bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of the fluorochrome 4'6-diamidino-2-phenylindole in genetic and developmental studies of chloroplast DNA.
Authors: Coleman A W
Published: May 2004

Use of the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) makes it possible to examine in situ the structure of chloroplast DNA (chDNA) with the fluorescence microscope. This simplifies the study of genetic and developmental changes in chloroplast DNA. Three examples are presented. (a) Wild-type Euglena gracilis B contains several chloroplast DNA nucleoids per chloroplast. A yellow mutant lacking functional chloroplasts is similar, but such nucleoids are absent in an aplastidic mutant strain known from biochemical studies to have lost its chDNA. (b) In vegetative cells of the giant-celled marine algae Acetabularia and Batophora, only about a quarter of the chloroplasts have even one discernible chloroplast DNA particle, and such particles vary in size, showing a 30-fold variation in the amount of DNA-bound DAPI fluorescence detected per chloroplast. By contrast, 98% of chloroplasts in developing Acetabularia cysts contain chDNA, with as many as nine nucleoids per chloroplast. (c) DAPI-stained chloroplasts of chromophyte algae display the peripheral ring of DNA expected from electron microscope studies. However, these rings are not uniform in thickness, but are necklace-like, with the appearance of beads on a string. Since the multiple nucleoids in plastids of chlorophyte algae also appear to be interconnected throughout the chloroplast, a common structural plan may underlie chDNA morphology in both groups of algae.
Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. I. Identification of surface proteins.
Authors: Willinger M , Frankel F R
Published: May 2004

To study the fate of external membrane proteins during phagocytosis, rabbit peritoneal neutrophils were labeled by enzymatic iodination. Iodine was incorporated into at least 13 proteins ranging in size from approximately 250,000 to 18,000 daltons as judged from autoradiography of gels after SDS-polyacrylamide gel electrophoresis of labeled cells. The major contractile proteins of neutrophils, actin and myosin, were not labeled when intact cells were iodinated but were labeled when homogenates of these cells were iodinated. Nine of the iodinated proteins were released by mild protease treatment of intact cells. A plasma membrane-rich fraction was isolated by density centrifugation. This fraction was enriched at least 10-fold for lactoperoxidase-labeled acid-insoluble proteins. It was enriched to the same extent for the presence of iodinated wheat germ agglutinin that had been bound to intact cells at 4 degrees C before homogenization. Analysis of SDS-polyacrylamide gel electrophoresis revealed that the proteins of this fraction were predominantly of high molecular weight. However, only 8 of the 13 proteins iodinated on intact cells were found in this fraction. The remaining five were enriched in a dense fraction containing nuclei, intact cells, and membranous vesicles, and may represent a specialized segment of the neutrophil cell surface.
Expression of microtubule networks in normal cells, transformed cells, and their hybrids.
Authors: Wolin S L , Kucherlapati R S
Published: May 2004

Microtubules play an important role in several cellular functions including cellular architecture and chromosome movement in cell division. Tubulin which polymerizes to form mictobules can be purified to homogeneity and used to raised antisera. Antisera prepared against porcine or chicken tubulin reacts well with mammalian tubulin. We have examined normal and transformed cells of mouse and human origin for microtubules by indirect immunofluorescence methods. Extensive networks of microtubules (MN) are easily detectable in normal and some transformed cells. The fixation procedure employed and the morphology and the cellular attachment properties seem to determine the ease of detection of MN in these cells. Cells derived from tumors and exhibiting several transformed phenotypes contained MN comparable to those of normal cells. Hybrids between transformed mouse cells and normal human cells were examined. They showed a variability in morphology, but all contained MN. These hybrids exhibited several transformed phenotypes. We conclude that in the cell lines we have examined there is no correlation between the transformed phenotypes and the organization of tubulin.
Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation
Authors: Newburger PE , Chovaniec ME , Greenberger JS , Cohen HJ
Published: May 2004

Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of β-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.
Helical substructure of neurofilaments isolated from Myxicola and squid giant axons.
Authors: Krishnan N , Kaiserman-Abramof I R , Lasek R J
Published: May 2004

Neurofilaments purified from invertebrate giant axons have been analyzed with the electron microscope. The neurofilaments have a helical substructure which is most easily observed when the neurofilaments are partially denatured with 0.5 M KCl or 2 M urea. When the ropelike structure comprising the neurofilaments untwists, two strands 4--5.5nm in diameter can be resolved. Upon further denaturation these strands break up into rod-shaped segments and subsequently these segments roll up into amorphous globular structures. Stained, filled densities can be resolved within the strand segments, and these resemble similar structures observed within the intact neurofilaments. The strands appear to consist of protofilaments 2--2.5 nm in diameter. These observations suggest that the neurofilament is a ropelike, helical structure composed of two strands twisted tightly around each other, and they su-port the filamentous rather than the golbular model of intermediate filament structure.
Identification of the subunit proteins of 10-nm neurofilaments isolated from axoplasm of squid and Myxicola giant axons.
Authors: Lasek R J , Krishnan N , Kaiserman-Abramof I R
Published: May 2004

Neurofilaments were isolated from the axoplasm of the giant axons of Myxicola infundibulum and squid. The axoplasm was fractionated by discontinuous sucrose gradient centrifugation and gel filtration on Sepharose 4B. The fractions were monitored for neurofilaments by electron microscopy. When isolated in the presence of chelating agents, the neurofilaments of Myxicola are composed almost entirely of protein subunits with mol wt of 150,000 and 160,000. Squid neurofilaments contain two major proteins with mol wt of 200,000 and 60,000. These proteins are compared with other intermediate filament proteins which have been reported in the literature.
Localization of submembranous cations to the leading end of human neutrophils during chemotaxis.
Authors: Cramer E B , Gallin J I
Published: May 2004

Potassium pyroantimonate was used to localize sites of bound cations in human neutrophils under conditions of random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis). The cells were placed in a standard chamber in which 0.45-micron micropore filters separated the cells from the stimulus (buffer, Escherichia coli endotoxin-activated serum or the synthetic chemotactic peptide N-formyl-Met-Leu-Phe). The small pore filters permitted pseudopod formation but impeded cell imgration through the filter. Cells examined under all conditions had electron-dense precipitates of antimonate salts in some granules. However, antimonate deposits were localized in the condensed chromatin of the nucleus during random migration and associated to a large extent with the uncondensed nuclear chromatin during chemokinesis and chemotaxis. Under conditions of chemokinesis deposition of antimonate procipitates appeared on the cytoplasmic side of the plasma membrane of neutrophils whereas under conditions of chemotaxis cation deposits beneath the cell membrane were localized to the pseudopods which were directed toward the chemoattractant. In addition to endotoxin-activated serum, concentrations of N-formyl-Met-Leu-Phe which caused neutrophil chemotaxis (10(-8) M) also caused cation deposition beneath the cell membrane at the leading end of the cell regardless of whether albumin was present in the incubation media. However, with higher concentrations of the synthetic peptide (10(-5) M) which caused granule release and were not chemotactic, submembranous cation deposition was not seen. EDTA (10 mM) and EGTA (10 mM) removed nuclear, granular, and submembranous cation deposits from neutrophils examined under conditions of chemotaxis. X-ray microprobe analysis of antimonate deposits revealed the possible presence of calcium but did not detect sodium or magnesium. The data indicate that chemotactic factors induce submembranous deposition of cations, most likely Ca++, which localize to the leading edge of cells exposed to a gradient of chemoattractant.
Localization of Na pumps in the tracheal epithelium of the dog.
Authors: Widdicombe J H , Basbaum C B , Yee J Y
Published: May 2004

Binding of [3H]ouabain by the dog's tracheal epithelium shows a nonspecific component depending linearly on ouabain concentration, and a specific saturable component with a Km of 10(-7) M. Control experiments showed that the tracer taken up was not trapped within the extracellular space nor bound to tissue collagen. Inhibition of the saturable uptake by high K, metabolic inhibition, low Na, and low temperature indicated that binding was to Na/K ATPase. One-sided exposures of tissue sheets to tracer showed that the submucosal side took up 10 X as much tracer as the luminal. Autoradiography localized tracer uptake under all conditions to the cells' basolateral membranes.
Electrically mediated fast polyspermy block in eggs of the marine worm, Urechis caupo.
Authors: Gould-Somero M , Jaffe L A , Holland L Z
Published: May 2004

Previous work has established that the polyspermy block in Urechis acts at the level of sperm-egg membrane fusion. (J. Exp. Zool. 196:105). Present results indicate that during the first 5--10 min after insemination the block is mediated by a positive shift in membrane potential (the fertilization potential) elicited by the penetrating sperm, since holding the membrane potential of the unfertilized egg positive by passing current reduces the probability of sperm entry, while progressively reducing the amplitude of the fertilization potential by decreasing external Na+ progressively enhances multiple sperm penetrations. Also, a normal fertilization potential is correlated with a polyspermy block even under conditions (pH 7) in which eggs do not develop. We have investigated the mechanism of the electrical polyspermy block by quantifying the relationship between sperm incorporation, membrane potential and ion fluxes. Results indicate that the polyspermy block is mediated by the electrial change per se and not by the associated fluxes of Na+, Ca++, and H+.
Increase of gap junctions between pancreatic B-cells during stimulation of insulin secretion.
Authors: Meda P , Perrelet A , Orci L
Published: May 2004

The development of gap junctions between pancreatic B-cells was quantitatively assessed in freeze-fracture replicas of isolated rat islets under different conditions of insulin secretion. The results show that in resting B-cells, gap junctions are small and scarce but that these junctions increase when insulin secretion is stimulated. Both a short (90 min) stimulation by glucose in vitro and a prolonged (2.5 d) stimulation by glibenclamide in vivo raise the number of gap junctions; in addition, the glibenclamide stimulation causes an increase in the size of individual gap junctions. As a consequence, the total area occupied by gap junctions on the B-cell membrane and the ratio of this area to the cell volume were found significantly increased in the latter condition. The slight increase of these values observed after the glucose stimulation did not reach significance. These data indicate a change of gap junctions during the secretory activity of the pancreatic B-cells. The possibility that the coupling of the cells is affected by the treatment is discussed.
Fate of plasma membrane during endocytosis. I. Uptake and processing of anti-plasma membrane and control immunoglobulins by cultured fibroblasts.
Authors: Schneider Y J , Tulkens P , de Duve C , Trouet A
Published: May 2004

The uptake and processing by cultured rat embryo fibroblasts of control rabbit immunoglobulins (C IgG) or IgG directed against plasma membrane constituents (anti-PM IgG), and labeled with fluorescein (F) or with radioactive acetate (A), have been investigated by cell fractionation and immunological techniques. Both F and A anti-PM IgGs become bound to the cell surface, by a process that is slow, but largely temperature-independent. In the presence of an excess of high-affinity antibodies, binding reaches an absolute limit which corresponds to extensive coating of the plasma membrane. The anti-PM IgGs remain attached to the membrane for at least several days, even at 37 degrees C, with no significant transfer to lysosomes or degradation. In contrast, C IgGs are handled very differently by the fibroblasts, and their fate is strikingly affected by the type of labeling used. AC IgG is taken up slowly, at a rate proportional to its concentration, and is subsequently broken down in what appears to be lysosomes. Part of the AC IgG also binds to the plasma membrane. FC IgG is taken up many times faster than AC IgG, though with the same strict linearity as a function of concentration. Most of the FC IgG taken up is stored in cytoplasmic granules which behave like lysosomes. For reasons that are not understood, only about half of the stored FC IgG can be broken down. Cells exposed simulatnaously to AC IgG and FC IgG, or to A anti-PM IgG and FC IgG, handle each type of IgG in its characteristic fashion. Kinetic analysis of these results indicates that Ac IgG could be taken up by fluid endocytosis, but that FC IgG must be interiorized by a selective mechanism, presumably adsorptive in nature. That anti-PM antibodies remain stably bound to the plasma membrane and do not interfere with the uptake of FC IgG is interpreted to indicate either that two distinct membrane domains are involved in the two phenomena, or that membrane patches coated with anti-PM IgG participate in endocytosis, and are recycled back to the cell surface after delivering their contents intracellularly.
Fate of plasma membrane during endocytosis. II. Evidence for recycling (shuttle) of plasma membrane constituents
Authors: Schneider Y , Tulkens P , de Duve C , Trouet A
Published: May 2004

Cultured rat embryo fibroblasts were first allowed to store for 24 h fluorescein-labeled goat immunoglobulins directed against rabbit immunoglobulins (F anti-R IgG), and were subsequently exposed for 24 h to [(3)H]acetylated rabbit immunoglobulins known to bind to the cell membrane either specifically (anti-plasma membrane IgG: A anti-PM IgG) or unspecifically (contol IgG: AC IgG). As a result of immunological interaction between the two antibodies (no effect was found if the cells had been preloaded with control goat FC IgG), a substantial portion of the stored F anti-R IgG was unloaded from its intracellular storage site, appearing in the medium in the form of soluble immune complexes with rabbit A IgG. Part of the unloaded F anti-R IgG also was recovered in association with the plasma membrane, but only when A anti-PM IgG was used. In addition, significant reverse translocation of AC IgG from plasma membrane to lysosomes or some related intracellular storage compartment was also observed. With A anti-PM IgG, this translocation was less marked and affecte at the same time the plasma membrane marker 5'- nucleotidase. Cells that had stored horseradish peroxidase (HRP) simultaneously with F anti-R IgG did not unload HRP when exposed to A anti-PM IgG.

These results support strongly, though not unequivocally, the concept that plasma membrane patches interiorized by endocytosis are recycled, or shuttled, back to the cell surface. In the framework of this concept, recycling antibody-coated membrane is taken to serve as vehicle for the selective intracellular capture and extracellular discharge of immunologically bound F anti-R IgG. The alternative explanation of regurgitation triggered off by immune complexes is considered less likely in view of the lack of HRP unloading.
Replication of mammalian DNA in vitro. Evidence for initiation from fiber autoradiography.
Authors: Hand R , Gautschi J R
Published: May 2004

We have used fiber autoradiography to examine the DNA product made in vitro in a lysed cell system. CHO cells were treated with 0.01% Brij-58 and the lysates were incubated at 30 degrees C in a complete reaction mixture for in vitro DNA synthesis with [3H]thymidine triphosphate ([3H]TTP) as the radioactive tracer. Fiber autoradiograms prepared from the DNA showed that it was synthesized on tandemly arranged replication units that were of average size of 20 micrometers, very similar to the size of units found in vivo. The rate of replication fork movement was 25--50% of the in vivo rate. More than 80% of forks stopped functioning by 15 min, and 95% stopped by 60 min. This suggests that synthesis is halted by premature terminations. Evidence for new initiations was provided by replication units with labeled origins in DNA synthesized in an in vitro reaction in which radioactivity was omitted for the first 10 min of incubation. This, plus the observations that the distance between initiation points (replication unit size) is not increased and that premature termination accounts largely for the cessation of synthesis, suggest that significant initiation takes place in this in vitro replication system.
Detection of gap junctions between the progeny of a canine macrophage colony-forming cell in vitro.
Authors: Porvaznik M , MacVittie T J
Published: May 2004

An in vitro monocyte-macrophage colony-forming cell (M-CFC) has been detected in canine bone marrow (BM). The colonies derived from these progenitor cells were similar to murine-derived M-CFC (MacVittie and Porvaznik, 1978, J. Cell Physiol. 97:305--314) colonies, since they showed a singular macrophage line of differentiation, a lag of 14--16 days before initiating colony formation, and they survived significantly longer in culture in the absence of colony-stimulating factor (CSF) than granulocyte-macrophage colony-forming cells (GM-CFC). Endotoxin (Salmonella typhosa lipopolysaccharide W)-stimulated dog serum was used as the CSF (7% vol/vol). Canine-derived M-CFC progeny were identified as macrophages on the basis of morphology, phagocytosis, and the presence of Fc receptors for IgG. Gap junctions were observed only in canine BM, M-CFC-derived colonies using freeze-fracture and lanthanum tracer techniques. They were not observed in any GM-CFC-derived colonies. The number of gap junctions observed in freeze-fracture replicas of BM, M-CFC-derived colonies (21 colonies from three different dogs) showed a significantly positive correlation (Kendall's tau = 0.70, P less than 0.001) with the size of the colony fracture plane area. Gap junctions were observed displaying hexagonal lattices of 9.3 nm +/- 0.08 (SE) particles with a center-to-center spacing of 10.4 nm +/- 1.0 (SE) on membrane P-fracture faces. On membrane E-fracture faces, highly ordered arrays of pits with 8.7 nm +/- 0.12 (SE) center-to-center spacing were observed. Arrays of both particles and pits were also observed in fracture-face breakthroughs within a gap junction. Thus, gap junctions can form in vitro between the cells of macrophage progeny of a canine M-CFC under appropriate growth conditions. The significance of this observation is that there may be a structural basis for cell-to-cell collaboration between BM macrophages and other capable cells that either pass into the tissue for modification or develop there into mature cell forms.
Localization of an endogenous lectin in chicken liver, intestine, and pancreas.
Authors: Beyer E C , Tokuyasu K T , Barondes S H
Published: May 2004

Extracts of adult chicken liver, pancreas, and intestine contain high levels of a lectin which appears to be identical to one previously purified from embryonic chick muscle. This lectin is virtually absent from adult muscle, but is highly concentrated in cells lining liver sinusoids, intestinal goblet cells, and the extracellular spaces surrounding pancreatic acini. These findings suggest that the lectin may play different roles in different tissues and at different times in the life of a chicken.
Quantitative biochemical analysis of microtubule content in normal and transformed 3T3 cells.
Authors: Eichhorn J H , Peterkofsky B
Published: May 2004

Microtubules in normal and transformed BALB 3T3 cells were preserved in a stabilizing medium and measured by a [3H]colchicine-binding tubulin assay, and compared to total cellular tubulin measured under nonstabilizing conditions. Essentially no change in tubulin or microtubule content was seen with changes in cell density or with changes in cellular morphology at various stages of growth of normal or transformed cells or induced by dibutyryl cAMP treatment of transformed cells. Of five cell lines transformed by a variety of agents, four had a significantly higher total tubulin content than untransformed 3T3 cells and all of them had an increased microtubule content. None of the transformed lines had a lower fraction of tubulin recoverable as sedimentable microtubules compared to untransformed cells, and in three of them this fraction was significantly higher. These results establish that microtubules are present in transformed cells to at least the extent (if not greater) than in normal cells but that there are variations in the total amount of tubulin and microtubules as well as the fraction of the total tubulin present as microtubules which are not strictly correlated with transformation or cell morphology.
Microtubule initiation at kinetochores and centrosomes in lysed mitotic cells. Inhibition of site-specific nucleation by tubulin antibody.
Authors: Pepper D A , Brinkley B R
Published: May 2004

A lysed cell system was developed to determine whether tubulin antibody can block the nucleation of exogenous tubulin at kinetochores and centrosomes. Mitotic PtK2 cells were pretreated with colcemid to remove all endogenous microtubules and were lysed with Triton X-100 in PIPES-EGTA-Mg++ buffer. This procedure left centrosomes, chromosomes, and kinetochores intact as determined by electron microscopy of thin-sectioned cells. Exposure of the lysed cells to phorphocellulose-purified tubulin dimers at 37 degrees C in the presence of 1 mM GTP resulted in site-specific nucleation of microtubules at centrosomes and kinetochores. Treatment of the lysed cell preparations with tubulin antibody before subsequent exposure to the exogenous tubulin resulted in almost complete blockage of microtubule nucleation, especially at kinetochores. Pretreatment of the lysed cell preparations with control antibody or buffer without antibody had no effect on the ability of centrosomes and kinetochores to initiate microtubule assembly. The implications of these results with respect to the molecular composition of centrosomes and kinetochores are discussed.
Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins.
Authors: Brown M S , Goldstein J L , Krieger M , Ho Y K , Anderson R G
Published: May 2004

Mouse peritoneal macrophages accumulate large amounts of cholesteryl ester when incubated with human low-density lipoprotein that has been modified by chemical acetylation (acetyl-LDL). This accumulation is related to a high-affinity cell surface binding site that mediates the uptake of acetyl-LDL by adsorptive endocytosis and its delivery to lysosomes. The current studies demonstrate that the cholesteryl ester accumulation can be considered in terms of a two-compartment model: (a) the incoming cholesteryl esters of acetyl-LDL are hydrolyzed in lysosomes, and (b) the resultant free cholesterol is re-esterified in the cytosol where the newly formed esters are stored as lipid droplets. The following biochemical and morphologic evidence supports the hydrolysis-re-esterification mechanism: (a) Incubation of macrophages with acetyl-LDL markedly increased the rate of cholesteryl ester synthesis from [14C]oleate, and this was accompanied by an increase in the acyl-CoA:cholesteryl acyltransferase activity of cell-free extracts. (b) When macrophages were incubated with reconstituted acetyl-LDL in which the endogenous cholesterol was replaced with [3H]-cholesteryl linoleate, the [3H]cholesteryl linoleate was hydrolyzed, and at least one-half of the resultant [3H]cholesterol was re-esterified to form [3H]cholesteryl oleate, which accumulated within the cell. The lysosomal enzyme inhibitor chloroquine inhibited the hydrolysis of the [3H]cholesteryl linoleate, thus preventing the formation of [3H]cholesteryl oleate and leading to the accumulation of unhydrolyzed [3H]cholesteryl linoleate within the cells. (c) In the electron microscope, macrophages incubated with acetyl-LDL had numerous cytoplasmic lipid droplets that were not surrounded by a limiting membrane. The time course of droplet accumulation was similar to the time course of cholesteryl ester accumulation as measured biochemically. (d) When acetyl-LDL was removed from the incubation medium, biochemical and morphological studies showed that cytoplasmic cholesteryl esters were rapidly hydrolyzed and that the resultant free cholesterol was excreted from the cell.
alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions.
Authors: Willingham M C , Maxfield F R , Pastan I H
Published: May 2004

Using transmission electron microscopy, we have studied the interaction of alpha 2 macroglobulin (alpha 2 M) with the surface of cultured fibroblasts. When cells were incubated for 2 h at 4 degrees C with ferritin-conjugated alpha 2 M, approximately 90% of the alpha 2 M was diffusely distributed on the cell surface, and the other 10% was concentrated in "coated" pits. A pattern of diffuse labeling with some clustering in "coated" pits was also obtained when cells were incubated for 5 min at 4 degrees C with alpha 2 M, fixed with glutaraldehyde, and the alpha 2 M was localized with affinity-purified, peroxidase-labeled antibody to alpha 2 M. Experiments in which cells were fixed with 0.2% paraformaldehyde before incubation with alpha 2 M showed that the native distribution of alpha 2 M receptors was entirely diffuse without significant clustering in "coated" pits. This indicates that some redistribution of the alpha 2 M-receptor complexes into clusters occurred even at 4 degrees C. In experiments with concanavalin A(Con A), we found that some of the Con A clustered in coated regions of the membrane and was internalized in coated vesicles, but much of the Con A was directly internalized in uncoated vesicles or pinosomes. We conclude that unoccupied alpha 2 M receptors are diffusely distributed on the cell surface. When alpha 2 M-receptor complexes are formed, they rapidly cluster in coated regions or pits in the plasma membrane and subsequently are internalized in coated vesicles. Because insulin and epidermal growth factor are internalized in the same structures as alpha 2 M (Maxfield, F.R., J. Schlessinger, Y. Schechter, I. Pastan, and M.C. Willingham. 1978. Cell, 14: 805--810.), we suggest that all peptide hormones, as well as other proteins that enter the cell by receptor-mediated endocytosis, follow this same pathway.
Spectrin rearrangement early in erythrocyte ghost endocytosis.
Authors: Hardy B , Bensch K G , Schrier S L
Published: May 2004

The endocytic vacuoles induced in white ghosts were found to be depleted of spectrin and therefore it was proposed that they arose from spectrin-free areas in the erythrocyte membrane. To follow changes in spectrin distribution during endocytosis, affinity-purified rabbit antispectrin antibodies were produced. Quantitative techniques were developed for the use of a highly specific 125I-F(ab')2 antispectrin, and these showed that before the appearance of vacuoles, as assessed by phase microscopy, there was a reproducible decrease in immunoreactive spectrin. To determine whether this spectrin decrease represented a local or diffuse spectrin loss or a spectrin rearrangement, morphologic studies were undertaken using transmission electron microscopy on samples treated with rabbit antispectrin and ferritin-conjugated goat anti-rabbit immunoglobulin. These studies showed that endocytosis was preceded by the creation of extensive spectrin-free areas separated by discrete spectrin-containing zones. Pretreatment of ghosts with alkaline phosphatase blocked all forms of endocytosis and prevented the creation of spectrin-free areas. Therefore, it is proposed that under the impetus of endocytosis inducers, phosphorylated spectrin is redistributed so that spectrin-free zones are created, and that endocytic vacuoles form and fuse in spectrin-free areas.
Intercellular adhesion as a function of the cell cycle traverse.
Authors: Hellerqvist C G
Published: May 2004

Intercellular adhesion is assumed to play an important role in a multitude of biological phenomena governing cellular behavior. The rate of intercellular adhesion as a function of the cell cycle traverse has been investigated using, in the monolayer assay, synchronized Chinese Hamster Ovary-K1 cells. Results obtained demonstrate that cells in G1 adhere to G1 cells at twice the rate that S cells adhere to each other. G1 cells adhere to S cells at an intermediate rate. The additive adhesiveness seen in G1 is abolished by brief trypsinization, suggesting that in G1 a qualitative or quantitative change occurs with respect to the presence or exposure of components involved in intercellular adhesion.
Immunocytochemical localization of amylase and chymotrypsinogen in the exocrine pancreatic cell with special attention to the Golgi complex.
Authors: Geuze J J , Slot J W , Tokuyasu K T
Published: May 2004

Affinity-purified, monospecific rabbit antibodies against rat pancreatic alpha-amylase and bovine pancreatic alpha-chymotrypsinogen were used for immunoferritin observations of ultrathin frozen sections of mildly fixed exocrine pancreatic tissue from secretion-stimulated (pilocarpine) rats and from overnight-fasted rats and guinea pigs. The labeling patterns for both antibodies were qualitatively alike: Labeling occurred in (a) the cisternae of the rough endoplasmic reticulum (RER) including the perinuclear cisterna, in (b) the peripheral area between the RER and cis-Golgi face, and (c) all Golgi cisternae, condensing vacuoles, and secretory granules. Labeling of cytoplasmic matrix was negligible. Structures that appeared to correspond to rigid lamellae were unlabeled. Differences in labeling intensities indicated that concentration of the zymogens starts at the boundary of the RER and cis-side of the Golgi complex. These data support the view that the Golgi cisternae are involved in protein processing in both stimulated and unstimulated cells and that Golgi cisternae and condensing vacuoles constitute a functional unit.
Changes in the random distribution of sialic acid at the surface of the myeloid sinusoidal endothelium resulting from the presence of diaphragmed fenestrae.
Authors: De Bruyn P P , Michelson S
Published: May 2004

Diaphragmed fenestrae (DF) are sites of increased vascular permeability. The anionic charge distribution at the luminal aspect of the DF of the endothelium of the bone marrow vessels has been studied after aldehyde fixation by means of colloidal iron (CI), native ferritin (NF), and polycationic ferritin (PCF). At pH 1.8, these cationic agents are bound by the nonmodified luminal endothelial cell surface but not at the sites of the DF. PCF was used over a pH range of 1.8--7.2 (CI is unstable at higher pH levels, whereas NF which has a pI of 4.5 is anionic above this point). PCF shows increased binding at the DF from pH 3.5 upwards. PCF binding at pH 1.8 at the nonmodified luminal cell surface is significantly diminished by neuraminidase treatment which, however, does not perceptibly reduce PCF binding at the higher pH levels. It is concluded that there are exposed sialic acid groups at the lunimal cell surface which are absent or significantly fewer at the sites of the DF, whereas other anionic materials possibly with a pKa higher than that of sialic acid (pKa 2.6) are present both at the DF and at the nonmodified endothelial cell surface.
Developmental program of murine erythroleukemia cells. Effect of the inhibition of protein synthesis.
Authors: Levenson R , Housman D
Published: May 2004

The relationship between protein synthesis and commitment to terminal erythroid differentiation by dimethylsulfoxide-treated murine erythroleukemia (MEL) cells has been studied. Treatment with cycloheximide blocks the commitment of MEL cells. The effects of cycloheximide are completely reversible, however. Treatment of MEL cells before commitment delays commitment for a period of time equal to the length of inhibitor treatment. Puromycin exerts a similar effect on the commitment of MEL cells. These results indicate that there is a continuous requirement for protein synthesis before the commitment event.
Contributions of lipids and proteins to the surface charge of membranes. An electron microscopy study with cationized and anionized ferritin.
Authors: Burry R W , Wood J G
Published: May 2004

The surface charge of cultured neurons was investigated with the electron microscope markers anionized ferritin (AF) and cationized ferritin (CF). To determine which membrane components could react with the markers, model reactions were used. Both protein-coated Sepharose beads and lipid vesicles were reacted at physiological pH. Results with these model reactions indicate that the following groups may contribute to the surface charge: acidic groups--the sialic acid of both glycoproteins and gangliosides, the carboxyl group of proteins, and the phosphates of phospholipids; basic groups--the amines of proteins. The effect of chemical fixation on the surface charge was investigated. Glutaraldehyde fixation was shown to increase the charge of neutral proteins but not by a mechanism involving unbound aldehydes. Glutaraldehyde fixation of phospholipid vesicles in the presence of CF showed that amine-containing phospholipids were cross-linked to CF. This cross-linkage was seen with the electron microscope as the clumping of CF and the burying of CF in the membrane. Paraformaldehyde fixation had a lesser effect on the charge of proteins but did react with phospholipids as did glutaraldehyde. It is concluded that at physiological pH: (a) most of the charged proteins and lipids on cell surface can contribute to the membrane surface charge, and (b) the membrane surface charge of cells can be greatly changed by chemical fixation.
Regional differentiation of the sperm surface as studied with 125I-diiodofluorescein isothiocyanate, an impermeant reagent that allows isolation of the labeled components.
Authors: Gabel C A , Eddy E M , Shapiro B M
Published: May 2004

The regional differentiation of the sperm surface has been studied with the aid of a novel covalent labeling technique that permits concurrent cytological, biochemical, and immunological analyses. For these studies isothiocyanate derivatives of fluorescein (FITC) and diiodofluorescein (IFC) were employed: the latter can be prepared with radioiodine to high specific activity (125IFC) and is an impermeant reagent for the erythrocyte surface. Sperm of sea urchin (Strongylocentrotus purpuratus), medaka )Oryzias latipes), and golden hamster bind the fluorescent chromophores with a nonuniform distribution, most of the fluorescence being associated with the midpiece. The radioactive derivative 125IFC permits an analysis of the proteins that are responsible for most of the binding. Additionally, 125 IFC-labeled sperm are capable of fertilizing eggs, as assessed by autoradiography. That IFC labels the surface of the sperm was inferred from the following: (a) the labeling of the surfaces of other cells by fluorescein isothiocyanate and its derivatives; (b) the agglutination of labeled sperm by antibodies directed against IFC; (c) the use of peroxidase-dependent immunocytochemical reaction using anti-IFC antibodies, with analysis by electron microscopy; and (d) extraction of labeled sea urchin sperm with Triton X-100 under conditions that preferentially solubilize the plasma membrane. The antiserum directed against IFC was used to isolate the labeled surface components from Triton X-100 extracts of whole sperm, by immunoprecipitation, with Staphylococcus-A protein serving as a coprecipitant. The results support previous data showing that the sperm surface is a heterogeneous mosaic of restricted domains, one notable zone being the midpiece, where common molecular properties may be shared by sperm with distinctly different morphologies. In addition, IFC-mediated covalent alteration of specific cell surface proteins may be used to label, to identify, and, with the use of anti-IFC antibodies, to isolate such proteins from other cellular constituents.
Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport
Authors: Schwab ME , Suda K , Thoenen H
Published: May 2004

The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals.
Rapid, sequential changes in surface morphology of PC12 pheochromocytoma cells in response to nerve growth factor
Authors: Connolly JL , Greene LA , Viscarello RR , Riley WD
Published: May 2004

The effect of nerve growth factor (NGF), a substance that promotes the differentiation and maintenance of certain neurons, was studied via scanning electron microscopy utilizing the PC12 clonal NGF-responsive pheochromocytoma cell line. After 2-4 d of exposure to NGF, these cells acquire many of the properties of normal sympathic neurons. However, by phase microscopy, no changes are discernible within the first 12-18 h. Since the primary NGF receptor appears to be a membrane receptor, it seemed likely that some of the initial responses to the factor may be surface related.

PC12 cells maintained without NGF are round to ovoid and have numerous microvilli and small blebs. After the addition of NGF, there is a rapidly initiated sequential change in the cell surface. Ruffles appear over the dorsal surface of the cells with 1 min, become prominent by 3 min, and almost disappear by 7 min. Microvilli, conversely, disappear as the dorsal ruffles become prominent. Ruffles are seen at the the periphery of cell at 3 min, are prominent on most of the cells by 7 min and are gone by 15 min. The surface remains smooth from 15 min until 45 min when large blebs appear. The large blebs are present on most cells at 2 h and are gone by 4 h. The surface remains relatively smooth until 6-7 h of NGF treatment, when microvilli reappear as small knobs. These microvilli increase in both number and length to cover the cell surface by 10 h.

These changes were not observed with other basic proteins, with α-bungarotoxin (which binds specifically to PC12 membranes), and were not affected by an RNA synthesis inhibitor that blocks initiation of neurite outgrowth. Changes in the cell surface architecture appear to be among the earlist NGF responses yet detected and may represent or reflect primary events in the mechanism of the factor's action.
Capping of exogenous Forssman glycolipid on cells.
Authors: Stern P L , Bretscher M S
Published: May 2004

When motile cells are incubated with Forssman glycolipid, the antigen is incorporated into the cells' plasma membranes. If cross-linked by antibody, the patched glycolipids cap. This process is sensitive to those drugs that are known to inhibit capping of protein antigens. The results support a flow mechanism for capping.
Transformation and motility of human platelets: details of the shape change and release reaction observed by optical and electron microscopy.
Authors: Allen R D , Zacharski L R , Widirstky S T , Rosenstein R , Zaitlin L M , Burgess D R
Published: May 2004

Blood platelets from 10 normal human subjects have been examined with a sensitive differential interference contrast (DIC) microscope. The entire transformation process during adhesion to glass is clearly visible and has been recorded cinematographically, including the disk to sphere change of shape, the formation of sessile protuberances, the extension and retraction of pseudopodia, and the spreading, ruffling, and occasional regression of the hyalomere. The exocytosis of intact dense bodies can be observed either by DIC microscopy, or by epifluorescence microscopy in platelets stained with mepacrine. Details of fluorescent flashes indicate that the dense bodies usually release their contents extracellularly, may do so intracytoplasmically under the influence of strong, short wavelength light on some preparations of mepacrine-stained platelets. The release of one or more dense bodies leaves a crater of variable size on the upper surface of the granulomere. Such craters represent the surface component of the open canalicular system and their formation and disappearance can be directly observed. Because these techniques permit quantitation of several parameters of motility which are not readily observable by other techniques, it is suggested that high extinction DIC microscope examination may become a rapid and useful method of studying congenital and acquired platelet disorders. Many features of platelet transformation have been confirmed and extended by scanning electron micrographs. These can in turn be interpreted by reference to time-lapse films of living platelets.
Restriction of patching of bound concanavalin A after incorporation of arachidonic acid into the plasma membrane of virally transformed fibroblasts.
Authors: Hill D J , Borysenko J Z
Published: May 2004

Topographical distribution of concanavalin A binding sites (CABS) was studied in two lines of virally transformed fibroblasts as a function of fatty acid composition. Fatty acid composition was manipulated by incubating cells in fatty acid, ATP, CoA, and delipidated fetal calf serum (FCS). VLM cells grown in medium containing 5% FCS have a clustered CABS distribution. Plasma membrane vesicles (PMVs) derived from these cells have an arachidonate content of 1.7%. Elevation of PMV arachidonate to 15.8% results in a marked restriction of CABS patching, while elevation to 6.8% is associated with intermediate restriction of patching. Restriction of patching is associated with increased microviscosity. CABS of Rous sarcoma virus-transformed chicken embryo fibroblasts (RSV-CEF) are also responsive to arachidonate enrichment medium. Whereas untreated cells have a clustered CABS distribution, cells incubated for 24 h in arachidonate enrichment medium have predominantly a dispersed CABS distribution. In both VLM cells and RSV-CEF, ATP, CoA, and delipidated FCS alone have no effect upon CABS mobility. Inhibition of CABS patching is also observed when aspirin is included in the arachidonate enrichment medium but not when the cells are incubated in prostaglandins, thus suggesting that the restriction of CABS mobility is not mediated by prostaglandins. Other fatty acids (palmitate, oleate, nonadecanoate) failed to restrict CABS movement. The inhibition of CABS mobility is independent of cell shape change.
pH regulates the polymerization of actin in the sea urchin egg cortex.
Authors: Begg D A , Rebhun L I
Published: May 2004

The state of actin in the isolated cortex of the unfertilized sea urchin egg can be controlled by experimentally manipulating the pH of the isolation medium. Cortices isolated at the pH of the unfertilized egg (6.5--6.7) do not contain filamentous actin, while those isolated at the pH of the fertilized egg (7.3--7.5) develop large numbers of microvilli which contain bundles of actin filaments. Cortices that are isolated at pH 6.5 and then transferred to isolation medium buffered at pH 7.5 also develop actin filaments. However, the filaments are not arranged in bundles and microvilli do not form. Although the cortical granules in cortices isolated at pH 6.5 discharge at a free Ca++ concentration of approximately 10 micrometer, actin polymerization is not induced by increasing the Ca++ concentration of the isolation medium. These results suggest that the increase in cytoplasmic pH which occurs following fertilization induces the polymerization of actin in the egg cortex.