bluuebunny/crossref_metadata_2025 huggingface dataset:
On gap junction structure.
Authors: Zampighi G , Corless J M , Robertson J D
Published: May 2004

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.
Polarization of endocytosis and receptor topography on cultured macrophages.
Authors: Walter R J , Berlin R D , Pfeiffer J R , Oliver J M
Published: May 2004

In the 1774.2 macrophage cell line, microtubule disassembly by colchicine causes the polarization of membrane functions ane structure. Colchicine-treated cells develop a bulge or protuberance that is bordered by microvillous membrane. The protuberance is the site of concanavalin A cap formation. The fluid pinocytosis of horseradish peroxidase and of fluorescein- and rhodamine-conjugated high molecular-weight dextrans, the adsorptive pinocytosis of concanavalin A, and the concentration and phagocytosis at 37 degrees C of a range of phagocytic particles (IgG- and complement-opsonized erythrocytes, complement-opsonized zymosan, latex shpres, albumin-stabilized oil droplets) are all similarly restricted to the protuberance. A reduction in the rate of dextran pinocytosis, determined by fluorimetry, and reductions in phagocytic rates for oil emulsion and IgG-opsonized erythrocytes accompany the polarization of endocytic activity in colchicine-trated 1774.2 macrophages. Membrane receptors for phagocytic particles are not confined to the protuberance but rather may display their own unique topographical asymmetry. The inherent topography of receptors was inferred from particle distribution under conditions that limit particle-receptor redistribution (after labeling at 4 degrees C or a very brief incubation at 37 degrees C). Under these restrictive conditions, latex binding sites were detected over the whole membrane whereas receptors for IgG-opsonized erythrocytes, aggregated IgG, complement-opsonized erythrocytes, and complement-opsonized zymosan were excluded from the protuberance. Thus, functional (endocytosis) and structural (inherent receptor distribution) analyses of membrane topography define different patterns of asymmetry in protuberant cells. The asymmetry induced in 1774.2 macrophages by colchicine is highly analogous to the functional and structural polarity of epithelial cells. Exploration of this analogy may provide insight into the development of polarized epithelia and, more generally, into mechanisms by which specialized areas of membrane are established.
Molecular organization of prolactin granules. III. Intracellular transport of sulfated glycosaminoglycans and glycoproteins of the bovine prolactin granule matrix
Authors: Giannattasio G , Zanini A , Rosa P , Meldolesi J , Margolis RK , Margolis RU
Published: May 2004

The intracellular transport of sulfated glycosaminoglycans (heparan sulfate and chondroitin sulfate) and glycoproteins of the prolactin (PRL) granule matrix, as well as that of PRL, was studied using a system of double-labeled bovine anterior pituitary slices. [(35)S]sulfate was used to label sulfated macromolecules and L-[(3)H]leucine to label PRL. In membraneless granules (isolated from a PRL granule fraction after solubilization of the membrane with Lubrol PX), sulfated glycosaminoglycans and glycoproteins were considerably labeled after a 15- min pulse, while the hormone was still unlabeled. During the chase incubation, the specific radioactivity of granule PRL and the various complex carbohydrate classes first increased, reaching a peak after approximately 40 min, and then began to decline. After 4 h of chase incubation the radioactivity remaining in granule PRL and sulfated complex carbohydrates was 50-60 percent of that observed at 40 min. Thus, in pituitary mammotrophs a pool of sulfated glycoproteins and glycosaminoglycans is transported intracellularly in parallel with PRL. This finding corroborates the previous conclusion (Zanini et al., 1980 J. Cell. Biol. 86:260-272) that sulfated macromolecules are structural components of the granule matrix. The discharge of labeled PRL and complex carbohydrates from the slices to the incubation medium was also investigated. [(35)S]-glycosaminoglycans and glycoproteins were released at a rapid rate during the first 30-40 min of chase incubation, when PRL granules had not yet attained maximum specific activities. By 40 min, their release tended to level off but the radioactivity accumulating in the incubation medium was still much larger (approximately a fourfold increase) than the losses observed concomitantly in PRL granules. These discharge kinetics contrast with that of [(3)H]PRL, which was not released during the 1st h of chase incubation but then began to accumulate at a high rate in the medium, in parallel with its decrease in granules. Dopamin (5 x 10(-7) M) strongly inhibited the release of labeled PRL but had no detectable effect on the release of labeled glycosaminoglycans and glycoproteins or on the discharge of (35)S-macromolecules as revealed by SDS polyacrylamide gel electrophoresis of incubation media. Thus the releases of PRL and sulfated macromolecules have different kinetics and can be dissociated from each other. These data indicate that much of the flycosaminoglycans and glycoproteins release form pituitary slices originates from sites other than PRL granules, and that at least part of the complex carbohydrates of the PRL granule matrix might not be released with the hormone but rather remains associated with the mammotroph cells after exocytosis.
The membrane proteins of the vacuolar system. II. Bidirectional flow between secondary lysosomes and plasma membrane.
Authors: Muller W A , Steinman R M , Cohn Z A
Published: May 2004

Lactoperoxidase covalently coupled to latex spheres (LPO-latex) has been used to selectively iodinate the phagolysome (PL) membrane within living macrophages, as discussed in the accompanying article. This procedure labeled approximately 24 polypeptides in the PL membrane; these were similar to those iodinatable on the external surface of the plasma membrane (PM). We now report on the translocation and fate of these proteins when the cells are returned to culture. TCA-precipitable radioactivity was lost from cells with biphasic kinetics. 20-50% of the cell-associated radiolabel was rapidly digested (t 1/2 approximately equal to 1 h) and recovered in the culture medium as monoiodotyrosine. 50-80% of the label was lost slowly from cells ( 1/2 approximately equal to 24-30 h). Quantitative analysis of gel autoradiograms showed that all radiolabeled proteins were lost at the same rate in both the rapid and slow phases of digestion. Within 15-30 min aftr labeling of the PL membrane, EM autoradiography revealed that the majority of the cell-associated grains, which at time 0 were associated with PL, were now randomly dispersed over the plasmalemma. At this time, analysis of PM captured by a second phagocytic load revealed the presence of all labeled species originally present in the PL membrane. This demonstrated the rapid, synchronous centrifugal flow of PL polypeptides to the cell surface. Evidence was also obtained for the continuous influx of representative samples of the PM into the PL compartment by way of pinocytic vesicles. This was based on the constant flow of fluid phase markers into latex-containing PL and on the internalization of all iodinatable PM polypeptides into this locus. These observations provide evidence for the continuous, bidirectional flow of membrane polypeptides between the PM and the secondary lysosome and represent an example of a membrane flow and recycling mechanism.
Biochemical analysis of actin in crane-fly gonial cells: evidence for actin in spermatocytes and spermatids--but not sperm.
Authors: Strauch A R , Luna E J , LaFountain J R
Published: May 2004

A biochemical assay employing DNase-I affinity chromatography, two-dimensional peptide analysis and SDS polyacrylamide gel electrophoresis was used to isolate, identify, and assess the amount of actin from gonial cells of the crane fly, Nephrotoma suturalis. Based on the analysis of cell homogenates under conditions in which all cellular actin is converted to the monomeric DNase-binding form, actin comprises approximately 1% of the total protein in homogenates of spermatocytes and spermatids. SDS gel analysis of mature sperm reveals no polypeptides with a molecular weight similar to that of actin. Under conditions that preserve native supramolecular states of actin, approximately 80% of the spermatocyte actin is in a sedimentable form whereas only approximately 30% of the spermatid actin is sedimentable. These differences could be meaningful with regard to strutural changes that occur during spermiogenesis. A comparative analysis of two-dimensional peptide maps of several radioiodinated actins reveals similarities among spermatocyte, spermatid, and human erythrocyte actins. The results suggest the general applicability of this approach to other cell types that contain limited amounts of actin.
DNA-mediated cotransfer of unlinked mammalian cell markers into mouse L cells.
Authors: Warrick H , Hsiung N , Shows T B , Kucherlapati R
Published: May 2004

Purified DNA from three different types of mammalian cells was precipitated with calcium phosphate and added to mouse L cells deficient in thymidine kinase (TK). Donor DNA was prepared from three cell lines: (a) mouse cells transfected with UV-inactivated herpes simplex virus (HSV) type 1, or a purified fragment of HSV carrying the TK gene (b) human HeLa cells, and (c( CHO, a cell line derived from Chinese hamster ovaries. Several hypoxanthine-aminopterin-thymidine resistant colonies were isolated from each experiment. The origin of the TK that is expressed in these cells was studied by polyacrylamide gel electrohporesis, isoelectric focusing, or heat stability. The TK in all instances was of the donor origin. To determine the extent of gene transfer we have assayed the CHO and HeLa DNA transfectants for galactokinase (GALK), a marker closely linked to TK, and 25 other isozymes representing a large number of different chromosomes. No cotransfer of GALK was observed, indicating that the size of the transferred DNA segment is limited. We observed that, in one instance, esterase-D, an unlinked marker of Chinese hamster origin, was transferred along with TK. These experiments indicate that nonselected markers can be transferred by this method, although at a low efficiency.
Localization and Biosynthesis of NADH-Cytochrome b(5) reductase, an integral membrane protein, in rat liver cells. III. Evidence for the independent insertion and turnover of the enzyme in various subcellular compartments
Authors: Borgese N , Pietrini G , Meldolesi J
Published: May 2004

The biosynthesis and turnover of rat liver NADH-cytochrome b(5) reductase was studied in in vivo pulse-labeling and long-term, double-labeling experiments. Rats under thiopental anesthesia were injected into the portal vein with [(3)H]L-leucine and sacrificed at various times after the injection. NADH-cytochrome b(5) reductase was extracted from liver cell fractions by cathepsin D-catalyzed cleavage and was then immunoadsorbed onto antireductase-bearing affinity columns in the presence of excess unlabeled rat serum. After elution of the enzyme from the columns with a pH-2.2 buffer, the amount of the reductase protein in the samples was determined by radioimmunoassay, and the radioactivity in reductase was determined on SDS polyacrylamide gel reductase bands. The specific radioactivity of the reductase extracted from the homogenate as well as from rough and smooth microsomal, mitochondrial, and Golgi fractions, estimated at the end of the pulse (10 min after the injection) and at various time points thereafter, remained approximately constant over a 6-h period. These data suggest tha tth eenzyme is independently inserted into the various membranes where it is located. Moreover, the specific radioactivity of the mitochondrial reductase was lower than that of the other fractions, suggesting that it turns over at a slower rate. The lower turnover rate of the mitochondrial enzyme was confirmed by long-term, double-labeling experiments carried out according to the technique of Arias et al. (J. Biol. Chem. 244: 3303-3315.). The relevance of these findings in relation to the understanding of membrane biogenesis and turnover is discussed.
Partial and complete detachment of neutrophils and eosinophils from schistosomula: evidence for the establishment of continuity between a fused and normal parasite membrane.
Authors: Caulfield J P , Korman G , Butterworth A E , Hogan M , David J R
Published: May 2004

Neutrophils and eosinophils adhering to the surface of schistosomula of Schistosoma mansoni have been partially or completely detached with hypertonic sucrose or by pipetting. The sucrose-treated neutrophils are attached only in areas where there are pentalaminar fusions between the neutrophil and tegumental membranes, suggesting that these fusions attach the cells to the parasites. Pipetting breaks many of the attached cells. In thin section, the tegumental membrane underlying these cells is seen to be pentalaminar. By freeze-fracture techniques, modified attachment areas are found. The edge zone often appears as a single strand of intramembrane particles (IMPs) on the P2 face and as a groove on the E2 face. The edge zone may also have large discontinuities, in which case it no longer separates membrane faces of unequal IMP density from one another. In addition, the IMPs on the IMP-rich areas become aggregated and surrounded by craters in the membrane. These experiments suggest that the fusions may be the mechanism by which the parasite acquires some host membrane components on its surface. On the other hand, eosinophil plasma membranes are seen adhering to a layer of electron-dense material on the parasite after the cells have been disrupted by pipetting. This suggests that eosinophils adhere to the parasite surface through their discharged granule material and not by membrane fusions.
Identification of membrane proteins mediating the interaction of human platelets
Authors: Phillips D , Jennings L , Edwards H
Published: May 2004

Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction. The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombin-activated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoproteins IIb (26 percent of the total in pre-extracted platelets) and III (14 percent), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation.
Localization of acetylcholine receptors and synaptic ultrastructure at nerve-muscle contacts in culture: dependence on nerve type
Authors: Cohen MW , Weldon PR
Published: May 2004

In cultures of xenopus myotomal muscle cells and spinal cord (SC) some of the nerve-muscle contacts exhibit a high density of acetylcholine receptors (AchRs [Anderson et al., 1977, J. Physiol. (Lond.). 268:731- 756,757-773]) and synaptic ultrastructure (Weldon and Cohen, 1979, J. Neurocytol. 8:239-259). We have examined whether similarly specialized contacts are established when the muscle cells are cultured with explants of xenopus dorsal root ganglia (DRG) or sympathetic ganglia (SG).

The outgrowth from the ganglionic explants contained neuronal and non- neuronal cell processes. Although both types of processes approached within 100 A of muscle cells, synaptic ultrastructure was rarely observed at these contacts. Because patches of postsynaptic ultrastructure also develop on noncontacted muscle cells, the very few examples of contacts with such specializations probably occurred by chance.

AChRs were stained with fluroscent α-bungarotoxin. More than 70 percent of the SC-contacted muscle cells exhibited a high receptor density along the path of contact. The corresponding values for DRG- and SG- contacted muscle cells were 10 and 6 percent. Similar values were obtained when the ganlionic and SC explants were cultured together in the same chamber. The few examples of high receptor density at ganglionic-muscle contacts resembled the characteristic receptor patches of noncontacted muscle cells rather than the narrow bands of high receptor density seen at SC-muscle contacts. In addition, more than 90 percent of these ganglionic- contacted muscle cells had receptor patches elsewhere, compared to less than 40 percent for the SC-contacted muscle cells.

These findings indicate that the SC neurites possess a specific property which is important for the establishment of synaptically specialized contacts with muscle and that this property is lacking in the DRG and SG neurites.
Cyclic 3',5'-AMP relay in Dictyostelium discoideum III. The relationship of cAMP synthesis and secretion during the cAMP signaling response.
Authors: Dinauer M C , MacKay S A , Devreotes P N
Published: May 2004

Refinement of a perfusion technique permitted the simultaneous measurement of cAMP-elicited [3H]cAMP secretion and intracellular [3H]cAMP levels in sensitive D. discoideum amoebae. These data were compared with measurements of the rate of [32P]cAMP synthesis by extracts of amoebae sonicated at different times during the cAMP signaling response. cAMP stimulation of intact cells led to a transient activation of adenylate cyclase, which was blocked if 10(-4) M NaN3 was added with the stimulus. During responses elicited by 10(-6) M cAMP, 10(-8) M cAMP, and an increment in cAMP from 10(-8) M to 10(-7) M, the rate of cAMP secretion was proportional to the intracellular cAMP concentration. Removal of a 10(-6) M cAMP stimulus 2 min after the initiation of the response led to a precipitous decline in intracellular cAMP. This decline was more rapid than could be accounted for by secretion alone, suggesting intracellular phosphodiesterase destruction of newly synthesized cAMP. Employing these data and a simple rate equation, estimates of the time-course of the transient activation of adenylate cyclase and the rate constants for cAMP secretion and intracellular phosphodiesterase activity were obtained. The calculated rate of cAMP synthesis rose for approximately 1 to 2 min, peaked, and declined to approach prestimulus levels after 3 to 4 min. This time-course agreed qualitatively with direct measurements of the time-course of activation, indicating that the activation of adenylate cyclase is a major in determining the time-course of the cAMP secretion response.
Cyclic 3', 5'-AMP relay in dictyostelium discoideum. IV. Recovery of the cAMP signaling response after adaptation to cAMP
Authors: Dinauer M , Steck TL , Devreotes P
Published: May 2004

In dictyoselium discoideum, an increase in extracellular cAMP activates adenylate cyclase, leading to an increase in intracellular cAMP and the rate of cAMP secretion. Cells adapt to any constant cAMP stimulus after several minutes, but still respond to an increase in the concentration of the stimulus. We have now characterized the decay of adaptation (deadaptation) after the removal of cAMP stimuli. Levels of adaptation were established by the perfusion of [(3)H]adenosine-labeled amoebae with a defined cAMP stimulus. After a variable recovery period, the magnitude of the signaling response to a second stimulus was measured; its attenuation was taken as a measure of residual adaption to the first stimulus. The level of adaptation established by the first stimulus depended on both its magnitude and duration. Deadaptation began as soon as the first stimulus was removed. The magnitude of the response to the second stimulus increased with the recovery time in a first-order fashion, with a t(1/2)=3-4 min for stimuli of 10(-8) M to 10(-5) M cAMP.

Responses to test stimuli, although reduced in magnitude, had an accelerated time-course when they closely followed a prior response that had not completely subsided. This effect is called priming; we believe it reveals a reversible, rate-limiting step that modulates the onset and termination of the signaling responses of amoebae that have not recently responded to a cAMP stimulus.

We have suggested that the cAMP signaling response is controlled by two antagonistic cellular processes, excitation and adaptation. The data reported here imply that both the rate of rise in the adaptation process and the final level reached depend on the occupancy of cAMP surface receptors and that the decay of adaptation when external cAMP is removed proceeds with first-order kinetics.
Banding and polarity of actin filaments in interphase and cleaving cells.
Authors: Sanger J M , Sanger J W
Published: May 2004

Heavy meromyosin (HMM) decoration of actin filaments was used to detect the polarity of microfilaments in interphase and cleaving rat kangaroo (PtK2) cells. Ethanol at -20 degrees C was used to make the cells permeable to HMM followed by tannic acid-glutaraldehyde fixation for electron microscopy. Uniform polarity of actin filaments was observed at cell junctions and central attachment plaques with the HMM arrowheads always pointing away from the junction or plaque. Stress fibers were banded in appearance with their component microfilaments exhibiting both parallel and antiparallel orientation with respect to one another. Identical banding of microfilament bundles was also seen in cleavage furrows with the same variation in filament polarity as found in stress fibers. Similarly banded fibers were not seen outside the cleavage furrow in mitotic cells. By the time that a mid-body was present, the actin filaments in the cleavage furrow were no longer in banded fibers. The alternating dark and light bands of both the stress fibers and cleavage furrow fibers are approximately equal in length, each measuring approximately 0.16 micrometer. Actin filaments were present in both bands, and individual decorated filaments could sometimes be traced through four band lengths. Undecorated filaments, 10 nm in diameter, could often be seen within the light bands. A model is proposed to explain the arrangement of filaments in stress fibers and cleavage furrows based on the striations observed with tannic acid and the polarity of the actin filaments.
Lens metabolic cooperation: a study of mouse lens transport and permeability visualized with freeze-substitution autoradiography and electron microscopy.
Authors: Goodenough D A , Dick J S , Lyons J E
Published: May 2004

Transport of metabolites is demonstrated between compartments of the adult mouse lens by freeze-substitution autoradiography. In vivo patterns of lysine incorporation are compared with in vitro patterns of lysine, glucose, uridine, and deoxyglucose incorporation. Intracellular and extracellular distributions of tritiated metabolites are determined by comparison of transported substrates with the nontransported molecules of similar molecular size: mannitol and sucrose. The permeability of the lens intercellular spaces is probed with Procion Yellow at the level of fluorescence microscopy, and with horseradish peroxidase at the electron microscope level. Freeze-fracture electron microscopy reveals gap junctions between epithelial cells, between lens fibers, and between epithelial cells and lens fibers. Zonulae occludentes (tight junctions) are not routinely observed between epithelial cells in the mouse. This latter result is subject to species variation, however, since zonulae occludentes are abundant between chicken epithelial cells. The permeability results suggest that the lens cells are capable of metabolic cooperation, mediated by an extensive gap junction network.
Contractile basis of ameboid movement. VII. The distribution of fluorescently labeled actin in living amebas.
Authors: Taylor D L , Wang Y L , Heiple J M
Published: May 2004

The technique of molecular cytochemistry has been used to follow the distribution of fluorescently labeled actin in living Chaos carolinensis and Amoeba proteus during ameboid movement and various cellular processes. The distribution of 5-iodoacetamidofluorescein-labeled actin was compared with that of Lissamine rhodamine B sulfonyl chloride-labeled ovalbumin microinjected into the same cell and recorded with an image intensification microscope system. Actively motile cells demonstrated a rather uniform distribution of actin throughout most of the cytoplasm, except in the tail ectoplasm and in plasma gel sheets, where distinct actin structures were observed. In addition, actin-containing structures were induced in the cortex during wound healing, concanavalin A capping, pinocytosis, and contractions elicited by phalloidin injections. The formation of distinct fluorescent actin structures has been correlated with contractile activities.
Immunocytochemical localization of coated vesicle protein in rodent nervous system.
Authors: Cheng T P , Byrd F I , Whitaker J N , Wood J G
Published: May 2004

Immunocytochemistry has been used to study the distribution of the major 180,000-mol wt protein of coated vesicles in rodent cerebellum. An antibody to the coat protein was prepared in rabbits and characterized by immunodiffusion and immunofixation of polyacrylamide gels. At the light microscope level the protein was primarily localized in punctate profiles surrounding Purkinje cells and within the cerebellar glomeruli. At the electron microscope level the punctate distribution was confined to presynaptic terminals of basket and Golgi II neurons as well as mossy fiber terminals of the glomeruli. This label was heaviest on the lattice coat of coated vesicles but, in addition, label was found within the presynaptic axoplasm and along the cytoplasmic surface of the plasmalemma. Coated vesicles in cell somata were labeled as well as the cytosol around groupings of these vesicles. These data suggest that there may be two forms (or more) of coated vesicle protein in neurons, a lattice form associated with coated vesicles and a soluble form associated with the cytoplasmic matrix.
Selective iodination and polypeptide composition of pinocytic vesicles.
Authors: Mellman I S , Steinman R M , Unkeless J C , Cohn Z A
Published: May 2004

We describe a method for the specific radioiodination of pinocytic vesicles (PVs) based upon the simultaneous endocytosis of lactoperoxidase (LPO) and glucose oxidase (GO). Initial experiments indicated that LPO was interiorized by the macrophage cell line J774 by fluid phase pinocytosis and without detectable binding to the plasma membrane (PM). Interiorization varied linearly with enzyme concentration and exposure time, was temperature dependent, and was undetectable at 4 degrees C. Employing EM cytochemistry, LPO activity was restricted to PVs after a 3- to 5-min pulse at 37 degrees C. These results formed the basis of the method for iodinating the luminal surface of PVs: 5-min exposure to both LPO and GO at 37 degrees C followed by washes and iodination (addition of 125I and glucose) at 4 degrees C. Enzyme-dependent incorporation of iodide into the polypeptides of both PV membrane and contents occurred. Several lines of evidence indicated that there was selective labeling of PV as opposed to PM. Iodination did not occur if the pinocytic uptake of LPO ad GO was inhibited by low temperature. EM autoradiography showed a cytoplasmic localization of grains, whereas a clear PM association was evident with surface labeling. LPO was iodinated only after PV labeling and was present within organelles demonstrating latency. After PV iodination, > 75% of several labeled membrane antigens could be immunoprecipitated by monoclonal antibodies only after cell lysis. In contrast, all labeled antigens were accessible to antibody on intact cells after surface labeling. The polypeptide compositions of PM and PV membrane were compared by SDS polyacrylamide gel electrophoresis and by quantitative immune precipitation using a panel of anti-J774 monoclonal antibodies. The electrophoretic profiles of iodinated proteins (15-20 bands) were strikingly similar in NP-40 lysates of both PV and PM iodinated cells. In addition, eight membrane antigens examined by immune precipitation, including the trypsin-resistant immunoglobulin (Fc) receptor and the H-2Dd histocompatibility antigen, were found to be iodinated to the same relative extents by both labeling procedures. We conclude that PV membrane is formed from a representative sample of PM polypeptide components.
Progesterone receptors in normal mammary gland: receptor modulations in relation to differentiation.
Authors: Haslam S Z , Shyamala G
Published: May 2004

The biological basis for the observed modulation in cytoplasmic progesterone receptors (PgR) of normal mammary gland occurring during mammary development was investigated. Specifically, the relative roles of hormones vs. differentiation on (a) the decrease in PgR concentration during pregnancy and lactation and (b) the loss of mammary responsiveness to estrogen during lactation were examined. PgR were measured using the synthetic progestin, R5020, as the ligand. The hormones estrogen and progesterone were tested in vivo for their effect of PgR concentration. Mammary gland differentiation was assessed morphologically and by measuring enzymatically active alpha-lactalbumin. These studies show that there is a stepwise decrease in PgR that occurs in two stages. The first decrease is completed by day 12 of pregnancy and the second decrease occurs only after parturition. There appears to be a hormonal basis for the first decrease and it appears to be caused by the negative effect of progesterone on estrogen-mediated increase in PgR. In direct contrast, the absence of PgR during lactation and the mammary tissue insensitivity to estrogenic stimulation of PgR were not related to the hormonal milieu of lactation but were directly related to the secretory state of the mammary gland and lactation per se.
Dynein-like Mg2+-ATPase in mitotic spindles isolated from sea urchin embryos (Strongylocentrotus droebachiensis).
Authors: Pratt M M , Otter T , Salmon E D
Published: May 2004

Two distinctly different ATPases have been reported to be endogenous to the mitotic apparatus: a Mg2+-ATPase resembling axonemal dynein, and a Ca2+-ATPase postulated to be bound in membranes. To examine the nature of the Mg2+-ATPase, we isolated membrane-free mitotic spindles from Stronglylocentrotus droebachiensis embryos by rapidly lysing these in a calcium-chelating, low-ionic-strength buffer (5 mM EGTA, 0.5 mM MgCl2, 10 mM PIPES, pH 6.8) that contained 1% Nonidet P-40. The fibrous isolated mitotic spindles closely resembled spindles in living cells, both in general morphology and in birefringence. In electron micrographs, the spindles were composed primarily of microtubules, free from membranes and highly extracted of intermicrotubular cytoplasmic ground substance. As analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the pelleted spindles contain 18% tubulin, variable amounts of actin (2-8%), and an unidentified protein of 55 kdaltons in a constant weight ratio to tubulin (1:2.5). The isolated spindles also contained two polypeptides, larger than 300 kdaltons, that comigrated with egg dynein polypeptides, and ATPase activity (0.02 mumol Pi/mg . min) that closely resembled both flagellar and egg dynein. The spindle Mg2+-ATPase showed a ratio of Ca2+-/Mg2+-ATPase = 0.85, had minimal activity in KCl and EDTA, and cleaved GTP at 35% of the rate of ATP. The Mg2+-ATPase was insensitive to ouabain or oligomycin. The spindle Mg2+-ATPase was inhibited by sodium vanadate but, like egg dynein, was less sensitive to vanadate than flagellar dynein. The spindle Mg2+-ATPase does not resemble the mitotic Ca2+-ATPase described by others. We propose that the spindle Mg2+-ATPase is egg dynein. Bound carbohydrate on the two high-molecular-weight polypeptides of both egg dynein and the spindle enzyme suggest that these proteins may normally associate with membranes in the living cell.
Definitive Evidence for the existence of tight junctions in invertebrates
Authors: Lane NJ , Chandler HJ
Published: May 2004

Extensive and unequivocal tight junctions are here reported between the lateral borders of the cellular layer that circumscribes the arachnid (spider) central nervous system. This account details the features of these structures, which form a beltlike reticulum that is more complex than the simple linear tight junctions hitherto found in invertebrate tissues and which bear many of the characteristics of vertebrate zonulae occludentes. We also provide evidence that these junctions form the basis of a permeability barrier to exogenous compounds. In thin sections, the tight junctions are identifiable as punctate points of membrane apposition; they are seen to exclude the stain and appear as election- lucent moniliform strands along the lines of membrane fusion in en face views of uranyl-calcium-treated tissues. In freeze-fracture replicas, the regions of close membrane apposition exhibit P-face (PF) ridges and complementary E-face (EF) furrows that are coincident across face transitions, although slightly offset with respect to one another. The free inward diffusion of both ionic and colloidal lanthanum is inhibited by these punctate tight junctions so that they appear to form the basis of a circumferential blood-brain barrier. These results support the contention that tight junctions exist in the tissues of the invertebrata in spite of earlier suggestions that (a) they are unique to vertebrates and (b) septate junctions are the equivalent invertebrate occluding structure. The component tight junctional 8- to 10-nm-particulate PF ridges are intimately intercalated with, but clearly distinct from, inverted gap junctions possessing the 13-nm EF particles typical of arthropods. Hence, no confusion can occur as to which particles belong to each of the two junctional types, as commonly happens with vertebrate tissues, especially in the analysis of developing junctions. Indeed, their coexistance in this way supports the idea, over which there has been some controversy, that the intramembrane particles making up these two junctional types must be quite distinct entities rather than products of a common precursor.
Induction of either contractile or structural actin-based gels in sea urchin egg cytoplasmic extract.
Authors: Kane R E
Published: May 2004

The gel formed by warming the 100,000 g supernate of isotonic extracts of sea urchin eggs to 40 degrees C is made up of actin and two additional proteins of mol wt of 58,000 and 220,000. Actin and 58,000 form a characteristic structural unit which has now been identified in the microvilli of the urchin egg and in the filopods of urchin coelomocytes. However, egg extract gels did not contract as those from other cell types do, and the aim of these experiments was to determine the reason for this lack of contraction. Although the extracts are dialyzed to a low ionic strength, myosin is present in soluble form and makes up approximately 1% of the protein of the extract. It becomes insoluble in the presence of high ATP concentrations at 0 degrees C, and the precipitate formed under these conditions consists almost entirely of myosin. This procedure provides a simple method of isolating relatively pure myosin without affecting other extract components and functions. Contraction will follow gelation in these extracts if the temperature and time of incubation used to induce actin polymerization are reduced to minimize myosin inactivation. At the optimal ATP and KCl concentration for contraction, the contracted material has an additional 250,000 component and contains very little 58,000. The conditions found to provide maximum gel yields favor the formation of the actin-58,000-220,000 structural gel, while reduced temperature and increase in KCl concentration results in a contractile gel whose composition is similar to those reported from amoeboid cell types. Both the structural protein cores found in the egg microvilli and a gel contraction related to the amoeboid motion which is seen in later urchin embryonic development can thus be induced in vitro in the same extract.
Apparent coordination of the biosynthesis of lipids in cultured cells: its relationship to the regulation of the membrane sterol:phospholipid ratio and cell cycling.
Authors: Cornell R B , Horwitz A F
Published: May 2004

The coordination of the syntheses of the several cellular lipid classes with one another and with cell cycle control were investigated in proliferating L6 myoblasts and fibroblasts (WI-38 and CEF). Cells cultured in lipid-depleted medium containing one of two inhibitors of hydroxymethylglutaryl-CoA reductase, 25-hydroxycholesterol or compactin, display a rapid, dose-dependent inhibition of cholesterol synthesis. Inhibition of the syntheses of each of the other lipid classes is first apparent after the rate of sterol synthesis is depressed severalfold. 24 h after the addition of the inhibitor, the syntheses of DNA, RNA, and protein also decline. The inhibition of sterol synthesis leads to a threefold reduction in the sterol:phospholipid ratio that parallels the development of proliferative and G1 cell cycle arrests and alterations in cellular morphology. All of these responses are reversed upon reinitiation of cholesterol synthesis or addition of exogenous cholesterol. A comparison of the timing of these responses with respect to the development of the G1 arrest indicates that the primary factor limiting cell cycling is the availability of cholesterol provided either from an exogenous source or by de novo synthesis. The G1 arrest appears to be responsible for the general inhibition of macromolecular synthesis in proliferating cells treated with 25-hydroxycholesterol. In contrast, the apparent coordinated inhibition of lipid synthesis is not a consequence of the G1 arrest but may in fact give rise to it. Sequential inhibition of lipid syntheses is also observed in cycling cells when the synthesis of choline-containing lipids is blocked by choline deprivation and is observed in association with G1 arrests caused by confluence or differentiation. In the nonproliferating cells, the syntheses of lipid and protein do not appear coupled.
Cholesterol availability modulates myoblast fusion.
Authors: Cornell R B , Nissley S M , Horwitz A F
Published: May 2004

The requirement of cholesterol for myoblast fusion has been linked to the primary step in the fusion process, calcium-dependent aggregation (recognition). Inhibition of cholesterol synthesis with 25-hydroxycholesterol or compactin in the absence of exogenous lipid dramatically inhibits calcium-mediated aggregation and concomitant fusion within several hours. Restimulating cholesterol synthesis or supplying exogenous cholesterol rapidly restores aggregation activity. Over this time period, however, the sterol:phospholipid ratio is unaltered, suggesting a local rather than a general membrane cholesterol requirement for the expression of aggregation activity. The aggregation response to a change in sterol availability occurs on a shorter time scale than that required to inhibit the synthesis of the protein(s) with aggregation activity; thus, the cholesterol-requiring step is posttranslational. We suggest that the assembly or maintenance of the aggregation activity depends on a continued local supply of cholesterol.
Localization of a 16,000-dalton fragment of the common precursor of adrenocorticotropin and beta-lipotropin in the rat and human pituitary gland.
Authors: Guy J , Leclerc R , Pelletier G
Published: May 2004

To clearly identify cells and organelles containing the common precursor (31,000 dalton) for both adrenocorticotropin (ACTH) and beta-lipotropin (beta-LPH), an immunohistochemical localization of a fragment (16,000 dalton) of the precursor that is not common to beta-LPH and ACTH was conducted in rat and human pituitary glands. With the help of specific antibodies that do not cross-react with beta-LPH and ACTH, the 16,000-dalton fragment was localized in the cells that also produce ACTH and beta-LPH in both the pars distalis and pars intermedia of the rat pituitary. At the electron microscope level, the secretory granules that contain ACTH were also stained for 16,000-dalton fragment. In the human pituitary, the 16,000-dalton fragment was also observed in all the secretory granules of lipocorticotrophs. These results suggest that, after enzymatic cleavage, fragment(s) of the common precursor and/or the whole common precursor remain packaged within the secretory granules with peptides of known activity.
Redistribution of membrane proteins in isolated mouse intestinal epithelial cells.
Authors: Ziomek C A , Schulman S , Edidin M
Published: May 2004

Single mouse intestinal epithelial cells (IEC) may be isolated by the use of a combination of methods used for the isolation of IEC from other species. Isolated cells remain viable for several hours. The membrane integral enzymes alkaline phosphatase and leucine aminopeptidase of isolated IEC are localized to the brush borders of IEC in tissue and in most newly isolated IEC. With time, both enzymes are found distributed over the entire cell surface. Redistribution appears to occur by diffusion in the plane of the membrane. It is slowed, but not blocked, if cells are maintained at 0 degrees C instead of at 37 degrees C, and it is not blocked by fixation in 0.5-3% paraformaldehyde. Drugs that alter cell membrane potential or that affect cell levels of ATP enhance the rate of redistribution of the enzymes.
Effect of microinjected N-ethylmaleimide-modified heavy meromyosin on cell division in amphibian eggs.
Authors: Meeusen R L , Bennett J , Cande W Z
Published: May 2004

N-Ethylmaleimide-modified heavy meromyosin (NEM-HMM) microinjected into amphibian eggs inhibits cytokinesis and the cortical contractions associated with wound closure. Injection of NEM-HMM into two-cell Rana pipiens embryos produces a zone of cleavage inhibition around the point of injection. Early furrows followed by time-lapse microcinematography are seen to slow and stop as they enter the NEM-HMM-injected zone. Arrested furrows slowly regress, leaving a large region of cytoplasm uncleaved. Few nuclei are found in these regions of cleavage inhibition. Wound closure is often inhibited by NEM-HMM, especially when this inhibitor is injected just beneath the egg cortex. We observe that the surface of an unfertilized Rana egg is covered with microvilli that disappear during the course of development. The surfaces of NEM-HMM-inhibited zones remain covered with microvilli and resemble the unfertilized egg surface.
Effects of antibodies against tubulin on the movement of reactivated sea urchin sperm flagella.
Authors: Asai D J , Brokaw C J
Published: May 2004

Antibodies binding to sea urchin flagellar outer-doublet tubulin have been isolated from rabbit sera by tubulin-affinity chromatography employing electrophoretically purified tubulin as the immobilized substrate. This procedure provides "induced" antitubulin antibody from immune sera and "spontaneous" antitubulin antibody from preimmune sera. These antitubulins were characterized in terms of their specificity, ability to bind to sea urchin axonemes, and effects on the motility of reactivated spermatozoa. Induced antitubulin antibody specifically reduced the bend angle and symmetry of the movement of demembranated reactivated spermatozoa without affecting the beat frequency. At identical concentrations, spontaneous antitubulin had no effect on motility. Affinity-purified induced antitubulins from three other rabbits all gave specific bend-angle inhibition, whereas their corresponding spontaneous antitubulins had no effect on the flagellar movement. The effects of antitubulin on microtubule sliding were examined by observing the sliding disintegration of elastase-digested axonemes induced by MgATP2+-. Affinity-purified induced antitubulin antibody, in quantities sufficient to completely paralyze reactivated flagella, did not inhibit microtubule sliding. The amplitude-inhibiting effect of induced antitubulin on reactivated spermatozoa may be caused by action on a mechanism responsible for controlling flagellar bending rather than by interference with the active sliding process. This is the first report of an antitubulin antibody having an inhibitory activity on microtubule-associated movement.