Different invasion phenotypes of Campylobacter isolates in Caco-2 cell monolayers.
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The pathogenesis of campylobacter enteritis is not well understood, but invasion into and translocation across intestinal epithelial cells may be involved in the disease process, as demonstrated for a number of other enteric pathogens. However, the mechanisms involved in these processes are not clearly defined for campylobacters. In this study, isolates were compared quantitatively in established assays with the enterocyte-like cell line, Caco-2, to determine the extent to which intracellular invasion contributes to translocation across epithelial cell monolayers, and whether isolates vary in this respect. Ten fresh Campylobacter isolates were compared and shown to differ in invasiveness by a factor of 10-fold by following their recovery from gentamicin-treated Caco-2 cells grown on nonpermeable tissue-culture wells. Four of these isolates with contrasting invasive ability were also shown to vary in their ability to translocate across Caco-2 cells grown on semipermeable Transwell inserts by a factor >10. However, translocation did not quantitatively correlate with the intracellular invasiveness of these isolates. Isolate no. 9752 was poorly invasive but had modest translocation ability, isolate no. 10392 was very invasive but did not translocate significantly and remained within the monolayer, isolate no. 9519 both translocated and invaded well, whereas, isolate no. 235 translocated very efficiently but was poorly invasive. Isolate no. 9519 also uniquely caused a transitory flattening of the Caco-2 cells and a possible drop in trans-epithelial electrical resistance (TEER) of the Transwell monolayers, whereas isolate no. 235 did not show these effects. Together these data demonstrate that there are significantly different 'invasion' phenotypes among Campylobacter strains involving different degrees of intracellular invasion, and either different rates of transcellular trafficking or, alternatively, paracellular trafficking. (+info)
A "release" protocol for isothermal titration calorimetry.
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Isothermal titration calorimetry (ITC) has become a standard method for investigating the binding of ligands to receptor molecules or the partitioning of solutes between water and lipid vesicles. Accordingly, solutes are mixed with membranes (or ligands with receptors), and the subsequent heats of incorporation (or binding) are measured. In this paper we derive a general formula for modeling ITC titration heats in both binding and partitioning systems that allows for the modeling of the classic incorporation or binding protocols, as well as of new protocols assessing the release of solute from previously solute-loaded vesicles (or the dissociation of ligand/receptor complexes) upon dilution. One major advantage of a simultaneous application of the incorporation/binding and release protocols is that it allows for the determination of whether a ligand is able to access the vesicle interior within the time scale of the ITC experiment. This information cannot be obtained from a classical partitioning experiment, but it must be known to determine the partition coefficient (or binding constant and stochiometry) and the transfer enthalpy. The approach is presented using the partitioning of the nonionic detergent C12EO7 to palmitoyloleoylphosphatidylcholine vesicles. The release protocol could also be advantageous in the case of receptors that are more stable in the ligand-saturated rather than the ligand-depleted state. (+info)
Effect of glutamine on the intestinal permeability changes induced by indomethacin in humans.
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BACKGROUND: Long-term non-steroidal anti-inflammatory drug (NSAID) intake may induce increased intestinal permeability, eventually resulting in enteropathy. Because increased permeability might be related to cell damage resulting from energy depletion, it was hypothesized that glutamine--the major energy source of the intestinal mucosal cell--might prevent permeability changes. METHODS: The 6-h urinary excretion of 51Cr-EDTA after an oral load of 51Cr-EDTA was used in this study as a measure for intestinal permeability. Healthy volunteers underwent a series of permeability tests: (i) basal test; (ii) test following NSAID (indomethacin); (iii) test following NSAID in combination with glutamine and/or misoprostol. RESULTS: The NSAID induced increased permeability in all volunteers. Pre-treatment with glutamine (3x7 g daily, 1 week before NSAID-dosing) did not prevent the NSAID-induced increase in permeability. Multiple doses of glutamine close in time to NSAID-dosing resulted in significantly lower permeability compared to the NSAID without glutamine. Co-administration of misoprostol with the multiple-dose scheme of glutamine resulted in a further reduction in the NSAID-induced increase in permeability. CONCLUSIONS: Glutamine decreases the permeability changes caused by NSAID-dosing when it is administered close in time, and misoprostol has a synergistic effect. (+info)
Biosynthesis and packaging of carboxypeptidase D into nascent secretory vesicles in pituitary cell lines.
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Metallocarboxypeptidase D (CPD) is a membrane-bound trans-Golgi network (TGN) protein. In AtT-20 cells, CPD is initially produced as a 170-kDa endoglycosidase H-sensitive glycoprotein. Within 30 min of chase, the CPD increases to 180 kDa and is resistant to endoglycosidase H as a result of carbohydrate maturation. CPD also undergoes an activation step required for binding to a substrate affinity resin. Blocking the protein exit from the endoplasmic reticulum inhibits the increase in molecular mass but not the step required for affinity column binding, suggesting that enzyme activation precedes carbohydrate maturation and that these reactions occur in distinct intracellular compartments. Only the higher molecular weight mature CPD enters nascent secretory vesicles, which bud from the TGN of permeabilized AtT-20 and GH3 cells. The budding efficiency of CPD into vesicles is 2-3-fold lower than that of endogenous proopiomelanocortin in AtT-20 cells or prolactin in GH3 cells. In contrast, the packaging of a truncated form of CPD, which lacks the cytoplasmic tail and transmembrane domain, was similar to that of proopiomelanocortin. Taken together, the results support the proposal that CPD functions in the TGN in the processing of proteins that transit the secretory pathway and that the C-terminal region plays a major role in TGN retention. (+info)
Bax, but not Bcl-xL, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations.
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Release of proteins through the outer mitochondrial membrane can be a critical step in apoptosis, and the localization of apoptosis-regulating Bcl-2 family members there suggests they control this process. We used planar phospholipid membranes to test the effect of full-length Bax and Bcl-xL synthesized in vitro and native Bax purified from bovine thymocytes. Instead of forming pores with reproducible conductance levels expected for ionic channels, Bax, but not Bcl-xL, created arbitrary and continuously variable changes in membrane permeability and decreased the stability of the membrane, regardless of whether the source of the protein was synthetic or native. This breakdown of the membrane permeability barrier and destabilization of the bilayer was quantified by using membrane lifetime measurements. Bax decreased membrane lifetime in a voltage- and concentration-dependent manner. Bcl-xL did not protect against Bax-induced membrane destabilization, supporting the idea that these two proteins function independently. Corresponding to a physical theory for lipidic pore formation, Bax potently diminished the linear tension of the membrane (i.e., the energy required to form the edge of a new pore). We suggest that Bax acts directly by destabilizing the lipid bilayer structure of the outer mitochondrial membrane, promoting the formation of a pore-the apoptotic pore-large enough to allow mitochondrial proteins such as cytochrome c to be released into the cytosol. Bax could then enter and permeabilize the inner mitochondrial membrane through the same hole. (+info)
Dual effect of alkylresorcinols, natural amphiphilic compounds, upon liposomal permeability.
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The effect of 5-n-alkylresorcinols, natural amphiphilic compounds, upon properties of phospholipid vesicles depends on their localization asymmetry. A significant increase of the bilayer permeability is observed when the title compounds are present only in the external medium. When these amphiphiles are preincorporated into the bilayer during its formation, the resulting liposomes effectively encapsulate water-soluble solutes which still remain in liposomes after 25 h. Additionally, the size of liposomes made of alkylresorcinol-phosphatidylcholine mixtures after eight cycles of freezing and thawing only (180-200 nm) is severalfold smaller than the size of vesicles prepared in a similar way from phospholipids only and the resulting liposomes are more homogeneous. These liposomes modified with alkylresorcinols are also stable during 40 day storage at both 4 degrees C and 20 degrees C, in contrast to control liposomes that already strongly aggregate after 10 days. (+info)
Polymersomes: tough vesicles made from diblock copolymers.
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Vesicles were made from amphiphilic diblock copolymers and characterized by micromanipulation. The average molecular weight of the specific polymer studied, polyethyleneoxide-polyethylethylene (EO40-EE37), is several times greater than that of typical phospholipids in natural membranes. Both the membrane bending and area expansion moduli of electroformed polymersomes (polymer-based liposomes) fell within the range of lipid membrane measurements, but the giant polymersomes proved to be almost an order of magnitude tougher and sustained far greater areal strain before rupture. The polymersome membrane was also at least 10 times less permeable to water than common phospholipid bilayers. The results suggest a new class of synthetic thin-shelled capsules based on block copolymer chemistry. (+info)
The effect of peroxisome proliferators on mitochondrial bioenergetics.
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Peroxisome proliferators are a group of structurally diverse chemicals that cause the proliferation of peroxisomes in rodents. The purpose of this investigation was to test the hypothesis that the shared effect of these compounds on peroxisome proliferation is mediated through a common inhibitory effect on mitochondrial bioenergetics. Freshly isolated rat liver mitochondria were energized with succinate. The effect of the chemicals on mitochondrial bioenergetics was analyzed by monitoring calcium-induced changes in membrane potential and swelling, as well as changes in mitochondrial respiration. Mitochondrial membrane potential was measured with a TPP(+)-sensitive electrode, and swelling was recorded spectrophotometrically. Mitochondrial oxygen uptake was monitored with a Clark-type oxygen electrode. Gemfibrozil and WY-14,643 induced the mitochondrial permeability transition as characterized by calcium-induced swelling and depolarization of membrane potential, both of which were inhibited by cyclosporine A. Fenofibrate, clofibrate, ciprofibrate and diethylhexyl phthalate, on the other hand, caused a direct dose-dependent depolarization of mitochondrial membrane potential. However, the mechanism of membrane depolarization varied among the test chemicals. Bezafibrate and trichloroethylene elicited no effect on succinate-supported mitochondrial bioenergetics. The results of this investigation demonstrate that although most, but not all, peroxisome proliferators interfere with mitochondrial bioenergetics, the specific biomolecular mechanism differs among the individual compounds. (+info)