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M J Costello

Publications and source records attributed to M J Costello.

At least 37 records · Page 2Linked to original sources
PubMed1989

Preparation of thin, fine-grained, tantalum metal replicas for freeze-fracture electron microscopy.

Two critical factors in the preparation of metal films on biological specimens are the type of metal used and the potentially damaging effects of radiant energy from the hot metal source. The excessive heating of surfaces is a major limitation to the replication of heat-sensitive aqueous specimens with refractory metals such as tungsten and tantalum, although these metals are known to form smaller grains and thinner films than the more commonly used platinum/carbon deposited under similar conditions. We describe here an electron gun designed for the evaporation of pure tantalum; surface heating is reduced through intermittent deposition controlled by varying the open/closed intervals of a fast shutter that operates in ultra-high vacuum. The effectiveness of the shutter was evaluated with a thin thermocouple in place of the specimen. The composition of the replicas was determined by x-ray microanalysis and by direct observation of the initial melting and subsequent evaporation of the tantalum bead supported on a tungsten rod that remained unchanged during the evaporation. The quality of the tantalum replicas was demonstrated with freeze-fracture replicas of reconstituted proteoliposomes and native membrane vesicles. With shutter intervals of 0.5 sec open and 1.0 sec closed, the surface heating was reduced enough to prevent unintentional etching and to preserve small pits complementary to protein particles in hydrophobic membrane surfaces and in ice.

Cell Membrane↗
PubMed1989

Distribution of gap junctions and square array junctions in the mammalian lens.

The morphology of membrane specializations of the cortex and nucleus of bovine lenses has been analyzed for both isolated membrane fractions and intact tissue fragments. Fractions of fiber cell membranes isolated from the outer cortex and the inner nucleus of lenses have been compared using x-ray diffraction, electron microscopy, SDS polyacrylamide gels and Western blots. Each fraction has distinctive structural characteristics. In x-ray experiments, the cortical fraction gives no sharp equatorial reflections (from the plane of the membrane), whereas the nuclear fraction gives sharp equatorial reflections which index on a square lattice of 6.6 nm. In thin-section electron micrographs, the cortical fraction is composed primarily of closed vesicles and flat membrane sheets, some of which contain pentalamellar structures similar in appearance to the 16-18 nm thick gap junctions found in other tissues. The nuclear fraction contains mostly undulating membrane pairs which often show 11-14 nm pentalamellar profiles and occasionally thicker junctions. In freeze-fracture images the cortical membranes display irregular clusters of intramembrane particles which resemble gap junctions, whereas the nuclear membranes contain numerous large square arrays with a 6.6 nm repeat and few irregular clusters or individual intramembrane particles. Images of fragments of intact lenses used in the membrane isolations give similar results; in the cortex the area covered by gap junctions is over 50 times the area covered by square lattices, whereas nuclear fiber cell membranes contain large square arrays. Thus, cortical and nuclear fiber cell membranes have quite different morphologies. In particular, the size of the square arrays of protein increases as the fiber cells mature. SDS polyacrylamide gels from cortical and nuclear fractions are similar in that they both contain MP26 as the major band. However, Western blot analysis shows increasing quantities of lower molecular weight, 25 kD and 22 kD, cleavage products as one progresses from the cortex to the nucleus. These data indicate that MP26 and/or its cleavage products form square crystalline arrays in nuclear fibers. The morphology of these arrays suggests a role for MP26 in cell-to-cell adhesion.

Animals↗
PubMed1988

Rapid induction of lorazepam dependence and reversal with flumazenil.

Benzodiazepine tolerance, dependence and withdrawal are well established clinical entities although the pharmacological basis for these are still unclear. Recent data suggest that the primary event may be a change in efficacy at the benzodiazepine receptor. The present study demonstrates the rapid development of tolerance and dependence to lorazepam, defines its pharmacology in more detail, and shows that it may be rapidly reversed by treatment with the benzodiazepine antagonist flumazenil. These observations argue in favour of a receptor efficacy change underlying benzodiazepine tolerance and withdrawal and suggest a potential pharmacological treatment for this common and disabling clinical problem.

Animals↗
PubMed1987

Purified lac permease and cytochrome o oxidase are functional as monomers.

Purified lac permease, the 46.5-kDa product of the lac Y gene that catalyzes lactose/H+ symport, or purified cytochrome o, a terminal oxidase of the Escherichia coli respiratory chain composed of four subunits with a composite molecular mass of 140 kDa, was reconstituted into proteoliposomes individually or in combination. The preparations were then examined by freeze-fracture electron microscopy employing conventional platinum/carbon replicas or by means of a new technique using thin tantalum replicas. In nonenergized proteoliposomes, both proteins appear to reconstitute as monomers based on (i) the variation of intramembrane particle density with protein concentration; (ii) the ratio of particles corresponding to each protein in proteoliposomes reconstituted with a known ratio of permease to oxidase; and (iii) the dimensions of the particles observed in tantalum replicas. The intramembrane particle diameters in tantalum replicas are about 20-25% smaller than those observed in conventional platinum/carbon replicas, indicating that the dimensions of the particles revealed with tantalum more accurately reflect the sizes of lac permease and cytochrome o. The diameters and heights of the permease and cytochrome o in tantalum replicas are 5.1 nm X 2.8 nm and 7.4 nm X 4.2 nm, respectively. Furthermore, a higher percentage of lac permease molecules exhibits a notch or cleft in tantalum replicas relative to platinum/carbon replicas. Importantly, the initial rate of lactose/H+ symport in proteoliposomes varies linearly with the ratio of lac permease to phospholipid, and no change is observed in either the size or distribution of lac permease molecules when the proteoliposomes are energized. The results taken as a whole provide a strong indication that both lac permease and cytochrome o reconstitute into proteoliposomes as monomers, that the permease does not dimerize in the presence of the H+ electrochemical gradient, and that both molecules are completely functional as monomers.

Carbon↗
PubMed1987

Structural features of the terminal loop region of frog retinal rod outer segment disk membranes: I. Organization of lipid components.

We have applied thin sectioning and freeze-fracture techniques to investigate the terminal loop structure of photoreceptive disks in frog retinal rod outer segments. Our studies of this region demonstrate a highly curved terminal loop bilayer that is continuous with both lamellar bilayers of the disk, and equivalent to them in dimensions and staining properties. Rhodopsin, however, appears to be excluded from this region of high curvature.

Animals↗
PubMed1987

Structural features of the terminal loop region of frog retinal rod outer segment disk membranes: II. Organization of the terminal loop complex.

In addition to a lipid bilayer component (Corless, Fetter, and Costello: J. Comp. Neurol. 257:1-8, '87), the terminal loop region of frog rod outer segment (ROS) disks displays a clustering of discrete elements referred to as the terminal loop complex. It consists of (1) semicircular or crescentic densities within the terminal loop, (2) linear interdisk densities spanning the cytoplasm near terminal loops, and (3) distinctive freeze-fracture particles associated with the terminal loop, located between 1 and 2. The linear interdisk densities are organized on a two-dimensional lattice that appears to ensheath completely the lamellar domains of all ROS disks. Indirect evidence is presented for a net axial alignment of intraloop densities. We interpret the large freeze-fracture particles of the terminal loop region to reflect transmembrane components that connect the interdisk and intraloop densities. Thus, we propose that the entire terminal loop (TL) complex is organized on a two-dimensional net. We further infer that each TL complex is organized as a dimeric unit and that such dimers interact axially and laterally to generate the observed lattice structure. It is suggested that one component of the terminal loop complex is the high molecular weight protein localized along the disk perimeter by Papermaster, Schneider, Zorn, and Kraehenbuhl (J. Cell. Biol. 78:415-425, '78).

Animals↗
PubMed1986

Freeze-fracture methods: preparation of complementary replicas for evaluating intracellular ice damage in ultrarapidly cooled specimens.

Thin biological specimen sandwiched between conductive metal foils and ultrarapidly frozen in the absence of chemical pretreatments are well suited for ultrastructural studies by the freeze-fracture technique. However, the roughness of the metal surfaces, together with the thinness of the specimen, produce highly irregular surfaces upon fracturing, and thus yield fragile metal replicas. The techniques described here for stabilizing the replicas, initially with an open mesh grid and finally with a Formvar film, provide a means for obtaining a high percentage of intact replicas from which complementary images can be prepared. With the aid of the montages, complementary regions on the replicas can be located at some later time, usually in less than 1 hr. The complementary images obtained are excellent for evaluating the factors which influence the quality and resolution of the replicas--such as heating and plastic deformation--and for describing the preservation of biological structures. Regions within the specimen which display unacceptable ice crystal growth or damage can be easily and confidently identified in complementary images.

Animals↗
PubMed1986

A procedure for obtaining complementary replicas of ultra-rapidly frozen sandwiched samples.

Complementary replicas of samples prepared for electron microscopy by the freeze-fracture/etch technique are extremely valuable in the interpretation of the exposed surfaces, the nature and location of the membrane fracture plane, and as an aid in the recognition of the potential artefacts of this technique. This paper describes a procedure for the preparation of complementary replicas of thin samples sandwiched between copper foil strips and frozen ultra-rapidly in the absence of chemical pretreatments. In this procedure, the copper foil support bearing the replica is floated on the surface of a chromic acid solution, resulting in the controlled dissolution of the copper metal. The replica which remains at the surface of the chromic acid is then stabilized against fragmentation during subsequent cleaning and rinsing steps by placing a 50 mesh gold grid on top of the replica. To minimize agitation of the replica/grid, all cleaning steps are performed in a single depression plate well. The clean replica/grid is picked up from below on a thin Formvar film, dried, and then separated from the extra film. Careful placement of the gold grid on the replicas and low magnification electron micrograph montages of the complementary grids facilitate the location of complementary regions and simplify examination of complementary specimen areas at higher magnification.

Animals↗
PubMed1985

Membrane specializations in mammalian lens fiber cells: distribution of square arrays.

Fragments of intact rat, pig and bovine lenses and isolated membranes from bovine lenses were examined by freeze-fracture-etch electron microscopy employing samples ultra-rapidly frozen in the absence of fixatives or cryoprotectants. Complementary replicas of the bovine outer cortex clearly display small patches of square arrays (6.6 nm repeat) as structures distinct from gap junctions in the same membranes. In the outer cortex square arrays appear in single membranes where the extracellular space is not thinned, whereas gap junctions, as in other tissues, only occur where the extracellular space is greatly attenuated. Square arrays in the middle cortex appear as patches of varying size surrounded by smooth membrane fracture faces. Within the undulating membranes of tongue-and-groove interdigitations in the inner cortex and nucleus, the square arrays are extensive and are located specifically on the regions which have convex curvature toward the cytoplasm. Square crystalline regions alternate with non-crystalline regions within each membrane of an undulating pair. Across the extracellular space, crystalline regions are matched with non-crystalline regions, thus making it unlikely that the square array participates in intercellular communication. Because the undulations occur in isolated membranes following urea washing to remove cytoplasmic and extrinsic membrane proteins, the square arrays probably play a crucial role in the formation of the undulations and in the maintenance of membrane curvature. X-ray diffraction experiments show reflections from the crystalline square array for all twenty of our preparations of isolated membranes. These x-ray experiments indicate that the square arrays observed by freeze-fracture are abundant in lens fiber cell membranes.

Animals↗
PubMed1984

Morphology of proteoliposomes reconstituted with purified lac carrier protein from Escherichia coli.

Proteoliposomes reconstituted with purified lac carrier protein from Escherichia coli were ultra-rapidly frozen and examined by freeze-fracture-etch electron microscopy. The proteoliposomes are greater than 95% unilamellar, and the majority are 30-150 nm in diameter. Fracture faces of proteoliposomes (at a protein:lipid molecular ratio of about 1:2500) display 7.0-nm diameter globular intramembrane particles uniformly distributed on convex and concave surfaces. Calculations of particle composition suggest that each intramembrane particle probably contains one or two molecules of the 46.5-kDa transmembranous lac carrier protein, depending on the correction factor for the thickness of the metal deposited to form the platinum/carbon replicas. Etched surfaces of the proteoliposomes are smooth. Incubation of the proteoliposomes with monoclonal antibody 4B1, which binds to an epitope in the lac carrier on the exterior of the proteoliposomes, dramatically alters the intramembrane particle distribution. After incubation with antibody, the convex (inner monolayer) fracture faces are nearly devoid of intramembrane particles, and an overall 4-fold reduction in the total number of intramembrane particles is observed.

Cell Membrane↗
PubMed1984

Selective contrast in electron microscopy of crystalline cytochrome oxidase.

We have used various techniques for preparation of specimens for electron microscopy in order to selectively contrast different regions of vesicle crystals of cytochrome c oxidase dimers. The results are consistent with a dimer composed of two y-shaped monomers [Fuller et al., J. Mol. Biol. 134 (1979) 305] aligned along one pair of arms with the other pair of arms approximately 70 A apart. The four arms of the monomers lie within and perpendicular to the lipid bilayer in which the dimer is embedded, and the arms protrude approximately 25 A from the lipid bilayer on the matrix side of the membrane. The cytoplasmic side domains of the two monomers split away from one another forming a large cleft in the dimer. Monovalent antibodies (Fab fragments) to subunit IV appear to bind to the two monomer arms which are closely apposed across the two-fold axis of the dimer.

Crystallography↗
PubMed1984

Inhibition of neutrophil activation by alpha1-acid glycoprotein.

We report that alpha1-acid glycoprotein (AAG), a naturally occurring human plasma protein and acute phase reactant of uncertain biological function, inhibits human neutrophil aggregation and superoxide anion generation induced by a variety of stimuli including zymosan treated serum, formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate. Inhibition was transient, directly proportional to the glycoprotein concentration and inversely proportional to the concentration of the stimulus added. Desialyzation, resulting in the removal of a substantial portion of the molecule's negative charge, did not alter the effectiveness of AAG. Removal of the penultimate galactose residues from desialyzed AAG resulted in a slight but significant reversal of inhibition, suggesting that the heteropolysaccharide units of AAG may be important for inhibition of cellular function. We therefore suggest that the acute phase glycoprotein AAG may be a significant modulator of neutrophil as well as platelet and lymphocyte function during inflammation.

Cell Aggregation↗
PubMed1983

The chromaffin granule as a model for membrane fusion: implications for exocytosis.

Rapid freeze/freeze-fracture and thin section electron micrographic studies of the Ca2+-promoted aggregation and fusion of isolated bovine adrenal medullary chromaffin granule membranes show that the granules undergo a series of morphological changes. The contact region becomes quite extensive and the membrane curvature changes radically at the edge of the contact site. The core material retracts away from the contact site leaving an electron lucent "stripe"; however, it remains adjacent to the membrane in the non-contact areas. The pentalaminar double membrane of the contact region often shows breaks. Close examination reveals that the two granule membranes have fused and become one continuous membrane. Rearrangement of large membrane associated particles (MAPs) can be seen by freeze fracture after Ca2+-promoted granule-granule contact. The broken pentalaminar septum becomes smaller and may break down into globular structures. These observations suggest a series of reactions in which the granules first form an encounter complex, then a stable complex. The membranes within the contact region undergo lateral displacement of the proteins and phase separations of the lipids, and then fuse. Analysis of the kinetics of turbidity and fluorescence changes during granule aggregation and fusion support the main postulates of the model. The initial events of aggregation are facilitated by putative recognition proteins and K+ will promote all activities except fusion. Recent observations that several soluble proteins (synexin, and albumin) will act as fusogens are discussed in terms of the relevance to exocytosis in vivo.

Animals↗
PubMed1982

The structure of junctions between lens fiber cells.

Junctions which have been isolated from bovine lens may correspond to the tongue-and groove interdigitations observed between intact cells. These junctions have an overall thickness of 13-14 nm, a striking undulating surface topology, and contain protein subunits of congruent to 27 000 daltons which are arranged in a 6.6-6.7-nm square lattice in the plane of each membrane. A similar square-lattice structure has been observed in junctions from fragments of intact rate lens. Junctions from both sources are structurally different from "typical' gap junctions.

Animals↗