Ligand-mediated tertiary structure changes of reconstituted P-glycoprotein. A tryptophan fluorescence quenching analysis.
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Ligand-dependent changes in accessibility of purified P-glycoprotein, functionally reconstituted in liposomes, were investigated by fluorescence measurements. Trp quenching experiments provided evidence that P-glycoprotein adopts different tertiary structures upon binding of drug substrates in the absence and presence of MgATP and its nonhydrolyzable analog, MgATPgammaS. Five anthracycline derivatives were tested as drug substrates: daunorubicin, 4'-epi-doxorubicin, iododoxorubicin, 4-demethoxy-daunorubicin, and methoxy-morpholino-doxorubicin. Among them, daunorubicin and 4'-epi-doxorubicin have been shown to be rejected outside the multidrug-resistant cells, whereas the three others have been shown to accumulate in multidrug-resistant cells overexpressing P-glycoprotein and therefore retain their cytotoxic activity. A small conformational change was associated with nucleotide binding and amplified after nucleotide hydrolysis. Different conformational states were adopted by P-glycoprotein upon the addition of the anthracycline derivatives in the absence and presence of MgATP or MgATPgammaS. These conformational changes are shown to be related to the nature of the antitumor agents and more precisely to their capacity to accumulate in resistant cells. These data also suggest that the cytotoxicity of iododoxorubicin and 4-demethoxy-daunorubicin is related to the fact they are not transported by P-glycoprotein. On the contrary, methoxy-morpholino-doxorubicin cytotoxicity may be explained in terms of its rapid reincorporation into the plasma membrane after being transported by P-glycoprotein. (+info)
Interactive binding to the two principal ligand binding sites of human serum albumin: effect of the neutral-to-base transition.
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The relationship between the two principal ligand binding sites, sites I and II, on human serum albumin (HSA) was quantitatively and qualitatively examined by equilibrium dialysis and fluorescence spectroscopy. Among the three subsite markers to site I, only the binding of dansyl-L-asparagine (DNSA), which is a subsite Ib marker (K. Yamasaki et al., Biochim. Biophys. Acta 1295 (1996) 147), was inhibited by the simultaneous binding of a site II ligand, such as ibuprofen and diazepam. This indicates that, in contrast to subsite Ib, subsites Ia and Ic do not strongly interact with site II. The thermodynamic characteristics for the coupling reaction between DNSA and ibuprofen and between DNSA and diazepam, which gave positive coupling free energies and negative values for both coupling enthalpy and entropy, indicated that the reaction process was entropically driven. Increase of pH from 6.5 to 8.2 caused an increase in coupling constant and entropy for the mutual antagonism between DNSA and the site II ligands on binding to HSA. The site II ligand-induced red-shift of lambda(max) and solvent accessibility of DNSA in subsite Ib were decreased when the albumin molecule was isomerized from the neutral (N) to the base (B) conformation in the physiological pH region. Based on these findings, we conclude that a 'competitive' like strong allosteric regulation exists for the binding of these two ligands to the N conformer, whereas for the B conformer this interaction can be classified as nearly 'independent'. Since the distance between Trp-214, which resides within the site I subdomain, and Tyr-411, which is involved in site II, is increased by 6 A during the N-B transition (N.G. Hagag et al., Fed. Proc. 41 (1982) 1189), we propose a mechanism for the pH-dependent antagonistic binding between subsite Ib and site II, which involves the transmission of ligand-induced allosteric effects from one site to another site, modified by changes in the spatial relationship of sites I and II caused by the N-B transition. (+info)
Insoluble gamma-tubulin-containing structures are anchored to the apical network of intermediate filaments in polarized CACO-2 epithelial cells.
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We have previously shown that a thin ( approximately 1 microm) layer of intermediate filaments located beneath the apical membrane of a variety of simple epithelial cells participates in the organization of apical microfilaments and microtubules. Here, I confirmed the apical distribution of gamma-tubulin-containing structures (potential microtubule-organizing centers) in CACO-2 cells and demonstrated perfect colocalization of centrosomes and nearly 50% of noncentrosomal gamma-tubulin with apical intermediate filaments, but not with apical F-actin. Furthermore, the antisense-oligonucleotide-mediated downregulation of cytokeratin 19, using two different antisense sequences, was more efficient than anticytoskeletal agents to delocalize centrosomes. Electron microscopy colocalization suggests that binding occurs at the outer boundary of the pericentriolar material. Type I cytokeratins 18 and 19 present in these cells specifically coimmunoprecipitated in multi-protein fragments of the cytoskeleton with gamma-tubulin. The size and shape of the fragments, visualized at the EM level, indicate that physical trapping is an unlikely explanation for this result. Drastic changes in the extraction protocol did not affect coimmunoprecipitation. These results from three independent techniques, indicate that insoluble gamma-tubulin-containing structures are attached to apical intermediate filaments. (+info)
Mortality patterns among workers exposed to acrylamide: 1994 follow up.
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OBJECTIVE: To update the mortality experience of a cohort of 8508 workers with potential exposure to acrylamide at three plants in the United States from 1984-94. METHODS: Analyses of standardised mortality ratios (SMR) with national and local rates and relative risk (RR) regression modelling were performed to assess site specific cancer risks by demographic and work history factors, and exposure indicators for acrylamide and muriatic acid. RESULTS: For the 1925-94 study period, excess and deficit overall mortality risks were found for cancer sites of interest: brain and other central nervous system (CNS) (SMR 0.65, 95% confidence interval (95% CI) 0.36 to 1.09), thyroid gland (SMR 2.11, 95% CI 0.44 to 6.17), testis and other male genital organs (SMR 0.28, 95% CI 0.01 to 1.59), and cancer of the respiratory system (SMR 1.10, 95% CI 0.99 to 1.22); however, none was significant or associated with exposure to acrylamide. A previously reported excess mortality risk of cancer of the respiratory system at one plant remained increased among workers with potential exposure to muriatic acid (RR 1.50, 95% CI 0.86 to 2.59), but was only slightly increased among workers exposed or unexposed to acrylamide. In an exploratory exposure-response analysis of rectal, oesophageal, pancreatic, and kidney cancer, we found increased SMRs for some categories of exposure to acrylamide, but little evidence of an exposure-response relation. A significant 2.26-fold risk (95% CI 1.03 to 4.29) was found for pancreatic cancer among workers with cumulative exposure to acrylamide > 0.30 mg/m3.years; however, no consistent exposure-response relations were detected with the exposure measures considered when RR regression models were adjusted for time since first exposure to acrylamide. CONCLUSION: The contribution of 1115 additional deaths and nearly 60,000 person-years over the 11 year follow up period corroborate the original cohort study findings of little evidence for a causal relation between exposure to acrylamide and mortality from any cancer sites, including those of initial interest. This is the most definitive study of the human carcinogenic potential of exposure to acrylamide conducted to date. (+info)
Adventures in membrane protein topology. A study of the membrane-bound state of colicin E1.
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The molecular aggregate size of the closed state of the colicin E1 channel was determined by fluorescence resonance energy transfer experiments involving a fluorescence donor (three tryptophans, wild-type protein) and a fluorescence acceptor (5-(((acetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (AEDANS), Trp-deficient protein). There was no evidence of energy transfer between the donor and acceptor species when bound to membrane large unilamellar vesicles. These experiments led to the conclusion that the colicin E1 channel is monomeric in the membrane-bound closed channel state. Experiments were also conducted to study the membrane topology of the closed colicin channel in membrane large unilamellar vesicles using acrylamide as the membrane-impermeant, nonionic quencher of tryptophan fluorescence in a battery of single tryptophan mutant proteins. Furthermore, additional fluorescence parameters, including fluorescence emission maximum, fluorescence quantum yield, and fluorescence decay times, were used to assist in mapping the topology of the closed channel. Results suggest that the closed channel comprises most of the polypeptide of the channel domain and that the hydrophobic anchor domain does not transverse the membrane bilayer but nonetheless is deeply embedded within the hydrocarbon core of the membrane. Finally, a model is proposed which features at least two states that are in rapid equilibrium with each other and in which one state is more heavily populated than the other. (+info)
Different adhesion properties of highly and poorly metastatic HT-29 colon carcinoma cells with extracellular matrix components: role of integrin expression and cytoskeletal components.
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Integrin-mediated tumour cell adhesion to extracellular matrix (ECM) components is an important step in the development of metastatic lesions. Thus, integrin expression and integrin-mediated adhesion of colon carcinoma cells to various ECM components was examined. Poorly (HT-29P) and highly (HT-29LMM) liver-metastatic colon carcinoma cells were used to study the rates of adhesion to collagen I (C I), collagen IV (C IV), laminin (LN), fibronectin (FN), or vitronectin (VN) in a static adhesion assay (10-120 min). Cells were untreated or treated with oligopeptides (RGD, GRGDS, YIGSR, RGES), anti-integrin antibodies, or colchicine, nocodazole, cycloheximide, acrylamide or cytochalasin D (to disrupt cytoskeletal structures). Both cell lines expressed similar patterns of integrin expression (alpha2, alpha3, ,alpha6, alphav, beta1, beta4, and beta5) by immunocytochemistry and immunoprecipitation. HT-29LMM cells showed significantly higher rates of adhesion to LN (P < 0.001) and FN (P < 0.001), but significantly poorer rates of adhesion to C I (P < 0.05) and C IV (P < 0.001) than HT-29P cells, respectively, adhesion to VN was insignificant. RGD and GRGDS inhibited HT-29LMM cell adhesion to FN only. Pretreatment with anti-beta, or anti-alpha2 integrin subunits suppressed adhesion to C I and C IV, and adhesion to LN was inhibited with anti-beta1 or anti-alpha6 integrin. Anti-beta1 or anti-alphav blocked adhesion to FN. Pretreatment of cells with cytochalasin D, cycloheximide or acrylamide inhibited adhesive interactions of both cell lines to the ECM components. In contrast, colchicine and nocodazole had no effect. The results demonstrate that adhesion of HT-29 cells to ECM is mediated, in part, by different integrins, depending on the substrate. Poorly and highly metastatic HT-29 cells possessed different patterns of adhesion to the various ECM substrates, but these differences were not due to different expression of integrin subunits. The results also suggested that the initial adhesion of poorly or highly metastatic HT-29 cells to ECM components requires, in part, the presence of native action and intermediate filaments, but not of microtubules. Thus the adhesion of tumour cells to ECM components may be dependent on signal transduction and assembly of microfilaments. (+info)
Stability, refolding and Ca2+ binding of pullulanase from the hyperthermophilic archaeon Pyrococcus woesei.
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The unfolding and refolding of the extremely heat-stable pullulanase from Pyrococcus woesei has been investigated using guanidinium chloride as denaturant. The monomeric enzyme (90 kDa) was found to be very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 4.86 +/- 0.29 M for intrinsic fluorescence and 4.90 +/- 0.31 M for far-UV CD changes. The unfolding process was reversible. Reactivation of the completely denatured enzyme (in 7.8 M guanidinium chloride) was obtained upon removal of the denaturant by stepwise dilution; 100% reactivation was observed when refolding was carried out via a guanidinium chloride concentration of 4 M in the first dilution step. Particular attention has been paid to the role of Ca2+ which activates and stabilizes this archaeal pullulanase against thermal inactivation. The enzyme binds two Ca2+ ions with a Kd of 0.080 +/- 0.010 microM and a Hill coefficient H of 1.00 +/- 0.10. This cation enhances significantly the stability of the pullulanase against guanidinium chloride-induced unfolding and the DeltaGH2OD increased from 6.83 +/- 0.43 to 8.42 +/- 0.55 kcal.mol-1. The refolding of the pullulanase, on the other hand, was not affected by Ca2+. (+info)
Synthesis of polyacrylamides N-substituted with PNA-like oligonucleotide mimics for molecular diagnostic applications.
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Two types of oligonucleotide mimics relative to peptide nucleic acids (PNAs) were tested as probes in nucleic acid hybridisation assays based on polyacrylamide technology. One type of mimic oligomers represented a chimera constructed of PNA and phosphono-PNA (pPNA) monomers, and the other one contained pPNA residues alternating with PNA-like monomers on the base of trans -4-hydroxy-L-proline (HypNA). A chemistry providing efficient and specific covalent attachment of these DNA mimics to acrylamide polymers using a convenient approach based on the co-polymerisation of acrylamide and some reactive acrylic acid derivatives with oligomers bearing 5'- or 3'-terminal acrylamide groups has been developed. A comparative study of polyacrylamide conjugates with oligonucleotides and mimic oligomers demonstrated the suitability and high potential of PNA-pPNA and HypNA-pPNA chimeras as sequence-specific probes in capture and detection of target nucleic acid fragments to serve current forms of DNA arrays. (+info)