bluuebunny/crossref_metadata_2025 huggingface dataset:
Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.
Authors: Niederman R , Amrein P C , Hartwig J
Published: May 2004

Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.
The structure of membrane crystals of the light-harvesting chlorophyll a/b protein complex.
Authors: Kühlbrandt W , Thaler T , Wehrli E
Published: May 2004

Membrane crystals of the light-harvesting chlorophyll a/b protein complex from pea chloroplasts were investigated using electron microscopy and image analysis. The membrane crystals formed upon precipitation of the detergent-solubilized complex with mono- and divalent cations in the presence of small amounts of Triton X-100. The crystalline fraction contained two polypeptides of 25,000 and 27,000 mol wt. Freeze-dried and freeze-etched specimens showed a periodic honeycomb structure on the surface of membrane crystals. Double replicas of freeze-fractured sheets showed a hexagonal lattice of particles on both fracture faces. Image analysis of negatively stained membrane crystals suggested that they had threefold rather than sixfold symmetry in projection. A projection map at 20-A resolution revealed two triangular structural units of opposite handedness per crystallographic unit cell. The structural units appeared to be inserted bidirectionally into the membrane, alternating in orientation perpendicular to the membrane plane.
Effect of cell shape change on the function and differentiation of rabbit mammary cells in culture
Authors: Haeuptle M , Suard YLM , Bogenmann E , Reggio H , Racine L , Kraehenbuhl J
Published: May 2004

We examined the role of cell shape, cytodifferentiation, and tissue topography on the induction and maintenance of functional differentiation in rabbit mammary cells grown as primary cultures on two-dimensional collagen surfaces or in three-dimensional collagen matrices. Mammary glands from mid-pregnant rabbits were dissociated into single cells, and epithelial cells were enriched by isopycnic centrifugation. Small spheroids of epithelial cells (approximately 50 cells) that formed on a rotary shaker were plated on or embedded in collagen gels. The cells were cultured for 1 d in serum-containing medium and then for up to 25 d in chemically defined medium. In some experiments, epithelial monolayers on gels were mechanically freed from the dishes on day 2 or 5. These gels retracted and formed floating collagen gels. On attached collagen gels, flat monolayers of a single cell type developed within a few days. The cells synthesized DNA until the achievement of confluence but did not accumulate milk proteins. No morphological changes were induced by prolactin (PRL). On floating gels, two cell types appeared in the absence of cell proliferation. The cells in direct contact with the medium became cuboidal and developed intracellular organelles typical of secretory cells. PRL-induced lipogenesis, resulting in large fat droplets filling the apical cytoplasm and accumulation of casein and α-lactalbumin in vesicles surrounding the fat droplets. We detected tranferrin in the presence or absence of PRL intracellularly in small vesicles but also in the collagen matrix in contact with the cell layer. The second cell type, rich in microfilaments and reminiscent of the myoepithelial cells, was situated between the secretory cell layer and the collagen matrix. In embedding gels, the cells formed hollow ductlike structures, which grew continuously in size. Secretory cells formed typical lumina distended by secretory products. We found few microfilament-rich cells in contact with the collagen gels. Storage and secretion of fat, caseins and alpha-lactalbumin required the presence of PRL, whereas the accumulation and vectorial discharge of transferrin was prolactin independent. There was no differentiation gradient between the tip and the cent of the outgrowth, since DNA synthesis and milk protein storage were random along the tubular structures.

These results indicate that establishment of functional polarity and induction of cytodifferentiation are influenced by the nature of the interaction of the cells with the collagen structure. The morphological differentiation in turn plays an important role in the synthesis, storage, and secretion of fat and milk proteins.
Proteoglycan and glycosaminoglycan synthesis in embryonic mouse salivary glands: effects of beta-D-xyloside, an inhibitor of branching morphogenesis.
Authors: Thompson H A , Spooner B S
Published: May 2004

The proteoglycans and glycosaminoglycans synthesized by embryonic mouse salivary glands during normal morphogenesis and in the presence of beta-xyloside, an inhibitor of branching morphogenesis, have been partially characterized. Control and rho-nitrophenyl-beta-D-xyloside-treated salivary rudiments synthesize proteoglycans that are qualitatively similar, based on mobility on Sepharose CL-4B under dissociative conditions and glycosaminoglycan composition. However, beta-xyloside inhibits total proteoglycan-associated glycosaminoglycan synthesis by 50%, and also stimulates synthesis of large amounts of free chondroitin (dermatan) sulfate. This free glycosaminoglycan accounts for the threefold stimulation of total glycosaminoglycan synthesis in beta-xyloside-treated cultures. Several observations suggest that the disruption of proteoglycan synthesis rather than the presence of large amounts of free glycosaminoglycan is responsible for the inhibition of branching morphogenesis. (a) We have been unable to inhibit branching activity by adding large amounts of chondroitin (dermatan) sulfate, extracted from beta-xyloside-treated cultures, to the medium of salivary rudiments undergoing morphogenesis. (b) In the range of 0.1-0.4 mM beta-xyloside, the dose-dependent inhibition of branching morphogenesis is directly correlated with the inhibition of proteoglycan synthesis. The stimulation of free glycosaminoglycan synthesis is independent of dose in this range, since stimulation is maximal even at the lowest concentration used, 0.1 mM. The data strongly suggest that the inhibition of branching morphogenesis is caused by the disruption of proteoglycan synthesis in beta-xyloside-treated salivary glands.
Construction of a synthetic messenger RNA encoding a membrane protein.
Authors: Rubenstein J L , Chappell T G
Published: May 2004

We have synthesized microgram quantities of a functional eucaryotic mRNA by in vitro transcription. For this purpose, we constructed a plasmid in which the Escherichia coli lactose promoter was 5' to the vesicular stomatitis virus (VSV) G protein gene (Rose, J. K., and C. J. Gallione, 1981, J. Virol., 39:519-528). This DNA served as the template in an in vitro transcription reaction utilizing E. coli RNA polymerase. The RNA product was capped using the vaccinia guanylyltransferase. A typical preparation of the synthetic G mRNA was equivalent to the amount of G mRNA that can be isolated from approximately 10(8) VSV-infected cells. This synthetic mRNA was translated by a wheat germ extract in the presence of microsomes, producing a polypeptide that was indistinguishable from G protein in its size, antigenicity, degree of glycosylation, and its membrane insertion. This technique should aid in identifying features needed by proteins for insertion into membranes.
The spectrin-related molecule, TW-260/240, cross-links the actin bundles of the microvillus rootlets in the brush borders of intestinal epithelial cells.
Authors: Glenney J R , Glenney P , Weber K
Published: May 2004

Previous studies have shown that molecules related to erythrocyte spectrin are present in the cortical cytoplasm of nonerythroid cells. We report here the localization by immunoelectron microscopy of one such molecule, TW-260/240, in the brush border of intestinal epithelial cells. Using highly specific antibodies against TW-260 and TW-240 as well as antibodies against fodrin, another spectrinlike molecule, we have found that the TW-260/240 molecules are displayed between rootlets at all levels of the terminal web. Occasionally, extended structures appear labeled suggestive of the fine filaments known to cross-link actin bundles. These results are in line with previous in vitro studies showing that TW-260/240 binds to, and cross-links, actin filaments. The results are discussed in terms of a model in which rootlets are immobilized in the terminal web in a matrix of TW-260/240.
Persistence of the mitogenic response to platelet-derived growth factor (competence) does not reflect a long-term interaction between the growth factor and the target cell.
Authors: Singh J P , Chaikin M A , Pledger W J , Scher C D , Stiles C D
Published: May 2004

Quiescent BALB/c-3T3 cells exposed briefly to platelet-derived growth factor (PDGF) become "competent" to replicate their DNA even if PDGF is removed from cell culture medium prior to the onset of DNA synthesis. We have suggested that persistence of the PDGF-induced competent state reflects a rapidly induced and relatively stable biochemical change within the target cells. Others suggest that the phenomenon reflects a long-term association between PDGF and its target cells or perhaps between PDGF and the cell culture dish. This controversy has been addressed (a) by examining the effect of anti-PDGF antibodies on PDGF-induced competence and (b) by studying the chemical fate of 125I-labeled PDGF. Anti-PDGF antibodies inactive both soluble and surface-bound PDGF. However, if quiescent 3T3 cells are exposed to PDGF for as little as 30 min, subsequent addition of these antibodies to the culture medium does not prevent the mitogenic response. Under conditions where the PDGF-induced competent state decays stochastically with a t1/2 of 18-20 h, cell-associated 125I-PDGF decays with a t1/2 of approximately 50 min. These data do not support the concept that persistence of the PDGF-induced competent state reflects a long-term association between PDGF and the target cells or between PDGF and the culture dish.
Reversible control of synaptic transmission in a single gene mutant of Drosophila melanogaster.
Authors: Koenig J H , Saito K , Ikeda K
Published: May 2004

Synaptic transmission of the single gene mutant, shibirets1 (shi), of Drosophila melanogaster is reversibly blocked by elevated temperature. The presynaptic mechanism of transmission was studied in the neuromuscular junction of the dorsal longitudinal flight muscle of this mutant. It was observed that when the temperature was raised to 29 degrees C in shi flies, the amplitude of the excitatory junction potential (EJP) greatly diminished, the frequency of spontaneously released miniature excitatory junction potentials (MEJP's) was greatly reduced, and almost complete vesicle depletion was observed. These conditions were reversible if the temperature was lowered to 19 degrees C. These data suggest that the block in transmission is a result of vesicle depletion. It is suggested that depletion occurs not as a result of excessive release of transmitter but rather as a result of a block in the recycling of vesicles, which causes depletion as exocytosis (transmitter release) proceeds normally.
Association of microtubule-associated protein 2 (MAP 2) with microtubules and intermediate filaments in cultured brain cells.
Authors: Bloom G S , Vallee R B
Published: May 2004

The classification of MAP 2 as a microtubule-associated protein is based on its affinity for microtubules in vitro and its filamentous distribution in cultured cells. We sought to determine whether MAP 2 is also able to bind in situ to organelles other than microtubules. For this purpose, primary cultures of rat brain cells were stained for immunofluorescence microscopy with a rabbit anti-MAP 2 antibody prepared in our laboratory, as well as with antibodies to vimentin, an intermediate filament protein, and to tubulin, the major subunit of microtubules. MAP 2 was present on cytoplasmic fibers in neurons and in a subpopulation of the flat cells present in the cultures. Our observations were concentrated on the flat cells because of their suitability for high-resolution immunofluorescence microscopy. Double antibody staining revealed co-localization of MAP 2 with both tubulin and vimentin in the flat cells. Pretreatment of the cultures with vinblastine resulted in the redistribution of MAP 2 into perinuclear cables that contained vimentin. Tubulin paracrystals were not stained by anti-MAP 2. In cells extracted with digitonin, the normal fibrillar distribution of MAP 2 was resistant to several treatments (PIPES buffer plus 10 mM Ca++, phosphate buffer at pH 7 or 9) that induced depolymerization of microtubules, but not intermediate filaments. Staining of the primary brain cells was not observed with preimmune serum nor with immune serum adsorbed prior to use with pure MAP 2. We detected MAP 2 on intermediate filaments not only with anti-MAP 2 serum, but also with affinity purified anti-MAP 2 and with a monoclonal anti-MAP 2 prepared in another laboratory. We conclude from these experiments that material recognized by anti-MAP 2 antibodies associates with both microtubules and intermediate filaments. We propose that one function of MAP 2 is to cross-link the two types of cellular filaments.
A domain-specific marker for the hepatocyte plasma membrane: localization of leucine aminopeptidase to the bile canalicular domain.
Authors: Roman L M , Hubbard A L
Published: May 2004

Indirect immunofluorescence was used to establish a domain-specific marker for hepatocyte plasma membranes. In frozen sections of fixed rat liver (0.5-4 microns), antibodies directed against rat intestinal leucine aminopeptidase (LAP) recognized an antigen that was restricted to the bile canalicular plasma membrane. Fluorescence was not observed on the sinusoidal or lateral membranes, and intracellular staining was not detected. The liver antigen was identified as LAP, based on its chemical similarity to intestinal LAP. First, immunoprecipitation experiments using trypsin-solubilized intestinal LAP (G-200 fraction, 91% pure) established a correlation between the loss of LAP enzyme activity from the soluble fraction and the appearance in the specific immunoprecipitates of polypeptides migrating on SDS PAGE between 110,000 and 130,000 daltons. The antigen precipitated from a detergent extract of liver plasma membranes had the same electrophoretic mobility. Second, the chymotryptic map of the major band in the liver immunoprecipitate was similar to that of purified intestinal LAP.
Cytoplasmic microtubule assembly-disassembly from endogenous tubulin in a Brij-lysed cell model.
Authors: Deery W J , Brinkley B R
Published: May 2004

We studied the characteristics of cytoplasmic microtubule reassembly from endogenous tubulin pools in situ using a Brij 58-lysed 3T3 cell system. Cells that were pretreated in vivo with colcemid retain endogenous tubulin in the depolymerized state after lysis. When lysed cells were removed from colcemid block and incubated in GTP-PIPES reassembly buffer at pH 6.9, microtubules repolymerized randomly throughout the cytoplasm, appeared to be free-ended and were generally not associated with the centrosomes. However, tubulin could be induced to polymerize in an organized manner from the centrosomes by increasing the pH to 7.6 in the presence of ATP and cAMP. Microtubules polymerized in ATP had significantly longer lengths than those assembled in GTP or UTP. When cells not treated with colcemid were lysed, the integrity of the cytoplasmic microtubule complex (CMTC) was maintained during subsequent incubation in reassembly buffer. However, in contrast to unlysed, living cells, microtubules of lysed cells were stable to colchicine. A significant fraction of the CMTC was stable to cold-induced disassembly whereas microtubules reassembled after lysis were extremely cold-sensitive. When cells not treated with colcemid were lysed and incubated in millimolar Ca++, microtubules depolymerized from their distal ends and a much reduced CMTC was observed. Ca++ reversal with EGTA rapidly resulted in a reformation of the CMTC apparently by elongation of Ca++ resistant microtubules.
Visualization of the binding, endocytosis, and transcytosis of low-density lipoprotein in the arterial endothelium in situ.
Authors: Vasile E , Simionescu M , Simionescu N
Published: May 2004

We investigated the interaction and transport of low-density lipoprotein (LDL) through the arterial endothelium in rat aorta and coronary artery, by perfusing in situ native, untagged human, and rat LDL. The latter was rendered electron-opaque after it interacted with the endothelial cell and was subsequently fixed within tissue. We achieved LDL electron-opacity by an improved fixation procedure using 3,3'-diaminobenzidine, and mordanting with tannic acid. The unequivocal identification of LDL was implemented by reacting immunocytochemically the perfused LDL with anti LDL-horseradish peroxidase conjugate. Results indicate that LDL is taken up and internalized through two parallel compartmented routes. (a) A relatively small amount of LDL is taken up by endocytosis via: (i) a receptor-mediated process (adsorptive endocytosis) that involved coated pits/vesicles, and endosomes, and, probably, (ii) a receptor-independent process (fluid endocytosis) carried out by a fraction of plasmalemmal vesicles. Both mechanisms bringing LDL to lysosomes supply cholesterol to the endothelial cell itself. (b) Most circulating LDL is transported across the endothelial cell by transcytosis via plasmalemmal vesicles which deliver LDL to the other cells of the vessel wall. Endocytosis is not enhanced by increasing LDL concentration, but the receptor-mediated internalization decreases at low temperature. Transcytosis is less modified by low temperature but is remarkably augmented at high concentration of LDL. While the endocytosis of homologous (rat) LDL is markedly more pronounced than that of heterologous (human) LDL, both types of LDL are similarly transported by transcytosis. These results indicate that the arterial endothelium possesses a dual mechanism for handling circulating LDL: by a high affinity process, endocytosis secures the endothelial cells' need for cholesterol; by a low-affinity nonsaturable uptake process, transcytosis supplies cholesterol to the other cells of the vascular wall, and can monitor an excessive accumulation of plasma LDL. Since in most of our experiments we used LDL concentrations above those found in normal rats, we presume that at low LDL concentrations saturable high-affinity uptake would be enhanced in relation to nonsaturable pathways.
Epithelial layer formation in differentiating aggregates of F9 embryonal carcinoma cells.
Authors: Grover A , Oshima R G , Adamson E D
Published: May 2004

F9 embryonal carcinoma (EC) cells, cultured in suspension in medium containing 5 X 10(-8) M retinoic acid, aggregate and differentiate into embryoid bodies with an outer layer of visceral endoderm cells that synthesize and secrete alphafetoprotein (AFP) (Hogan, B. L. M., A. Taylor, and E. Adamson, 1981, Nature (Lond.). 291:235-237). Here we analyze the formation of the outer layer of cells as a model for epithelial differentiation. Three morphological phases are described, but analyses of cell numbers and the synthetic rates of some proteins, as well as the appearance of markers of visceral endoderm and basement membrane, show that the formation of the outer layer occurs as an orderly progression of multiple events. The markers used to follow the ontogeny of epithelial layer formation include SSEA-1, l, and i blood group antigens, laminin, fibronectin, type IV collagen, cytoskeletal intermediate filament proteins (vimentin, Endo A, and B), and AFP. The onset of epithelium formation occurs between the third and fourth day of culture, but its function is maximally expressed only when it is well organized. We found the rate of AFP secretion to be a measure of the proper alignment and maturity of the epithelium which occurs at the seventh or eighth day. This model of epithelium formation may help to explain how similar processes occur during embryogenesis.
Synaptonemal complexes are integral components of the isolated mouse spermatocyte nuclear matrix.
Authors: Ierardi L A , Moss S B , Bellvé A R
Published: May 2004

Synaptonemal complexes (SCs) have been isolated as integral components of the nuclear matrix from purified mouse pachytene spermatocytes. These nuclear synaptonemal complex-matrices are prepared by extracting Triton X-100-treated nuclei with low (0.2 M) and high (1.0 or 2.0 M) NaCl, DNase I, and RNase A to remove 85% of the nuclear proteins, 97% of the RNA, and 99% of the DNA. Studies with the light and electron microscopes indicate that these matrices, while lacking a distinct lamina, contain nuclear pores interconnected by a fiber network, residual nucleoli, and interchromatin fibers. In addition, the pachytene spermatocyte matrices contain residual XY heterochromatin and the principal components of the SCs, including two lateral elements, a central element, a presumptive centromere, and attachment plaques. These SCs are preserved within the matrix and retain their structural association with the pore-fiber complex, even when subjected to strong dissociating conditions. Nuclear matrices from pachytene spermatocytes and spermatids (steps 1-8), when analyzed by SDS PAGE, contain an array of polypeptides distinct from those of mouse liver nuclear matrices. Proteins of spermatogenic matrices range in Mr from 8,000 to approximately 150,000. The prominent lamina proteins (Mr approximately 60,000-70,000) of somatic nuclear matrices are either absent or represent only a minor part of the spermatogenic matrix. The polypeptide composition of the pachytene spermatocyte and spermatid matrices are similar, although minor quantitative and qualitative differences are evident. These observations suggest that the SC constituents may consist of a heterogeneous group of proteins present in low proportion relative to total matrix proteins, or they may be retained, but in a different form, within the spermatid matrix.
Immunoelectron microscopic studies of desmin (skeletin) localization and intermediate filament organization in chicken cardiac muscle.
Authors: Tokuyasu K T , Dutton A H , Singer S J
Published: May 2004

We studied the localization of desmin (skeletin), the major protein subunit of muscle-type intermediate filaments, in adult chicken cardiac muscle by high resolution immunoelectron microscopic labeling of ultrathin frozen sections of the intact fixed tissues. We carried out single labeling for desmin and double labeling for both desmin and either vinculin or alpha-actinin. In areas removed from the intercalated disk membranes, we observed desmin labeling between adjacent Z-bands in every interfibrillar space. Where these spaces were wide and contained mitochondria, convoluted strands of desmin labeling bridged between the periphery of neighboring Z-bands and the mitochondria. The intermediate filaments appeared to be organized in a more three-dimensional manner within the interfibrillar spaces of cardiac as compared to skeletal muscle. Near the intercalated disks, desmin labeling was intense within the interfibrillar spaces, but was completely segregated from the microfilament attachment sites (fascia adherens) where vinculin and alpha-actinin were localized. Desmin therefore appears to play no role in the attachment of microfilaments to the intercalated disk membrane. We discuss the role of intermediate filaments in the organization of cardiac and skeletal striated muscle in the light of these and other results.
U1 small nuclear ribonucleoprotein studied by in vitro assembly.
Authors: Wieben E D , Madore S J , Pederson T
Published: May 2004

The small nuclear RNAs are known to be complexed with proteins in the cell (snRNP). To learn more about these proteins, we developed an in vitro system for studying their interactions with individual small nuclear RNA species. Translation of HeLa cell poly(A)+ mRNA in an exogenous message-dependent reticulocyte lysate results in the synthesis of snRNP proteins. Addition of human small nuclear RNA U1 to the translation products leads to the formation of a U1 RNA-protein complex that is recognized by a human autoimmune antibody specific for U1 snRNP. This antibody does not react with free U1 RNA. Moreover, addition of a 10- to 20-fold molar excess of transfer RNA instead of U1 RNA does not lead to the formation of an antibody-recognized RNP. The proteins forming the specific complex with U1 RNA correspond to the A, B1, and B2 species (32,000, 27,000, and 26,000 mol wt, respectively) observed in previous studies with U1 snRNP obtained by antibody-precipitation of nuclear extracts. The availability of this in vitro system now permits, for the first time, direct analysis of snRNA-protein binding interactions and, in addition, provides useful information on the mRNAs for snRNP proteins.
Localization of GTP-stimulated core glycosylation to fused microsomes.
Authors: Paiement J , Bergeron J J
Published: May 2004

Purified rough microsomes from liver maximally incorporated N-acetyl-[3H]glucosamine into endogenous acceptors from UDP-N-acetyl-[3H]glucosamine substrate, providing the associated ribosomes were removed and 0.5 mM GTP was added. These conditions also led to the coalescence of microsomes into large fused membranes. By measurement of membrane profiles on electron micrographs, a correlation was observed between GTP-stimulated glycosylation and microsomal membrane length (r2 = 0.92). Membrane fusion was not observed in the absence of GTP, with sugar transfer inhibited by greater than 90% for acid-resistant acceptors (protein), and approximately 50% for acid-labile acceptors (lipid-linked intermediates). When radiolabeled acceptors were localized by electron microscope radioautography, high concentrations of silver grains (83 grains/100 microns membrane length) were observed over fused membranes with lower grain densities observed over unfused membranes in the same preparation (20 grains/100 microns). These studies directly link microsomal membrane fusion to GTP-stimulated core glycosylation. The observations extend the suggestion of Godelaine et al. (1979, Eur. J. Biochem. 96:17-26) that physiological levels of GTP promote the translocation of substrate across endoplasmic reticulum membranes which, we propose, occurs via a membrane fusion phenomenon.
Monensin inhibits intracellular dissociation of asialoglycoproteins from their receptor.
Authors: Harford J , Wolkoff A W , Ashwell G , Klausner R D
Published: May 2004

Treatment of short-term monolayer cultures of rat hepatocytes with the proton ionophore, monensin, abolishes asialoglycoprotein degradation, despite little effect of the drug on either surface binding of ligand or internalization of prebound ligand. Centrifuging cell homogenates on Percoll density gradients indicates that, as a result of monensin treatment, ligand does not enter lysosomes but sediments instead in a lower density subcellular fraction that is likely an endocytic vesicle. Analyzing the degree of receptor association of intracellular ligand revealed that monensin prevents the dissociation of the receptor-ligand complex that normally occurs subsequent to endocytosis. The weak base, chloroquine, also blocks this intracellular dissociation. Evidence from sequential substitution experiments is presented, indicating that monensin and chloroquine act at the same point in the sequence of events leading to ligand dissociation. These data are discussed in terms of a pH-mediated dissociation of the receptor-ligand complex within a prelysosomal endocytic vesicle.
Membrane-associated phosphoproteins in Plasmodium berghei-infected murine erythrocytes.
Authors: Wiser M F , Wood P A , Eaton J W , Sheppard J R
Published: May 2004

Normal and Plasmodium berghei (NYU-2 strain)-infected murine erythrocytes display substantially different patterns of plasma membrane phosphoproteins phosphorylation. Intact erythrocytes (normal and parasite infected) incubated with 32Pi and isolated washed erythrocyte plasma membranes incubated with gamma-32P-ATP were analyzed for phosphoproteins by SDS PAGE and autoradiography. Two new phosphoproteins of molecular weight 45,000 (pp45) and 68,000 (pp68), which are absent in normal erythrocyte membranes, are associated with the membranes of infected erythrocytes subjected to both intact-cell and isolated-membrane phosphorylation conditions. Two-dimensional gel electrophoresis indicates that pp45 and pp68 are of parasite origin. Partial or complete proteolytic digestion reveals that pp45 is phosphorylated at similar amino acid residues both in intact cells and in isolated membranes. The pp45 phosphoprotein can be detected at as low as 3% parasitemia and its phosphorylation is not affected by 10 microM cAMP, 1 mM Ca2+, or 5 mM EGTA. Extraction of isolated washed plasma membranes with 0.5% Triton X-100 or 0.1 M NaOH indicates that pp45 is detergent insoluble and only partially extractable with NaOH, suggesting that pp45 is closely associated with the host erythrocyte plasma membrane.
Cytoskeletal components of the vertebrate neuromuscular junction: vinculin, alpha-actinin, and filamin.
Authors: Bloch R J , Hall Z W
Published: May 2004

We have used immunocytochemical methods to investigate the cytoskeletal constituents of the vertebrate neuromuscular junction. Specific, affinity-purified antibodies to three cytoskeletal proteins, vinculin, alpha-actinin, and filamin, bound to neuromuscular junctions in sections of normal rat, mouse, chick, and Xenopus muscles. All three antibodies bound to the synaptic regions of denervated rat muscle fibers, indicating that the proteins recognized by these antibodies are associated with postsynaptic structures. The three proteins are present at the neuromuscular junction in muscle fibers of embryonic and neonatal animals, and therefore, may play an important role in its differentiation.
Isolation of Chinese hamster ovary cell mutants requiring the continuous presence of taxol for cell division.
Authors: Cabral F R
Published: May 2004

Chinese hamster ovary (CHO) cell mutants resistant to the cytotoxic effects of taxol and requiring the drug for normal growth were isolated in a single step. One of these mutant cell lines, Tax-18, fails to divide in the absence of taxol; instead, the cells become larger, rounder, flatter, and multinucleated. Analysis by flow cytometry indicates that during taxol deprivation there is an accumulation of cells in G2 + M phase but that the cells are able to leak through the block in the absence of cell division and further increase their DNA content beyond the tetraploid amount. This interpretation is confirmed by karyotype analysis and by time-lapse studies that show cells rounded for mitosis two to five times longer than in wild-type cultures or in Tax-18 cultures grown in taxol. The cells finally attempt to undergo cytokinesis, fail, and spread out again, but as larger cells than before. Tax-18 has a normal growth rate and morphology when grown in taxol even at concentrations three to five times below the selecting concentration of the drug. The cells, however, have increased sensitivity to microtubule-disrupting drugs such as colcemid, griseofulvin, and D2O. The mutation for taxol auxotrophy behaves recessively in somatic cell hybridization experiments, and the phenotypic reversion rate is approximately 10(-5) in a nonmutagenized population. Both alpha- and beta-tubulin are present in apparently normal amounts and with normal electrophoretic mobilities on two-dimensional gels. The results suggest that Tax-18 lacks a factor necessary for mitosis and that taxol may be able to substitute for this factor.
The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies.
Authors: Nelson W G , Sun T T
Published: May 2004

The keratins are a highly heterogeneous group of proteins that form intermediate filaments in a wide variety of epithelial cells. These proteins can be divided into at least seven major classes according to their molecular weight and their immunological reactivity with monoclonal antibodies. Tissue-distribution studies have revealed a correlation between the expression of specific keratin classes and different morphological features of in vivo epithelial differentiation (simple vs. stratified; keratinized vs. nonkeratinized). Specifically, a 50,000- and a 58,000-dalton keratin class were found in all stratified epithelia but not in simple epithelia, and a 56,500- and a 65-67,000-dalton keratin class were found only in keratinized epidermis. To determine whether these keratin classes can serve as markers for identifying epithelial cells in culture, we analyzed cytoskeletal proteins from various cultured human cells by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The 56,500- and 65-67,000-dalton keratins were not expressed in any cultured epithelial cells examined so far, reflecting the fact that none of them underwent morphological keratinization. The 50,000- and 58,000-dalton keratin classes were detected in all cultured cells that originated from stratified squamous epithelia, but not in cells that originated from simple epithelia. Furthermore, human epidermal cells growing as a monolayer in low calcium medium continued to express the 50,000- and 58,000-dalton keratin classes. These findings suggest that the 50,000- and 58,000-dalton keratin classes may be regarded as "permanent" markers for stratified squamous epithelial cells (keratinocytes), and that the expression of these keratin markers does not depend on the process of cellular stratification. The selective expression of the 50,000- and 58,000-dalton keratin classes, which are synthesized in large quantities on a per cell basis, may explain the high keratin content of cultured keratinocytes.
Inhibition of DNA synthesis in SV3T3 cultures by isolated 3T3 plasma membranes.
Authors: Peterson S W , Lerch V
Published: May 2004

3T3 plasma membranes were added to subconfluent cultures of SV3T3 cells in the presence of fusogens. If this protocol results in the introduction into the SV3T3 cell membrane of 3T3 plasma membrane components responsible for density-dependent inhibition of growth, then the SV3T3 cell cultures would be expected to show decreased rates of DNA synthesis as they approach confluence. Results of these experiments indicate that rates of DNA synthesis in SV3T3 cultures so treated were as much as 63% less than in untreated controls. This effect could not be attributed to the fusogens or to the 3T3 plasma membranes alone. This growth-inhibitory effect is specific for 3T3 membranes and is not observed when SV3T3 plasma membranes are fused with SV3T3 cell cultures. These data support the hypothesis that one aspect of the loss of density-dependent inhibition of growth in SV3T3 cells is a deletion or alteration in plasma membrane components and, further, that density-dependent inhibition of growth can be in part restored to SV3T3 cell cultures by fusing the cells with 3T3 plasma membranes.
Proximodistal degeneration of C-fibers detached from their perikarya.
Authors: Cancalon P
Published: May 2004

Degeneration was followed in the garfish olfactory nerve after removal of the mucosa containing the cell bodies. Degeneration, as measured by a decrease in the weight of consecutive 3-mm nerve segments, spreads at constant velocity from the site of injury toward the synaptic area. The proximodistal degeneration is temperature dependent and progresses from 0.3 mm/d at 10 degrees C to 13.0 mm/d at 35 degrees C. Between 14 and 35 degrees C, the velocity increases linearly with temperature. At all the temperatures investigated, these proximodistal degeneration velocities are identical to the rates of slow intraaxonal flow measured in axons detached from their cell bodies, or to the rates measured in regenerating fibers, and, except at 10 degrees C, are 3.3 times faster than the rate of slow flow in intact nerves. These results were confirmed by light and electron microscopy. We hypothesize that the collapse and subsequent degeneration of the axons is the result of a proximodistal depletion of cytoskeletal elements no longer provided by the cell body to the axon by slow intraaxonal flow. A significant number of axons disappeared rapidly from the nerve before the arrival of the slow degenerative wave. From studies by other groups, this rapid degeneration may be the result of a lack of rapidly transported, mainly membranous components.
Maturation-promoting factor induces nuclear envelope breakdown in cycloheximide-arrested embryos of Xenopus laevis.
Authors: Miake-Lye R , Newport J , Kirschner M
Published: May 2004

We have studied the effect of maturation-promoting factor (MPF) on embryonic nuclei during the early cleavage stage of Xenopus laevis development. When protein synthesis is inhibited by cycloheximide during this stage, the embryonic cell cycle arrests in an artificially produced G2 phase-like state, after completion of one additional round of DNA synthesis. Approximately 100 nuclei can be arrested in a common cytoplasm if cytokinesis is first inhibited by cytochalasin B. Within 5 min after injection of MPF into such embryos, the nuclear envelope surrounding each nucleus disperses, as determined histologically or by immunofluorescent staining of the nuclear lamina with antilamin antiserum. The breakdown of the nuclear envelope occurs at levels of MPF comparable to or slightly lower than those required for oocyte maturation. Amplification of MPF activity, however, does not occur in the arrested egg as it does in the oocyte. These results suggest that MPF can act to advance interphase nuclei into the first events of mitosis and show that the nuclear lamina responds rapidly to MPF.
Reconstitution of cytochrome f/b6 and CF0-CF1 ATP synthetase complexes into phospholipid and galactolipid liposomes.
Authors: Mörschel E , Staehelin L A
Published: May 2004

Cytochrome f/b6 and ATP synthetase (CF0-CF1) complexes from spinach chloroplasts have been reconstituted into liposomes prepared from soybean phospholipids and purified spinach galactolipids. Freeze-fracture analysis revealed homogeneous populations of particles spanning the lipid bilayers with their elongated axes perpendicular to the membrane plane. The lipid composition of the liposomes had no effect on the size of the reconstituted complexes, the average diameter of cytochrome f/b6 complex measuring 8.5 nm, and of the CF0 base piece of the ATP synthetase, 9.5 nm. When reconstituted cytochrome f/b6 complexes were cross-linked by means of antibodies prepared against the whole complex, the thus aggregated particles formed either hexagonal or square arrays. In both instances the center-to-center spacing of the particles was 8.3 nm, thereby suggesting that this value could be closer to the real diameter of the complexes than the one obtained from measuring individual particles. Assuming an ellipsoidal shape for these particles, and using a measured height of 11 nm, a molecular weight of approximately 280,000 could be calculated for the reconstituted cytochrome f/b6 complex, consistent with a dimeric configuration. In many instances the crystalline sheets of antibody-aggregated cytochrome f/b6 complexes were found to be free in the buffer solution; apparently the antibody-induced strains caused the sheet-like aggregates to pop out of the liposomal membranes. Agglutination studies of inside-out and right-side-out thylakoid vesicles revealed the antigenic determinants of the cytochrome f and cytochrome b6 polypeptides to be exposed on the inner thylakoid surface and to be present in stacked and unstacked membrane regions. The molecular weight calculated from the size of freeze-fractured CF0 base pieces was over twice the value determined by x-ray scattering data. This discrepancy may be caused by significant lipid domains within the base piece, or by an unusual fracturing behavior of the base piece in reconstituted liposomes.
G2 cell cycle arrest induced by glycopeptides isolated from the bovine cerebral cortex.
Authors: Charp P A , Kinders R J , Johnson T C
Published: May 2004

The ability of glycopeptides, isolated from bovine cerebral cortex, to alter cell division was studied by cell-cycle analyses. The results showed that glycopeptides arrested baby hamster kidney (BHK)-21 cells and Chinese hamster ovary (CHO) cells in the G2 phase of the cell cycle. Upon removal of the growth inhibition from arrested BHK-21 cells, the mitotic index in colchicine-treated cultures increased from 5 to 40% within 6 h and the increase in mitotic activity was accompanied by a complete doubling of all arrested cells within this 6-h time period. Determination of DNA content in growth-arrested BHK-21 cells showed that growth-arrested cells contained about twice the DNA of control cell cultures. Although CHO cells treated in a like manner with growth inhibitor could not be arrested for the same length of time as BHK-21 cells (18 h vs. 72 h before initiation of escape) and to the same degree (60% of the cell population vs. 99% of BHK-21 cells), the escape kinetics of CHO cells did indicate a G2 arrest. Approximately 3.5 h after escape began, CHO cell numbers in treated cultures attained the cell numbers found in control cultures. This rapid growth phase occurring in less than 4 h indicated that the growth inhibitor induced a G2 arrest-point in CHO cells that was not lethal since the entire arrested cell population divided.
Acanthamoeba discriminates internally between digestible and indigestible particles.
Authors: Bowers B , Olszewski T E
Published: May 2004

The capacity of Acanthamoeba to distinguish nutritive yeast particles from non-nutritive plastic beads during phagocytosis was investigated. When cells were allowed to phagocytose yeast to capacity, endocytosis stopped and subsequent presentation of particles (either yeast or beads) did not result in further uptake. By contrast, when cells were allowed to phagocytose plastic beads to capacity and a second dose of particles was presented (either yeast or beads), the cells exocytosed the internal particles and took up new ones. Yeast rendered indigestible by extensive chemical cross-linking were taken up at rates similar to those of untreated yeast, but, like beads, they were exocytosed when a second dose of particles was presented. The results show that an internal distinction is made between vacuoles containing yeast and vacuoles containing plastic beads, and they are consistent with the hypothesis that the presence within the vacuoles of material capable of being digested prevents exocytosis.
Alteration of membrane barrier in stripped rough microsomes from rat liver on incubation with GTP: its relevance to the stimulation by this nucleotide of the dolichol pathway for protein glycosylation.
Authors: Godelaine D , Beaufay H , Wibo M , Ravoet A M
Published: May 2004

The membrane barrier of stripped rough microsomes from rat liver is markedly altered on incubation with GTP at 37 degrees C: after 30 min the structure-linked latency of mannose-6-phosphatase was considerably reduced, and esterase and nucleoside diphosphatase were partly released into the suspension medium. This phenomenon was already maximal with 30 microM GTP and was specific for this nucleotide. Similar conditions enhance the dolichol-mediated glycosylation of protein in microsomes incubated with uridine diphosphate N-acetylglucosamine and guanosine diphosphate mannose (Godelaine, D., H. Beaufay, M. Wibo, and A. Amar-Costesec, 1979, Eur. J. Biochem., 96:17-26; Godelaine, D., H. Beaufay, and M. Wibo, 1979, Eur. J. Biochem., 96:27-34). The GTP-induced permeability and glycosylation activities evolved in parallel in rough microsomes subjected to various treatments to detach the ribosomes and were maximal after removal of congruent to 60% of the RNA. In addition, GTP had no effect of this type in smooth microsome subfractions. Triton X-100, in spite of complex inhibitory effects on glycosylation reactions, mimicked the action of GTP by increasing the amount of microsomal dolichylphosphate that reacts with uridine diphosphate N-acetylglucosamine and by enhancing synthesis of dolichylpyrophosphoryl-chitobiose at concentrations greater than 2 mg/ml. Thus, GTP may activate dolichol-mediated glycosylation reactions in stripped microsomes by lowering the permeability barrier that prevents access of sugar nucleotides to the inner aspect of the membrane. The genuine role of GTP in the functioning of the endoplasmic reticulum membrane in situ remains unknown. Because GTP seems to act only on rough microsomes, we hypothesize that this role is somehow related to biosynthesis of protein by the rough endoplasmic reticulum.
Functional activity of enucleated human polymorphonuclear leukocytes.
Authors: Roos D , Voetman A A , Meerhof L J
Published: May 2004

Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells.