Heteropore populations of bullfrog alveolar epithelium

Diffusional fluxes of a large number of hydrophilic solutes and water across bullfrog (Rana catesbeiana) alveolar epithelium were measured in the Ussing-type flux chamber. Lungs were isolated from double-pithed animals and studied as flat sheets. Radioactive solutes and water were added to the upstream reservoir, and the rate of change of downstream reservoir radioactivity was monitored. A permeability coefficient was estimated for each substance from a linear relationship between radiotracer concentration in the downstream reservoir and time. These permeability data were used to analyze the equivalent water-filled pore characteristics of the alveolar epithelial barrier. The data reveal that the alveolar epithelium is best characterized by two distinct pore populations rather than by a single homogeneous pore population. The large-pore population consists of pores with a radius of about 5 nm and occupies 4% of the available pore area. The small-pore population consists of pores with a radius of about 0.5 nm and occupies 96% of the available pore area. The number of small pores to large pores is 2.68 X 10(3). After the alveolar surface was damaged by acid, a large-pore population with a radius of about 27 nm was seen, allowing nearly free diffusion of solutes. A major implication of the presence of two populations of pores in the alveolar epithelium is that hydrostatically driven bulk water flow occurs predominantly through the large pores, while osmotically driven bulk water flow takes place predominantly through the small pores. As a result, in general, hydrostatic and osmotic gradients may not be equally effective driving forces for water flow across this tissue.     

Basal and amiloride-induced short-circuit current across isolated toad skin (Bufo arenarum)

We have previously demonstrated that amiloride (amil) addition to the isolated ventral pelvic (VPel) skin of Bufo arenarum toad induces negative short-circuit current values, which are equivalent to the isotopically measured net chloride transport. In the present work, we found that exposure of various regions of toad skin to amil yielded different values of short-circuit current (aSCC): negative aSCC was found in the VPel and ventral pectoral skin, while those of the dorsal one were not different from zero. The distinct values of aSCC found show a regional difference in the active chloride absorption, probably related to postural adaptations. A possible role of this adaptation would be related to chloride participation in the saline balance of the animals, or the maintenance of epithelial integrity.     

Structure of the major membrane protein complex from urinary bladder epithelial cells by cryo-electron crystallography.

Numerous protein plaques cover the apical surface of mammalian urinary bladder epithelial cells. These plaques contain four integral membrane proteins, called uroplakins, which form a well-ordered array of hexameric complexes. The 3D structure of these naturally occurring 2D crystals was studied by cryo-electron-crystallographic methods using a slow-scan charged-coupled device (CCD) camera to record the electron micrographs. A 1.2 nm projection map calculated from untilted crystals shows that each hexamer comprises a ring of six inner and six outer domains at a radius of 5.7 nm and 9.2 nm respectively. The 3D structure shows that the mass is distributed strongly asymmetrically with respect to the membrane, with most of the mass protruding from the luminal face. Both domains in the asymmetric unit traverse the membrane and protrude from the membrane on the cytoplasmic side. On the luminal side, the two domains are bridged forming a stretched arc. The total thickness of the complex is about 13.2 nm. A model of the urothelial plaque reveals that contacts between the hexamers are much less extended than within the hexamers.     

Cysteine scanning mutagenesis of alpha4, a putative pore-lining helix of the Bacillus thuringiensis insecticidal toxin Cry1Aa

Helix alpha4 of Bacillus thuringiensis Cry toxins is thought to line the lumen of the pores they form in the midgut epithelial cells of susceptible insect larvae. To define its functional role in pore formation, most of the alpha4 amino acid residues were replaced individually by a cysteine in the Cry1Aa toxin. The toxicities and pore-forming abilities of the mutated toxins were examined, respectively, by bioassays using neonate Manduca sexta larvae and by a light-scattering assay using midgut brush border membrane vesicles isolated from M. sexta. A majority of these mutants had considerably reduced toxicities and pore-forming abilities. Most mutations causing substantial or complete loss of activity map on the hydrophilic face of the helix, while most of those having little or only relatively minor effects map on its hydrophobic face. The properties of the pores formed by mutants that retain significant activity appear similar to those of the pores formed by the wild-type toxin, suggesting that mutations resulting in a loss of activity interfere mainly with pore formation.     

Expression of the HB9 homeobox gene concomitant with proliferation accompanying epidermal stratification during development of chick embryonic tarsometatarsal skin

A homeobox gene, HB9, has been isolated from the tarsometatarsal skin of 13-day-old chick embryos using a degenerate RT-PCR-based screening method. In situ hybridization analysis revealed that, during development of chick embryonic skin, the HB9 gene was expressed in epidermal basal cells of the placodes, but not in those of interplacodes, and in the dermal cells under the placodes at 9 days before addition of an intermediate layer by proliferation of the basal cells in the placodes. With the onset of epidermal stratification, the direction of the basal cell mitosis changed, with the axis becoming vertical to the epidermal surface. Placodes and interplacodes form outer and inner scales, respectively, after they have elongated distally (Tanaka S, Kato Y (1983b) J Exp Zool 225: 271–283). During scale ridge elongation at 12–15 days, HB9 was strongly expressed in the epidermis of the outer scale face, where the cell proliferation is more active than in the epidermis of the inner scale face; hence, stratification of the outer scale face is more prominent than that of the inner scale face. After 16 days, when mitotic activity in the epidermal basal cells decreases and the thickness of the epidermis is maintained at a constant level, the HB9 expression decreases with the onset of epidermal keratinization. These results suggest that HB9 may be involved in the proliferation of the epidermal basal cells that accompanies epidermal stratification.     

Detection of a large cation-selective channel in nuclear envelopes of avian erythrocytes.

To determine whether the nuclear envelope of eukaryotic cells has the capability to regulate ion fluxes, we have used the patch-clamp technique to detect ion channels in this membrane system. Since possible sites for ion channels in the nuclear envelope include not only the nuclear pores, but also both the inner and outer nuclear membranes, we have patched giant liposomes composed of phosphatidylcholine and nuclear envelope fragments isolated from mature avian erythrocytes. A large, cation-selective channel with a maximum conductance of approximately 800 pS in symmetrical 100 mM KCl was detected. This channel is a possible candidate for a nuclear pore.     

Effects of cadmium on Na+ transport in the isolated skin of the toad Pleurodema thaul

Cadmium ions applied to either (outer or inner) surface of the isolated toad skin dose-dependently increased the short-circuit current (SCC), the potential difference (V) and the active sodium conductance (G(Na)) in the concentration range 0.07-0.50mM. Maximal stimulatory effect was over 30% with an EC(50) of about 0.1mM. The effect of the highest concentration used (0.75mM) decreased considerably, and when it was applied to the inner surface (10 experiments), induced between 30% and 40% inhibition of the electric parameters in four experiments. Pretreatment with amiloride inverted the stimulatory effect of externally applied Cd(2+), suggesting competitive action on the apical Na(+) channel. The effect of noradrenaline (NA) was increased after outer application of Cd(2+) and decreased after inner application of the metal: the latter effect might be due to cadmium inhibition of the activity of Na(+),K(+)-ATPase. On the other hand, pretreatment with amiloride was followed by partial although transient reversal of its effects by serosal Cd(2+), which might be explained by action of cadmium on cytoplasmic lysine residues concerned with Na(+) channel gating. The amiloride test showed that the increment of the electric parameters was due principally to stimulation of the driving potential for Na(+) (V-E(Na(+))) and that inhibition was accompanied by a reduction in the V-E(Na(+)) and by a significant decrease in skin resistance indicating possible disruption of membrane or cell integrity. These data are in favor of the possibility that externally applied Cd(2+) activates toad skin ion transport, partly by increasing apical sodium conductance and also by stimulating the V-E(Na(+)), and that internally applied Cd(2+), with easier access to membrane and cellular constituents, may inhibit the sodium pump.     

Structure, surface charge, and self-assembly of the S-layer lattice from Bacillus coagulans E38-66.

In freeze-etched preparations, whole cells from Bacillus coagulans E38-66 exhibited an oblique S-layer lattice (a = 9.4 nm; b = 7.4 nm; gamma = 80 degrees). The three-dimensional structure of the crystalline array was characterized by optical and computer image analysis. The lattice showed two distinctly shaped types of pores. In vitro self-assembly of isolated subunits yielded flat sheets and open-ended cylinders composed of two back-to-back monolayers. Unlike whole cells, in vitro self-assembly products were capable of binding polycationized ferritin (pI, approximately 11). This showed that only the inner S-layer face adhering to the peptidoglycan-containing layer in whole cells was net negatively charged. S-layer monomers and/or oligomers were capable of generating a closed monolayer with oblique symmetry on poly-L-lysine-coated supports. The monolayer had a typical crazy paving appearance, with numerous crystal boundaries. The handedness of the oblique lattice and ability to bind polycationized ferritin revealed that the subunits had bound with the outer, not net negatively charged face to the poly-L-lysine-coated supports. Carbodiimide-activated carboxyl groups on either cell wall fragments or self-assembly products could covalently bind high-molecular-weight nucleophiles such as ferritin. This confirmed the location of negatively charged carboxyl groups on the outermost surface of both S-layer faces. The difference in pH optimum for carbodiimide activation indicated a preponderance of alpha- and beta-carboxyl groups on the inner S-layer face and a preponderance of beta- and gamma-carboxyl groups on the outer S-layer face.     

Octagonal nuclear pores

Negative staining of isolated nuclear envelopes by phosphotungstate shows that the nuclear pores are octagonal rather than circular. Pores of the same shape and approximately the same width, 663 ± 5 A, were demonstrated in the newt, Triturus, the frog, Rana, and the starfish, Henricia. The outer and inner diameters of the annulus associated with each pore are respectively greater and less than the width of the pore itself. For this reason surface views of the envelope, unless negatively stained, fail to show the true dimensions of the pores.     

Further characterization of the outer membrane ofSerratia marcescens and an oxacilline-sensitive mutant: Surface carbohydrates and outer membrane lipid composition

Whole cells and isolated outer membranes ofSerratia marcescens and an oxacilline-sensitive mutant could be agglutinated only with concanavalin A and wheat germ agglutinin. Different amounts of carbohydrates were accessible to the lectins in the two strains, and different amounts of carbohydrate seemed to be exposed at the outer cell surface and the inner side of the outer membrane. The fatty acid composition of the isolated outer membrane revealed small but significant quantitative difference. The increased sensitivity towards various antibacterial agents was thought to be the result of an altered hydrophobic-hydrophilic interaction of the outer membrane components.     

Inner but Not Outer Membrane Leaflets Control the Transition from Glycosylphosphatidylinositol-anchored Influenza Hemagglutinin-induced Hemifusion to Full Fusion

Cells that express wild-type influenza hemagglutinin (HA) fully fuse to RBCs, while cells that express the HA-ectodomain anchored to membranes by glycosylphosphatidylinositol, rather than by a transmembrane domain, only hemifuse to RBCs. Amphipaths were inserted into inner and outer membrane leaflets to determine the contribution of each leaflet in the transition from hemifusion to fusion. When inserted into outer leaflets, amphipaths did not promote the transition, independent of whether the agent induces monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In contrast, when incorporated into inner leaflets, positive curvature agents led to full fusion. This suggests that fusion is completed when a lipidic fusion pore with net positive curvature is formed by the inner leaflets that compose a hemifusion diaphragm. Suboptimal fusion conditions were established for RBCs bound to cells expressing wild-type HA so that lipid but not aqueous dye spread was observed. While this is the same pattern of dye spread as in stable hemifusion, for this “stunted” fusion, lower concentrations of amphipaths in inner leaflets were required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation and enlargement of fusion pores induced by HA. We propose that after the HA-ectodomain induces hemifusion, the transmembrane domain causes pore formation by conferring positive spontaneous curvature to leaflets of the hemifusion diaphragm.     

Cytoplasmic and cell surface structure of purified liver gap junctions revealed by freeze-drying

After freeze-drying of purified liver gap junction plaques and vesicles the structural features of the inner and outer aspects of purified gap junctions were investigated. No structural details were seen on the cytoplasmic side of the connexions whereas on the cell surface side the connexions were organized in a paracrystalline pattern and exhibited a central depression or pore. We conclude that the central pore through each connexion varies in diameter along its length and that the closing site is located near the cytoplasmic face.     

Synthetic multifunctional pores that open and close in response to chemical stimulation

Studies on synthetic multifunctional pores with external and internal active sites for ligand gating and noncompetitive blockage are presented, with emphasis on the contribution of external ligands to the characteristics of pore. A comparison between different synthetic multifunctional pores reveals that the location of functional groups in rigid-rod beta-barrel pores is precisely reflected in the function: molecular recognition at the outer barrel surface results in pore opening, while molecular recognition at the inner barrel surface results in pore closing. Negligible nonspecific leakage, disappearance of pH gating, inhibition of intervesicular pore transfer, and maybe also the flickering of currents of single open pores characterize external ligands as adhesive cushions that liberate the pore from lateral pressure exerted by the surrounding membrane. Refined molecular models show good agreement with pore design and experimental facts with regard to function.     

Scanning electron microscopy of chick epiblast expansion on the vitelline membrane. Cell-substrate interactions

Cell-substrate interactions have been studied by examining migrating edge cells of the expanding chick extraembryonic epiblast on their normal substrate and in culture. Scanning electron microscopy shows that the outer face of the vitelline membrane is a random meshwork of fibrils (80 nm diam). The inner face, which is the normal substrate of epiblast expansion, is composed of a random branched system of fibers (400 nm diam) overlain by a network of fibrils (40 nm diam). The epiblast edge in situ has radially oriented filopodia (20 μm long, 200 nm diam.), frequently extending from broad lamellipodia. Blastoderms cultured on the inner face of unincubated vitelline membrane expand at a normal rate but display ruffles as well as filopodia and lamellipodia. When the blastoderm is cultured on the outer membrane face there is no expansion, but cells leave the edge and migrate across the membrane. In these cultures, ruffles are observed on the ventral epiblast face. Absence of the mass of yolk in culture appears to permit or provoke the observed ruffling. Comparison of dissociated epiblast edge cells and skin epithelial cells, cultured on glass and on the vitelline membrane inner face, indicates that epiblast cells remain flattened and display characteristic filopodia on both substrates, whereas skin cells display ruffles on the vitelline membrane but are flattened on glass. The mode of migration of epiblast edge cells seems to be more dependent on intrinsic factors than that of skin cells.     

Protective role of epithelium in the guinea pig airway

We developed an in vitro system to assess the role of the epithelium in regulating airway tone using the intact guinea pig trachea (J. Appl. Physiol. 64: 466-471, 1988). This method allows us to study the response of the airway when its inner epithelial surface or its outer serosal surface is stimulated independently. Using this system we evaluated how the presence of intact epithelium can affect pharmacological responsiveness. We first examined responses of tracheae with intact epithelium to histamine, acetylcholine, and hypertonic KCl when stimulated from the epithelial or serosal side. We then examined the effect of epithelial denudation on the responses to these agonists. With an intact epithelium, stimulation of the inner epithelial side always caused significantly smaller changes in diameter than stimulation of the outer serosal side. After mechanical denudation of the epithelium, these differences were almost completely abolished. In the absence of intact epithelium, the trachea was 35-fold more sensitive to histamine and 115-fold more sensitive to acetylcholine when these agents were applied to the inner epithelial side. In addition, the presence of an intact epithelium almost completely inhibited any response to epithelial side challenge with hypertonic KCl. These results indicate that the airway epithelial layer has a potent protective role in airway responses to luminal side stimuli, leading us to speculate that changes in airway reactivity measured in various conditions including asthma may result in part from changes in epithelial function.     

早寒武世梅树村期软舌螺壳孔沟系统的初步研究

对滇东会泽雨碌、寻甸摆宰早寒武世梅树村期软舌螺壳质微细结构的研究表明,软舌螺壳的孔沟系统十分发育,壳的外层由分叉与合并呈纵向排列的长纤束组成,内层由横向分布的粒柬组成。在外层纤柬之间和纤柬内分布有大量的三角形或长椭圆形的孔洞,这些孔洞的长轴平行于壳的生长方向;在内层粒束之间或粒柬内有横向分布的长沟。内外层的沟孔可以相通。在一些软舌螺纵向和横向的切片中,均发现早期磷酸岩化作用中已被磷质颗粒充填的孔沟形态,这些孔沟分布相当密集,基本垂直壳面,几乎通达壳壁的顶底。多孔性的壳既反映了软舌螺对其特殊的生理和生态功能的要求,也反映了早期带壳动物的原始性和它们在早期生物矿化作用中的特殊地位。     

Morphology and ontogeny of single-walled multiporous sensilla of hemimetabolous insects

Comparative morphological investigations were made to determine the common organization plan of single-walled multiporous sensilla. The development of multiporous chemoreceptive sensilla of Gryllus, Oncopeltus and Lepisma follows the same path. Each chemoreceptive sensillum is associated with four types of enveloping cell. During ontogeny, enveloping cell 1 secretes the dendritic sheath. Enveloping cell 4 builds the connection of the hair base with the antennal cuticle. In Gryllus and Oncopehus, enveloping cells 2 and 3 build the hair shaft, the wall pores and pore tubules in nearly equal parts. Enveloping cells 2 and 3 lie side by side in the hair process, in which enveloping cell 2 produces the inner part, enveloping cell 3 the outer part of the hair shaft. In Lepisma the predominant part of the hair shaft with the wall pores is formed by the doubled enveloping cells 3. Interpreting our findings and the literature data, a new proposal is given for the homology of the enveloping cells. In singlewalled chemoreceptors, enveloping cell 1 is considered as thecogen and enveloping cell 4 as tormogen cell. Enveloping cell 2 is interpreted as inner trichogcn cell and enveloping cell 3 as outer trichogen cell.     

A study of the perturbation effects of the local anesthetic procaine on human erythrocyte and model membranes and of modifications of the sodium transport in toad skin

The interaction of the local anesthetic procaine with human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), isolated toad skins, and molecular models is described. The latter consisted of phospholipid multilayers built-up of dimyristoylphosphatidylcholine (DMPC) and of dimyristoylphosphatidylethanolamine (DMPE), representatives of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Optical and scanning electron microscopy of human erythrocytes revealed that procaine induced the formation of stomatocytes. Experiments performed on IUM at 37 degrees C by fluorescence spectroscopy showed that procaine interacted with the phospholipid bilayer polar groups but not with the hydrophobic acyl chains. X-ray diffraction indicated that procaine perturbed DMPC structure to a higher extent when compared with DMPE, its polar head region being more affected. Electrophysiological measurements disclosed a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of procaine to isolated toad skin, reflecting inhibition of active ion transport.     

Water transport across isolated term human amnion

Summary The hydrodynamic permeability of normal term human amnion is measured using pressure-driven bulk flows. The permeability coefficient is found to vary widely, variations between tissues taken from different subjects being significantly greater than those from samples taken from one subject. No correlation is observed between this coefiicient and either tissue thickness or the diffusional permeability coefficient measured using tritiated water; it is, however, found to be very sensitive to epithelial damage.The results indicate that the bulk transport of water through amnion is largely controlled by the amniotic epithelium alone. This contrasts with water diffusion which is a function of total membrane thickness. The two permeability coefficients cannot therefore be employed to formulate an equivalent pore model of the whole tissue. An equivalent pore model of the epithelial layer only is considered and the results assessed in the light of other evidence bearing on the structure of amnion. It is concluded that the epithelial layer is intersected by a large number of pores with radius 10 to 30 Å, and a smaller number of much broader pores.     

Conformational change of the hexagonally packed intermediate layer of Deinococcus radiodurans monitored by atomic force microscopy.

Both surfaces of the hexagonally packed intermediate (HPI) layer of Deinococcus radiodurans were imaged in buffer solution by atomic force microscopy. When adsorbed to freshly cleaved mica, the hydrophilic outer surface of the HPI layer was attached to the substrate and the hydrophobic inner surface was exposed to the stylus. The height of a single HPI layer was 7.0 nm, while overlapping edges of adjacent single layers adsorbed to mica had a height of 14.7 nm. However, double-layered stacks with inner surfaces facing each other exhibited a height of 17.4 nm. These stacks exposed the outer surface to the stylus. The different heights of overlapping layers and stacks are attributed to differences in the interaction between inner and outer surfaces. At high resolution, the inner surface revealed a protruding core with a central pore connected by six emanating arms. The pores exhibited two conformations, one with and the other without a central plug. Individual pores were observed to switch from one state to the other.