RESUMO
BACKGROUND: In cases of immunodeficiency, a systemic infection may be revealed by atypical symptoms, particularly those involving the skin. PATIENTS AND METHODS: The present case describes a 19-year-old male with X-linked hypogammaglobulinemia, or Bruton agammaglobulinemia, treated with intravenous immunoglobulin G antibodies. Over a 6-week period, the patient developed recurrent plaques in both legs, first on one and then on the other, without fever. Blood cultures were repeated and the fifth pair proved positive for Campylobacter jejuni. An abdominal scan showed appendicitis without intestinal signs. The patient was treated with azithromycin for 2 weeks, which resulted in full recovery from the skin lesions. DISCUSSION: Campylobacter bacteremia infections are severe and carry a 15% mortality rate at 30 days. The majority of affected patients present humoral immunodeficiency. The literature contains reports of 10 patients with C. jejuni-associated cellulitis, of whom 6 presented hypogammaglobulinemia. We postulate that the cutaneous manifestations were caused by septic metastases. The immunoglobulin replacement therapy mainly comprised IgG antibodies; IgA and IgM antibodies appear to play a key role in the response to C. jejuni infection, which could explain the susceptibility observed. The American guidelines recommend blood and skin cultures in patients with cellular immune defects. We suggest that this recommendation be extended to patients with humoral immunodeficiency.
Assuntos
Agamaglobulinemia/complicações , Infecções por Campylobacter/diagnóstico , Celulite (Flegmão)/microbiologia , Doenças Genéticas Ligadas ao Cromossomo X/complicações , Antibacterianos/uso terapêutico , Apendicite/microbiologia , Azitromicina/uso terapêutico , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Infecções por Campylobacter/tratamento farmacológico , Celulite (Flegmão)/tratamento farmacológico , Humanos , Masculino , Adulto JovemRESUMO
Radiomics in neuroimaging uses fully automatic segmentation to delineate the anatomical areas for which radiomic features are computed. However, differences among these segmentation methods affect radiomic features to an unknown extent. A scan-rescan dataset (n = 46) of T1-weighted and diffusion tensor images was used. Subjects were split into a sleep-deprivation and a control group. Scans were segmented using four segmentation methods from which radiomic features were computed. First, we measured segmentation agreement using the Dice-coefficient. Second, robustness and reproducibility of radiomic features were measured using the intraclass correlation coefficient (ICC). Last, difference in predictive power was assessed using the Friedman-test on performance in a radiomics-based sleep deprivation classification application. Segmentation agreement was generally high (interquartile range = 0.77-0.90) and median feature robustness to segmentation method variation was higher (ICC > 0.7) than scan-rescan reproducibility (ICC 0.3-0.8). However, classification performance differed significantly among segmentation methods (p < 0.001) ranging from 77 to 84%. Accuracy was higher for more recent deep learning-based segmentation methods. Despite high agreement among segmentation methods, subtle differences significantly affected radiomic features and their predictive power. Consequently, the effect of differences in segmentation methods should be taken into account when designing and evaluating radiomics-based research methods.
Assuntos
Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Encéfalo/diagnóstico por imagem , Humanos , Processamento de Imagem Assistida por Computador/métodos , Neuroimagem , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: In endothelial cells, nitric oxide (NO) is produced by the endothelial isoform of nitric oxide synthase (eNOS), which is localized in the cholesterol-rich plasmalemmal microdomains involved in signal transduction, known as caveolae. The present study was undertaken to evaluate the effect of hypercholesterolemia and fatty streak formation on the endothelial caveolae and on endothelial function, and attempted to determine to what extent the caveolae were involved in endothelium-derived NO production. METHODS AND RESULTS: We first studied the effect of atheroma on endothelial NO production. Fatty streak infiltrated aorta of cholesterol-fed New Zealand White rabbits demonstrated an impairment of acetylcholine-induced relaxation and nearly normal calcium ionophore A23187-induced maximal relaxation. The abundance of caveolae in the endothelium covering the fatty streak, as well as their 'grape-like' clustering, appeared to be decreased. We therefore investigated the effect, on endothelial NO production, of the cholesterol-binding agents 2-hydroxypropyl-beta-cyclodextrin (hp-beta-CD) and filipin, known to alter caveolae structure and/or function. Treatment with either hp-beta-CD (2%) or filipin (4 microg/ml) did not affect contraction to phenylephrine or relaxant responses to A23187 or to the NO donor sodium nitroprusside. In contrast, both treatments impaired acetylcholine-induced relaxation. Cultured bovine aortic endothelial cells (BAEC) similarly treated with hp-beta-CD demonstrated a 50% decrease of total cellular cholesterol and a decreased abundance of caveolae as well as their 'grape-like' clustering. Cholesterol depletion decreased the bradykinin-induced transient peak of free intracellular calcium and subsequent receptor-stimulated NO production (assessed using reporter cells rich in soluble guanylyl cyclase), whereas that elicited by A23187 remained unaltered. CONCLUSION: Fatty streak deposit is associated with a decrease in caveolae 'transductosomes' abundance which appears to represent a novel mechanism of endothelial dysfunction.
Assuntos
Doenças da Aorta/fisiopatologia , Arteriosclerose/fisiopatologia , Membrana Celular/fisiologia , Endotélio Vascular/fisiopatologia , beta-Ciclodextrinas , 2-Hidroxipropil-beta-Ciclodextrina , Animais , Aorta Torácica/efeitos dos fármacos , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Técnicas de Cultura de Células , Membrana Celular/ultraestrutura , Ciclodextrinas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/ultraestrutura , Filipina/farmacologia , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Masculino , Microscopia Eletrônica , Contração Muscular/efeitos dos fármacos , Óxido Nítrico/biossíntese , CoelhosAssuntos
Tecnologia de Alimentos , Lipidômica , Oxisteróis/farmacologia , Esteróis/farmacologia , Pesquisa Biomédica/tendências , Congressos como Assunto , Comportamento Cooperativo , Tecnologia de Alimentos/métodos , Tecnologia de Alimentos/tendências , Humanos , Internacionalidade , Itália , Lipidômica/métodos , Lipidômica/tendências , Oxisteróis/química , Oxisteróis/metabolismo , Esteróis/química , Esteróis/metabolismoRESUMO
A new family of benzyl-phenoxy-ethanamine derivatives has been assayed for trypanocidal activity. Using tritiated morpholino-benzyl-phenoxy-ethanamine as a probe, it is shown that this ligand is able to bind specifically to a protein contained in extracts of Trypanosoma equiperdum. The binding is saturable and of high affinity (KD = 4 nM: Bmax = 200 fmol (mg protein)-1). The in vitro activities of the investigated compounds against this parasite correlate with their affinities to the putative binding site. Moreover, using an azido functionalized morpholino-benzyl-phenoxyethanamine as photoprobe a major M(r) = 40,000 protein was specifically revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. This molecular weight corresponds with the previously observed value determined for the antioestrogen binding site protein of rat liver which has been shown to specifically bind antioestrogens of the triphenylethylene family and phenoxyethanamine derivatives.
Assuntos
Proteínas de Transporte/metabolismo , Etanolaminas/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma/metabolismo , Marcadores de Afinidade , Animais , Ligação Competitiva , Proteínas de Transporte/química , Antagonistas de Estrogênios/metabolismo , Cinética , Proteínas de Protozoários/química , Tripanossomicidas/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma/efeitos dos fármacosRESUMO
The antiestrogen binding site (AEBS) is a membranous protein complex that has been shown to be intimately linked with the antiproliferative and antiretroviral effects of certain antiestrogenic compounds such as tamoxifen (Tx). Various specific ligands of AEBS derived from benzylphenoxy ethanamine and a new benzoyl structure were synthesized either by modification of the aminoether side chain or by halogen substitution at the meta-, ortho-, and para position on the benzoyl group. Using the MCF-7 cellular strain and its RTx6 variant (a clone selected for its antigrowth resistance to tamoxifen), it was shown that under high drug concentrations the cytotoxicity of the ligands was directly correlated with their affinity for AEBS. In agreement with previous observations made on triphenylethylenic ligands, modification of the basic ethanamine side chain modulated the ligand affinities. Chloride in meta increased ligand efficacy, whereas chloride substitution in ortho and para decreased it. Effects on AEBS-positive MCF-7 cells were drug concentration- and time-dependent, whereas they were unspecific on the AEBS-negative RTx6 cell line. These cytotoxic effects were confirmed in the absence of estrogen receptor on human AEBS-positive uterine cervix cell carcinoma HeLa cells, but were non-specific on rat fibroblastic AEBS-negative (low concentration) NRK cells. The cytotoxicities of these ligands are related to their affinities for AEBS.
Assuntos
Antineoplásicos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Antagonistas de Estrogênios/farmacologia , Tamoxifeno/análogos & derivados , Animais , Ligação Competitiva , Linhagem Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células HeLa/metabolismo , Humanos , Ratos , Receptores de Estrogênio/análiseRESUMO
We compared the anti-proliferative properties of 4-benzylphenoxy-N ethyl morpholine (morpho-BPE) and trifluopromazine (TFP) on both the human breast cancer cell lines, MCF7, and its tamoxifen-resistant variant RTx6. We found that the calmodulin antagonist trifluopromazine (TFP) which bound ABS weakly, inhibited MCF7 cell growth but did not follow the relationship observed for diphenylmethane derivatives between MCF7-inhibitory potencies and their Ki. Regarding the tamoxifen-resistant RTx6 cells, TFP but not morpho-BPE induced inhibition of the proliferation. Using a tritiated derivative of morpho-BPE, two distinct binding sites could be demonstrated. Indeed, a low affinity binding site was present in both cell lines whereas a high affinity binding site was mainly found in MCF7 cells although being in lower concentration (less than 10%) in RTx6 cells. Both tamoxifen and TFP displaced morpho-BPE from the two binding sites. The uptake and efflux of the tritiated drug were similar in the two cell lines. The drug did not appear to be metabolized. We concluded that TFP and morpho-BPE belong to distinct classes of molecules and that ABS mediates the anti-proliferative action of diphenylmethane derivatives but not the inhibitory effect of the calmodulin antagonist TFP.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Morfolinas/farmacologia , Promazina/análogos & derivados , Receptores de Droga , Receptores de Estrogênio/fisiologia , Sítios de Ligação , Divisão Celular/efeitos dos fármacos , Resistência a Medicamentos , Humanos , Cinética , Promazina/farmacologia , Tamoxifeno/metabolismo , Tamoxifeno/farmacologia , Trítio , Células Tumorais CultivadasRESUMO
1-Benzyl-4-(N-2-pyrrolidinylethoxy)benzene (PBPE) is a cytotoxic derivative of the antitumoral drug tamoxifen. PBPE binds with high-affinity and specificity to the microsomal antiestrogen-binding site (AEBS). PBPE, as well as some other high-affinity AEBS ligands, shares structural features with high-affinity and selective sigma receptor ligands in the N-(arylethyl)-N-alkyl-2-(1-pyrrolidinyl)ethylamine class, such as BD1008, which are cytotoxic against tumoral cells. Based on these structural and pharmacological similitudes, we set out to examine whether AEBS and sigma receptors could be related binding sites. We showed that BD1008 had a high affinity for AEBS. However, prototypical sigma receptor ligands were very low-affinity competitors on AEBS. Surprisingly, AEBS ligands displayed a high affinity for sigma-1 and sigma-2 receptor subtypes, showing that AEBS and sigma receptor-binding sites were not mutually exchangeable. Moreover, phenytoin, which is an allosteric modulator of sigma-1 receptor, was a competitive inhibitor of [3H]tamoxifen on AEBS. These results suggest that the tamoxifen-binding site on AEBS and the sigma ligand-binding site on sigma receptors were not identical but related entities. We also showed here that the high-affinity and specific AEBS ligands also bound sigma receptors with high affinity. Moreover, the compounds that were capable of displacing tamoxifen from AEBS were cytotoxic against tumoral cells but not against the AEBS-deficient cell line Rtx-6. These results confirm that AEBS and sigma receptors might belong to the same family of proteins, and that the tamoxifen-binding site might be involved in the cytotoxicity of AEBS ligands and some classes of sigma compounds.
Assuntos
Antineoplásicos Hormonais/farmacologia , Compostos Benzidrílicos/farmacologia , Receptores sigma/metabolismo , Tamoxifeno/farmacologia , Regulação Alostérica , Animais , Sítios de Ligação , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Ligantes , Masculino , Fenitoína/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Receptor Sigma-1RESUMO
We previously reported evidence for two subpopulations of several classes of steroid receptors that could be distinguished by their requirement of a low molecular weight factor (Mr=700-3000 Da) for binding to nonspecific, calf thymus DNA-cellulose [Cavanaugh, A. H. and Simons Jr., S. S., Journal of Steroid Biochemistry and Molecular Biology, 48, 433-446 (1994)]. This factor appeared to be enriched in (NH4)2SO4 precipitates of nuclear extracts. Using human progesterone receptors (PRs) and biologically active DNA sequences in a modified avidin/biotin-coupled DNA (ABCD) binding assay, we now report a factor-mediated increase in PR binding to specific DNA sites that was indistinguishable from that seen with nonspecific sites. The main advantages of this modified assay are that both kinetic and equilibrium binding of receptor-steroid complexes to DNA can be directly monitored in solution. The ability of either Sephadex G-50 chromatography or sodium arsenite to prevent that binding which is increased by added factor supported the existence of PR subpopulations that are independent of the acceptor DNA sequence. The factor was found, surprisingly, to be low concentrations (> or = 5 mM) of (NH4)2SO4, which anomalously is partially excluded from Sephadex G-10 columns, and can be mimicked by some salts but not sodium arsenite. Kinetic analyses demonstrated that the mechanism of action of salt was to accelerate the rate of binding of PR. Salt also had a much greater effect on the nonspecific binding of PR, such that the ratio of specific to nonspecific DNA binding was greatest at elevated salt concentrations (approximately 75 mM) that afforded submaximal levels of PR binding to specific DNA sites. Further analysis of the DNA-bound receptors revealed that the smaller, A-form of PR is preferentially bound to specific DNA sequences both in the presence and in the absence of various salt concentrations. Thus, the differences in DNA binding of PR +/- salt do not correlate with the preferential binding of A or B isoform. The unequal behavior of PR subpopulations and/or isoforms for binding to specific DNA sequences offers added mechanisms for selective transcriptional regulation of genes in intact cells.
Assuntos
DNA/genética , DNA/metabolismo , Receptores de Progesterona/metabolismo , Esteroides/metabolismo , Sulfato de Amônio/farmacologia , Animais , Arsenitos/farmacologia , Sequência de Bases , Sítios de Ligação/genética , Bovinos , Linhagem Celular , Estabilidade de Medicamentos , Humanos , Técnicas In Vitro , Cinética , Metanossulfonato de Metila/análogos & derivados , Metanossulfonato de Metila/farmacologia , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Ratos , Receptores de Progesterona/classificação , Receptores de Progesterona/genética , Sais/farmacologiaRESUMO
Glycine receptor antagonists have been proposed to have multiple therapeutic applications, including the treatment of stroke, epilepsy, and anxiety. The present study compared the biochemical and behavioral profiles of two strychnine-insensitive glycine receptor antagonists, MDL 100,458 (3-(benzoylmethylamino)-6-chloro-1H-indole-2- carboxylic acid) and MDL 102,288 (5,7-dichloro-1,4-dihydro-4-[[[4- [(methoxycarbonyl)amino]phenyl]sulfonyl]imino]-2-quinolinecarboxylic acid monohydrate). Both compounds potently inhibited [3H]glycine binding to rat cortical/hippocampal membranes (Ki = 136, 167 nM, respectively) without showing significant activity in 18 other receptor binding assays. In an in vitro functional assay, both compounds completely antagonized N-methyl-D-aspartate (NMDA)-stimulated cGMP accumulation in rat cerebellar slices. However, in contrast to their equipotency in the glycine receptor assay, MDL 100,458 was approximately 6-fold more potent than MDL 102,288 in the cGMP assay (IC50 values = 1.25, 7.8 microM, respectively). Behavioral tests demonstrated that MDL 102,288 and MDL 100,458 exhibited strikingly different in vivo profiles. MDL 100,458 antagonized audiogenic seizures in DBA/2J mice (ED50 = 20.8 mg/kg i.p.), whereas MDL 102,288 was without effect in the dose range tested (ED50 > 300 mg/kg i.p.). Central nervous system penetration did not appear to account for this difference. For example, MDL 102,288 was not active following direct intracerebroventricular administration (ED50 > 16 micrograms; vs. 0.78 microgram for MDL 100,458). In a test of anxiolytic activity, MDL 102,288 reduced separation-induced ultrasonic vocalizations in rat pups (ED50 = 6.3 mg/kg i.p.) whereas MDL 100,458 was only weakly active (ED50 = 80.8 mg/kg i.p.). Furthermore, the anxiolytic effect of MDL 102,288 was selective in that it occurred at doses that did not produce motoric disruption as measured by an inclined-plane test (ED50 > 160 mg/kg; therapeutic index > 25.4). In contrast, the anxiolytic activity of MDL 100,458 was non-selective in that it occurred at doses that also produced motoric disruption (ED50 = 57.7 mg/kg; therapeutic index = 0.7). Thus, two glycine receptor antagonists which have similar in vitro binding profiles as selective ligands for the strychnine-insensitive glycine receptor, demonstrate different in vitro and in vivo functional profiles. The reason for these differences is not clear, though one possibility could be that the compounds may act on different NMDA receptor subtypes. These data support the possibility that different glycine receptor antagonists may have different therapeutic targets.
Assuntos
Glicinérgicos/farmacologia , Indóis/farmacologia , Quinolonas/farmacologia , Receptores de Glicina/antagonistas & inibidores , Estimulação Acústica , Animais , Animais Recém-Nascidos , Ansiedade de Separação/psicologia , Ligação Competitiva/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Glicina/metabolismo , Camundongos , Camundongos Endogâmicos DBA , Ratos , Ratos Sprague-Dawley , Receptores de Droga/efeitos dos fármacos , Receptores de Droga/metabolismo , Receptores de Glutamato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsões/induzido quimicamente , Convulsões/psicologia , Vocalização Animal/efeitos dos fármacosRESUMO
Several diphenylmethane derivatives have been synthesized with variable affinities for Anti-estrogen Binding Sites (ABS) but not for the estrogen receptor. Using these molecules as probes it is shown that their binding affinities for ABS correlate with their abilities to inhibit the growth of MCF-7 human breast cancer cells. In contrast they have no influence on the proliferation of tamoxifen-resistant variant cells (RTx6) in which ABS are undetectable. These data support the conclusion that ABS has a functional role in the anti-proliferative effect of triphenylethylene anti-estrogens and structurally related compounds.
Assuntos
Compostos Benzidrílicos , Antagonistas de Estrogênios/metabolismo , Receptores de Droga , Receptores de Estrogênio/metabolismo , Ligação Competitiva , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Estradiol/metabolismo , Humanos , Cinética , Relação Estrutura-AtividadeRESUMO
We have recently reported that cytostatic concentrations of the microsomal antiestrogen-binding site (AEBS) ligands, such as PBPE (N-pyrrolidino-(phenylmethyphenoxy)-ethanamine,HCl) and tamoxifen, induced differentiation characteristics in breast cancer cells through the accumulation of post-lanosterol intermediates of cholesterol biosynthesis. We show here that exposure of MCF-7 (human breast adenocarcinoma cell line) cells to higher concentrations of AEBS ligands triggered active cell death and macroautophagy. Apoptosis was characterized by Annexin V binding, chromatin condensation, DNA laddering and disruption of the mitochondrial functions. We determined that cell death was sterol- and reactive oxygen species-dependent and was prevented by the antioxidant vitamin E. Macroautophagy was characterized by the accumulation of autophagic vacuoles, an increase in the expression of Beclin-1 and the stimulation of autophagic flux. We established that macroautophagy was sterol- and Beclin-1-dependent and was associated with cell survival rather than with cytotoxicity, as blockage of macroautophagy sensitized cells to AEBS ligands. These results show that the accumulation of sterols by AEBS ligands in MCF-7 cells induces apoptosis and macroautophagy. Collectively, these data support a therapeutic potential for selective AEBS ligands in breast cancer management and shows a mechanism that explains the induction of autophagy in MCF-7 cells by tamoxifen and other selective estrogen receptor modulators.
Assuntos
Antineoplásicos Hormonais/farmacologia , Apoptose , Autofagia , Neoplasias da Mama/metabolismo , Colesterol/metabolismo , Moduladores de Receptor Estrogênico/farmacologia , Etilaminas/toxicidade , Pirrolidinas/toxicidade , Tamoxifeno/farmacologia , Sítios de Ligação , Diferenciação Celular , Feminino , Humanos , Ligantes , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas , Vitamina E/farmacologiaRESUMO
Splenectomized mice are more resistant than normal mice to infection by Listeria monocytogenes. The nature of splenic regulation of cell mediated immunity to Listeria was investigated. Splenectomized mice were reconstituted with normal syngeneic spleen cells and normal plasma from Listeria-stimulated normal donors to determine if suppression of resistance in normal mice was cellular or humoral. Mice receiving spleen cells showed no decreased resistance, but mice receiving plasma showed decreased resistance as determined from bacterial numbers in the liver. The suppressive effect was associated with plasma components having a molecular weight less than 10,000. The data suggest that a suppressor factor is produced by spleen associated cells in response to stimulation of the cell-mediated immune system.
Assuntos
Imunidade Celular , Listeriose/imunologia , Baço/imunologia , Animais , Feminino , Dose Letal Mediana , Masculino , Camundongos , Esplenectomia , Linfócitos T Reguladores/imunologiaRESUMO
A low affinity (Kd = 30 nM), large capacity (Bmax = 2.6 pmol/g tissue) estrogen binding site was photolabeled from estradiol-stimulated rat uterus cytosol. To maximize levels of this binding site and reduce those of the type I binding site, ovariectomized rats were injected with high doses of estradiol (10 micrograms per day) for four days with the last injection two hours before sacrifice. This treatment depleted type I estrogen receptors from the cytosol (by 90%) and raised levels of type II sites in the nucleus without affecting cytosolic type II levels. The type II estradiol binding sites were distinguished from the type I sites on the basis of their dissociation kinetics, pH-sensitivity and their behavior towards potassium chloride, somatostatin, sodium thiocyanate, sulfhydryl reagents and ammonium sulfate precipitation. These type II binding sites could be covalently photolabeled with tritiated estrone. A molecular weight of 43 kDa was found on SDS PAGE.
Assuntos
Citosol/metabolismo , Estrona/metabolismo , Útero/metabolismo , Marcadores de Afinidade , Animais , Sítios de Ligação , Feminino , Concentração de Íons de Hidrogênio , Ratos , Ratos EndogâmicosRESUMO
Published ultraviolet (uv) inactivation data and in vitro transcription studies have suggested that vesicular stomatitis virus (VSV) leader RNA was solely responsible for the inhibition of host cell RNA synthesis by this virus. Since no protein product is encoded in leader RNA, this conclusion implied that no protein synthesis should be required for this effect. Therefore, the inhibitory activity of VSV was examined in the presence of the protein synthesis inhibitors, cycloheximide, pactamycin, and emetine. Protein synthesis inhibitors are known not to interfere with VSV primary transcription, but in their presence viral replication and amplification of transcription do not take place. Although at 39 degrees the VSV mutant tsG22 could undergo only primary transcription, maximum inhibition of host cell RNA synthesis took place. However, in the presence of the protein synthesis inhibitors the VSV mutant was no longer able to interfere with host cell RNA synthesis. These results could not be explained by a change in the concentration of intracellular leader RNA which remained unaltered by the drugs. Similar results were also obtained with wild-type VSV in the presence of cycloheximide. Upon removal of the drug, inhibition of host cell RNA synthesis was reestablished in parallel with the restoration of protein synthesis. It is concluded that protein synthesis is required for the inhibitory activity of VSV, presumably because the active inhibitory complex is a nucleoprotein containing leader RNA and either a cellular protein or the viral N protein. The cellular protein would have to be in limiting supply since de novo protein synthesis was required for the inhibition to take place.
Assuntos
Transformação Celular Viral , Biossíntese de Proteínas , RNA/genética , Transcrição Gênica , Vírus da Estomatite Vesicular Indiana/genética , Animais , Linhagem Celular , Cricetinae , Cicloeximida/farmacologia , Emetina/farmacologia , Rim , Cinética , Mutação , Pactamicina/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA Viral/isolamento & purificação , Sorotipagem , Transcrição Gênica/efeitos dos fármacos , Vírion/genéticaRESUMO
The anti-estrogen binding site (ABS) is an apparently ubiquitous component of cells that has been shown to be intimately linked with the antiproliferative effects of certain antiestrogenic compounds, like tamoxifen, which is currently used for the treatment of breast cancer. However, the identification and in vitro study of this novel protein has been hampered to date by a lack of convenient probes that will efficiently label the molecule in nonpurified preparations. Thus, using a selective ABS ligand (4-benzylphenoxy-N-ethylmorpholine, MBPE) as starting material, we synthesized a photosensitive azido derivative, [(2-azido-4-benzyl)phenoxy]N-ethylmorpholine (azido-MBPE) that can be prepared in a tritiated form. Azido-MBPE has a high affinity for ABS (Kd = 3 nM), identical to that of tamoxifen, and covalently labels 5 and 12% of membrane-bound and detergent-solubilized ABS, respectively. Its incorporation is selectively and competitively inhibited by other ABS ligands (tamoxifen greater than nitromifen greater than hydroxytamoxifen). [3H]Azido-MBPE potently photolabels either membrane-bound or detergent-solubilized ABS as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions revealing specific photoincorporation in a protein band of Mr = 40,000. This molecular weight is approximately two times lower than what we observed previously for ABS preparations studied under nondenaturing conditions and postlabeled with [3H]tamoxifen (Mr = 80,000-110,000). In chromatofocusing experiments with photolabeled ABS, a single specifically labeled protein fraction migrating with a pI of 6.4 was found to exhibit a Mr of 40,000 when subsequently electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. These results indicate that [3H]azido-MBPE is a specific high affinity probe of ABS that will prove useful in the ultimate identification of this protein.
Assuntos
Marcadores de Afinidade/metabolismo , Fígado/metabolismo , Microssomos Hepáticos/metabolismo , Morfolinas/metabolismo , Receptores de Droga , Receptores de Estrogênio/metabolismo , Tamoxifeno/metabolismo , Animais , Sítios de Ligação , Feminino , Membranas Intracelulares/metabolismo , Cinética , Ovariectomia , Ratos , Ratos Endogâmicos , Receptores de Estrogênio/isolamento & purificaçãoRESUMO
We previously demonstrated that, in addition to the estrogen receptor, the Antiestrogen Binding Site (ABS) is also a potent mediator of the antitumorous activity of the clinical drug tamoxifen. Because of report discrepancies in the binding parameters of rat liver ABS we first attempted to improve binding study conditions. In this way buffer, protein concentration, methodology for bound/free ligand separation and phospholipidic ratio were determined. This work was used to evaluate the Stoke radius (4.4 S) and isoelectric point (pH = 6.6) of the protein in its native state. These studies constituted the obligatory transition from rat liver to pure ABS protein.
Assuntos
Fígado/química , Receptores de Droga/isolamento & purificação , Animais , Neoplasias da Mama/química , Feminino , Focalização Isoelétrica , Especificidade de Órgãos/fisiologia , Fosfolipídeos/análise , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Homologia de Sequência de Aminoácidos , Solubilidade , Ultracentrifugação , Útero/químicaRESUMO
Our knowledge of the biological role of the antiestrogen binding site ABS in the antitumoral activity of tamoxifen, will be increased with the determination of its coding gene sequence. To this end our team has for some time attempted to purify this membranous protein. In this work we report the purification to homogeneity of ABS from rat liver in a six step succession. Specific photolabeling with a tritiated photoprobe, solubilization of rat liver microsomes, chromatofocusing of the labeled proteins, preparative electrophoresis on polyacrylamide gel, and two consecutive high performance liquid chromatography separations on C4 hydrophobic resin produced 2.5 micrograms of pure ABS by silver stain analysis of SDS-PAGE. The NH2-terminal residue of the protein appears to be blocked, which hinders the Edman degradation method for obtention of the whole protein sequence.
Assuntos
Fígado/metabolismo , Receptores de Droga/isolamento & purificação , Animais , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Ratos , Receptores de Droga/metabolismo , Solubilidade , Água/químicaRESUMO
Inhibition of host cellular protein synthesis by vesicular stomatitis virus (VSV) has been suggested to be primarily the result of competition for ribosomes between cellular and viral mRNAs (H. F. Lodish and M. Porter, J. Virol., 36:719-733, 1980; Lodish and Porter, J. Virol. 38:504-517, 1981). This hypothesis was investigated by regulating the extent of VSV mRNA synthesis through the use of defective interfering particles. Although intracellular VSV mRNA concentrations decreased by as much as a factor of 14 at high multiplicities of infection of defective interfering particles, the inhibition of host cell protein synthesis by VSV decreased by a maximum of only 10%. The data also indicated that under these conditions the protein-synthesizing capacity of the cells was not exhausted. We concluded that competition for cellular ribosomes could not have been the major factor in the inhibition of host cell protein synthesis by VSV. This conclusion was further supported by inhibition data obtained with VSV mutants. The ts G22 mutant, defective in replication but not in primary transcription, inhibited host protein synthesis at the nonpermissive temperature (39 degrees C) to the same extent as did wild-type virus, even though it generated only 30 to 50% of the amount of viral mRNA as did wild-type virus. Conversely, in infections with the R1 mutant, which did not inhibit host cell protein synthesis, the amount of total and polysome-bound viral mRNA was indistinguishable from that obtained in infections by wild-type virus.
Assuntos
Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Vírus da Estomatite Vesicular Indiana/fisiologia , Animais , Linhagem Celular , Cricetinae , Vírus Defeituosos/fisiologia , Rim , Mutação , Ribossomos/metabolismo , Temperatura , Vírus da Estomatite Vesicular Indiana/genéticaRESUMO
New compounds have been synthesized based on the structure of the anti-tumoral drug tamoxifen and its diphenylmethane derivative, N,N-diethyl-2-[(4-phenyl-methyl)-phenoxy]-ethanamine, HCl (DPPE). These new compounds have no affinity for the estrogen receptor (ER) and bind with various affinity to the anti-estrogen binding site (AEBS). Compounds 2, 10, 12, 13, 20a, 20b, 23a, 23b, 29 exhibited 1.1-69.5 higher affinity than DPPE, and compounds 23a and 23b have 1.2 and 3.5 higher affinity than tamoxifen. Three-dimensional structure analysis, performed using the intersection of the van der Waals volume occupied by tamoxifen in its crystallographic state and the van der Waals volume of these new compounds in their calculated minimal energy conformation, correlated well with their pKi for AEBS (r = 0.84, P<0.0001, n = 18). This is the first structure-affinity relationship (SAR) ever reported for AEBS ligands. Moreover in this study we have reported the synthesis of new compounds of higher affinity than the lead compounds and that are highly specific for AEBS. Since these compounds do not bind ER they will be helpful to study AEBS mediated cytotoxicity. Moreover our study shows that our strategy is a new useful guide to design high affinity and selective ligands for AEBS.