Search PubMed⌕ Search

Biomedical subjects

G Zampighi

Publications and source records attributed to G Zampighi.

At least 19 recordsLinked to original sources
PubMed1990

Cell isolation and genotoxicity assessment in gastric mucosa.

It has been claimed that in vitro digestion of in vivo DNA-labeled gastric mucosa is suitable for evaluation of genotoxic effects of drugs or chemicals. This method was then used to show that omeprazole (a novel antiulcer drug) was potentially genotoxic. In this study we have examined the method used and the interaction of omeprazole and its derivatives with purified DNA. The method was shown to enrich for dividing cells (6.92 +/- 0.693%, N = 43, 2-hr labeling) in the digest from the intact tissue and was therefore unsuitable for estimating unscheduled DNA synthesis in the gastric mucosa induced by chemicals or drugs including omeprazole. It was further shown that neither omeprazole or its acid-activated product, a cationic sulfenamide, were able to react with isolated purified DNA from either a prokaryote (E. coli) or a eukaryote (salmon sperm). Hence any conclusions using this method attributing acute genotoxic effects to any chemical are based on unrecognized artifacts of the technique and are unsound. In addition, these results negate the suggestion that omeprazole or its gastric metabolites are genotoxic.

Animals↗
PubMed1990

Properties of channels reconstituted from the major intrinsic protein of lens fiber membranes.

Detergent-solubilized plasma membrane protein of either adult bovine or calf lens and high-performance liquid chromatography-purified major intrinsic protein (MIP) of the lens were reconstituted into unilamellar vesicles and planar lipid bilayers. Freeze-fracture studies showed that the density of intramembrane particles in the vesicles was proportional to the protein/lipid ratio. At high ratios, these particles crystallized into tetragonal arrays as does MIP in lens fibers. Channels induced by either purified MIP or detergent-solubilized protein had essentially identical properties. The conductance of multichannel membranes was maximal near 0 mV and decreased to 0.49 +/- 0.08 of the maximum value at voltages greater than 80 mV. The dependence of the conductance on voltage was well fit by a two-state Boltzmann distribution. Voltage steps greater than 30 mV elicited an ohmic current step followed by a slow (seconds) biexponential decrease. The amplitudes and time constants depended on the magnitude but not the sign of the voltage. Steps from 100 mV to voltages less than 30 mV caused the channels to open exponentially with a millisecond time constant. Analysis of latency to first closure after a voltage step gave nearly the same time constants as multichannel kinetics. Single-channel conductance is proportional to salt concentration from 0.1 to 1.0 M in KCl. In 0.1M KCl, the channel had two preferred conductance states with amplitudes of 380 and 160 pS, as well as three additional substates. Multi- and single-channel data suggest that the channel has two kinetically important open states. The channel is slightly anion selective. The properties of the channel do not vary appreciably from pH 7.4 to 5.8 or from pCa 7 to 2. We propose that a channel with these properties could contribute to maintenance of lens transparency and fluid balance.

Animals↗
PubMed1988

Structural characteristics of gap junctions. I. Channel number in coupled and uncoupled conditions.

Gap junctions between crayfish lateral axons were studied by combining anatomical and electrophysiological measurements to determine structural changes associated during uncoupling by axoplasmic acidification. In basal conditions, the junctional resistance, Rj, was approximately 60-80 k omega and the synapses appeared as two adhering membranes; 18-20-nm overall thickness, containing transverse densities (channels) spanning both membranes and the narrow extracellular gap (4-6 nm). In freeze-fracture replicas, the synapses contained greater than 3 X 10(3) gap junction plaques having a total of approximately 3.5 X 10(5) intramembrane particles. "Single" gap junction particles represented approximately 10% of the total number of gap junction particles present in the synapse. Therefore, in basal conditions, most of the gap junction particles were organized in plaques. Moreover, correlations of the total number of gap junction particles with Rj suggested that most of the junctional particles in plaques corresponded to conducting channels. Upon acidification of the axoplasm to pH 6.7-6.8, the junctional resistance increased to approximately 300 k omega and action potentials failed to propagate across the septum. Morphological measurements showed that the total number of gap junction particles in plaques decreased approximately 11-fold to 3.1 X 10(4) whereas the number of single particles dispersed in the axolemmae increased significantly. Thin sections of these synapses showed that the width of the extracellular gap increased from 4-6 nm in basal conditions to 10-20 nm under conditions where axoplasmic pH was 6.7-6.8. These observations suggest that single gap junction particles dispersed in the synapse most likely represent hemi-channels produced by the dissasembly of channels previously arranged in plaques.

Animals↗
PubMed1986

Crystallization of the gastric H,K-ATPase.

Crystalline arrays of the gastric H,K-ATPase were obtained in membrane preparations from hog and rabbit gastric mucosa. The lattice was formed rapidly in a medium containing K+, vanadate, Mg2+, and dimethyl sulfoxide at pH 6.0-6.9 in imidazole buffer from 4 to 22 degrees C. The crystal lattice exhibited P2 symmetry, and the unit cell dimension (a = 5.6, b = 11, and c = 10 nm) could accommodate 2 polypeptides of mass 116-129 kDa. In addition, the isolated preparation contained previously undescribed long cylindrical structures 16 nm thick. These structures consisted of a central core 6-7 nm wide from which particles spaced 5.5 nm apart protruded symmetrically.

Adenosine Triphosphatases↗
PubMed1986

One-dimensional crystals of (Na+ + K+)-ATPase dimers.

Preparations of purified (Na+ + K+)-ATPase contain both fragments of membranes and long and undulating cylindrical structures. These structures have been described as edgeways of membrane fragments. We have analyzed these structures using negative staining, thin sectioning and freeze-fracture-etch electron microscopy and describe their structure for the first time. Each cylinder is 12-19 nm in width and is comprised of an unstained core from which rows of distinct particles spaced 5-6 nm apart project on both sides. Each cylindrical structure was interpreted as a linear polymer of (alpha beta)2 dimers of (Na+ + K+)-ATPase molecules. Therefore, the particles that project from both sides are the cytoplasmic domains of the molecules of the enzyme, whereas the membrane-spanning domains form the unstained core of the cylinder. From considerations of the packing of the dimers in the cylinder we conclude that the cross-sectional area of the cytoplasmic domain should be larger than that of the membrane-spanning domain. Our results are consistent with the hypothesis that the (alpha beta) protomer is the native state of the enzyme. The (alpha beta)2 dimers observed in the fractions are the result of a secondary aggregation process occurring during the purification procedure.

Animals↗
PubMed1985

Intercellular fibrillar skeleton in the basal interdigitations of kidney tubular cells.

The tubular cells from the thick ascending limb of the loop of Henle in rabbit kidney medulla contain in their basal-lateral surfaces a complex system of interdigitations. Within these interdigitations, the plasma membranes are separated by extracellular spaces of relatively constant width that contain a previously undescribed fibrillar system. The structural organization and distribution of this intercellular fibrillar skeleton was studied using freeze-fracture etch and then section electron microscopy. The skeleton is comprised of discrete strands with a density of 300 to 400 per micron 2 evenly distributed along the entire basal-lateral region. Each strand has the shape of a brace and it is constructed from up to eight finer filaments each having a width of about 2 nm. The filaments are tightly joined together along their shafts for about 30 nm but they separate at both ends for about 10 nm before contacting the external surface of the plasma membrane. We propose that this intercellular fibrillar skeleton is responsible for maintaining the wide (about 50 nm) and uniform plasma membrane separation along the entire length of the basal-lateral region of the tubular cells of the thick ascending limb.

Animals↗
PubMed1984

Structural organization of (Na+ + K+)-ATPase in purified membranes.

The structural organization of crystalline, membrane-bound (Na+ + K+)-ATPase was studied by negative staining and thin sectioning. The enzyme molecules were induced to form crystalline arrays within fragments of membrane by incubation in defined ionic conditions. The enzyme remained fully active after crystallization. Negative staining and computer processing of images of the crystalline specimens identified two discrete crystalline arrays. The dimensions of the unit cell of one of the arrays were large enough to accommodate an alpha beta protomer; those of the other array, an (alpha beta)2 diprotomer . Thin sections of the crystalline fraction contained a unique membrane complex that was formed from two apposed plasma membranes. The paired membranes in this complex were separated by a center-to-center space of 15 nm containing evenly spaced septa that connected the membrane surfaces; the overall thickness of the entire structure was 22-25 nm. The agglutinin from Ricinus communis, a lectin that binds to the carbohydrate moiety of the beta-subunit of (Na+ + K+)-ATPase, decorated the free surfaces of the complex. Therefore, this complex of paired membranes is the result of interactions between the cytoplasmic domains of the enzyme. From measurements of the dimensions of these structures, we estimate the overall length of the enzyme to be approximately 11.5 nm along the axis perpendicular to the plane of the membrane, and the molecular protrudes more (approximately 5 nm) on the cytoplasmic surface than on the extracytoplasmic surface (approximately 2 nm).

Animals↗
PubMed1980

On the structure of the hemocyanin channel in lipid bilayers.

Keyhole limpet hemocyanin has been shown by others (Alvarez, O., Diaz, E. and Latorre, R. (1975) Biochim. Biophys. Acta 389, 444-448) to form single conductance channels in black lipid membranes. In an attempt to visualize how the large (300 A) water-soluble hemocyanin molecule interacts with lipid bilayers, we have examined hemocyanin in the presence of lipids with the electron microscope. We find that incubation of lipids with keyhole limpet hemocyanin produces a characteristic 70 A in diameter, ring-shaped particle or annulus associated with the bilayer. This annulus, which appears to be quite distinct from previously observed aggregated and dissociated forms of hemocyanin, may be responsible for the channel formation in black lipid membranes.

Animals↗
PubMed1980

On gap junction structure.

We have studied the stain distribution within rat liver gap junctions for specimens prepared by thin sectioning and negative staining. Pools of stain molecules exist in two specific locations with respect to the distinctive morphological units (connexons) of the junction. One pool of stain surrounds the connexons and is restricted to the extracellular space in the gap between the adjacent plasma membranes. The other pool of stain is located along in the central axis of each connexon, measures 1-2 nm in diameter and 4-5 nm in length, and is restricted to the gap region. On rare occasions, barely discernible linear densities seem to extend from this latter pool of stain and traverse the entire width of the junction. The data indicate the existence of a hydrophilic cavity along the central axis of te connexon which, in most instances, is restricted to the gap region. However, the precise depth to which this cavity may further extend along the connexon axis is still uncertain.

Animals↗
PubMed1978

Fine structure of the electrotonic synapse of the lateral giant axons in a crayfish (Procambarus clarkii).

The electrotonic synapse of lateral giant axons of the crayfish was studied by conventional thin sectioning. The most prominent membrane specialization observed in this synaptic region is the communicating junction. It is characterized by a close apposition of the two axonal membranes which are separated by a 4--5 nm wide gap. Other characteristics of the junction are an array of particles spaced about 20--22 nm apart and a row of vesicles symmetrically arranged at the cytoplasmic leaflets of each membrane. The communicating junction does not cover the entire surface of the electrotonic synapse. Indeed, we have found other specializations such as: finger-like Schwann cell processes extending between synaptic membranes, saccular invaginations of one synaptic membrane into its axon, and coated vesicles continuous with one of the membranes. In addition, large vesicular pieces of the communicating junctions, with their accompanying vesicles, appeared to extend deeply inside the axoplasm. The morphological appearance of the communicating junction is found to be different from the one reported for mammalian maculae communicans such as liver or heart muscle. This is surprising because, regardless of their morphological differences, both junctions seem to transmit electrotonically.

Animals↗
PubMed1976

Regular structures in unit membranes. III. Further observations on the particulate component of the suckling rat ileum endocytic membrane complex.

Further morphological observations on the particulate components decorating the lumenal surfaces of membranes of the endocytic complex of the epithelial cells of the suckling rat ileum are presented. The particles each measure approximately 7.5 nm across and give the appearance of the capital letter H in frontal view. They consist of the enzyme n-acetyl-beta-glucosaminidase (NAG). They are arranged in rows called "decorated strips" with the symmetrical lateral bars in register and spaced approximately 14.5 nm apart. Decorated strips lie side-by-side in the external (lumenal) surface of the membrane. They are parallel and sometimes spaced approximately 14.5 nm apart making an orthogonal lattice. The lateral spacing between the decorated strips under certain conditions is reduced and sometimes there is shear between the adjacent ones. Occasionally, shear is present within the decorated strips themselves, with slight displacement of the two sides of each H-shaped particle. A purified preparation of these membranes has been studied by electron microscopy using thin sectioning, negative stain, Markham translation and optical diffraction computer image reconstruction methods. The individual particles comprising the array can be seen in the membrane surface in profile view when dried in a pool of negative stain. They appear either triangular or diamond-shaped in such views. If triangular, they appear to consist of three domains at the corners of an equilateral triangle. One side of each triangular figure is parallel to the membrane surface but separated from it by a dense band of negative stain approximately 2 nm thick that runs along the surface of the membrane. Sometimes a fourth symmetrical domain is visible within this dense band, giving a diamond-shaped figure. This fourth domain connects the particle to the membrane. Thus, each H-shaped particle is a double structure, with each half in profile view appearing as a diamond figure of four symmetrical domains. Each H-shaped particle is believed to consist of either two or four molecules of NAG.

Animals↗
PubMed1976

Regular structures in unit membranes. II. Morphological and biochemical characterization of two water-soluble membrane proteins isolated from the suckling rat ileum.

Specialized plasma membranes from the endocytic complex of ileal epithelial cells of suckling rats were isolated by differential flotation. Thin-section and negative-stain electron microscopy showed the luminal surfaces of these membranes to be covered by an ordered array of particles 14.5-nm separations in long rows. This particulate coating was released from the membrane surfaces by 10 mM CaCl2 and recovered free of membranes after dialysis against 0.5 mM EGTA and high-speed centrifugation. Two proteins were resolved by gel filtration to be in supernate: n-acetyl-beta-glucosaminidase and a filamentous protein which attaches n-acetylglucosaminidase to the membrane surface thereby providing bidirectionality to the array of enzyme. We believe that the filamentous protein has not been previously described. Therefore we have called it ligatin from the latin ligare, which translates "to bind together". Furthermore, we suggest that the membranes of the endocytic complex contain sites for the extracellular digestion of carbohydrate moieties in the maternal milk.

Animals↗
PubMed1975

On the connection between the transverse tubules and the plasma membrane in frog semitendinosus skeletal muscle. Are caveolae the mouths of the transverse tubule system?

The transverse tubular system (TTS) of skeletal muscle fibers represents the morphological basis for the inward spread of conduction of the electrical signal that triggers muscle contraction. A historical account of the main steps contributing to the elucidation of the structure and function of the TSS has been presented by Huxley (1971). While the localization of the TSS and its association with the sarcoplasmic reticulum (SR) is well documented; there is still a need further to develop our knowledge of the morphology of the connection between the TSS and the plasma membrane. It is generally believed that the TSS opens directly to the extracellular space and that there is continuity between its membrane and the sarcolemma. However, direct observation of such a connection has been clearly shown only for the myotome of fish (Franzini-Armstrong and Porter, 1964). In other muscle fibers, only indirect evidence of the connection has been provided by experiments showing penetration of extracellular tracers into the TSS. These extracellular markers were also observed inside another membrane-bounded compartment consisting of round profiles named "caveolae" (Yamada, 1955) or "pinocytotic vesicles" (Ashurst, 1969). The present study deals with the communication between the TTS, caveolae, and plasma membrane (Peachey, 1965); Ezerman and Ishikawa, 1967; Schiaffino and Margreth, 1968; and Rayns et al., 1968). A detailed study of the caveolae compartment was undertaken with ruthenium red as an electron-dense tracer. As a result of this study, we propose that in certain species the caveolae compartment represents the transitional region in the connection between the TSS and the sarcolemma.

Animals↗
PubMed1973

Fine structure of the synaptic discs separated from the goldfish medulla oblongata.

Synaptic discs are structures localized in the club ending synapses on the Mauthner cell lateral dendrite of the goldfish medulla oblongata. The synaptic discs present a hexagonal array of particles approximately 8.5 nm center-to-center when observed in en face view. This lattice covers the entire surface Divalent cations are important in the stabilization of this particular hexagonal array of particles When a synaptic disc-rich fraction is treated with chelating agents (EDTA or EGTA), definite changes occur in the hexagonal lattice. First, the synaptic membranes show zones without particles interspersed with zones covered with the hexagonal array of particles Second, the synaptic discs break down and a new structure characterized by two parallel dense bands (7 nm each), separated by a 4 nm gap, is observed. The negative stain fills the gap region showing striations spaced approximately 10 nm center-to-center crossing the gap, but it does not penetrate the dense bands This "double band" structure is interpreted as an edge on view of a fragment of the synaptic membrane complex. Further treatment of this fraction with a chelating agent plus 0.3% deoxycholate produces an increase in the number of double band structures. However, EDTA plus Triton X-100 (a treatment known to produce solubilization of membrane proteins) never shows such double band structure An ordered material was observed associated with the cytoplasmic leaflets of the double bands This material consists of rows of beads approximately 4 nm in diameter and spaced at intervals of approximately 7 nm. Each of these beads is joined to the band by a thin stalk.

Animals↗