Transepithelial transport of maternal antibody: purification of IgG receptor from newborn rat intestine

In newborn rats, passive immunity is acquired from the mother by selective transport across the gut wall of immunoglobulin (IgG) present in colostrum and milk. Ultrastructural and physiologic studies of this mechanism have shown that the binding and uptake of IgG exhibits saturation kinetics and stereochemical specificity consistent with it being a receptor-mediated process. We report here the isolation and purification of a protein from membranes of neonatal rat enterocytes that binds immunoglobulins. The basis of our purification procedure is the extraction of this IgG-binding protein from isolated membranes in the absence of detergents and its biospecific elution from an IgG affinity column. This purified protein consists of two similar polypeptides of 52,000 and 48,000 Mr. The interaction of this purified protein with immunoglobulin is isotype dependent, with specificity for IgG and its Fc fragment, and pH dependent, with optimal binding at the intraluminal pH of 6.0. This intestinal IgG-binding protein is found in enterocytes of the proximal intestine during the early postnatal period, but is absent after weaning when transport of IgG ceases. Our results suggest that this purified intestinal IgG-binding protein functions in the transepithelial transport of IgG in rat neonates.     

Evidence for a primary endocytic vesicle involved in synaptic vesicle biogenesis

The regulated release of neurotransmitters at synapses is mediated by the fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane. Continuous synaptic activity relies on the constant recycling of synaptic vesicle proteins into newly formed synaptic vesicles. At least two different mechanisms are presumed to mediate synaptic vesicle biogenesis at the synapse as follows: direct retrieval of synaptic vesicle proteins and lipids from the plasma membrane, and indirect passage of synaptic vesicle proteins through an endosomal intermediate. We have identified a vesicle population with the characteristics of a primary endocytic vesicle responsible for the recycling of synaptic vesicle proteins through the indirect pathway. We find that synaptic vesicle proteins colocalize in this vesicle with a variety of proteins known to recycle from the plasma membrane through the endocytic pathway, including three different glucose transporters, GLUT1, GLUT3, and GLUT4, and the transferrin receptor. These vesicles differ from "classical" synaptic vesicles in their size and their generic protein content, indicating that they do not discriminate between synaptic vesicle-specific proteins and other recycling proteins. We propose that these vesicles deliver synaptic vesicle proteins that have escaped internalization by the direct pathway to endosomes, where they are sorted from other recycling proteins and packaged into synaptic vesicles.     

Molecular basis of synaptic vesicle cargo recognition by the endocytic sorting adaptor stonin 2

Synaptic transmission depends on clathrin-mediated recycling of synaptic vesicles (SVs). How select SV proteins are targeted for internalization has remained elusive. Stonins are evolutionarily conserved adaptors dedicated to endocytic sorting of the SV protein synaptotagmin. Our data identify the molecular determinants for recognition of synaptotagmin by stonin 2 or its Caenorhabditis elegans orthologue UNC-41B. The interaction involves the direct association of clusters of basic residues on the surface of the cytoplasmic domain of synaptotagmin 1 and a β strand within the μ–homology domain of stonin 2. Mutation of K783, Y784, and E785 to alanine within this stonin 2 β strand results in failure of the mutant stonin protein to associate with synaptotagmin, to accumulate at synapses, and to facilitate synaptotagmin internalization. Synaptotagmin-binding–defective UNC-41B is unable to rescue paralysis in C. elegans stonin mutant animals, suggesting that the mechanism of stonin-mediated SV cargo recognition is conserved from worms to mammals.     

Evidence for two endocytic transport pathways in plant cells

Intracellular trafficking of endocytic vesicles in eukaryotes varies with the nature of the cargo molecules and the targeted organelle, and proceeds through an intricate network of internal endosomal compartments. However, the path for fluid-phase endocytosis (FPE), the internalization of external solutes from the apoplast via plasmalemma generated vesicles, remains unresolved despite some indication of a direct transport route to the vacuole. To test this hypothesis, we made use of the membrane-impermeable Na-dependent fluorescent marker Coro-Na in combination with the fluorescent membrane marker FM 4-64 and confocal laser scanning microscopy. When protoplasts from sweet lime juice cells were incubated in Na-free solution, FM 4-64, Coro-Na, and 200 mM sucrose, two distinct types of labeled vesicles were evident. A set of vesicles (1 μm in diameter) was intensely labeled with Coro-Na and to a lesser extent with FM 4-64, whereas the second type of 1–7 μm structures appeared exclusively labeled with FM 4-64. These data demonstrate the parallel functioning of two endocytic pathways in plant cells. In one system, a set of small endocytic vesicles merge with the endosome, whereas a separate set of vesicles fuse to form larger vesicles independent from the endosome. Although it is likely that both vesicle systems eventually contribute to solutes reaching the vacuole, given their size (1–7 μm), and based on previous observations of endocytic vesicle formation protruding from the plasmalemma and merging with the vacuole, we conclude that these latter vesicles constitute the primary FPE vesicle system.     

Cytoskeletal control of alanine transport across the rat and turtle small intestine

Procaine inhibited significantly (P less than 0.01) alanine accumulation in the rat intestinal strips in a concentration-dependent pattern, whereas it showed no effect on alanine uptake by the turtle intestinal cells. Colchicine and Vinca alkaloids at 5 X 10(-4) and 1.5 X 10(-6) M respectively caused a significant inhibition (P less than 0.01) of intracellular alanine concentration in the rat with no effect noticed in the turtle. Unidirectional influx of alanine across the brush border membrane of the rat was significantly (P less than 0.01) reduced in the presence of procaine, colchicine and vincristine in the preincubation medium. The same drugs did not show any effect on alanine influx into the turtle small intestine. Electron microscopy showed major structural alterations in the cytoskeletal organization of the turtle intestine in response to procaine, colchicine or vincristine treatment. It is proposed that microtubular system may participate in the overall transport mechanism of alanine across the small intestine.     

The components of taurine transport across the rat small intestine A kinetic study

Summary The transport of taurine across adult Sprague-Dawley rat small intestine was studied in vitro using small intestinal strips. The kinetics of the transport mechanism were investigated under both steady-state and influx conditions. Our findings were compatible with the presence of two distinct transport mechanisms; a linear non-carrier mediated component and a saturable carrier mediated component, with almost equal contribution from each. The mediated component was found to be largely Na⁺-dependent and exhibited marked inhibition by B-alanine and structurally related sulfur amino acids.     

Evidence for active sodium transport across alveolar epithelium of isolated rat lung

We have previously presented evidence that cultured alveolar epithelial cell monolayers actively transport sodium from medium to substratum, a process that can be inhibited by sodium transport blockers and stimulated by beta-agonists. In this study, the isolated perfused rat lung was utilized in order to investigate the presence of active sodium transport by intact adult mammalian alveolar epithelium. Radioactive tracers (22Na and [14C]sucrose) were instilled into the airways of isolated Ringer-perfused rat lungs whose weight was continuously monitored. The appearance of isotopes in the recirculated perfusate was measured, and fluxes and apparent permeability-surface area products were determined. A pharmacological agent (amiloride, ouabain, or terbutaline) was added to the perfusate during each experiment after a suitable control period. Amiloride and ouabain resulted in decreased 22Na fluxes and a faster rate of lung weight gain. Terbutaline resulted in increased 22Na flux and a more rapid rate of lung weight loss. [14C]sucrose fluxes were unchanged by the presence of these pharmacological agents. These data are most consistent with the presence of a regulable active component of sodium transport across intact mammalian alveolar epithelium that leads to removal of sodium from the alveolar space, with anions and water following passively. Regulation of the rate of sodium and fluid removal from the alveolar space may play an important role in the prevention and/or resolution of alveolar pulmonary edema.     

Inhibition of sugar active transport across rat intestine in vivo by cadmium

Galactose absorption by rat jejunum perfused in vivo is inhibited by 0.5 mM Cd2+. This effect is explained by impairment of the phlorizin-sensitive sugar transport system, as Cd does not modify the absorption of L-sorbose or that of galactose in the presence of 0.5 mM phlorizin. Cd inhibition is observed as early as in the 1st minute, does not increase by previous exposure of the mucosa to the metal and does not decrease after washing with saline solution, but it is entirely reversed by EDTA or dithioerythritol. Results agree with a Cd2+ binding to HS- groups of membrane proteins at the brush border, appertaining or functionally related to the phlorizin-sensitive and Na+ dependent transport system for sugars.     

Action of mercury on sugar transport across rat small intestine, in vivo

Absorption of galactose from in vivo perfused rat jejunum was inhibited by 0.1-0.5 mM Hg2+. A few minutes' delay was required for maximal inhibition values. The effects remained after saline solution washing but were in part reversed by EDTA and in higher proportion by dithioerythritol. Absorption inhibition could be ascribed to impairment of the sugar-Na phlorizin-sensitive cotransport component: The passive apparently diffusional component that remains under 0.5 mM phlorizin and absorption of L-sorbose were unaffected by the metal. Hg action is explained as due to its binding to thiol and perhaps other chemical groups of proteins, at different depths in the membrane, which are directly or indirectly related to the sugar transport system.     

ESR study of proton transport across phospholipid vesicle membranes

A new method for measuring the rates of proton transfer through bilayer phospholipid membranes using pH-sensitive nitroxyl radicals is suggested. The pH-sensitive alkylating radical was covalently bound to glutathione. This modified glutathione is pH sensitive at pH 1.5-4.5 and does not penetrate across phospholipid membranes. Using ESR this probe was applied to register the kinetics of pH variations inside large unilamellar phospholipid vesicles after creation of a transmembrane proton gradient. In the acidic region (pH approximately 3) the main mechanism of transmembrane proton transfer is that via transport of a proton in the form of an undissociated acid. The membrane permeability coefficients have been determined for a series of acids (HCl, HClO4, HNO3, upper estimate for H2SO4). Taking into account that imidazoline and imidazolidine nitroxyl radicals can be used as pH probes in a wide range of pH, the present method can be developed for measuring the rates of transmembrane proton transfer in neutral and alkaline media.     

Rab5 and Rab7 control endocytic sorting along the axonal retrograde transport pathway

Vesicular pathways coupling the neuromuscular junction with the motor neuron soma are essential for neuronal function and survival. To characterize the organelles responsible for this long-distance crosstalk, we developed a purification strategy based on a fragment of tetanus neurotoxin (TeNT H(C)) conjugated to paramagnetic beads. This approach enabled us to identify, among other factors, the small GTPase Rab7 as a functional marker of a specific pool of axonal retrograde carriers, which transport neurotrophins and their receptors. Furthermore, Rab5 is essential for an early step in TeNT H(C) sorting but is absent from axonally transported vesicles. Our data demonstrate that TeNT H(C) uses a retrograde transport pathway shared with p75(NTR), TrkB, and BDNF, which is strictly dependent on the activities of both Rab5 and Rab7. Therefore, Rab7 plays an essential role in axonal retrograde transport by controlling a vesicular compartment implicated in neurotrophin traffic.     

The effect of local anesthetics on calcium transport across the rat and turtle small intestine

Procaine at different concentrations enhanced significantly (P less than 0.01) calcium accumulation in rat intestinal cells, whereas the same concentrations of procaine inhibited significantly (P less than 0.01) calcium uptake by the turtle small intestine. Unidirectional calcium influx across the rat small intestine was significantly enhanced (P less than 0.001) by the presence of procaine in the preincubation medium. However, procaine had no effect on calcium influx across the turtle intestinal cells. The cell water content and the cell volume were not altered by preincubating the intestinal tissues with procaine in both animals.     

SNX4 coordinates endosomal sorting of TfnR with dynein-mediated transport into the endocytic recycling compartment

SNX-BAR proteins are a sub-family of sorting nexins implicated in endosomal sorting. Here, we establish that through its phox homology (PX) and Bin-Amphiphysin-Rvs (BAR) domains, sorting nexin-4 (SNX4) is associated with tubular and vesicular elements of a compartment that overlaps with peripheral early endosomes and the juxtanuclear endocytic recycling compartment (ERC). Suppression of SNX4 perturbs transport between these compartments and causes lysosomal degradation of the transferrin receptor (TfnR). Through an interaction with KIBRA, a protein previously shown to bind dynein light chain 1, we establish that SNX4 associates with the minus end-directed microtubule motor dynein. Although suppression of KIBRA and dynein perturbs early endosome-to-ERC transport, TfnR sorting is maintained. We propose that by driving membrane tubulation, SNX4 coordinates iterative, geometric-based sorting of the TfnR with the long-range transport of carriers from early endosomes to the ERC. Finally, these data suggest that by associating with molecular motors, SNX-BAR proteins may coordinate sorting with carrier transport between donor and recipient membranes.     

Analysis of the endocytic pathway upon intracellular transport of IgG molecules through Fc receptors

The uniformly distributed Fc receptors (FcRs) on the surface of many cell types are involved in a variety of immune reactions by non-specifically facilitating the entry of antigen-specific IgG molecules to the cell. Such reactions may be beneficial to the organism when foreign antigens are involved, or harmful in cases of self antigens and viruses. In order to avoid the IgG-mediated self antigen presentation or viral infection in autoimmunity and viral attack respectively, we attempt in this study to inhibit the intracellular transport of antibodies. This blockage, however, implies: efficacy of inhibition, inability of de novo exocytosis of the internalised antibody and finally maintenance of normal cell growth and morphology. We thus concentrate our interest on the endocytic pathway followed by a neutralising antibody in murine trophoblast cells where we try to inhibit antibody intracellular transport by various agents according to the criteria set above. In our model-system, IFN-gamma, upon induction of FcRs, facilitates endocytosis of the anti-p21ras antibody which blocks in turn the IFN-gamma-induced surface class II major histocompatibility complex (MHC) expression. Using various intracellular transport inhibitors, we study the required conditions by which these compounds cancel the inhibitory action of anti-p21ras and allow induction of class II MHC molecules by IFN-gamma. The effectiveness of the inhibitors in a ranking order is shown as following: monodansyl cadaverine > didansyl cadaverine > pepstatin A > leupeptin > NH4Cl > brefeldin A > ZPCK > TPCK. From these inhibitors, only brefeldin A, leupeptin, pepstatin and ZPCK do not allow exocytosis of the antibody in the culture medium and only didansyl cadaverine, pepstatin and leupeptin maintain cell viability and morphology. However, by sequential elimination based on this study's established criteria, only pepstatin A and leupeptin are shown to be effective inhibitors to specific antibody intracellular transport, protecting also the cell's viability and physiology.     

Changes in sialic acid and fucose contents of enterocytes across the crypt-villus axis in developing rat intestine

Alterations in sialic acid and fucose contents of different populations of epithelial cells have been studied in suckling and adult rat intestine. The progression of cells from crypt base to villus tip is associated with an increase in sialic acid and a decrease in fucose levels of the cells in adult rats. In suckling pups, sialic acid is uniformly distributed along the length of villi, and fucose is richly (P less than 0.01) present in cryptic cells compared to that at the villus tip. Adult-type changes in sialylation and fucosylation of enterocytes across the crypt-villus axis were precociously produced by cortisone administration to suckling pups. Thyroxine treatment was less effective in influencing the glycosylation process in rat intestine.