Saturable stimulation of fatty acid transport through model cytoplasm by soluble binding protein.
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To better define the role of soluble binding proteins in the cytoplasmic transport of amphipathic molecules, we measured the diffusional mobility of a fluorescent long-chain fatty acid, 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate (NBD-stearate), through model cytoplasm as a function of soluble binding protein concentration. Diffusional mobilities were correlated with the partition of the fatty acid between membrane and protein binding sites. Cytoplasm was modeled as a dense suspension of liposomes, and albumin was used as a model binding protein. Albumin saturably increased NBD-stearate mobility through the membrane suspension approximately eightfold. Fatty acid mobility in the absence of albumin was identical to the mobility of the membrane vesicles (1.99 +/- 0.33 x 10(-8) cm(2)/s), whereas the mobility at saturating concentrations was identical to the mobility of albumin (1.65 +/- 0.12 x 10(-7) cm(2)/s). The protein concentration producing half-maximal stimulation of NBD-stearate diffusion (42.8 +/- 0.3 microM) was unexpectedly greater than that required to solubilize half of the NBD-stearate (17.9 +/- 3.0 microM). These results support a proposed mechanism for cytoplasmic transport of small amphipathic molecules in which aqueous diffusion of the protein-bound form of the molecule largely determines the transport rate. However, slow interchange of fatty acid between the binding protein and membranes also appears to influence the transport rate in this model system. (+info)
Mechanisms of pyrazinamide resistance in mycobacteria: importance of lack of uptake in addition to lack of pyrazinamidase activity.
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Mycobacteria are known to acquire resistance to the antituberculous drug pyrazinamide (PZA) through mutations in the gene encoding pyrazinamidase (PZase), an enzyme that converts PZA into pyrazinoic acid, the presumed active form of PZA against bacteria. Additional mechanisms of resistance to the drug are known to exist but have not been fully investigated. Among these is the non-uptake of the pro-drug, a possibility investigated in the present study. The uptake mechanism of PZA, a requisite step for the activation of the pro-drug, was studied in Mycobacterium tuberculosis. The incorporation of [14C]PZA by the bacilli was followed in both neutral and acidic environments since PZA activity is known to be optimal at acidic pH. By using a protonophore (carbonyl cyanide m-chlorophenylhydrazone; CCCP), valinomycin, arsenate and low temperature, it was shown that an ATP-dependent transport system is involved in the uptake of PZA. Whilst the structurally analogous compound nicotinamide inhibited the transport system of PZA, other structurally related compounds such as pyrazinoic acid, isoniazid and cytosine did not. Acidic conditions were also without effect. Based on diffusion experiments in liposomes, it was found that PZA diffuses rapidly through membrane bilayers, faster than glycerol, whilst the presence of OmpATb, the porin-like protein of M. tuberculosis, in proteoliposomes slightly increased the diffusion of the drug. This finding may explain why the cell wall mycolate hydrophobic layer does not represent the limiting step in the diffusion of PZA, as judged from comparative experiments using a M. tuberculosis strain and its isogenic mutant elaborating 40% less covalently linked mycolates. PZase activity, and PZA uptake and susceptibility in different mycobacterial species were compared. M. tuberculosis, a naturally PZA-susceptible species, was the only species that exhibited both PZase activity and PZA uptake; no such correlation was observed with the four naturally resistant species examined. Mycobacterium smegmatis possessed a functional PZase but did not take up PZA; the reverse was true for the PZase-negative strain of Mycobacterium avium used, with PZA uptake comparable to that of M. tuberculosis. Mycobacterium bovis BCG and Mycobacterium kansasii exhibited neither a PZase activity nor PZA uptake. These data clearly demonstrate that one of the mechanisms of resistance to PZA resides in the failure of strains to take up the drug, indicating that susceptibility to PZA in mycobacteria requires both the presence of a functional PZase and a PZA transport system. No correlation was observed between the occurrence and cellular location of PZase and of nicotinamidase in the strains examined, suggesting that one or both amides can be hydrolysed by other mycobacterial amidases. (+info)
Determinants of paclitaxel penetration and accumulation in human solid tumor.
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The present study examined the determinants of the penetration and accumulation of [(3)H]paclitaxel (12-12,000 nM) in three-dimensional histocultures of patient tumors and of a human xenograft tumor in mice. The results showed 1) significant and saturable drug accumulation in tumors, 2) extensive drug retention in tumors, and 3) a slower penetration but a more extensive accumulation in the xenograft tumor compared with patient tumors. Drug penetration was not rate-limited by drug diffusion from medium through the matrix supporting the histocultures. The difference in the expression of the mdr1 P-glycoprotein did not fully account for the difference in the drug accumulation in xenograft and patient tumors. Autoradiography and imaging were used to evaluate the spatial relationship between tumor architecture, tumor cell distribution, and drug distribution as a function of time and initial drug concentration in culture medium. The tumor cell density and the kinetics of drug-induced apoptosis were also evaluated. The results indicate that a high tumor cell density is a barrier to paclitaxel penetration and that the apoptotic effect of paclitaxel enhances its penetration in solid tumor. These factors are responsible for the time- and concentration-dependent drug penetration rate, with drug penetration confined to the periphery until apoptosis and reduction of epithelial cell density occurred at 24 h, after which time paclitaxel penetrated the inner parts of the tumor. (+info)
Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy.
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We report the application of confocal imaging and fluorescence correlation spectroscopy (FCS) to characterize chemically well-defined lipid bilayer models for biomembranes. Giant unilamellar vesicles of dilauroyl phosphatidylcholine/dipalmitoyl phosphatidylcholine (DLPC/DPPC)/cholesterol were imaged by confocal fluorescence microscopy with two fluorescent probes, 1, 1'-dieicosanyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C(20)) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3 -phosphoc holine (Bodipy-PC). Phase separation was visualized by differential probe partition into the coexisting phases. Three-dimensional image reconstructions of confocal z-scans through giant unilamellar vesicles reveal the anisotropic morphology of coexisting phase domains on the surface of these vesicles with full two-dimensional resolution. This method demonstrates by direct visualization the exact superposition of like phase domains in apposing monolayers, thus answering a long-standing open question. Cholesterol was found to induce a marked change in the phase boundary shapes of the coexisting phase domains. To further characterize the phases, the translational diffusion coefficient, D(T), of the DiI-C(20) was measured by FCS. D(T) values at approximately 25 degrees C ranged from approximately 3 x 10(-8) cm(2)/s in the fluid phase, to approximately 2 x 10(-9) cm(2)/s in high-cholesterol-content phases, to approximately 2 x 10(-10) cm(2)/s in the spatially ordered phases that coexist with fluid phases. In favorable cases, FCS could distinguish two different values of D(T) in a region of two-phase coexistence on a single vesicle. (+info)
Quantitative evaluation of alternative mechanisms of blood and testes disposition of di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate in rats.
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Di(2-ethylhexyl) phthalate (DEHP), a commercially important plasticizer, induces testicular toxicity in laboratory animals at high doses. After oral exposure, most of the DEHP is rapidly metabolized in the gut to mono(2-ethylhexyl) phthalate (MEHP), which is the active metabolite for induction of testicular toxicity. To quantify the testes dose of MEHP with various routes of exposure and dose levels, we developed a physiologically based pharmacokinetic (PBPK) model for DEHP and MEHP in rats. Tissue:blood partition coefficients for DEHP were estimated from the n-octanol: water partition coefficient, while partition coefficients for MEHP were determined experimentally using a vial equilibration technique. All other parameters were either found in the literature or estimated from blood or tissue levels following oral or intravenous exposure to DEHP or MEHP. A flow-limited model failed to adequately simulate the available data. Alternative plausible mechanisms were explored, including diffusion-limited membrane transport, enterohepatic circulation, and MEHP ionization (pH-trapping model). In the pH-trapping model, only nonionized MEHP is free to become partitioned into the tissues, where it is equilibrated and trapped as ionized MEHP until it is deionized and released. All three alternative models significantly improved predictions of DEHP and MEHP blood concentrations over the flow-limited model predictions. The pH-trapping model gave the best predictions with the largest value of the log likelihood function. Predicted MEHP blood and testes concentrations were compared to measured concentrations in juvenile rats to validate the pH-trapping model. Thus, MEHP ionization may be an important mechanism of MEHP blood and testes disposition in rats. (+info)
Biophysical characterization of lithostathine. Evidences for a polymeric structure at physiological pH and a proteolysis mechanism leading to the formation of fibrils.
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Lithostathine is a calcium carbonate crystal habit modifier. It is found precipitated under the form of fibrils in chronic calcifying pancreatitis or Alzheimer's disease. In order to gain better insight into the nature and the formation of fibrils, we have expressed and purified recombinant lithostathine. Analytical ultracentrifugation and quasi-elastic light scattering techniques were used to demonstrate that lithostathine remains essentially monomeric at acidic pH while it aggregates at physiological pH. Analysis of these aggregates by electron microscopy showed an apparently unorganized structure of numerous monomers which tend to precipitate forming regular unbranched fibrils. Aggregated forms seem to occur prior to the apparition of fibrils. In addition, we have demonstrated that these fibrils resulted from a proteolysis mechanism due to a specific cleavage of the Arg(11)-Ile(12) peptide bond. It is deduced that the NH(2)-terminal undecapeptide of lithostathine normally impedes fiber formation but not aggregation. A theoretical model explaining the formation of amyloid plaques in neurodegenerative diseases or stones in lithiasis starting from lithostathine is described. Therefore we propose that lithostathine, whose major function is unknown, defines a new class of molecules which is activated by proteolysis and is not involved in cytoskeleton nor intermediate filament functions. (+info)
Trafficking of endothelial nitric-oxide synthase in living cells. Quantitative evidence supporting the role of palmitoylation as a kinetic trapping mechanism limiting membrane diffusion.
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To examine endothelial nitric-oxide synthase (eNOS) trafficking in living endothelial cells, the eNOS-deficient endothelial cell line ECV304 was stably transfected with an eNOS-green fluorescent protein (GFP) fusion construct and characterized by functional, biochemical, and microscopic analysis. eNOS-GFP was colocalized with Golgi and plasma membrane markers and produced NO in response to agonist challenge. Localization in the plasma membrane was dependent on the palmitoylation state, since the palmitoylation mutant of eNOS (C15S/C26S eNOS-GFP) was excluded from the plasma membrane and was concentrated in a diffuse perinuclear pattern. Fluorescence recovery after photobleaching (FRAP) revealed eNOS-GFP in the perinuclear region moving 3 times faster than the plasmalemmal pool, suggesting that protein-lipid or protein-protein interactions are different in these two cellular domains. FRAP of the palmitoylation mutant was two times faster than that of wild-type eNOS-GFP, indicating that palmitoylation was influencing the rate of trafficking. Interestingly, FRAP of C15S/C26S eNOS-GFP but not wild-type eNOS-GFP fit a model of protein diffusion in a lipid bilayer. These data suggest that the regulation of eNOS trafficking within the plasma membrane and Golgi are probably different mechanisms and not due to simple diffusion of the protein in a lipid bilayer. (+info)
Conductances, diffusion and streaming potentials in the rat proximal tubule.
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1. Transtubular potential differences and specific resistances were measured in rat proximal tubules by means of single and double barrelled glass micro-electrodes. 2. Tip localization was made by observation of effective resistance changes measured with double barrelled micro-electrodes upon passage of oil droplets, and by perfusion with choline C1. 3. Mean early proximal p.d.s. of the order of -1 to -2 mV, and late values of +0-5 to +1mV were found. Mean specific resistances ranged from 12 to 15 omega cm2. 4. Diffusion potentials and single ion relative conductances were evaluated, perfusing the lumen with solutions differing only with respect to one salt concentration. Na and K conductances were similar and greater than those of C1. 5. Luminal and peritubular perfusions with hypotonic solutions showed the occurrence of streaming potentials in this structure suggesting the existence of pores lined with negative charges. The effective diameter of these pores appeared to be reduced by hypotonic perfusion, as evidenced by a significant increase in resistance, indicating that the main ion path across this structure is represented by intercellular spaces. (+info)