Statistical evidence for two major proteins in freeze-fractured gastric parietal cell tubulovesicles and canaliculus

In a previous work, resting and acid-secreting rabbit gastric mucosa were freeze-fractured and shadowed at 45° with Pt-C. The shadow widths of proteic particles of tubulovesicle and canaliculus membranes were measured and compared. It was concluded that the frequency distributions of widths are significantly different in resting and secreting membranes and that each distribution accounts for several subpopulations of homogenous particles. In the present study, an attempt is made to describe the experimental distributions as a mixture of those of two major proteins, say A and B and their aggregates (AA, AB and BB). The modelling, although simple, gave a very satisfactory statistical fit between observed and computed distributions. The comparison of parameters calculated from histamine and ranitidine experimental data further improves the fits and finally, component A accounts for 69% of the particles. Most replica of A particles are heart-shaped and the median shadow widths are 6.1 and 6.8 nm in canaliculus and tubulovesicles respectively. The component B accounts for 31% of the particles. They mainly appear as small barrels and the median shadow widths are 8.8 and 10.3 nm in canaliculus and tubulovesicles respectively. According to calculated parameters and observed particle replica, the onset of secretion does not change the relative ratio of proteins but changes their shapes. Component A should be the (H⁺, K⁺)ATPase whereas debate on the identity of B is wide open.     

Correlative statistical analysis and computer modelling of intramembraneous particle distributions in human erythrocyte membranes.

The planar distribution of intramembranous particles on the P faces of freeze-fractured human erythrocyte membranes is characterized by radial distribution, angular distribution and differential density distribution analysis. Various degrees of intramembranous particle aggregation induced by spectrin removal and low pH are differentiated through computation. Random hard disk models with various disk diameters are built for comparison studies. In all samples, the 80 +/- 10 A particles are found to have a preferred neighboring distance of 100 +/- 10 A, but no preferred angular relation is found between neighboring particles. A pattern recognition process using both radial and density distribution analyses reveals that none of the particle distributions observed may be regarded as random. The fact that the particle distributions observed are neither even nor random suggests that factors other than long range electrostatic force alone are involved in determining the particle distribution.     

Ultrastructure of the sodium pump: Comparison of thin sectioning, negative staining, and freeze-fracture of purified, membrane-bound (Na+, K+)-ATPase

Purified (Na+, K+)-ATPase was studied by electron microscopy after thin sectioning, negative staining, and freeze-fracturing, particular emphasis being paid to the dimensions and frequencies of substructures in the membranes. Ultrathin sections show exclusively flat or cup-shaped membrane fragments which are triple-layered along much of their length and have diameters of 0.1-0.6 μm. Negative staining revealed a distinct substructure of particles with diameters between 30 and 50 A and with a frequency of 12,500 +/- 2,400 (SD) per μm(2). Comparisons with sizes of the protein components suggest that each surface particle contains as its major component one large catalytic chain with mol wt close to 100,000 and that two surface particles unite to form the unit of (Na+,K+)-ATPase which binds one molecule of ATP or ouabain. The further observations that the surface particles protrude from the membrane surface and are observed on both membrane surfaces in different patterns and degrees of clustering suggest that protein units span the membrane and are capable of lateral mobility. Freeze-fracturing shows intramembranous particles with diameters of 90-110 A and distributed on both concave and convex fracture faces with a frequency of 3,410 +/- 370 per μm(2) and 390 +/- 170 per μm(2), respectively. The larger diameters and three to fourfold smaller frequency of the intramembranous particles as compared to the surface particles seen after negative staining may reflect technical differences between methods, but it is more likely that the intramembranous particle is an oliogomer composed of two or even more of the protein units which form the surface particles.     

Electron microscopic study of troponin

Troponin and its components or fragments were observed in an electron microscope by the use of the rotary shadowing technique. In freshly prepared troponin with low viscosity, globular particles were mainly observed. The size of the long axis of the particles was 13.2 +/- 1.3 nm and the size perpendicular to the long axis was 9.5 +/- 1.2 nm. The mean axial ratio was 1.4 +/- 0.3. Most of the particles observed in a stored troponin preparation, having a higher viscosity than that of fresh troponin, had a globular head with a thin tail, with the total length of 25.4 +/- 1.4 nm (head-tail type particles). The axial size of the globular portion was 8.3 +/- 1.2 nm and the tail length was 17.1 +/- 1.6 nm. Observation of various particles during the transitional stages indicated that, in the globular particles, the tail region of head-tail type particle was associated along the globular head region. Troponin T was a filamentous particle with 16.9 +/- 1.5 nm length. The 26K fragment of troponin T, which was devoid of the N-terminal 45 residues from troponin T, was a filamentous particle with the length of 14.4 +/- 1.3 nm. Troponin T1, one of two chymotryptic subfragments of troponin T, was a filamentous particle of 11.6 +/- 1.4 nm length. Troponin C.T in the presence of Ca2+ was a particle with a globular head (7 nm in size) and a tail of about 17 nm length. The Fab fragment of anti-troponin T1 formed regular transverse striations along the thin filament of rabbit skeletal muscle with a 38 nm period.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of size of polystyrene particles on their retention within the rat peritoneal compartment, and on their interaction with rat peritoneal macrophages in vitro

Polystyrene particles (size range 300 nm-3 microns diameter) were radioiodinated and their capture by rat peritoneal macrophages measured in vitro. For unmodified particles, most efficient accumulation was observed using a diameter of 600 nm (Endocytic Index (E.I.) = 16.4 +/- 2.9 microliters/10(6) cells/h). Particles (3 microns diameter) which had been modified to become more hydrophilic by hydroxymethylation showed an increased rate of capture (E.I. = 136.6 +/- 91.2 microliters/10(6) cells/h). Following intraperitoneal administration to rats, unmodified 3 micron particles showed selective accumulation in the omentum (18.4% injected dose/g), and this was increased for the hydroxymethylated bead (35.3% dose/g). The smaller (800 nm) particles were better able to leave the peritoneal compartment. Radiolabelled particles isolated from a peritoneal wash after 5 h were mostly cell-associated (72-86%, depending on the type of particle).     

Assessing the accuracy of routine photon correlation spectroscopy analysis of heterogeneous size distributions

The aim of the current study was to investigate the ability of a fixed-angle routine photon correlation spectrometer (PCS) to resolve bimodal size distributions. The focus was on dispersions consisting of a majority of smaller and a minority of bigger particles. Monodisperse latex beads of sizes from 21 to 269 nm were measured first as single-size dispersions and then with various binary blends. For single-size dispersions, the mean diameters obtained were as indicated by the manufacturer, except for 21- and 34-nm particles, which were somewhat smaller. PCS analysis of blends of 21+102-nm and 34+102-nm particles resulted in bimodal distributions with particle diameters of the 2 peaks in the expected magnitude down to critical blending ratios of 0.002% and 0.08% of bigger particles, respectively. At these ratios, PCS results became inconsistent, and an increased number of monomodal results and/or high residuals were seen. For 21+102-nm blends, at even smaller ratios (0.001%), more consistent results were obtained again with predominantly monomodal distributions in the size range of the smaller particles (ie, the bigger particles were neglected). PCS analysis of blends of 21+269-nm particles yielded bimodal distributions with diameters within the expected magnitude as long as the content of bigger particles did not exceed 0.005%. Above this ratio, predominantly monomodal results with mean diameters in the magnitude of the bigger particles were obtained (ie, the smaller particles were neglected). In conclusion, a routine PCS instrument can resolve bimodal size distributions of colloidal dispersions only at certain ratios of the 2 subpopulations. Both low and high ratios lead to 1 of the 2 subpopulations being neglected. Phone: Not Forwarded; Fax: Not Forwarded;     

Hydrodynamic diameters of murine mammary, Rous sarcoma, and feline leukemia RNA tumor viruses: studies by laser beat frequency light-scattering spectroscopy and electron microscopy.

We have studied purified preparations of murine mammary tumor virus (MuMTV), Rous sarcoma virus (RSV; Prague strain), and feline leukemia virus (FeLV) by laser beat frequency light-scattering spectroscopy, ultra-centrifugation, and electron microscopy. The laser beat frequency light-scattering spectroscopy measurements yield the light-scattering intensity, weighted diffusion coefficients. The corresponding average hydrodynamic diameters, as calculated from the diffusion coefficients by the Stokes-Einstein equation for MuMTV, RSV, and FeLV, respectively, are: 144 +/- 6 nm, 147 +/- 7 nm, and 168 +/- 6 nm. Portions of the purified RSV and MuMTV preparations, from which light-scattering samples were obtained, and portions of the actual FeLV light-scattering samples were examined by negatively stained, catalase crystal-calibrated electron microscopy. The light-scattering intensity weighted averages of the electron micrograph size distributions were calculated by weighing each size by its theoretical relative scattering intensity, as obtained from published tables computed according to the Mie scattering theory. These averages and the experimentally observed hydrodynamic diameters agreed to within +/- 5%, which is the combined experimental error in the electron microscopic and light-scattering techniques. We conclude that the size distributions of singlet particles observed in the electron micrographs are statistically true representations of the sedimentation-purified solution size distributions. The sedimentation coefficients (S20, w) for MuMTV, RSV, and FeLV, respectively, are: 595 +/- 29S, 689 +/- 35S, and 880 +/- 44S. Virus partial specific volumes were taken as the reciprocals of the buoyant densities, determined in sucrose density gradients. The Svedberg equation was used to calculate particle weights from the measured diffusion and sedimentation coefficients. The particle weights for MuMTV, RSV, and FeLV, respectively, are: (3.17 +/- 0.32) x 10(8), (4.17 +/- 0.42) x 10(8), and (5.50 +/- 0.55) x 10(8) daltons.     

Gravitational and magnetic activation methods applied to cultured cells (LK 35.2)

Monoclonal antibody 10.2-16 is directed toward the mouse class II major histocompatibility complex gene product 1-Ak expressed on the cell line LK35.2. Instead of activating cells by fluorophor we used (acrylamide-coated) heavy and magnetic microspheres of 0.6 micron in radius. These microspheres are chemically coupled (carbodiimide method) with the antibody toward the surface antigen. The cells are observed through a microscope with horizontal alignment, as they sediment in a (temperature controlled) tube with square cross-section. Stokes Law allows the determination of the density of cells (first alone) using viscosity and density of Dulbecco's modified Eagle's Medium together with the observed mean sedimentation velocity (66 microns/min) and a mean diameter of 10 microns. We found a density of 1.0558 +/- 0.0028 g/cm3 at 10 degrees C. Independently, thinly coated, heavy (and magnetizable) microspheres with the cited antibody are attached to cells and observed likewise. The increased sedimentation velocity permits us to show that the cells were fully covered with microspheres (290 per cell). A magnetic field gradient opposing gravity moved these cells against gravity with two different mean velocities, 340 microns/min and 850 microns/min. The higher velocity resulted in 290 particles per cell, the lower one in 130 particles per cell. The limits for the expansion of this method to smaller particle sizes (down to 10 nm) are evaluated.     

The platinum-carbon replication of a homogenous population of gold particles makes log-normal distributions of shadow widths and lengths

Gold particles were prepared, dried on grids and shadowed at 45° with a 1.2 nm platinum-carbon (Pt-C) film using the shadowing conditions previously described for the freeze-fracture of gastric parietal cell membranes. The particle diameters and the particle shadow widths and lengths were measured using an image analysis system. Statistical analysis of 2000 diameters, shadow widths and shadow lengths indicated that a homogenous population of particles had a normal frequency distribution of diameters (mean diameter 14.5 ± 1.5 nm) and that the Pt-C shadowing transformed that normal curve into a log-normal frequency distribution of shadow widths. The frequency distribution of shadow lengths was log-normal too. We conclude that a statistical partition of experimental frequency distributions of particle shadow widths and lengths of natural membranes to determine the number and parameters of individual components should involve log-normal subdistributions rather than normal ones.     

Intracellular Ca2+ shift and signal transduction from the tubulovesicular portion of gastric parietal cells during gastrin stimulation or Ca2+ ionophore treatment: comparison between luminescent and fluorescent probes, and electron probe X-ray microanalyzer

In gastrin-stimulated, aequorin-loaded parietal cells from guinea pig gastric mucosa, a rapid but transient increase in the cytosolic free Ca2+ concentration ([Ca2+]i), owing to Ca2+ released from the store(s), and a more prolonged Ca2+ entry from outside the cells were observed. However, there was a little increase in [Ca2+]i when similar measurements were assessed by quin 2 or fura-2 in physiological saline. However, depletion or elimination of Na+ from the incubation medium caused a significant increase in the [Ca2+]; response to gastrin as measured by quin 2. These findings suggest that aequorin and quin 2 (or fura-2) provide information about different aspects of Ca2+ homeostasis and that there is an inhomogeneity of [Ca2+]i in the cytoplasm during gastrin stimulation. By the gastrin stimulation, the intracellular Ca2+ gradients were shifted from the unidentified portion(s) to the restricted apical cytoplasm, as determined by electron probe X-ray microanalysis. Therefore, localization and identification of the source of intracellular Ca2+ as a pool were determined by an X-ray microanalyzer. In the resting state, the tubulovesicle had high Ca2+ concentration compared with the level in the apical cytoplasm. Cells treated with the Ca2+ ionophore ionomycin had a decreased tubulovesicular Ca2+ level, followed by a reciprocal increase in area of the canalicular membrane. The secretory canaliculus in stimulated cells had lower Ca2+ or higher K+ and Cl- concentrations than that of tubulovesicles or cytoplasm in the resting state, respectively. These findings suggest that the Ca2+ pool of the parietal cell is in the tubulovesicles and (or) luminal cell membrane and that the Ca2+ released from the store(s) may mediate a flow of K+ or Cl- into the secretory canaliculus.     

Chromatin fibers are left-handed double helices with diameter and mass per unit length that depend on linker length

Four classes of models have been proposed for the internal structure of eukaryotic chromosome fibers--the solenoid, twisted-ribbon, crossed-linker, and superbead models. We have collected electron image and x-ray scattering data from nuclei, and isolated chromatin fibers of seven different tissues to distinguish between these models. The fiber diameters are related to the linker lengths by the equation: D(N) = 19.3 + 0.23 N, where D(N) is the external diameter (nm) and N is the linker length (base pairs). The number of nucleosomes per unit length of the fibers is also related to linker length. Detailed studies were done on the highly regular chromatin from erythrocytes of Necturus (mud puppy) and sperm of Thyone (sea cucumber). Necturus chromatin fibers (N = 48 bp) have diameters of 31 nm and have 7.5 +/- 1 nucleosomes per 10 nm along the axis. Thyone chromatin fibers (N = 87 bp) have diameters of 39 nm and have 12 +/- 2 nucleosomes per 10 nm along the axis. Fourier transforms of electron micrographs of Necturus fibers showed left-handed helical symmetry with a pitch of 25.8 +/- 0.8 nm and pitch angle of 32 +/- 3 degrees, consistent with a double helix. Comparable conclusions were drawn from the Thyone data. The data do not support the solenoid, twisted-ribbon, or supranucleosomal particle models. The data do support two crossed-linker models having left-handed double-helical symmetry and conserved nucleosome interactions.     

Freeze-fracture studies on unmyelinated axolemma of rat cervical sympathetic trunk: correlation with saxitoxin binding

The density and diameter distributions of intramembranous particles (IMPs) within unmyelinated axolemma from rat cervical sympathetic trunk were examined with freeze-fracture electron microscopy. The axolemma displays a highly asymmetrical partitioning of IMPs with ca. 1200 IMPs microns-2 on P-faces and ca. 100 IMPs microns-2 on E-faces. Particle sizes (diameters) are unimodally distributed on both fracture faces, with a range from 2.4 nm to 15.6 nm. Approximately 16% of the particles on P-faces and 28% of particles on E-faces are of a large (greater than 9.6 nm) diameter. On both fracture faces, the IMPs appear to be randomly distributed; no aggregations of particles were observed. The results indicate that there are ca. 230 large IMPs microns-2 of unmyelinated axolemma from rat cervical sympathetic trunk. The density of these IMPs is similar to the density of saxitoxin binding sites on unmyelinated axolemma from rat cervical sympathetic trunk (Pellegrino et al. 1984 (Brain Res. 305, 357-360)), which suggests that many of the large diameter particles may be the morphological correlate of voltage-sensitive Na+ channels.     

Alteration of pial vessel responses to blood pressure changes in rats after hypoxia

Previous studies in newborn lamb have shown impairment of cerebral blood flow autoregulation after hypoxia followed by reoxygenation. The present study was done to see if such a phenomenon existed in the adult rat and if it could be demonstrated at the level of the pial arterioles. Using an open cranial window preparation, we assessed the changes in pial vessel diameter during blood pressure alterations induced by hemorrhage and reinfusion of blood, before and after 30 s of hypoxia, in 15 male Sprague-Dawley rats. Mean diameters of pial arteries in the study group of rats were 128 +/- 54 microns before hypoxia and 141 +/- 61 microns after normoxia following hypoxia. The corresponding diameters in rats serving as time controls were 136 +/- 52 and 138 +/- 52 microns. Slopes of pial vessel diameters as a function of mean arterial blood pressures decreased significantly (p less than 0.05) after hypoxia from -0.86 +/- 0.45 to 0.03 +/- 0.66 (mean +/- SD). In the control rats not subjected to hypoxia, the slopes remained unchanged over a similar time period (-0.60 +/- 0.16 and -0.42 +/- 0.19). The negative slopes indicate that pial vessels dilate during hypotension and constrict during hypertension. Such vascular responses may play a role in autoregulation of cerebral blood flow. We found that a relatively brief period of hypoxia can cause a long-lasting impairment of vascular responses even after restoration of normoxia. These findings are consistent with a previous report of persistent impairment of cerebral blood flow autoregulation after a brief period of hypoxia.     

Oleic acid allows more apoprotein A-1 to bind with higher affinity to large emulsion particles saturated with cholesterol

Apoprotein (apo) A-1 binding to large triolein-rich emulsion particles saturated with cholesterol has been examined as a function of the oleic acid content. Six emulsion systems were formed containing 0.3-1.0% (by weight) oleic acid, 82.9-86.3% triolein, 10.6-7.2% egg yolk phosphatidylcholine, and 6.7-5.5% cholesterol. The average emulsion particle diameters calculated from these lipid compositions ranged between 84 and 116 nm. Negative stain electron microscopy of an emulsion containing 1% oleic acid showed a polydisperse population of only large spherical particles with a mean diameter of 116 +/- 54 nm. The calculated cholesterol concentrations of the particles surface and core for the six emulsions were 43.3 +/- 1.1 and 5.6 +/- 0.2 mol%, respectively, and were rather constant. Therefore, when the surface oleic acid concentrations increased from 2.6 to 10.1 mol%, the phospholipid concentration decreased from 55.1 to 45.9 mol%. In the core, oleic acid increased at the expense of triolein. In the range studied a nearly 4-fold increase in the surface oleic acid content produces a similar increase in the binding capacity (N) and reduces the dissociation constant (Kd). The changes in the Kd and N values were linearly dependent on the surface oleic acid concentration. These data show that oleic acid allows more apoA-1 to bind with higher affinity to large emulsion particles saturated with cholesterol.     

A freeze-fracture study of postembryonic differentiation of latissimus dorsi muscles of the chicken

The differentiation of fiber type characteristics in the anterior (ALD) and posterior (PLD) latissimus dorsi muscles is examined by the freeze-fracture technique in 1-, 7- and 30-day-old chicks. Several characteristics of plasma membrane (caveolae, rectilinear arrays, intramembranous particles) and sarcoplasmic reticulum which show fiber type differences in the adult ALD and PLD muscles are compared in the developmental stages. The caveolar density in the ALD fibers is about 20/microns2 at 1 day increasing to about 37/microns2 at 30 days, whereas in the PLD fibers it remains at about 20/microns2 during this period. The distribution of the caveolae in the two muscles is different from the beginning; in the ALD fibers the caveolae are distributed throughout the plasma membrane and in PLD fibers they are patterned into clusters overlying the I band regions. The density of intramembranous particles of 1-day ALD and PLD plasma membranes appears similar, but by 7 days the particle counts in the sarcolemma of the ALD muscle are about twice as numerous as those in the PLD muscle. The rectilinear arrays are virtually absent in the ALD muscle, whereas in the PLD muscle their density is about 10/microns2 at 1 day and about 20/microns2 at 7 days. Already at 1 day posthatching the SR in ALD and PLD fibers has the adult configuration, i.e., an open irregular network in ALD fibers and periodically arranged tubules with triadic expansions in the PLD fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological Variants of Coliphage P1

Lysates of P1 from all hosts tested contained at least three morphological variants with respect to head size. These were termed "big" (P1B), "small" (P1S), and "minute" (P1M). Since successive clonings of plaques isolated on many different hosts failed to change the proportions of the variants, we concluded that the production of variants was a function of the P1 genome rather than that of the host. In the electron microscope, the heads appeared to be icosadeltahedra, having face-to-face head diameters of 86 +/- 2 nm, 65 +/- 2 nm, and 47 +/- 2 nm. Assuming the head capsids to be composed of the same protein subunits, these diameters were compatible with T = 16, 9, and 4 with a lattice constant (intercapsomere distance) of 12 to 13 nm. The tails of all variants were morphologically indistinguishable. Each consisted of a hollow tail tube surrounded by a contractile sheath attached to the head by means of a "head-neck connector" which could be a specialized vertex capsomere. In CsCl gradients, a number of bands were observed. One band contained the majority of P1B particles and 99% of the plaque-forming units. Two other bands contained P1S particles whose densities suggested a content of about 40 and 60% of the complete P1B genome. The less dense of these two bands also contained defective P1B particles with a calculated content of only 60% of the complete genome. The P1S particles tested injected their deoxyribonucleic acid (DNA) into host cells and killed them. Genetic markers contained in this band could be rescued by infectious P1B particles, confirming the evidence of Ikeda and Tomizawa that this fraction contains P1 DNA.     

Freeze-fracture study of the zymogen granule membrane of pancreas: two novel types of intramembrane particles

The ultrastructure of the zymogen granule (ZG) membrane has been observed in vitro by rapid freezing and freeze-fracture techniques. Unidirectional shadowing of the plasmic fracture (PF) leaflet of the intact granule reveals a relatively smooth surface uniformly studded by intramembrane particles (IMP; 360 microns2) their diameters ranging from 5 to 18 nm (mean = 10.2 nm) but does not allow a clear visualization of the particles on the external fracture (EF) leaflet. Indeed, rotary shadowing reveals that the EF leaflet presents a highly textured subparticle background with a significantly lower frequency of IMP (44 microns2) showing diameters from 9 to 18 nm and a shift to larger IMP (mean = 12.3 nm). Two hitherto undescribed types of IMP are found on both leaflets of the membrane: first a population of 13-nm particles with an electron-lucent center or "pore", the most frequent type on the EF face (26%), is a second population of large IMP (15 nm) characterized by a large "pore" (5.0 nm diameter) subdivided by a delicate cross-shaped structure. In alkaline conditions, pH 8.2, ZG lysis occurs rapidly and membrane ghosts thus obtained were rapidly frozen or suspended in dextran and filtered immediately. Transmission electron microscopy (TEM) shows many opened ghosts with adhering amorphous material and numerous small vesicles near or still attached to openings in the ghosts. Freeze-fracture preparations show that granule lysis is accompanied by major alterations of membrane ultrastructure; the subparticle background on the EF leaflet is now visible only as a cap or linear crest at one pole of the ghosts. These two newly formed zones are demarcated by a row of 13-nm particles, whereas the other IMP are confined to the subparticle background. Some images suggest that the subparticle background and 13-nm IMP necklace give rise to vesicles, some of them occasionally attached to the ghosts. The subparticle background on the EF leaflet shows a complementary imprint on the PF leaflet which is similarly modified. This study shows the presence of hitherto undescribed types of IMP and also demonstrates alterations of certain domains of zymogen granule membranes that occur at the moment of lysis, associated with a redistribution of different particle populations.     

Membrane modifications in chick osteoclasts revealed by freeze-fracture replicas

Remarkable differences among various membranes of bone cells became evident by examination of freeze-fracture replicas. In osteoclasts, three types of intramembranous particles (IMPs) were identified based on their size and shape: two sizes of isolated globular particles (8 and 12 nm in diameter) and rod-shaped, linear aggregates (8 x 30 nm in dimension). Furthermore, the density and distribution pattern of these IMPs enabled us to distinguish three different domains of membranes of osteoclasts including ruffled border, clear zone, and basolateral regions, as were also observed in thin sections. The highest density of IMPs was 3,500-4,000/microns2 in the ruffled border membrane, and these IMPs included linear aggregates among the usual globular particles. Linear aggregated particles were also observed in the membrane of cytoplasmic vesicles in the vicinity of the ruffled border region, but not in this membrane in other bone cells. In attached osteoclasts, the distribution patterns and densities of IMPs in each ruffled-finger and -plate were extremely variable, from closely to the loosely packed membrane particles. Focal aggregates of membrane particles were also frequently encountered. An important outcome of the present study was the finding that the presence of linear aggregated particles proved to be an additional criterion for distinguishing membrane domains in freeze-replicas of osteoclasts. The surface of the clear zone membrane was not smooth in profile, but revealed a number of eminences that were almost free of particles. Basolateral membranes exhibited a particle density of 2,400/microns2. Globular particles were homogeneously scattered in random fashion on their exposed fracture faces. In some cases, aggregates of IMPs on the basolateral membranes were encountered. In comparison with the ruffled fingers, microprojections from the basolateral surface showed a lesser density of IMPs and were devoid of rod-shaped or linear aggregated particles. Differences between osteoblasts and osteocytes were apparent in the density and the size of IMPs. The membranes of osteoblasts and osteocytes contained the same types of globular particles as seen in osteoclasts. Various sizes of gap junctions were located only on basolateral membranes of the osteoblasts. In contrast, no cellular junctions were observed between osteoclasts and any other type of cells.     

Association of syntaxin 3 and vesicle-associated membrane protein (VAMP) with H+/K(+)-ATPase-containing tubulovesicles in gastric parietal cells.

H+/K(+)-ATPase is the proton pump in the gastric parietal cell that is responsible for gastric acid secretion. Stimulation of acid secretion is associated with a reorganization of the parietal cells resulting in the incorporation of H+/K(+)-ATPase from a cytoplasmic membrane pool, the tubulovesicle compartment, into the apical canalicular membrane. To better characterize the role of membrane trafficking events in the morphological and physiological changes associated with acid secretion from parietal cells, we have characterized the expression and localization of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in these cells. Each of the six different SNARE proteins examined [syntaxins 1 through 4 of 25-kDa synaptosome-associated protein, and vesicle-associated membrane protein] were found to be expressed in parietal cells. Furthermore, two of these SNAREs, vesicle-associated membrane protein and syntaxin 3, were associated with H+/K(+)-ATPase-containing tubulovesicles while the remainder were excluded from this compartment. The expression of syntaxin 1 and synaptosome-associated protein of 25 kDa in parietal cells, two SNAREs previously thought to be restricted to neuroendocrine tissues, suggests that parietal cells may utilize membrane trafficking machinery that is similar to that utilized for regulated exocytosis in neurons. Furthermore, the localization of syntaxin 3, a putative target membrane SNARE, to the tubulovesicle compartment indicates that syntaxin 3 may have an alternative function. These observations support a role for intracellular membrane trafficking events in the regulated recruitment of H+/K(+)-ATPase to the plasma membrane after parietal cell stimulation.     

Miniaturization of asymmetrical flow field-flow fractionation and application to studies on lipoprotein aggregation and fusion

Asymmetrical flow field-flow fractionation (AsFlFFF), a technique that provides direct measurement of particle size and diffusion coefficient, is converted into miniaturized scale. In comparison with conventional AsFlFFF, the separation of proteins in miniaturized AsFlFFF is achieved within shorter time periods, with smaller sample amounts, and with lower mobile phase consumption. Minimization of the overloading and optimization of the separation efficiency are prerequisites to good results. Miniaturized AsFlFFF is applied to the measurement of particle sizes of high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL). The average hydrodynamic diameters at pH 7.4 in 8.5mM phosphate buffer containing 1mM EDTA and 150 mM NaCl are 8.6+/-0.5, 11.2+/-0.2, 22.1+/-0.7, and 48.9+/-7.5 nm for subgroups HDL3, HDL2, LDL, and VLDL, respectively. In addition, the effect of different factors on the aggregation and fusion of LDL particles is studied. LDL particle sizes are unaffected by the addition of up to 300 mM NaCl and by an increase of the carrier solution pH from 3.2 to 7.4, but treatment of LDL with alpha-chymotrypsin, sphingomyelinase, or copper sulfate leads to the formation of aggregated and fused LDL particles.