bluuebunny/crossref_metadata_2025 huggingface dataset:
Actin in the photoreceptor connecting cilium: immunocytochemical localization to the site of outer segment disk formation.
Authors: Chaitin M H , Schneider B G , Hall M O , Papermaster D S
Published: May 2004

Actin has been localized in Rana pipiens retinas that were fixed and embedded in aldehyde cross-linked BSA. Thin sections were reacted sequentially with (a) affinity-purified antiactin antibodies induced in rabbits; (b) biotinyl-sheep anti-rabbit antibodies; and (c) avidin-ferritin conjugates. As expected, antiactin labeling density was high in the apical pigment epithelial cell processes and in the calycal processes of photoreceptors. Actin was also localized in a new site. The connecting cilium that joins the inner and outer segments of both rods and cones was heavily labeled by antiactin at its outer segment (OS), or distal, end. In this region of the cilium, the plasma membrane evaginates to form new OS disks and these basal disks were labeled in some instances. Below the new disks in rods, the cytoplasm of liplike expansions of the distal cilium was also heavily labeled. The plasma membrane and interior of the connecting cilium and the remainder of the OS were unlabeled. These findings suggest that actin may participate in the vectorial transport of opsin and other intrinsic membrane proteins that are incorporated into newly forming OS disks. The results also implicate actin in the membrane expansion involved with OS disk formation.
Transport of protein between cytoplasmic membranes of fused cells: correspondence to processes reconstituted in a cell-free system.
Authors: Rothman J E , Urbani L J , Brands R
Published: May 2004

Mixed monolayers containing vesicular stomatitis virus-infected Chinese hamster ovary clone 15B cells (lacking UDP-N-acetylglucosamine transferase I, a Golgi enzyme) and uninfected wild-type Chinese hamster ovary cells were formed. Extensive cell fusion occurs after the monolayer is exposed to a pH of 5.0. The vesicular stomatitis virus encoded membrane glycoprotein (G protein) resident in the rough endoplasmic reticulum (labeled with [35S]methionine) or Golgi complex (labeled with [3H]palmitate) of 15B cells at the time of fusion can reach Golgi complexes from wild-type cells after fusion; G protein present in the plasma membrane cannot. Transfer to wild-type Golgi complexes is monitored by the conversion of G protein to an endoglycosidase H-resistant form upon arrival, and also demonstrated by immunofluorescence microscopy. G protein in the Golgi complex of the 15B cells at the time of fusion exhibits properties vis a vis its transfer to an exogenous Golgi population identical to those found earlier in a cell-free system (Fries, E., and J. E. Rothman. 1981. J. Cell Biol., 90: 697-704). Specifically, pulse-chase experiments using the in vivo fusion and in vitro assays reveal the same two populations of G protein in the Golgi complex. The first population, consisting of G protein molecules that have just received their fatty acid, can transfer to a second Golgi population in vivo and in vitro. The second population, entered by G protein approximately 5 min after its acylation, is unavailable for this transfer, in vivo and in vitro. Presumably, this second population consists of those G-protein molecules that had already been transferred between compartments within the 15B Golgi population, in an equivalent process before cell fusion or homogenization for in vitro assays. Evidently, the same compartment boundary in the Golgi complex is detected by these two measurements. The surprisingly facile process of glycoprotein transit between Golgi stacks that occurs in vivo may therefore be retained in vitro, providing a basis for the cell-free system.
Intercompartmental transport in the Golgi complex is a dissociative process: facile transfer of membrane protein between two Golgi populations.
Authors: Rothman J E , Miller R L , Urbani L J
Published: May 2004

The transfer of the vesicular stomatitis virus-encoded glycoprotein (G protein) between Golgi populations in fused cells (Rothman, J. E., L. J. Urbani, and R. Brands. 1984. J. Cell Biol. 99:248-259) is exploited here to study and to help define the compartmental organization of the Golgi stack and to characterize the mechanism of intercompartmental transport. We find that G protein that has just received its peripheral N-acetylglucosamine in the Golgi complex of one cell is efficiently transferred to the Golgi complex of another cell to receive galactose (Gal). Remarkably, this transport occurs at the same rate between these two compartments whether they are present in the same or different Golgi populations. Therefore, a dissociative (presumably vesicular) transport step moves G protein from one part of the Golgi in which N-acetylglucosamine is added to another in which Gal is added. Minutes later, upon receiving Gal, the same G protein molecules are very poorly transferred to an exogenous Golgi population after cell fusion. Therefore, once this intercompartmental transfer has already taken place (before fusion), it cannot take place again (after fusion); i.e., transport across the compartment boundary in the Golgi complex that separates the sites of N-acetylglucosamine and Gal incorporation is a vectorial process. We conclude that transfers between Golgi cisternae occur by a stochastic process in which transport vesicles budding from cisternae dissociate, can diffuse away, and then attach to and fuse with the appropriate target cisterna residing in the same or in a different stack, based on a biochemical pairing after a random encounter. Under these circumstances, a transported protein would almost always randomize among stacks with each intercisternal transfer; it would not progress systematically through a single stack. Altogether, our studies define three sequential compartments in the Golgi stack.
Monoclonal antibodies prepared against Dictyostelium actin: characterization and interactions with actin.
Authors: Simpson P A , Spudich J A , Parham P
Published: May 2004

Three mouse monoclonal antibodies, Act I, Act II, and Act IV, against actin from the cellular slime mold Dictyostelium discoideum, have been made and characterized. All three antibodies are IgG1 and share the following properties: They form stable complexes with monomeric Dictyostelium actin, which prevents polymerization of the actin into filaments. On addition to preformed actin filaments, they cause a reduction in filament size and in the viscosity of the actin solution. They cross-react strongly with actins from the lower eucaryotes Physarum and Acanthamoeba, but not with alpha-actins from rabbit and human muscle or beta- and gamma-actins from human erythrocytes and a human B lymphoid cell line. Act II and Act IV recognize a similar antigenic determinant that is topographically distinct from that identified by Act I. In protein immunoblotting, only Act I bound strongly to Dictyostelium actin. Analysis of actin fragments with this technique showed that amino acids 13 to about 50 are required for Act I binding to actin. A comparison of the amino acid sequences of actins from lower eucaryotes and higher vertebrates implicates threonine 41 as a critical residue in the Act I antigenic site. The properties of Act II and Act IV suggest that they recognize antigenic sites involving the NH2-terminal six residues.
Targeting of beta-glucuronidase to lysosomes in mannose 6-phosphate receptor-deficient MOPC 315 cells.
Authors: Gabel C A , Kornfeld S
Published: May 2004

The murine plasma cell line MOPC 315 efficiently targets newly synthesized acid hydrolases to lysosomes in spite of a marked deficiency in the level of the mannose 6-phosphate receptor (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). To better understand the routing of lysosomal enzymes in this cell line, pulse-chase experiments were performed with [2-3H]mannose and [35S]methionine followed by immunoprecipitation of beta-glucuronidase and IgA. By 3 h of chase, essentially all of the newly synthesized beta-glucuronidase had undergone proteolytic processing, suggesting that the molecules had reached lysosomes. At this time 30% of the pulse-labeled IgA was still intracellular. The oligosaccharides on the intracellular IgA were of the high mannose-type, while the secreted IgA contained processed, complex-type oligosaccharides. This indicates that the intracellular IgA was still in the endoplasmic reticulum or an early region of the Golgi complex when all of the beta-glucuronidase had reached lysosomes. Therefore, beta-glucuronidase and IgA must exit from the endoplasmic reticulum or the early Golgi complex at different rates, a finding that is inconsistent with bulk phase movement of these proteins from the endoplasmic reticulum to the trans Golgi complex. The addition of the ionophore monensin greatly slows the rate of IgA secretion from MOPC 315 cells and the molecules secreted have incompletely processed oligosaccharides. In contrast, monensin only slightly delays the transport of newly synthesized beta-glucuronidase to lysosomes and causes no significant alteration in the extent of oligosaccharide phosphorylation, a process that appears to occur in the early (cis) Golgi complex. However, the labeled beta-glucuronidase was deficient in sialylated, phosphorylated hybrid oligosaccharides whose biosynthesis requires the action of late stage oligosaccharide processing enzymes assumed to be localized in the trans Golgi complex.
Differentiation of murine erythroleukemia cells results in the rapid repression of vimentin gene expression.
Authors: Ngai J , Capetanaki Y G , Lazarides E
Published: May 2004

We show that vimentin filaments are present in undifferentiated Friend murine erythroleukemia cells, but are lost progressively to undetectable levels by 96 h of dimethyl sulfoxide-mediated differentiation. The amount of newly synthesized cytoskeletal vimentin is decreased dramatically by 24 h of induction, and is paralleled by a rapid loss of vimentin mRNA (approximately 25-fold reduction at 96 h). Hence, disappearance of vimentin filaments in these cells appears to be regulated at the level of vimentin mRNA abundance. On the other hand, the levels of actin synthesis and actin mRNA remain essentially unchanged. The kinetics of vimentin mRNA reduction during dimethyl sulfoxide-mediated differentiation, and the levels of vimentin mRNA observed in the presence of hexamethylene-bisacetamide or hemin as inducers suggest that the cessation of vimentin expression precedes, but may be associated with commitment to terminal differentiation. Our results demonstrate the dynamic regulation of vimentin expression in mammalian erythropoiesis.
Communication between receptors for different ligands on a single cell: ligation of fibronectin receptors induces a reversible alteration in the function of complement receptors on cultured human monocytes.
Authors: Wright S D , Licht M R , Craigmyle L S , Silverstein S C
Published: May 2004

Receptors for the third component of complement (C3) on cultured human monocytes (MO) bind ligand-coated particles but do not initiate phagocytosis. The function of these receptors, however, is altered dramatically after MO attach to surfaces coated with fibronectin (FN) or after MO are exposed to phorbol esters. FN and phorbol esters "activate" C3 receptors such that they promote vigorous phagocytosis. Here we show that activation of C3 receptors requires the continuous presence of FN or phorbol esters and is rapidly reversible when these stimuli are removed. Activation does not change the number or distribution of C3 receptors on the surface of MO. We conclude that the function of C3 receptors is regulated by reversible reactions that are initiated by ligation of a different class of receptors on the surface of the same cell.
Phorbol esters rapidly stimulate amiloride-sensitive Na+/H+ exchange in a human leukemic cell line.
Authors: Besterman J M , Cuatrecasas P
Published: May 2004

The human, leukemic cell line, HL-60, undergoes differentiation in response to tumor-promoting phorbol esters. Recent studies have implicated stimulation of a Na+/H+ antiporter as an initial event in cellular differentiation and/or proliferation. The effects of phorbol esters on Na+-dependent H+ efflux from HL-60 cells were studied by pH-stat titration. Tumor-promoting phorbol diesters, but not the inactive parent alcohol, stimulated Na+-dependent H+ efflux in a rapid (within 1 min at 37 degrees C) and reversible manner. Stimulation was dependent on the concentration of extracellular sodium; lithium could substitute for sodium, but choline could not. Stimulation was dependent on the activity of extracellular protons and was inhibited completely by amiloride. The concentrations of phorbol diesters at which we observed half-maximal stimulation of Na+-dependent H+ efflux are very similar to the Kd reported in the literature for binding of these phorbol diesters to the phorbol ester receptor and the Km for phorbol diester activation of protein kinase C. Overall characterization of basal and phorbol ester-stimulated H+ efflux indicate that stimulation of a Na+/H+ antiporter constitutes a primary event in phorbol ester interaction with HL-60 cells.
Spectrin immunofluorescence distinguishes a population of naturally capped lymphocytes in situ.
Authors: Repasky E A , Symer D E , Bankert R B
Published: May 2004

Immunofluorescence analysis of mammalian lymphocytes using antiserum directed against chicken erythrocyte alpha-spectrin revealed a lymphocyte population in which spectrin antigen was arranged in the form of a discrete cap (hereafter referred to as capped lymphocytes). This subset could be easily distinguished from other lymphocytes in which the spectrin antigen was diffusely distributed near the plasma membrane (noncapped lymphocytes). The subset of capped lymphocytes could be visualized in situ and in isolated cells in the absence of added ligand. Using frozen sections of lymphoid organs that were fixed in formaldehyde prior to the immunofluorescence procedure, capped lymphocytes were found in characteristic locations depending on the tissue examined. In the thymus, the major population of medullary lymphocytes were capped whereas cortical lymphocytes were mostly noncapped. In Peyer's patches, capped lymphocytes were interspersed with noncapped lymphocytes throughout the tissue. In the spleen, capped lymphocytes were concentrated in the periarterial lymphoid sheath of the white pulp and in lymph nodes they were found predominantly in the paracortical and cortical regions. Capped lymphocytes were not visible in the thymus until just before birth and did not appear in the spleen until 3 d after birth. When lymphocytes were isolated from lymphoid organs, fixed in formaldehyde and prepared for immunofluorescence, capped and noncapped lymphocytes were still identifiable and present in the same relative proportions as seen in situ. Results identical to those described above are obtained using antisera directed against guinea pig fodrin. Natural capping of proteins previously shown to co-migrate with a variety of cell surface macromolecules after cross-linking may be a new means of identifying various stages of lymphocyte activation or differentiation.
Kinetics and intermediates of marginal band reformation: evidence for peripheral determinants of microtubule organization.
Authors: Miller M , Solomon F
Published: May 2004

The microtubules of the mature erythrocyte of the chicken are confined to a band at the periphery. Whole-mount electron microscopy after extraction reveals that the number of microtubules in each cell is almost the same. All the microtubules can be depolymerized by incubation in the cold, and the marginal band can be quantitatively and qualitatively reformed by return to 39 degrees C. These properties allow the reformation of the marginal band to be treated as an in vivo microtubule assembly reaction. The kinetics of this reaction and the intermediates detected during reformation suggest a mechanism of microtubule organization that is distinct from that observed in other cell types. Apparently only one or two growing microtubule ends are available for assembly--assembly is only detected at the cell periphery, even at early times--and there is no evidence of the participation of a microtubule-organizing center.
Thyrotropin-releasing hormone increases cytosolic free Ca2+ in clonal pituitary cells (GH3 cells): direct evidence for the mobilization of cellular calcium.
Authors: Schlegel W , Wollheim C B
Published: May 2004

Changes in the cytosolic free Ca2+ concentration following cell surface receptor activation have been proposed to mediate a wide variety of cellular responses. Using the specific Ca2+ chelator quin2 as a fluorescent intracellular probe, we measured the Ca2+ levels in the cytosol of clonal rat pituitary cells, GH3 cells. We demonstrate that thyrotropin-releasing hormone (TRH) at nanomolar concentrations leads to a rapid and transient increase in cytosolic Ca2+. This increase was found to occur in Ca2+-free media in the presence of EGTA, thus at extracellular Ca2+ levels that are below the cytosolic concentrations, and was not prevented by verapamil, a Ca2+ channel blocker. Depolarization of GH3 cells with K+, which can mimic the action of TRH on prolactin release, increased cytosolic Ca2+ levels only in the presence of free extracellular Ca2+, and this increase could be blocked by verapamil. These data show that the mobilization of intracellular Ca2+ due to TRH action that has been proposed by previous studies actually leads to an increase in cytosolic free Ca2+. The kinetic features of this response emphasize the key role of cytosolic free Ca2+ in stimulus-secretion coupling.
Peripheral proteins of postsynaptic membranes from Torpedo electric organ identified with monoclonal antibodies.
Authors: Froehner S C
Published: May 2004

Highly purified postsynaptic membranes from Torpedo electric organ contain the acetylcholine receptor as well as other proteins. To identify synapse-specific components, we prepared monoclonal antibodies (mabs) to proteins extracted from the membranes with either lithium diiodosalicylate or alkaline treatment. 10 mabs specific for three different proteins were obtained. Seven mabs reacted with a major 43,000-mol-wt protein (43K protein). This protein is composed of isoelectric variants (pl = 7.2-7.8) and each of the mabs reacted with all of the variants. Analysis of these mabs by competition for binding to 43K protein and by reaction with proteolytic fragments of 43K protein in immunoblots showed that they recognize at least five different epitopes. Two mabs reacted with a protein of 90,000 mol wt (90K protein) and one with a protein of 58,000 mol wt composed of isoelectric variants (pl = 6.4-6.7) (58K protein). The 43K and 58K proteins appeared to co-purify with the receptor-containing membranes while the 90K protein did not. Immunofluorescence experiments indicated that the anti-43K mabs bind to the innervated face of Torpedo electrocytes and that a component related to the 43K protein is found at the rat neuromuscular junction. The anti-58K mab stained the innervated face, although rather weakly, while the anti-90K mabs reacted intensely with the non-innervated membrane. Thus, the 43K protein and possibly also the 58K protein are synaptic components while the 90K protein is predominantly nonsynaptic.
Somatostatin discriminates between the intracellular pathways of secretory and membrane proteins.
Authors: Green R , Shields D
Published: May 2004

Somatostatin is a14-amino acid peptide hormone that inhibits the secretion of a variety of other polypeptide hormones, including growth hormone. Here we describe an experimental system used to determine whether somatostatin can discriminate in its inhibition between secretory and plasma membrane proteins. Growth hormone-secreting cells (GH3) were infected with vesicular stomatitis virus and pulse-chased with [35S]methionine to follow the simultaneous intracellular transit of growth hormone and the viral membrane glycoprotein, G protein. Secretion of growth hormone was monitored by immunoprecipitation of chase media, while appearance of G protein on the plasma membrane was detected by cell surface labeling and virus purification. In the presence of somatostatin (10 micrograms/ml), the secretion of growth hormone was inhibited by 80%. In contrast, G protein appeared on the plasma membrane with slightly enhanced kinetics. When cells were treated with the ionophore monensin (0.2 microM), there was a dramatic inhibition of both the secretion of growth hormone and the incorporation of G protein into plasma membranes. Our results on the differential effect of somatostatin provide evidence for sorting of secretory and membrane proteins into distinct compartments in the secretory pathway. The data further suggest that this sorting event occurs late in the Golgi complex or after proteins exit from that organelle.
The influence of light on cone disk shedding in the lizard, Sceloporus occidentalis.
Authors: Bernstein S A , Breding D J , Fisher S K
Published: May 2004

The lizard, Sceloporus occidentalis has an all-cone retina. In lizards maintained on a 12-h light:12-h dark (12L:12D) cycle, a burst of cone outer segment (COS) shedding occurs 2 h after light offset (1400 h circadian time) (Young, R.W., 1977, J. Ultrastruct. Res. 61:172-72). In this investigation, we studied the effect of different lighting regimes on the pattern of cone disk shedding in this species. When lizards entrained to a 12L:12D cycle are kept in constant darkness (DD), the shedding peak is advanced approximately 2 h and the magnitude of shedding is reduced to 30% of control. COS increased in mean length from 12 micron in controls to 14 micron after one cycle in DD and maintained this length during a second cycle in DD. In constant light (LL), disk shedding was damped to approximately 10% of control values. Shedding synchrony in LL was also perturbed and therefore cyclic shedding bursts could not be distinguished. During LL there was a much larger increase in COS mean length than in DD. After one cycle of LL, COS length was 15 micron and after two cycles COS length exceeded 17 micron. When lizards entrained to 12L:12D are shifted to a 6L:18D regimen, the first shedding cycle is biphasic. The first peak of 5% shedding occurs 2 h after light offset whereas a second larger peak (13%) occurs according to the entrained schedule (1400 h). This manipulation separates out a dark-triggered and circadian shedding component, which is normally superimposed in lizards entrained to a 12L:12D cycle. When entrained lizards are placed in 36 h of LL followed by light offset, the peak shedding response after light offset is double the control response (53% vs. 27%). After 30 h of LL (lights off 90 degrees out of phase), there is a biphasic shedding response similar to the 6L:18D regimen although this time the dark-triggered shedding component is greater in magnitude then the circadian component. COS turnover is estimated by extrapolating from COS mean length increases during LL. From this method we obtained a 2.7-micron increase in COS length during each day in LL. If COS growth is not augmented during LL, this would yield a 4-5-d turnover time for the average 12.5-micron COS.
Analysis of the role of microfilaments and microtubules in acquisition of bipolarity and elongation of fibroblasts in hydrated collagen gels.
Authors: Tomasek J J , Hay E D
Published: May 2004

Fibroblasts in situ reside within a collagenous stroma and are elongate and bipolar in shape. If isolated and grown on glass, they change from elongate to flat shape, lose filopodia, and acquire ruffles. This shape change can be reversed to resemble that in situ by suspending the cells in hydrated collagen gels. In this study of embryonic avian corneal fibroblasts grown in collagen gels, we describe for the first time the steps in the acquisition of the elongate shape and analyze the effect of cytoskeleton-disrupting drugs on filopodial activity, assumption of bipolarity, and cell elongation within extracellular matrix. We have previously shown by immunofluorescence that filopodia contain actin but not myosin and are free of organelles. The cell cortex is rich in actin and the cytosol, in myosin. By using antitubulin, we show in the present study that microtubules are aligned along the long axis of the bipolar cell body. The first step in assumption of the elongate shape is extension of filopodia by the round cells suspended in collagen, and this is not significantly affected by the drugs we used: taxol to stabilize microtubules; nocodazole to disassemble microtubules; and cytochalasin D to disrupt microfilaments. The second step, movement of filopodia to opposite ends of the cell, is disrupted by cytochalasin, but not by taxol or nocodazole. The third step, extension of pseudopodia and acquisition of bipolarity similarly requires intact actin, but not microtubules. If fibroblasts are allowed to become bipolar before drug treatment, moreover, they remain so in the presence of the drugs. To complete the fourth step, extensive elongation of the cell, both intact actin and microtubules are required. Retraction of the already elongated cell occurs on microtubule disruption, but retraction requires an intact actin cytoskeleton. We suggest that the cell interacts with surrounding collagen fibrils via its actin cytoskeleton to become bipolar in shape, and that microtubules interact with the actin cortex to bring about the final elongation of the fibroblast.
Calcium and magnesium contents and volume of the terminal cisternae in caffeine-treated skeletal muscle.
Authors: Yoshioka T , Somlyo A P
Published: May 2004

(a) The effects of caffeine on the composition and volume of the terminal cisternae (TC) of the sarcoplasmic reticulum (SR) in frog skeletal muscle were determined with rapid freezing, electron microscopy, and electron probe analysis. (b) Caffeine (5 mM) released approximately 65% of the Ca content of the TC in 1 min and 84% after 3 min. The release of Ca from the TC was associated with a highly significant increase in its Mg content. This increase in Mg was not reduced by valinomycin. There was also a small increase in the K content of the TC at 1 min, although not after 3 min of caffeine contracture. (c) On the basis of the increase in Mg content during caffeine contracture and during tetanus (Somlyo, A. V., H. Gonzalez-Serratos, H. Shuman, G. McClellan, and A. P. Somlyo, 1981, J. Cell Biol., 90:577-594), we suggest that both mechanisms of Ca release are associated with an increase in the Ca and Mg permeability of the SR membranes, the two ions possibly moving through a common channel. (d) There was a significant increase in the P content of the TC during caffeine contracture, while in tetanized muscle (see reference above) there was no increase in the P content of the TC. (e) Mitochondrial Ca content was significantly increased (at 1 and at 3 min) during caffeine contracture. Valinomycin (5 microM) blocked this mitochondrial Ca uptake. (f) The sustained Ca release caused by caffeine in situ contrasts with the transient Ca release observed in studies of fragmented SR preparations, and could be explained by mediation of the caffeine-induced Ca release by a second messenger produced more readily in intact muscle than in isolated SR. (g) The TC were not swollen in rapidly frozen, caffeine-treated muscles, in contrast to the swelling of the TC observed in conventionally fixed, caffeine-treated preparation, the latter finding being in agreement with previous studies. (h) The fractional volume of the TC in rapidly frozen control (resting) frog semitendinosus muscles (approximately 2.1%) was less than the volume (approximately 2.5%) after glutaraldehyde-osmium fixation.
Comparison of prohormone-processing activities in islet microsomes and secretory granules: evidence for distinct converting enzymes for separate islet prosomatostatins.
Authors: Noe B D , Debo G , Spiess J
Published: May 2004

In previous work we have examined the nature of converting enzymes for proinsulin, proglucagon, and prosomatostatin-I (PSS-I) in secretory granules isolated from anglerfish islets. The purpose of the present study was to extend the examination of precursor conversion to islet microsomes and to compare prohormone processing, including that of PSS-I and prosomatostatin-II (PSS-II), in islet secretory granules and microsomes. Microsomes (rough endoplasmic reticulum [RER] and Golgi complex) and secretory granules were prepared from anglerfish islets by differential and discontinuous density-gradient centrifugation. Microsomes were further fractionated into Golgi- and RER-enriched subfractions. Lysed secretory granule or microsome preparations were incubated in the presence of a mixture of radioactively labeled islet prohormones. Extracts of products generated were subjected to analysis by gel filtration and high-pressure liquid chromatography. Accuracy of product cleavage was monitored by comparing high-pressure liquid chromatography retention times from the radiolabeled in vitro conversion products with the retention times of labeled products from tissue extracts. All converting activity in microsomes was found to be similar to that in granules in that it had a pH optimum near pH 5 and was inhibited by p-chloromercuribenzoate. No significant differences in the converting activity of Golgi complex- and RER-enriched subfractions of microsomes was observed. The proinsulin, proglucagon, and PSS-II converting-enzymes, which were found in islet secretory granules, were also present and membrane-associated in islet microsomes. However, converting activity for PSS-I was displayed only in secretory granules. This suggests that two or more separate enzymes are involved in processing PSS-I and PSS-II, and that these enzymes have either differential distribution or differential activity in RER/Golgi complex and secretory granules. The demonstration of converting enzyme activity in islet microsomes supports the proposal that these enzymes may be synthesized at the RER and are internalized along with the prohormones.
Immunocytochemical studies of quaking mice support a role for the myelin-associated glycoprotein in forming and maintaining the periaxonal space and periaxonal cytoplasmic collar of myelinating Schwann cells.
Authors: Trapp B D , Quarles R H , Suzuki K
Published: May 2004

The myelin-associated glycoprotein (MAG) is an integral membrane glycoprotein that is located in the periaxonal membrane of myelin-forming Schwann cells. On the basis of this localization, it has been hypothesized that MAG plays a structural role in (a) forming and maintaining contact between myelinating Schwann cells and the axon (the 12-14-nm periaxonal space) and (b) maintaining the Schwann cell periaxonal cytoplasmic collar of myelinated fibers. To test this hypothesis, we have determined the immunocytochemical localization of MAG in the L4 ventral roots from 11-mo-old quaking mice. These roots display various stages in the association of remyelinating Schwann cells with axons, and abnormalities including loss of the Schwann cell periaxonal cytoplasmic collar and dilation of the periaxonal space of myelinated fibers. Therefore, this mutant provides distinct opportunities to observe the relationships between MAG and (a) the formation of the periaxonal space during remyelination and (b) the maintenance of the periaxonal space and Schwann cell periaxonal cytoplasmic collar in myelinated fibers. During association of remyelinating Schwann cells and axons, MAG was detected in Schwann cell adaxonal membranes that apposed the axolemma by 12-14 nm. Schwann cell plasma membranes separated from the axolemma by distances greater than 12-14 nm did not react with MAG antiserum. MAG was present in adaxonal Schwann cell membranes that apposed the axolemma by 12-14 nm but only partially surrounded the axon and, therefore, may be actively involved in the ensheathment of axons by remyelinating Schwann cells. To test the dual role of MAG in maintaining the periaxonal space and Schwann cell periaxonal cytoplasmic collar of myelinated fibers, we determined the immunocytochemical localization of MAG in myelinated quaking fibers that displayed pathological alterations of these structures. Where Schwann cell periaxonal membranes were not stained by MAG antiserum, the cytoplasmic side of the periaxonal membrane was "fused" with the cytoplasmic side of the inner compact myelin lamella and formed a major dense line. This loss of MAG and the Schwann cell periaxonal cytoplasmic collar usually resulted in enlargement of the 12-14-nm periaxonal space and ruffling of the apposing axolemma. In myelinated fibers, there was a strict correlation between the presence of MAG in the Schwann cell periaxonal membrane and (a) maintenance of the 12-14-nm periaxonal space, and (b) presence of the Schwann cell periaxonal cytoplasmic collar.(ABSTRACT TRUNCATED AT 400 WORDS)
Surface charge distribution on the endothelial cell of liver sinusoids.
Authors: Ghitescu L , Fixman A
Published: May 2004

The topography of the charged residues on the endothelial cell surface of liver sinusoid capillaries was investigated by using electron microscopic tracers of different size and charge. The tracers used were native ferritin (pl 4.2-4.7) and its cationized (pl 8.4) and anionized (pl 3.7) derivatives, BSA coupled to colloidal gold (pl of the complex 5.1), hemeundecapeptide (pl 4.85), and alcian blue (pl greater than 10). The tracers were either injected in vivo or perfused in situ through the portal vein of the mouse liver. In some experiments, two tracers of opposite charge were sequentially perfused with extensive washing in between. The liver was processed for electron microscopy and the binding pattern of the injected markers was recorded. The electrostatic nature of the tracer binding was assessed by perfusion with high ionic strength solutions, by aldehyde quenching of the plasma membrane basic residues, and by substituting the cell surface acidic moieties with positively charged groups. Results indicate that the endothelial cells of the liver sinusoids expose on their surface both cationic and anionic residues. The density distribution of these charged groups on the cell surface is different. While the negative charge is randomly and patchily scattered all over the membrane, the cationic residues seem to be accumulated in coated pits. The charged groups co-exist in the same coated pit and bind the opposite charged macromolecule. It appears that the fixed positive and negative charges of the coated pit glycocalyx are mainly segregated in space. The layer of basic residues is located at 20-30-nm distance of the membrane, while most of the negative charges lie close to the external leaflet of the plasmalemma.
Concentrations of high-mobility-group proteins in the nucleus and cytoplasm of several rat tissues.
Authors: Kuehl L , Salmond B , Tran L
Published: May 2004

Nuclear and cytoplasmic fractions were isolated from various tissues of the rat by a nonaqueous technique. The high-mobility-group (HMG) proteins were extracted from these fractions with acid and separated by one- and two-dimensional PAGE. The concentrations of high-mobility-group proteins HMG1, HMG2, and HMG17 in the nucleus and cytoplasm were then estimated from the staining intensities of the electrophoretic bands. The cytoplasmic concentrations of these proteins were very low--usually less than 1/30 of those present in the corresponding nuclear fractions. For the tissues studied (liver, kidney, heart, and lung), the concentrations of HMG proteins in the nucleus did not differ significantly from one tissue to another. Averaged over the four tissues investigated, there were 0.28 molecule of HMG1, 0.18 molecule of HMG2, and 0.46 molecule of HMG17 per nucleosome. These values are considerably higher than those that have been reported previously.
High density of transmembrane glycoproteins on the flagellar surface of boar sperm cells.
Authors: Aguas A P , Pinto da Silva P
Published: May 2004

Membrane halves of boar sperm flagella were produced by freeze-fracture and labeled in situ with concanavalin A and wheat germ agglutinin; the lectins were visualized with protein-gold complexes. Concanavalin A and wheat germ agglutinin binding sites partition with both protoplasmic and exoplasmic halves of the membrane. A high density of lectin marking was found on protoplasmic membrane halves; we conclude that the label corresponds to transmembrane glycoproteins that, on freeze-fracture, are dragged across the outer (exoplasmic) half of the phospholipid bilayer. Our demonstration of numerous transmembrane proteins in sperm flagella offers the structural setting for previous models on flagellar surface motility that postulate accessibility of motile membrane components to the submembranous cytoskeleton.
Changes in distribution of nuclear matrix antigens during the mitotic cell cycle.
Authors: Chaly N , Bladon T , Setterfield G , Little J E , Kaplan J G , Brown D L
Published: May 2004

We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts. Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence. For comparison, lamins and histones were localized using human autoimmune antibodies. At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components. Antibody P1 stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus. Antibody I1 detected an antigen distributed as fine granules throughout the nuclear interior. Monoclonals PI1 and PI2 stained both the nuclear periphery and interior, with some characteristic differences. During mitosis, P1 and I1 were chromosome-associated, whereas PI1 and PI2 dispersed in the cytoplasm. Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order. With antibody I1, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to I1 may be involved in chromatin/chromosome higher-order organization throughout the cell cycle. Antibodies PI1 and PI2 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase. Antibody PI2 also detected antigen associated with the spindle poles.
Drug-induced dispersal of transcribed rRNA genes and transcriptional products: immunolocalization and silver staining of different nucleolar components in rat cells treated with 5,6-dichloro-beta-D-ribofuranosylbenzimidazole.
Authors: Scheer U , Hügle B , Hazan R , Rose K M
Published: May 2004

Upon incubation of cultured rat cells with the adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), nucleoli reversibly dissociate into their substructures, disperse throughout the nuclear interior, and form nucleolar "necklaces". We have used this experimental system, which does not inhibit transcription of the rRNA genes, to study by immunocytochemistry the distribution of active rRNA genes and their transcriptional products during nucleolar dispersal and recovery to normal morphology. Antibodies to RNA polymerase I allow detection of template-engaged polymerase, and monoclonal antibodies to a ribosomal protein (S1) of the small ribosomal subunit permit localization of nucleolar preribosomal particles. The results show that, under the action of DRB transcribed rRNA, genes spread throughout the nucleoplasm and finally appear in the form of several rows, each containing several (up to 30) granules positive for RNA polymerase I and argyrophilic proteins. Nucleolar material containing preribosomal particles also appears in granular structures spread over the nucleoplasm but its distribution is distinct from that of rRNA gene-containing granules. We conclude that, although transcriptional units and preribosomal particles are both redistributed in response to DRB, these entities retain their individuality as functionally defined subunits. We further propose that each RNA polymerase-positive granular unit represents a single transcription unit and that each continuous array of granules ("string of nucleolar beads") reflects the linear distribution of rRNA genes along a nucleolar organizer region. Based on the total number of polymerase I-positive granules we estimate that a minimum of 60 rRNA genes are active during interphase of DRB-treated rat cells.
Basal lamina formation by cultured microvascular endothelial cells.
Authors: Kramer R H , Bensch K G , Davison P M , Karasek M A
Published: May 2004

The production of a basal lamina by microvascular endothelial cells (MEC) cultured on various substrata was examined. MEC were isolated from human dermis and plated on plastic dishes coated with fibronectin, or cell-free extracellular matrices elaborated by fibroblasts, smooth muscle cells, corneal endothelial cells, or PF HR9 endodermal cells. Examination of cultures by electron microscopy at selected intervals after plating revealed that on most substrates the MEC produced an extracellular matrix at the basal surface that was discontinuous, multilayered, and polymorphous. Immunocytochemical studies demonstrated that the MEC synthesize and deposit both type IV collagen and laminin into the subendothelial matrix. When cultured on matrices produced by the PF HR9 endodermal cells MEC deposit a subendothelial matrix that was present as a uniform sheet which usually exhibited lamina rara- and lamina densa-like regions. The results indicate that under the appropriate conditions, human MEC elaborate a basal lamina-like matrix that is ultrastructurally similar to basal lamina formed in vivo, which suggests that this experimental system may be a useful model for studies of basal lamina formation and metabolism.
Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus.
Authors: Sleight R G , Pagano R E
Published: May 2004

We have examined the internalization and degradation of a fluorescent analog of phosphatidylcholine after its insertion into the plasma membrane of cultured Chinese hamster fibroblasts. 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD-PC) was incorporated into the cell surface by liposome-cell lipid transfer at 2 degrees C. The fluorescent lipid remained localized at the plasma membrane as long as the cells were kept at 2 degrees C; however, when the cells were warmed to 37 degrees C, internalization of some of the fluorescent lipid occurred. Most of the internalized C6-NBD-PC accumulated in the Golgi apparatus although a small amount was found randomly distributed throughout the cytoplasm in punctate fluorescent structures. Internalization of the fluorescent lipid at 37 degrees C was blocked by the presence of inhibitors of endocytosis. Incubation of cells containing C6-NBD-PC at 37 degrees C resulted in a rapid degradation of the fluorescent lipid. This degradation occurred predominantly at the plasma membrane. The degradation of C6-NBD-PC resulted in the release of NBD-fatty acid into the medium. We have compared the internalization of the fluorescent lipid with that of a fluorescent protein bound to the cell surface. Both fluorescent lipid and protein remained at the plasma membrane at 2 degrees C and neither were internalized at 37 degrees C in the presence of inhibitors of endocytosis. However, when incubated at 37 degrees C under conditions that permit endocytosis, the two fluorescent species appeared at different intracellular sites. Our data suggest that there is no transmembrane movement of C6-NBD-PC and that the fluorescent probe reflects the internalization of the outer leaflet of the plasma membrane lipid bilayer. The results are consistent with the Golgi apparatus as being the primary delivery site of phospholipid by bulk membrane movement from the plasma membrane.
Monoclonal antibodies demonstrate limited structural homology between myosin isozymes from Acanthamoeba.
Authors: Kiehart D P , Kaiser D A , Pollard T D
Published: May 2004

We used a library of 31 monoclonal and six polyclonal antibodies to compare the structures of the two classes of cytoplasmic myosin isozymes isolated from Acanthamoeba: myosin-I, a 150,000-mol-wt, globular molecule; and myosin-II, a 400,000-mol-wt molecule with two heads and a 90-nm tail. This analysis confirms that myosin-I and -II are unique gene products and provides the first evidence that these isozymes have at least one structurally homologous region functionally important for myosin's role in contractility. Characterization of the 23 myosin-II monoclonal antibody binding sites by antibody staining of one-dimensional peptide maps and solid phase, competitive binding assays demonstrate that they bind to at least 15 unique sites on the myosin-II heavy chain. The antibodies can be grouped into six families, whose members bind close to one another. None of the monoclonal antibodies bind to myosin-II light chains and polyclonal antibodies against myosin-II light or heavy chain bind only to myosin-II light or heavy chains, respectively: no antibody binds both heavy and light chains. Six of eight monoclonal antibodies and one of two polyclonal sera that react with the myosin-I heavy chain also bind to determinants on the myosin-II heavy chain. The cross-reactive monoclonal antibodies bind to the region of myosin-II recognized by the largest family of myosin-II monoclonal antibodies. In the two papers that immediately follow, we show that this family of monoclonal antibodies to myosin-II binds to the myosin-II tail near the junction with the heads and inhibits both the actin-activated ATPase of myosin-II and contraction of gelled cytoplasmic extracts of Acanthamoeba cytoplasm. Further, this structurally homologous region may play a key role in energy transduction by cytoplasmic myosins.
Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.
Authors: Kiehart D P , Pollard T D
Published: May 2004

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.