpG). Collectively, our data suggest that macrophage ABCA1 dampens inflammation by reducing MyD88-dependent TLRs trafficking to lipid rafts by selective reduction of FC content in lipid rafts.
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Dyslipidemia induces opposing effects on intrapulmonary and extrapulmonary host defense through divergent TLR response phenotypes.
J. Immunol.
PUBLISHED: 06-25-2010
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Dyslipidemia influences innate immune responses in the bloodstream, but whether and how pulmonary innate immunity is sensitive to circulating lipoproteins is largely unknown. To define whether dyslipidemia impacts responses to bacteria in the airspace and, if so, whether differently from its effects in other tissues, airspace, bloodstream, and i.p. responses to LPS and Klebsiella pneumoniae were investigated using murine models of dyslipidemia. Dyslipidemia reduced neutrophil (PMN) recruitment to the airspace in response to LPS and K. pneumoniae by impairing both chemokine induction in the airspace and PMN chemotaxis, thereby compromising pulmonary bacterial clearance. Paradoxically, bacteria were cleared more effectively from the bloodstream during dyslipidemia. This enhanced systemic response was due, at least in part, to basal circulating neutrophilia and basal TLR4/MyD88-dependent serum cytokine induction and enhanced serum cytokine responses to systemically administered TLR ligands. Dyslipidemia did not globally impair PMN transvascular trafficking to, and host defense within all loci, because neutrophilia, cytokine induction, and bacterial clearance were enhanced within the infected peritoneum. Peritoneal macrophages from dyslipidemic animals were primed for more robust TLR responses, reflecting increased lipid rafts and increased TLR4 expression, whereas macrophages from the airspace, in which cholesterol was maintained constant during dyslipidemia, had normal responses and rafts. Dyslipidemia thus imparts opposing effects upon intra- and extrapulmonary host defense by inducing tissue-divergent TLR response phenotypes and dysregulating airspace/blood compartmental levels of PMNs and cytokines. We propose that the airspace is a "privileged" site, thereby uniquely sensitive to dyslipidemia.
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Discovery of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amine aurora kinase inhibitors.
J. Med. Chem.
PUBLISHED: 05-14-2010
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Through cell-based screening of our kinase-directed compound collection, we discovered that a subset of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amines were potent cytotoxic agents against cancer cell lines, suppressed mitotic histone H3 phosphorylation, and caused aberrant mitotic phenotypes. It was subsequently established that these compounds were in fact potent inhibitors of aurora A and B kinases. It was shown that potency and selectivity of aurora kinase inhibition correlated with the presence of a substituent at the aniline para-position in these compounds. The anticancer effects of lead compound 4-methyl-5-(2-(4-morpholinophenylamino)pyrimidin-4-yl)thiazol-2-amine (18; K(i) values of 8.0 and 9.2 nM for aurora A and B, respectively) were shown to emanate from cell death following mitotic failure and increased polyploidy as a consequence of cellular inhibition of aurora A and B kinases. Preliminary in vivo assessment showed that compound 18 was orally bioavailable and possessed anticancer activity. Compound 18 (CYC116) is currently undergoing phase I clinical evaluation in cancer patients.
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Near infrared-fluorescent and magnetic resonance imaging molecular probe with high T1 relaxivity for in vivo multimodal imaging.
Chem. Commun. (Camb.)
PUBLISHED: 04-13-2010
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A new gadolinium chelating NIR fluorescent molecular probe increases T(1) relaxivity of water protons, facilitating combined optical and magnetic resonance imaging.
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Discovery and characterization of 2-anilino-4- (thiazol-5-yl)pyrimidine transcriptional CDK inhibitors as anticancer agents.
Chem. Biol.
PUBLISHED: 03-06-2010
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The main difficulty in the development of ATP antagonist kinase inhibitors is target specificity, since the ATP-binding motif is present in many proteins. We introduce a strategy that has allowed us to identify compounds from a kinase inhibitor library that block the cyclin-dependent kinases responsible for regulating transcription, i.e., CDK7 and especially CDK9. The screening cascade employs cellular phenotypic assays based on mitotic index and nuclear p53 protein accumulation. This permitted us to classify compounds into transcriptional, cell cycle, and mitotic inhibitor groups. We describe the characterization of the transcriptional inhibitor class in terms of kinase inhibition profile, cellular mode of action, and selectivity for transformed cells. A structural selectivity rationale was used to optimize potency and biopharmaceutical properties and led to the development of a transcriptional inhibitor, 3,4-dimethyl-5-[2-(4-piperazin-1-yl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one, with anticancer activity in animal models.
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Design, synthesis, and evaluation of 2-methyl- and 2-amino-N-aryl-4,5-dihydrothiazolo[4,5-h]quinazolin-8-amines as ring-constrained 2-anilino-4-(thiazol-5-yl)pyrimidine cyclin-dependent kinase inhibitors.
J. Med. Chem.
PUBLISHED: 02-12-2010
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Following the recent discovery and development of 2-anilino-4-(thiazol-5-yl)pyrimidine cyclin dependent kinase (CDK) inhibitors, a program was initiated to evaluate related ring-constrained analogues, specifically, 2-methyl- and 2-amino-N-aryl-4,5-dihydrothiazolo[4,5-h]quinazolin-8-amines for inhibition of CDKs. Here we report the rational design, synthesis, structure-activity relationships (SARs), and cellular mode-of-action profile of these second generation CDK inhibitors. Many of the analogues from this chemical series inhibit CDKs with very low nanomolar K(i) values. The most potent compound reported in this study inhibits CDK2 with an IC(50) of 0.7 nM ([ATP] = 100 microM). Furthermore, an X-ray crystal structure of 2-methyl-N-(3-(nitro)phenyl)-4,5-dihydrothiazolo[4,5-h]quinazolin-8-amine (11g), a representative from the chemical series in complex with cyclin A-CDK2, is reported, confirming the design rationale and expected binding mode within the CDK2 ATP binding pocket.
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Formation of a mast cell synapse: Fc epsilon RI membrane dynamics upon binding mobile or immobilized ligands on surfaces.
J. Immunol.
PUBLISHED: 12-30-2009
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Fc epsilonRI on mast cells form a synapse when presented with mobile, bilayer-incorporated Ag. In this study, we show that receptor reorganization within the contacting mast cell membrane is markedly different upon binding of mobile and immobilized ligands. Rat basophilic leukemia mast cells primed with fluorescent anti-DNP IgE were engaged by surfaces presenting either bilayer-incorporated, monovalent DNP-lipid (mobile ligand), or chemically cross-linked, multivalent DNP (immobilized ligand). Total internal reflection fluorescence imaging and electron microscopy methods were used to visualize receptor reorganization at the contact site. The spatial relationships of Fc epsilonRI to other cellular components at the synapse, such as actin, cholesterol, and linker for activation of T cells, were also analyzed. Stimulation of mast cells with immobilized polyvalent ligand resulted in typical levels of degranulation. Remarkably, degranulation also followed interaction of mast cells, with bilayers presenting mobile, monovalent ligand. Receptors engaged with mobile ligand coalesce into large, cholesterol-rich clusters that occupy the central portion of the contacting membrane. These data indicate that Fc epsilonRI cross-linking is not an obligatory step in triggering mast cell signaling and suggest that dense populations of mobile receptors are capable of initiating low-level degranulation upon ligand recognition.
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An improved synthesis of a fluorescent gabapentin-choline conjugate for single molecule detection.
Tetrahedron Lett.
PUBLISHED: 08-14-2009
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Voltage-gated calcium ion channels are comprised of pore-forming ?(1) and auxiliary ?(2)?, ? and ? subunits. They are important molecular devices involved in a variety of cell functions. Fluorescently labeled acylcholine analogues are important in studies such as ion channel regulation. Cy3-3-acetylcholine has recently been synthesized for single molecule detection studies; albeit in an extremely low overall yield (0.06 %). In this work, an alternative route to that used in the previous Cy3-3-acetylcholine synthesis was developed with a 90 % yield at a significantly lower material cost.
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Radioactivity-synchronized fluorescence enhancement using a radionuclide fluorescence-quenched dye.
J. Am. Chem. Soc.
PUBLISHED: 06-12-2009
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We demonstrate the first evidence of radioactivity-synchronized fluorescence quenching of a near-infrared light-emitting dye by a radionuclide, (64)Cu, and subsequent fluorescence enhancement upon (64)Cu decay to the daughter isotopes (64)Ni and (64)Zn. The dynamic switch from high radioactivity and low fluorescence to low radioactivity and high fluorescence is potentially useful for developing complementary multimodal imaging and detection platforms for chemical, environmental, and biomedical applications as well as for unraveling the mechanisms of metal-induced dynamic fluorescence changes.
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Physiologic upper limit of pore size in the blood-tumor barrier of malignant solid tumors.
J Transl Med
PUBLISHED: 04-27-2009
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The existence of large pores in the blood-tumor barrier (BTB) of malignant solid tumor microvasculature makes the blood-tumor barrier more permeable to macromolecules than the endothelial barrier of most normal tissue microvasculature. The BTB of malignant solid tumors growing outside the brain, in peripheral tissues, is more permeable than that of similar tumors growing inside the brain. This has been previously attributed to the larger anatomic sizes of the pores within the BTB of peripheral tumors. Since in the physiological state in vivo a fibrous glycocalyx layer coats the pores of the BTB, it is possible that the effective physiologic pore size in the BTB of brain tumors and peripheral tumors is similar. If this were the case, then the higher permeability of the BTB of peripheral tumor would be attributable to the presence of a greater number of pores in the BTB of peripheral tumors. In this study, we probed in vivo the upper limit of pore size in the BTB of rodent malignant gliomas grown inside the brain, the orthotopic site, as well as outside the brain in temporalis skeletal muscle, the ectopic site.
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Synthesis and characterization of a new fluorogenic substrate for alpha-galactosidase.
Anal Bioanal Chem
PUBLISHED: 04-09-2009
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Alpha-galactosidase A hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins in lysosomes. Mutations in alpha-galactosidase cause lysosomal accumulation of the glycosphingolipid, globotriaosylceramide, which leads to Fabry disease. Small-molecule chaperones that bind to mutant enzyme proteins and correct their misfolding and mistrafficking have emerged as a potential therapy for Fabry disease. We have synthesized a red fluorogenic substrate, resorufinyl alpha-D-galactopyranoside, for a new alpha-galactosidase enzyme assay. This assay can be measured continuously at lower pH values, without the addition of a stop solution, due to the relatively low pK(a) of resorufin (approximately 6). In addition, the assay emits red fluorescence, which can significantly reduce interferences due to compound fluorescence and dust/lint as compared to blue fluorescence. Therefore, this new red fluorogenic substrate and the resulting enzyme assay can be used in high-throughput screening to identify small-molecule chaperones for Fabry disease.
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Synthesis and pharmacological characterization of [(125)I]MRS1898, a high-affinity, selective radioligand for the rat A(3) adenosine receptor.
Purinergic Signal.
PUBLISHED: 04-01-2009
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A known selective agonist of the A(3) adenosine receptors (AR), MRS1898 [(1R,2R,3S,4R,5S)-4-{2-chloro-6-[(3-iodophenylmethyl)amino]purin-9-yl}-1-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2,3-diol], was synthesized in radioactive form and characterized pharmacologically. This agonist ligand series, based on nucleoside analogues containing a rigid, bicyclic ring system in place of the ribose moiety, was selected for radiolabeling due to its high A(3)AR affinity across species, with nanomolar binding at both rat and human A(3)ARs. The radioiodination of MRS1898 on its N (6)-3-iodobenzyl substituent was accomplished in 76% radiochemical yield by iododestannylation of a 3-(trimethylstannyl)benzyl precursor. [(125)I]MRS1898 bound to the rat A(3)AR with a K(d) value of 0.17 +/- 0.04 nM and a B(max) value of 0.66 +/- 0.15 pmol/mg protein. The competition binding profiles for other agonists and antagonists obtained with this radioligand are similar to those previously obtained with other radioligands. The advantages of [(125)I]MRS1898 compared with previously used radioligands are primarily its high selectivity and affinity for the rat A(3)AR and also its facile synthesis and radiochemical stability; however, a relatively high level of nonspecific binding presents a limitation. Thus, we have introduced the first selective radioligand for the rat A(3)AR.
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A new resorufin-based alpha-glucosidase assay for high-throughput screening.
Anal. Biochem.
PUBLISHED: 02-16-2009
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Mutations in alpha-glucosidase cause accumulation of glycogen in lysosomes, resulting in Pompe disease, a lysosomal storage disorder. Small molecule chaperones that bind to enzyme proteins and correct the misfolding and mistrafficking of mutant proteins have emerged as a new therapeutic approach for the lysosomal storage disorders. In addition, alpha-glucosidase is a therapeutic target for type II diabetes, and alpha-glucosidase inhibitors have been used in the clinic as alternative treatments for this disease. We have developed a new fluorogenic substrate for the alpha-glucosidase enzyme assay, resorufin alpha-d-glucopyranoside. The enzyme reaction product of this new substrate emits at a peak of 590 nm, reducing the interference from fluorescent compounds seen with the existing fluorogenic substrate, 4-methylumbelliferyl-alpha-D-glucopyranoside. Also, the enzyme kinetic assay can be carried out continuously without the addition of stop solution due to the lower pK(a) of the product of this substrate. Therefore, this new fluorogenic substrate is a useful tool for the alpha-glucosidase enzyme assay and will facilitate compound screening for the development of new therapies for Pompe disease.
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In vivo fluorescence lifetime imaging monitors binding of specific probes to cancer biomarkers.
PLoS ONE
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One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the "image and treat" concept, especially for early evaluation of the efficacy of the therapy.
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Synthesis of ApoSense compound [18F]2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoic acid ([18F]NST732) by nucleophilic ring opening of an aziridine precursor.
Nucl. Med. Biol.
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The small molecule 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoic acid (NST732) is a member of the ApoSense family of compounds, capable of selective targeting, binding and accumulation within cells undergoing apoptotic cell death. It has application in molecular imaging and blood clotting particularly for monitoring antiapoptotic drug treatments. We are investigating a fluorine-18-radiolabeled analog of this compound for positron emission tomography studies.
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