bluuebunny/crossref_metadata_2025 huggingface dataset:
Light-regulated translation of chloroplast proteins. I. Transcripts of psaA-psaB, psbA, and rbcL are associated with polysomes in dark-grown and illuminated barley seedlings
Authors: Klein RR , Mason HS , Mullet JE
Published: May 2004

We have previously observed (Klein, R. R., and J. E. Mullet, 1986, J. Biol. Chem. 261:11138-11145) that translation of two 65-70-kD chlorophyll a-apoproteins of Photosystem I (gene products of psaA and psaB) and a 32-kD quinone-binding protein of Photosystem II (gene product of psbA) was not detected in plastids of dark-grown barley seedlings even though transcripts for these proteins were present. In the present study it was found that nearly all of the psaA-psaB transcripts in plastids of dark-grown plants were associated with membrane-bound polysomes. Membrane-associated polysomes from plastids of dark-grown plants synthesized the 65-70-kD chlorophyll a-apoproteins at low levels when added to a homologous in vitro translation extract capable of translation elongation. However, when etioplast membranes were disrupted with detergent, in vitro synthesis of the 65-70-kD chlorophyll a-apoproteins increased to levels observed with polysomes of plastids from illuminated plants. These results suggest that synthesis of the chlorophyll a-apoproteins of Photosystem I is arrested on membrane-bound polysomes at the level of polypeptide chain elongation. In addition to the selective activation of chlorophyll a-apoprotein translation, illumination also caused an increase in chloroplast polysomes (membrane-associated and stromal) and induced a recruitment of psbA and rbcL transcripts into chloroplast polysomes. These results indicate that in conjunction with the selective activation of chlorophyll a-apoprotein elongation, illumination also caused a general stimulation of chloroplast translation initiation.
A stochastic model for leukocyte random motility and chemotaxis based on receptor binding fluctuations
Authors: Tranquillo RT , Lauffenburger DA , Zigmond SH
Published: May 2004

Two central features of polymorphonuclear leukocyte chemosensory movement behavior demand fundamental theoretical understanding. In uniform concentrations of chemoattractant, these cells exhibit a persistent random walk, with a characteristic "persistence time" between significant changes in direction. In chemoattractant concentration gradients, they demonstrate a biased random walk, with an "orientation bias" characterizing the fraction of cells moving up the gradient. A coherent picture of cell movement responses to chemoattractant requires that both the persistence time and the orientation bias be explained within a unifying framework. In this paper, we offer the possibility that "noise" in the cellular signal perception/response mechanism can simultaneously account for these two key phenomena. In particular, we develop a stochastic mathematical model for cell locomotion based on kinetic fluctuations in chemoattractant/receptor binding. This model can simulate cell paths similar to those observed experimentally, under conditions of uniform chemoattractant concentrations as well as chemoattractant concentration gradients. Furthermore, this model can quantitatively predict both cell persistence time and dependence of orientation bias on gradient size. Thus, the concept of signal "noise" can quantitatively unify the major characteristics of leukocyte random motility and chemotaxis. The same level of noise large enough to account for the observed frequency of turning in uniform environments is simultaneously small enough to allow for the observed degree of directional bias in gradients.
Mouse T lymphoma cells contain a transmembrane glycoprotein (GP85) that binds ankyrin
Authors: Kalomiris EL , Bourguignon LY
Published: May 2004

In this study we have used complementary biochemical and immunological techniques to establish that the lymphoma GP85 membrane glycoprotein is a transmembrane protein with a cytoplasmic domain that binds directly to ankyrin, a molecule known to link the membrane to the cytoskeleton. The evidence supporting our conclusion that the GP85 is a transmembrane glycoprotein is as follows: (a) GP85 can be surface-labeled with Na 125I and contains wheat germ agglutinin-binding sites, indicating that it has an extracellular domain; (b) GP85 can be phosphorylated by intracellular kinases, indicating that it has an intracellular domain; and (c) GP85 can be successfully incorporated into phospholipid vesicles, indicating the existence of a hydrophobic domain in the molecule. Further studies show that GP85 displays immunological cross-reactivity with the lymphocyte Pgp-1 (differentiation-specific) membrane glycoprotein, and with the erythrocyte anion transport membrane protein, band 3. Immunocytochemical studies indicate that an ankyrin-like protein accumulates underneath the lymphoma GP85 cap structure, suggesting an association of the ankyrin-like protein and GP85. This relationship has been further confirmed by the following results of binding and reconstitution experiments: (a) purified GP85 binds directly to an ankyrin-Sepharose column; (b) purified GP85 inserts into phospholipid vesicles in both the normal (right side out) and reversed (inside out) orientation (and with only the reversed configuration permits binding of ankyrin to GP85); and (c) cleavage of GP85 with trypsin yields a 40-kD peptide fragment that is part of the cytoplasmic domain and contains the ankyrin binding site(s). Based on these findings, we suggest that the lymphoma GP85 transmembrane glycoprotein contains a cytoplasmic domain that is directly involved in linking ankyrin to the cytoskeleton. This transmembrane linkage may play a pivotal role in receptor capping and cell activation in lymphocytes.
Inositol 1,4,5-trisphosphate-induced calcium release and guanine nucleotide-binding protein-mediated periodic calcium rises in golden hamster eggs
Authors: Miyazaki S
Published: May 2004

Periodic increases in intracellular free calcium occur upon fertilization of golden hamster eggs (Miyazaki et al. 1986. Dev. Biol. 118:259-267). To investigate the underlying mechanism, inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides were microinjected into the egg while Ca2+ transients were monitored by aequorin luminescence and/or hyperpolarization in the membrane potential, which indicates the exact timing and spatial distribution of the Ca2+ rise. Injection of IP3 induced an immediate Ca2+ transient of 13-18 s in the entire egg. The critical concentration of IP3 was 80 nM in the injection pipette (2 nM in the egg, assuming uniform distribution); the effect was all-or-none. The Ca2+ rise occurred even in Ca-free external medium. Injection of 5 mM GTP or 0.33 mM guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) (calculated intracellular concentration, 200 or 12 microM, respectively) caused a similar Ca2+ transient with a delay of 160-200 s. More than 50 microM GTP gamma S produced recurring and attenuating Ca2+ transients in a local area of the cytoplasm, with an initial delay of 25-40 s and intervals of 45-60 s. In Ca-free medium the first one to two Ca2+ transients occurred but succeeding ones were absent. Preinjection of guanosine-5'-O-(2-thiodiphosphate) inhibited the occurrence of both GTP gamma S-induced and sperm-induced Ca2+ transients in a dose-dependent manner. Neither pertussis nor cholera toxins had effect. It was proposed that sperm-egg interaction activates a GTP-binding protein that stimulates production of IP3, causing the first one to two Ca releases from internal stores, and also stimulates a pathway for elevation of Ca2+ permeability in the plasma membrane, thereby sustaining the repeated Ca2+ releases.
Actin filaments, stereocilia, and hair cells of the bird cochlea. V. How the staircase pattern of stereociliary lengths is generated
Authors: Tilney LG , Tilney MS , Cotanche DA
Published: May 2004

The stereocilia on each hair cell are arranged into rows of ascending height, resulting in what we refer to as a "staircase-like" profile. At the proximal end of the cochlea the length of the tallest row of stereocilia in the staircase is 1.5 micron, with the shortest row only 0.3 micron. As one proceeds towards the distal end of the cochlea the length of the stereocilia progressively increases so that at the extreme distal end the length of the tallest row of the staircase is 5.5 micron and the shortest row is 2 micron. During development hair cells form their staircases in four phases of growth separated from each other by developmental time. First, stereocilia sprout from the apical surfaces of the hair cells (8-10-d embryos). Second (10-12-d embryos), what will be the longest row of the staircase begins to elongate. As the embryo gets older successive rows of stereocilia initiate elongation. Thus the staircase is set up by the sequential initiation of elongation of stereociliary rows located at increased distances from the row that began elongation. Third (12-17-d embryos), all the stereocilia in the newly formed staircase elongate until those located on the first step of the staircase have reached the prescribed length. In the final phase (17-d embryos to hatchlings) there is a progressive cessation of elongation beginning with the shortest step and followed by taller and taller rows with the tallest step stopping last. Thus, to obtain a pattern of stereocilia in rows of increasing height what transpires are progressive go signals followed by a period when all the stereocilia grow and ending with progressive stop signals. We discuss how such a sequence could be controlled.
Mapping of the microvillar 110K-calmodulin complex: calmodulin-associated or -free fragments of the 110-kD polypeptide bind F-actin and retain ATPase activity
Authors: Coluccio LM , Bretscher A
Published: May 2004

The 110K-calmodulin complex isolated from intestinal microvilli is an ATPase consisting of one polypeptide chain of 110 kD in association with three to four calmodulin molecules. This complex is presumably the link between the actin filaments in the microvillar core and the surrounding cell membrane. To study its structural regions, we have partially cleaved the 110K-calmodulin complex with alpha-chymotrypsin; calmodulin remains essentially intact under the conditions used. As determined by 125I-calmodulin overlays, ion exchange chromatography, and actin-binding assays, a 90-kD digest fragment generated in EGTA remains associated with calmodulin. The 90K-calmodulin complex binds actin in an ATP-reversible manner and decorates actin filaments with an arrow-head appearance similar to that found after incubation of F-actin with the parent complex; binding occurs in either calcium- or EGTA-containing buffers. ATPase activity of the 90-kD digest closely resembles the parent complex. In calcium a digest mixture containing fragments of 78 kD, a group of three at approximately 40 kD, and a 32-kD fragment (78-kD digest mixture) is generated with alpha-chymotrypsin at a longer incubation time; no association of these fragments with calmodulin is observed. Time courses of digestions and cyanogen bromide cleavage indicate that the 78-kD fragment derives from the 90-kD peptide. The 78-kD mixture can also hydrolyze ATP. Furthermore, removal of the calmodulin by ion exchange chromatography from this 78-kD mixture had no effect on the ATPase activity of the digest, indicating that the ATPase activity resides on the 110-kD polypeptide. The 78 kD, two of the three fragments at approximately 40 kD, and the 32-kD fragments associate with F-actin in an ATP-reversible manner. Electron microscopy of actin filaments after incubation with the 78-kD digest mixture reveals coated filaments, although the prominent arrowhead appearance characteristic of the parent complex is not observed. These data indicate that calmodulin is not required either for the ATPase activity or the ATP-reversible binding of the 110K-calmodulin complex to F-actin. In addition, since all the fragments that bind F-actin do so in an ATP-reversible manner, the sites required for F-actin binding and ATP reversibility likely reside nearby.
Genomic organization and biosynthesis of secreted and cytoplasmic forms of gelsolin
Authors: Kwiatkowski DJ , Mehl R , Yin HL
Published: May 2004

Gelsolin is an actin regulatory protein which is unique among vertebrates in that it is found as both an intrinsic cytoplasmic protein and as a secreted plasma protein. We demonstrate that plasma and cytoplasmic gelsolins are derived by alternative transcriptional initiation sites and message processing from a single gene 70 kb long, containing at least 14 exons. Their message and amino acid sequences are identical except at the 5' end/NH2 termini. The cytoplasmic-specific 5' sequence is derived from two exons that encode untranslated sequence, while the plasma message-specific 5' sequence is derived from a single exon that encodes untranslated sequence, the signal peptide, and the first 21 residues of the plasma protein. The two transcriptional initiation sites are separated by greater than or equal to 32 kb. Biosynthetic and RNase protection studies indicate that a number of cell types make both plasma and cytoplasmic gelsolin in widely varying amounts and ratios.
Cell surface proteoglycan binds mouse mammary epithelial cells to fibronectin and behaves as a receptor for interstitial matrix
Authors: Saunders S , Bernfield M
Published: May 2004

The proteoglycan (PG) on the surface of NMuMG mouse mammary epithelial cells consists of at least two functional domains, a membrane-intercalated domain which anchors the PG to the plasma membrane, and a trypsin-releasable ectodomain which bears both heparan and chondroitin sulfate chains. The ectodomain binds cells to collagen types I, III, and V, but not IV, and has been proposed to be a matrix receptor. Because heparin binds to the adhesive glycoproteins fibronectin, an interstitial matrix component, and laminin, a basal lamina component, we asked whether the cell surface PG also binds these molecules. Cells harvested with either trypsin or EDTA bound to fibronectin; binding of trypsin-released cells was inhibited by the peptide GRGDS but not by heparin, whereas binding of EDTA-released cells was inhibited only by a combination of GRDS and heparin, suggesting two distinct cell binding mechanisms. In the presence of GRGDS, the EDTA-released cells bound to fibronectin via the cell surface PG. Binding via the cell surface PG was to the COOH-terminal heparin binding domain of fibronectin. In contrast with the binding to fibronectin, EDTA-released cells did not bind to laminin under identical assay conditions. Liposomes containing the isolated intact cell surface PG mimic the binding of whole cells. These results indicate that the mammary epithelial cells have at least two distinct cell surface receptors for fibronectin: a trypsin-resistant molecule that binds cells to the sequence RGD and a trypsin-labile, heparan sulfate-rich PG that binds cells to the COOH-terminal heparin binding domain. Because the cell surface PG binds cells to the interstitial collagens (types I, III, and V) and to fibronectin, but not to basal lamina collagen (type IV) or laminin, we conclude that the cell surface PG is a receptor on epithelial cells specific for interstitial matrix components.
Developmental expression of 2ar (osteopontin) and SPARC (osteonectin) RNA as revealed by in situ hybridization
Authors: Nomura S , Wills AJ , Edwards DR , Heath JK , Hogan BL
Published: May 2004

2ar has been identified as a gene inducible by tumor promoters and growth factors in a variety of cultured mouse cell lines (Smith, J. H., and D. T. Denhardt. 1987. J. Cell. Biochem. 34:13-22). Sequence analysis shows that it codes for mouse osteopontin, an RGDS-containing, phosphorylated, sialic acid-rich Ca++-binding protein originally isolated from bone (Oldberg, A., A. Franzen, and D. Heinegard. 1986. Proc. Natl. Acad. Sci. USA. 83:8819-8823; Prince, C. W., T. Oosawa, W. T. Butler, M. Tomana, A. S. Brown, and R. E. Schrohenloer. 1987. J. Biol. Chem. 262:2900-3907.). In this paper we use Northern blot analysis and in situ hybridization to localize expression of 2ar during mouse embryogenesis. 2ar RNA is first detected in developing limb bones and calvaria at 14.5 d p.c., in a population of cells distinct from those expressing SPARC (osteonectin). High levels of 2ar expression are also seen in the bone marrow-derived granulated metrial gland cells of the deciduum and placenta, and in a number of epithelial tissues, including embryonic and postnatal kidney tubules, uterine epithelium and sensory epithelium of the embryonic ear. The temporal and spatial pattern of 2ar expression seen in vivo suggests that the protein plays a wider role than previously realized, in processes which are not confined to bone development.
Regulation of the synthesis and activity of urokinase plasminogen activator in A549 human lung carcinoma cells by transforming growth factor-beta
Authors: Keski-Oja J , Blasi F , Leof EB , Moses HL
Published: May 2004

Transforming growth factor-beta (TGF beta) is a regulator of cellular proliferation which can alter the proteolytic activity of cultured cells by enhancing the secretion of endothelial type plasminogen activator inhibitor and affecting the secretion of plasminogen activators (PAs) in cultured fibroblastic cells. We used the TGF beta-responsive malignant human lung adenocarcinoma cell line A549 to study the relationships between the known TGF beta-induced growth inhibition and the effects of TGF beta on the secretion of PA activity by A549 cells. PA activity was quantitated by caseinolysis assays, and characterized by urokinase mRNA analysis, immunoprecipitation, and zymography assays. PA-inhibitor production was observed in autoradiograms of SDS-polyacrylamide gels and reverse zymography assays. It was found that TGF beta enhanced the production of PA activity by these cells, in accordance with an enhancement of urokinase mRNA levels. A concomitant stimulation of type 1 PA-inhibitor production was also observed in A549 cells in response to TGF beta. In contrast to the observations of A549 cells, TGF beta caused a decrease in the expression of both urokinase and the tissue-type PA mRNA in human embryonic WI-38 lung fibroblasts indicating opposite regulation of the expression of PAs in these cells. The results suggest that TGF beta may play a role in the regulation of the invasive, proteolytically active phenotype of certain lung carcinoma cells.
Neuron-glia cell adhesion molecule interacts with neurons and astroglia via different binding mechanisms
Authors: Grumet M , Edelman GM
Published: May 2004

The neuron-glia cell adhesion molecule (Ng-CAM) is present in the central nervous system on postmitotic neurons and in the periphery on neurons and Schwann cells. It has been implicated in binding between neurons and between neurons and glia. To understand the molecular mechanisms of Ng-CAM binding, we analyzed the aggregation of chick Ng-CAM either immobilized on 0.5-micron beads (Covaspheres) or reconstituted into liposomes. The results were correlated with the binding of these particles to different types of cells as well as with cell-cell binding itself. Both Ng-CAM-Covaspheres and Ng-CAM liposomes individually self-aggregated, and antibodies against Ng-CAM strongly inhibited their aggregation; the rate of aggregation increased approximately with the square of the concentration of the beads or the liposomes. Much higher rates of aggregation were observed when the ratio of Ng-CAM to lipid in the liposome was increased. Radioiodinated Ng-CAM on Covaspheres and in liposomes bound both to neurons and to glial cells and in each case antibodies against Ng-CAM inhibited 50-90% of the binding. Control preparations of fibroblasts and meningeal cells did not exhibit significant binding. Adhesion between neurons and glia within and across species (chick and mouse) was explored in cellular assays after defining markers for each cell type, and optimal conditions of shear, temperature, and cell density. As previously noted using chick cells (Grumet, M., S. Hoffman, C.-M. Chuong, and G. M. Edelman. 1984 Proc. Natl. Acad. Sci. USA. 81:7989-7993), anti-Ng-CAM antibodies inhibited neuron-neuron and neuron-glia binding. In cross-species adhesion assays, binding of chick neurons to mouse astroglia and binding of mouse neurons to chick astroglia were both inhibited by anti-Ng-CAM antibodies. To identify whether the cellular ligands for Ng-CAM differed for neuron-neuron and neuron-glia binding, cells were preincubated with specific antibodies, the antibodies were removed by washing, and Ng-CAM-Covasphere binding was measured. Preincubation of neurons with anti-Ng-CAM antibodies inhibited Ng-CAM-Covasphere binding but similar preincubation of astroglial cells did not inhibit binding. In contrast, preincubation of astroglia with anti-astroglial cell antibodies inhibited binding to these cells but preincubation of neurons with these antibodies had no effect. Together with the data on Covaspheres and liposome aggregation, these findings suggested that Ng-CAM-Covaspheres bound to Ng-CAM on neurons but bound to different molecules on astroglia.(ABSTRACT TRUNCATED AT 400 WORDS)
Molecular forms, binding functions, and developmental expression patterns of cytotactin and cytotactin-binding proteoglycan, an interactive pair of extracellular matrix molecules
Authors: Hoffman S , Crossin KL , Edelman GM
Published: May 2004

Cytotactin is an extracellular matrix protein that is found in a restricted distribution and is related to developmental patterning at a number of neural and non-neural sites. It has been shown to bind specifically to other extracellular matrix components including a chondroitin sulfate proteoglycan (cytotactin-binding [CTB] proteoglycan) and fibronectin. Cell binding experiments have revealed that cytotactin interacts with neurons and fibroblasts. When isolated from brain, both cytotactin and CTB proteoglycan contain the HNK-1 carbohydrate epitope. Here, specific antibodies prepared against highly purified cytotactin and CTB proteoglycan were used to correlate the biochemical alterations and modes of binding of these proteins with their differential tissue expression as a function of time and place during chicken embryo development. It was found that, during neural development, both the levels of expression of cytotactin and CTB proteoglycan and of the molecular forms of each molecule varied, following different time courses. In addition, a novel Mr 250,000 form of cytotactin was detected that contained chondroitin sulfate. The intermolecular binding of cytotactin and CTB proteoglycan and the binding of cytotactin to fibroblasts were characterized further and found to be inhibited by EDTA, consistent with a dependence on divalent cations. Unlike the molecules from neural tissue, cytotactin and CTB proteoglycan isolated from non-neural tissues such as fibroblasts lacked the HNK-1 epitope. Nevertheless, the intermolecular and cellular binding activities of cytotactin isolated from fibroblast culture medium were comparable to those of the molecule isolated from brain, suggesting that the HNK-1 epitope is not directly involved in binding. Binding experiments involving enzymatically altered molecules that lack chondroitin sulfate suggested that this glycosaminoglycan is also not directly involved in binding. Although they clearly formed a binding couple, the spatial distributions of cytotactin and CTB proteoglycan in the embryo were not always coincident. They were similar in tissue sections from the cerebellum, gizzard, and vascular smooth muscle. In contrast, CTB proteoglycan was present in cardiac muscle where no cytotactin is present, and it was seen in cartilage throughout development unlike cytotactin, which was present only in immature chondrocytes. Cell culture experiments were consistent with the previous conclusion that cytotactin was specifically synthesized by glia, whereas CTB proteoglycan was specifically synthesized by neurons.(ABSTRACT TRUNCATED AT 400 WORDS)
Meiotic chromosome behavior in spread preparations of yeast.
Authors: Dresser M E , Giroux C N
Published: May 2004

Chromosome behavior in meiosis is well characterized from cytological and genetic descriptions but little is known of the underlying molecular mechanisms, largely because no one experimental system has been developed to support an integrated application of modern cytological, genetic, and molecular biological methods. To combine efficient analyses of meiotic chromosome structure and function in a single organism, we have extended to yeast methods for making spread preparations of nuclei. Features of yeast meiosis that parallel meiosis in large eukaryotes, such as bouquet formation and prophase chromosome condensation that occurs in concert with synaptonemal complex formation, are evident for the first time. The ability to analyze large numbers of nuclei at the light and electron microscopes in preparations amenable to a variety of cytological and immunocytological techniques will facilitate the description of meiosis at the molecular level in yeast.
Rhizobium fix genes mediate at least two communication steps in symbiotic nodule development.
Authors: Putnoky P , Grosskopf E , Ha D T , Kiss G B , Kondorosi A
Published: May 2004

To identify bacterial genes involved in symbiotic nodule development, ineffective nodules of alfalfa (Medicago sativa) induced by 64 different Fix-mutants of Rhizobium meliloti were characterized by assaying for symbiotic gene expression and by morphological studies. The expression of leghemoglobin and nodulin-25 genes from alfalfa and of the nifHD genes from R. meliloti were monitored by hybridizing the appropriate DNA probes to RNA samples prepared from nodules. The mutants were accordingly divided into three groups. In group I none of the genes were expressed, in group II only the plant genes were expressed and in group III all three genes were transcribed. Light and electron microscopical analysis of nodules revealed that nodule development was halted at different stages in nodules induced by different group I mutants. In most cases nodules were empty lacking infection threads and bacteroids or nodules contained infection threads and a few released bacteroids. In nodules induced by a third mutant class bacteria were released into the host cells, however the formation of the peribacteroid membrane was not normal. On this basis we suggest that peribacteroid membrane formation precedes leghemoglobin and nodulin-25 induction, moreover, after induction of nodulation by the nod genes at least two communication steps between the bacteria and the host plants are necessary for the development of the mature nodule. By complementing each mutant of group I with a genomic R. meliloti library made in pLAFRl, four new fix loci were identified, indicating that several bacterial genes are involved in late nodule development.
Murine fetal liver macrophages bind developing erythroblasts by a divalent cation-dependent hemagglutinin.
Authors: Morris L , Crocker P R , Gordon S
Published: May 2004

During mammalian development the fetal liver plays an important role in hematopoiesis. Studies with the macrophage (M phi)-specific mAb F4/80 have revealed an extensive network of M phi plasma membranes interspersed between developing erythroid cells in fetal liver. To investigate the interactions between erythroid cells and stromal M phi, we isolated hematopoietic cell clusters from embryonic day-14 murine fetal liver by collagenase digestion and adherence. Clusters of erythroid cells adhered to glass mainly via M phi, 94% of which bound 19 +/- 11 erythroblasts (Eb) per cell. Bound Eb proliferated vigorously on the surface of fetal liver M phi, with little evidence of ingestion. The M phi could be stripped of their associated Eb and the clusters then reconstituted by incubation with Eb in the presence of divalent cations. The interaction required less Ca++ than Mg++, 100 vs. 250 microM for half-maximal binding, and was mediated by a trypsin-sensitive hemagglutinin on the M phi surface. After trypsin treatment fetal liver M phi recovered the ability to bind Eb and this process could be selectively inhibited by cycloheximide. Inhibition tests showed that the Eb receptor differs from known M phi plasma membrane receptors and fetal liver M phi did not bind sheep erythrocytes, a ligand for a distinct M phi hemagglutinin. We propose that fetal liver M phi interact with developing erythroid cells by a novel nonphagocytic surface hemagglutinin which is specific for a ligand found on Eb and not on mature red cells.
Fc receptor-mediated phagocytosis occurs in macrophages at exceedingly low cytosolic Ca2+ levels.
Authors: Di Virgilio F , Meyer B C , Greenberg S , Silverstein S C
Published: May 2004

Cytosolic free Ca2+ ([Ca2+]i) homeostasis was investigated in mouse peritoneal macrophages and in the macrophage-like cell line J774. [Ca2+]i measurements were performed in both cells in suspension and cells in monolayers loaded with either quin2 or fura-2. Resting [Ca2+]i was 110-140 and 85-120 nM for cell suspensions and monolayers, respectively. There were no significant differences in [Ca2+]i between the two macrophage populations whether quin2 or fura-2 were used as Ca2+ indicators. Addition of heat-aggregated IgG, IgG-coated erythrocyte ghosts, or a rat monoclonal antibody (2.4G2) directed against mouse Fc receptor II induced a rise in [Ca2+]i. This [Ca2+]i increase was consistently observed in J774 and peritoneal macrophage suspensions and in J774 macrophage monolayers; in contrast it was observed inconsistently in peritoneal macrophages in monolayer cultures. The increase in [Ca2+]i induced by ligation of Fc receptors was inhibited totally in macrophages in suspension and by 80% in macrophages in monolayers by a short preincubation of macrophages with PMA; however, phagocytosis itself was unaffected. The effect of reducing cytosolic Ca2+ to very low concentrations on Fc receptor-mediated phagocytosis was also investigated. By incubating macrophages with high concentrations of quin2/AM in the absence of extracellular Ca2+, or by loading EGTA into the cytoplasm, the [Ca2+]i was buffered and clamped to 1-10 nM. Despite this, the phagocytosis of IgG-coated erythrocytes proceeded normally. These observations confirm the report of Young et al. (Young, J. D., S. S. Ko, and Z. A. Cohn. 1984. Proc. Natl. Acad. Sci. USA. 81:5430-5434) that ligation of Fc receptors causes Ca2+ mobilization in macrophages. However, these results confirm and extend the findings of McNeil et al. (McNeil, P. L., J. A. Swanson, S. D. Wright, S. C. Silverstein, and D. L. Taylor. 1986. J. Cell Biol. 102:1586-1592) that a rise in [Ca2+]i is not required for Fc receptor-mediated phagocytosis; and they provide direct evidence that Fc receptor-mediated phagocytosis occurs normally even at exceedingly low [Ca2+]i.
Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. II. Morphological analysis.
Authors: Pasdar M , Nelson W J
Published: May 2004

Biochemical analysis of the kinetics of assembly of two cytoplasmic plaque proteins of the desmosome, desmoplakins I (250,000 Mr) and II (215,000 Mr), in Madin-Darby canine kidney (MDCK) epithelial cells, demonstrated that these proteins exist in a soluble and insoluble pool, as defined by their extract ability in a Triton X-100 high salt buffer (CSK buffer). Upon cell-cell contact, there is a rapid increase in the capacity of the insoluble pool at the expense of the soluble pool; subsequently, the insoluble pool is stabilized, while proteins remaining in the soluble pool continue to be degraded rapidly (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677-685). In this paper, we have sought to determine the spatial distribution of the soluble and insoluble pools of desmoplakins I and II, and their organization in the absence and presence of cell-cell contact by using differential extraction procedures and indirect immunofluorescence microscopy. In the absence of cell-cell contact, two morphologically and spatially distinct patterns of staining of desmoplakins I and II were observed: a pattern of discrete spots in the cytoplasm and perinuclear region, which is insoluble in CSK buffer; and a pattern of diffuse perinuclear staining, which is soluble in CSK buffer, but which is preserved when cells are fixed in 100% methanol at -20 degrees C. Upon cell-cell contact, in the absence or presence of protein synthesis, the punctate staining pattern of desmoplakins I and II is cleared rapidly and efficiently from the cytoplasm to the plasma membrane in areas of cell-cell contact (less than 180 min). The distribution of the diffuse perinuclear staining pattern remains relatively unchanged and becomes the principal form of desmoplakins I and II in the cytoplasm 180 min after induction of cell-cell contact. Thereafter, the relative intensity of staining of the diffuse pattern gradually diminishes and is completely absent 2-3 d after induction of cell-cell contact. Significantly, double immunofluorescence shows that during desmosome assembly on the plasma membrane both staining patterns coincide with a subpopulation of cytokeratin intermediate filaments. Taken together with the preceding biochemical analysis, we suggest that the assembly of desmoplakins I and II in MDCK epithelial cells is regulated at three discrete stages during the formation of desmosomes.
Relation between the organization of spectrin and of membrane lipids in lymphocytes.
Authors: Del Buono B J , Williamson P L , Schlegel R A
Published: May 2004

In lymphocytes, the cytoskeletal protein spectrin exhibits two organizational states. Because the plasma membrane lipids of lymphocytes also display two organizational states, it was asked whether there is a relation between the organization of spectrin and of membrane lipids. When mouse thymocytes were stained with merocyanine 540 (MC540), a fluorescent lipophilic probe that binds preferentially to loosely packed, disorganized lipid bilayers, some cells fluoresced brightly and some only dimly or not at all. When the same population was stained for spectrin by indirect immunofluorescence, the spectrin in some cells was uniformly distributed, while in others it was concentrated in a unipolar aggregate. Techniques enriching for mature thymocytes selected for cells displaying low MC540 fluorescence and aggregated spectrin, the same characteristics found in peripheral blood lymphocytes. Flow cytometric sorting of thymocytes based on MC540 phenotype simultaneously sorted them by spectrin phenotype. Finally, treatment with agents that alter the distribution of spectrin caused mature lymphocytes to display high MC540 fluorescence and uniform spectrin. Thus, a relation exists between the organizational states of spectrin and of membrane lipids in lymphocytes: aggregated spectrin is found in cells with tightly organized membrane lipids, uniform spectrin in those with loosely organized lipids. Spectrin may thus be involved in modulating membrane lipid organization in lymphocytes as it is in erythrocytes. Since loosely organized lipids may promote adhesion of blood cells to reticuloendothelial cells, spectrin may thereby be involved in transducing an internally generated adhesion signal to the lymphocyte surface.
Expression of the gene for main intrinsic polypeptide (MIP): separate spatial distributions of MIP and beta-crystallin gene transcripts in rat lens development.
Authors: Yancey S B , Koh K , Chung J , Revel J P
Published: May 2004

The main intrinsic polypeptide (MIP) is the major protein present in the lens fiber cell membrane and is the product of a gene which, as far as is known, is expressed only in the lens. We have used in situ hybridization and immunofluorescence microscopy to characterize the expression of this gene during the course of development in the rat. At progressive stages of lens morphogenesis, we find that synthesis of the protein is closely tied to the accumulation of MIP mRNA in cells that are committed to terminal differentiation, first in the elongating presumptive primary lens fibers and later in the secondary fibers as they differentiate from the anterior epithelial cells. The transcripts accumulate in the basal cytoplasm of the primary fibers and in the cytoplasm which surrounds the cell nucleus in the secondary fibers. We have compared this pattern of expression with that of a gene for a cytoplasmic protein, beta-crystallin beta-A1/A3. In sharp contrast to the localized concentrations seen for the MIP mRNA, beta-A1/A3 transcripts are relatively uniformly distributed throughout the cytoplasm. Neither MIP nor crystallin gene appears to be transcriptionally active in the undifferentiated epithelial cell, but transcripts from the beta-A1/A3 gene appear earlier in fiber cell differentiation than do those from the gene for MIP.
Cytoskeleton-associated plectin: in situ localization, in vitro reconstitution, and binding to immobilized intermediate filament proteins.
Authors: Foisner R , Leichtfried F E , Herrmann H , Small J V , Lawson D , Wiche G
Published: May 2004

The association and interaction of plectin (Mr 300,000) with intermediate filaments and filament subunit proteins were studied. Immunoelectron microscopy of whole mount cytoskeletons from various cultured cell lines (rat glioma C6, mouse BALB/c 3T3, and Chinese hamster ovary) and quick-frozen, deep-etched replicas of Triton X-100-extracted rat embryo fibroblast cells revealed that plectin was primarily located at junction sites and branching points of intermediate filaments. These results were corroborated by in vitro recombination studies using vimentin and plectin purified from C6 cells. Filaments assembled from mixtures of both proteins were extensively crosslinked by oligomeric plectin structures, as demonstrated by electron microscopy of negatively stained and rotary-shadowed specimens as well as by immunoelectron microscopy; the binding of plectin structures on the surface of filaments and cross-link formation occurred without apparent periodicity. Plectin's cross-linking of reconstituted filaments was also shown by ultracentrifugation experiments. As revealed by the rotary-shadowing technique, filament-bound plectin structures were oligomeric and predominantly consisted of a central globular core region of 30-50 nm with extending filaments or filamentous loops. Solid-phase binding to proteolytically degraded vimentin fragments suggested that plectin interacts with the helical rod domain of vimentin, a highly conserved structural element of all intermediate filament proteins. Accordingly, plectin was found to bind to the glial fibrillar acidic protein, the three neurofilament polypeptides, and skin keratins. These results suggest that plectin is a cross-linker of vimentin filaments and possibly also of other intermediate filament types.
Modulation of desmin intermediate filament assembly by a monoclonal antibody.
Authors: Ip W
Published: May 2004

We have used a monoclonal antibody against desmin to examine the assembly of intermediate filaments (IF) from their building blocks, the tetrameric protofilaments. The antibody, designated D76, does not cross react with any other IF proteins (Danto, S.I., and D.A. Fischman. 1984. J. Cell Biol. 98:2179-2191). It binds to a region amino-terminal to cys-324 of avian desmin that is resistant to chymotrypsin and trypsin digestion, and in the electron microscope appears to bind to the ends of tetrameric protofilaments. In combination, these findings suggest that the epitope of the antibody resides at the amino-terminal end of the alpha-helical rod domain. Preincubation of desmin protofilaments with an excess of D76 antibodies blocks their subsequent assembly into IF. In the presence of sub-stoichiometric amounts of antibodies, IF are assembled from protofilaments but they are morphologically aberrant in that (a) they are capped by IgG molecules at one or both ends; (b) they are unraveled to varying degree, revealing a characteristic right-handed helical arrangement of sub-filamentous strands of different diameters. The antibody binds only to the ends but not along the length of desmin IF. The most straightforward explanation for this is that the epitope resides in a part of the desmin molecule that becomes buried within the core of the filament upon polymerization and is therefore inaccessible to the antibody.
Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.
Authors: Boukamp P , Petrussevska R T , Breitkreutz D , Hornung J , Markham A , Fusenig N E
Published: May 2004

In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long-term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation.
Different forms of microtubule-associated protein 2 are encoded by separate mRNA transcripts.
Authors: Garner C C , Matus A
Published: May 2004

Brain microtubule-associated protein 2 (MAP2) consists of a pair of high molecular mass (280 kD) polypeptides, MAP2a and MAP2b, and a recently identified 70-kD protein, MAP2c, which is antigenically related to these high molecular mass MAP2's. Using cDNA clones we have analyzed the expression of these three proteins at the nucleic acid level. cDNA probes selective for the high molecular mass MAP2's a and b identified only a 9-kb mRNA, whereas a probe for sequence common to all three MAP2 isoforms, a, b, and c, recognized the 9-kb transcript and additionally a 6-kb mRNA. Southern blot analysis with cDNA probes indicated that there is only one MAP2 gene from which these two distinct mRNAs are derived. The 70-kD MAP2c protein is much more abundant in neurons of developing brain than those of adult tissues. Similarly the expression of the 6-kb MAP2c-related mRNA, is much greater in neonatal than adult rat brain, indicating that the developmental expression of MAP2 is determined by transcriptional regulation from a single MAP2 gene.
Identification of a polyphosphoinositide-modulated domain in gelsolin which binds to the sides of actin filaments.
Authors: Yin H L , Iida K , Janmey P A
Published: May 2004

Gelsolin is a Ca2+- and polyphosphoinositide-modulated actin-binding protein which severs actin filaments, nucleates actin assembly, and caps the "barbed" end of actin filaments. Proteolytic cleavage analysis of human plasma gelsolin has shown that the NH2-terminal half of the molecule severs actin filaments almost as effectively as native gelsolin in a Ca2+-insensitive but polyphosphoinositide-inhibited manner. Further proteolysis of the NH2-terminal half generates two unique fragments (CT14N and CT28N), which have minimal severing activity. Under physiological salt conditions, CT14N binds monomeric actin coupled to Sepharose but CT28N does not. In this paper, we show that CT28N binds stoichiometrically and with high affinity to actin subunits in filaments, suggesting that it preferentially recognizes the conformation of polymerized actin. Analysis of the binding data shows that actin filaments have one class of CT28N binding sites with Kd = 2.0 X 10(-7) M, which saturates at a CT28N/actin subunit ratio of 0.8. Binding of CT28N to actin filaments is inhibited by phosphatidylinositol 4,5-bisphosphate micelles. In contrast, neither CT14N nor another actin-binding domain located in the COOH-terminal half of gelsolin form stable stoichiometric complexes with actin along the filaments, and their binding to actin monomers is not inhibited by PIP2. Based on these observations, we propose that CT28N is the polyphosphoinositide-regulated actin-binding domain which allows gelsolin to bind to actin subunits within a filament before serving.
Mode of fibroblast growth enhancement by human interleukin-1.
Authors: Singh J P , Adams L D , Bonin P D
Published: May 2004

Previous studies have demonstrated that interleukin-1 (IL-1) stimulates fibroblast growth (Schmidt, J. A., S. B. Mizel, D. Cohn, and I. Green. 1982. J. Immunol. 128:2177-2182) and binds to specific, high affinity receptors of BALB/c3T3 cells (Bird, T. A., and J. Saklatval. 1986. Nature (Lond.). 324:263-265, 266-268). We have investigated the mechanism of fibroblast growth stimulation by IL-1. Addition of fibroblast growth factor derived from platelets (PDGF) to a quiescent culture of BALB/c3T3 cells produced 8-10-fold increase in DNA synthesis during 24-h incubation. The cellular action of PDGF was mediated through competence induction and required synergistic action of plasma-derived factors for full mitogenic activity. When tested at a wide range of concentrations (0.1-100 pM), natural IL-1 or recombinant IL-1 produced only a maximum of 5-10% of DNA synthesis elicited in response to PDGF or serum. Induction of DNA synthesis required continuous presence of IL-1 and did not exhibit synergism with plasma. Competence induction and mitogenic stimulation by PDGF was associated with early induction of proteins P32, P38, P46-48, P75, and changes in cytoskeletal organization. Examination of these early cellular changes showed that IL-1 did not produce similar induction of cellular proteins and the morphological changes associated with growth stimulation. These results suggest that the mode of IL-1 action on BALB/c3T3 was not through competence induction. When IL-1 was added to cells rendered competent by brief exposure to PDGF, 10-15% additional DNA synthesis occurred during the first 24 h. Extended incubation of PDGF-treated cells in the presence of IL-1 revealed that the stimulation by IL-1 occurred predominantly during the subsequent cycle of DNA replication, wherein DNA synthesis reached three- to fivefold higher than the untreated cultures. We conclude (a) IL-1 alone is not a potent mitogen for BALB/c3T3 cells, and does not bring cells out of the growth arrest Go phase, (b) treatment with PDGF renders the cells more responsive to IL-1, (c) part of the IL-1 action on competent cells may be characterized as progression inducing activity, further, (d) our results indicate that action of IL-1 on PDGF-treated cells produces sustained DNA synthesis for an extended period, perhaps by preventing the entry of cells into growth arrest Go phase.