bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the development and maintenance of epithelial cell surface polarity with monoclonal antibodies.
Authors: Herzlinger D A , Ojakian G K
Published: May 2004

We examined epithelial cell surface polarity in subconfluent and confluent Madin-Darby canine kidney (MDCK) cells with monoclonal antibodies directed against plasma membrane glycoproteins of 35,000, 50,000, and 60,000 mol wt. The cell surface distribution of these glycoproteins was studied by immunofluorescence and immunoelectron microscopy. At the ultrastructural level, the electron-dense reaction product localizing all three glycoproteins was determined to be uniformly distributed over the apical and basal cell surfaces of subconfluent MDCK cells as well as on the lateral surfaces between contacted cells; however, after formation of a confluent monolayer, these glycoproteins could only be localized on the basal-lateral plasma membrane. The development of cell surface polarity was followed by assessing glycoprotein distribution with immunofluorescence microscopy at selected time intervals during growth of MDCK cells to form a confluent monolayer. These results were correlated with transepithelial electrical resistance measurements of tight junction permeability and it was determined by immunofluorescence that polarized distributions of cell surface glycoproteins were established just after electrical resistance could be detected, but before the development of maximal resistance. Our observations provide evidence that intact tight junctions are required for the establishment of the apical and basal-lateral plasma membrane domains and that development of epithelial cell surface polarity is a continuous process.
Ca2+-dependent binding of severin to actin: a one-to-one complex is formed.
Authors: Giffard R G , Weeds A G , Spudich J A
Published: May 2004

Severin is a protein from Dictyostelium that severs actin filaments in a Ca2+-dependent manner and remains bound to the filament fragments (Brown, S. S., K. Yamamoto, and J. A. Spudich , 1982, J. Cell Biol., 93:205-210; Yamamoto, K., J. D. Pardee , J. Reidler , L. Stryer , and J. A. Spudich , 1982, J. Cell Biol. 95:711-719). Further characterization of the interaction of severin with actin suggests that it remains bound to the preferred assembly end of the fragmented actin filaments. Addition of severin in molar excess to actin causes total disassembly of the filaments and the formation of a high-affinity complex containing one severin and one actin. This severin -actin complex does not sever actin filaments. The binding of severin to actin, measured directly by fluorescence energy transfer, requires micromolar Ca2+, as does the severing and depolymerizing activity reported previously. Once bound to actin in the presence of greater than 1 microM Ca2+, severin is not released from the actin when the Ca2+ is lowered to less than 0.1 microM by addition of EGTA. Tropomyosin, DNase I, phalloidin, and cytochalasin B have no effect on the ability of severin to bind to or sever actin filaments. Subfragment 1 of myosin, however, significantly inhibits severin activity. Severin binds not only to actin filaments, but also directly to G-actin, as well as to other conformational species of actin.
Appearance of chemotactic responsiveness to elastin peptides by developing fetal bovine ligament fibroblasts parallels the onset of elastin production.
Authors: Mecham R P , Griffin G L , Madaras J G , Senior R M
Published: May 2004

We studied chemotaxis to elastin peptides by bovine ligamentum nuchae fibroblasts to determine whether there is a developmental association between chemotactic responsiveness to elastin and expression of the elastin phenotype. Undifferentiated ligament cells demonstrate chemotactic responsiveness to platelet-derived growth factor and fibronectin, known chemoattractants for fibroblasts, but do not show chemotaxis to elastin peptides. After matrix-induced differentiation, however, young cells display a positive chemotactic response to elastin that persists even after the cells are removed from the matrix substratum. Matrix-induced chemotaxis to elastin could be inhibited selectively by incorporation of bromodeoxyuridine into DNA of undifferentiated cells before (but not after) contact with inducing matrix. These results show that the appearance of chemotaxis to elastin peptides parallels the onset of elastin synthesis and suggests that the acquisition of chemotactic responsiveness to elastin and expression of the elastin phenotype are affected by the same inducing elements or processes and may be closely coupled in development.
A localized surface protein of guinea pig sperm exhibits free diffusion in its domain.
Authors: Myles D G , Primakoff P , Koppel D E
Published: May 2004

Using the technique of fluorescence redistribution after photobleaching, we are studying the cellular mechanisms involved in localizing surface molecules to particular domains. A number of antigens localized to discrete surface regions have been identified with monoclonal antibodies on guinea pig sperm cells ( Primakoff , P., and D. G. Myles , 1983, Dev. Biol., 98:417-428). One of these monoclonal antibodies, PT-1, binds exclusively to the posterior tail region of the sperm cell surface. PT-1 recognizes an integral membrane protein that in complex with n-octyl-beta-D-glucopyranoside has a sedimentation coefficient of 6.8S in sucrose density gradients. Fluorescence redistribution after photobleaching measurements reveal that within its surface domain the PT-1 antigen diffuses rapidly (D = 2.5 X 10(-9) cm2/s) and completely (greater than 90% recovery after bleaching). These results rule out for this membrane protein all models that invoke immobilization as a mechanism for maintaining localization. We propose that the mechanism for localization of the PT-1 antigen may be a barrier to diffusion at the domain boundary.
Phosphoinositide metabolism and the morphology of human erythrocytes.
Authors: Ferrell J E , Huestis W H
Published: May 2004

ATP-depleted human erythrocytes lose their smooth discoid shape and adopt a spiny, crenated form. This shape change coincides with the conversion of phosphatidylinositol-4,5-bisphosphate to phosphatidylinositol and phosphatidic acid to diacylglycerol. Both crenation and lipid dephosphorylation are accelerated by iodoacetamide, and both are reversed by nutrient supplementation. The observed changes in lipid populations should shrink the membrane inner monolayer by 0.6%, consistent with estimates of bilayer imbalance in crenated cells. These observations suggest that metabolic crenation arises from a loss of inner monolayer area secondary to the degradation of phosphatidylinositol-4,5-bisphosphate and phosphatidic acid. A related process, crenation after Ca2+ loading, appears to arise from a loss inositides by a different pathway.
Density of newly synthesized plasma membrane proteins in intracellular membranes II. Biochemical studies.
Authors: Quinn P , Griffiths G , Warren G
Published: May 2004

Using two independent methods, incorporation of radioactive amino-acid and quantitative immunoblotting, we have determined that the rate of synthesis of each of the Semliki Forest virus (SFV) proteins in infected baby hamster kidney (BHK) cells is 1.2 X 10(5) copies/cell/min. Given the absolute surface areas of the endoplasmic reticulum and Golgi complex presented in the companion paper (Griffiths, G., G. Warren, P. Quinn , O. Mathieu - Costello , and A. Hoppeler , 1984, J. Cell Biol. 98:2133-2141), and the approximate time spent in these organelles during their passage to the plasma membrane (Green J., G. Griffiths, D. Louvard , P. Quinn , and G. Warren 1981, J. Mol. Biol. 152:663-698), the mean density of each viral protein in these organelles can be calculated to be 90 and 750 molecules/micron 2 membrane, respectively. In contrast, we have determined that the density of total endogenous integral membrane proteins in these organelles is approximately 30,000 molecules/micron 2 so that the spike proteins constitute only 0.28 and 2.3% of total membrane protein in the endoplasmic reticulum and Golgi, respectively. Quantitative immunoblotting was used to give direct estimates of the concentrations of one of the viral membrane protein precursors (E1) in subcellular fractions; these agreed closely with the calculated values. The data are discussed with respect to the sorting of transported proteins from those endogenous to the intracellular membranes.
Immunocytochemical analysis of intermediate filaments in embryonic heart cells with monoclonal antibodies to desmin.
Authors: Danto S I , Fischman D A
Published: May 2004

Monoclonal antibodies ( McAbs ) have been generated against a preparation of intermediate filament proteins (IFP) from adult chicken gizzard. Two antibodies, D3 and D76 , have been characterized in detail. They bind specifically to desmin but recognize different epitopes. In the adult chicken, both McAbs produced equivalent immunofluorescent staining patterns, reacting in frozen sections with all forms of muscle tissue, including vascular smooth muscle, but with no other tissue types. In isolated skeletal myofibrils and in longitudinal frozen sections of cardiac and skeletal muscle, desmin was detected with both McAbs at the Z-band and in longitudinally-oriented filament bundles between myofibrils. In contrast to these results in the adult, the intermediate filaments (IF) of embryonic cardiac myocytes in primary cultures were decorated only with McAb D3, whereas McAb D76 was completely unreactive with these cells. Similarly, frozen sections through the heart at early stages of embryonic chick development (Hamburger-Hamilton stages 17-18) revealed regions of myocytes, identified by double immunofluorescence with myosin-specific McAbs , that were unstained with McAb D76 even though similar regions were stained by McAb D3. That McAb D76 reacted with desmin in all adult cardiac myocytes but not with all embryonic heart cells indicates that embryonic and adult cardiac IF are immunologically distinct and implies a conversion in IF immunoreactivity during cardiac development.
Variants deficient in phagocytosis of latex beads isolated from the murine macrophagelike cell line J774.
Authors: Takasaki S , Emling F , Leive L
Published: May 2004

Variants that lack the ability to ingest latex beads were isolated from the mouse macrophagelike cell line J774. Carboxylated latex beads were derivatized with polylysine and then daunomycin by a carbodiimide method. Cells that ingested such beads were killed; variants that survived were isolated. Variants were detected at very low frequency and only after nitrosoguanidine mutagenesis. Of 11 independent isolates, 10 showed a lowered rate of uptake of polystyrene beads (without daunomycin). All of these proved normal in rate and extent of Fc-mediated phagocytosis. There was essentially no change in sensitivity to free daunomycin in the variants compared to the parent. These results support the previous hypothesis that there are differences in the metabolic routes of receptor-mediated and nonspecific phagocytosis.
Mouse myeloma cells that make short immunoglobulin heavy chains: pleiotropic effects on glycosylation and chain assembly.
Authors: Kenter A L , Warren T , Shields D , Birshtein B K
Published: May 2004

Two variants in immunoglobulin heavy chain production, derived from the MPC 11 mouse myeloma cell line, make short heavy (H) chains with identical precise deletions of the CH3 domain. The CH3 domain is expressed in the H chain mRNA from both variants. Although in vitro translation of this mRNA produces one H chain species, deleted heavy chains are secreted as heavy-light (HL) and H2L2 moieties in contrast to MPC 11, which secretes only H2L2 . The heavy chains of HL apparently contain more carbohydrate (CHO+) than do the H chains of H2L2 , and inhibition of N-linked glycosylation results in the secretion of relatively more H2L2 . Here we present evidence suggesting that (a) the absence of the CH3 domain has led to conformational changes in these molecules, (b) these changes permit posttranslational glycosylation, and (c) unrestrained glycosylation can frequently yield unusual CHO+ structures that make complete assembly unlikely.
The role of intermolecular disulfide bonding in deposition of GP140 in the extracellular matrix.
Authors: Carter W G
Published: May 2004

Human WI-38 fibroblasts in cultures synthesized at least three molecular forms of the major, extracellular matrix glycoprotein (GP), GP140: (a) cytoplasmic GP140 (1.2 ng of GP140/micrograms of cell protein) was detergent-soluble, underglycosylated, and possessed detectable levels of intermolecular disulfide bonding; (b) matrix GP140 (3.6 ng of GP140/micrograms of cell protein) was detergent-insoluble, more highly glycosylated and polymerized by intermolecular disulfide bonding, and co-distributed in the extracellular matrix with fibronectin; and (c) released GP140 (2 ng of GP140/micrograms of cell protein per 24 h) was recovered in the conditioned culture media and lacked intermolecular disulfide bonding. Cytoplasmic GP140 was the immediate biosynthetic precursor of the matrix form of GP140. In addition, various human adult and fetal tissues contained a form of GP140 that resembled the fibroblast matrix GP140 in the degree of intermolecular disulfide bonding, relative molecular mass, and immunological reactivity. Analysis of the sequence of events in assembly of GP140 and fibronectin in the extracellular matrix detected the following: (a) fibronectin was first to appear in the extracellular matrix; (b) GP140 accumulated in the cytoplasm, then deposited in the extracellular matrix and co-aligned with the established fibronectin; and (c) maturation of the extracellular matrix proceeded by continued intermolecular disulfide bonding. To evaluate possible roles for intermolecular disulfide bonding in cell interactions, a unique assay system was utilized based on the ability of labeled cells to incorporate radioactive matrix components into a biotinylated exogenous matrix. Precipitation of the biotinylated matrix from extracts of the cultures using avidin indicated: (a) disulfide bonding of radioactive GP140 and fibronectin into the exogenous biotinylated matrix required cell contact with the matrix. The newly deposited GP140 and fibronectin derived from the cells and not from GP140 and fibronectin present in the conditioned culture media. (b) Pro-alpha 1 and Pro-alpha 2 procollagens, present in the culture media, bound to the exogenous matrix in a noncovalent manner and were independent of cell contact. (c) SV40 transformed cells (WI-38 VA13) synthesized released form GP140 but did not deposit GP140 into the biotinylated matrix.
Induction of chondrogenesis in limb mesenchymal cultures by disruption of the actin cytoskeleton.
Authors: Zanetti N C , Solursh M
Published: May 2004

Cell shape is known to influence the chondrogenic differentiation of cultured limb bud mesenchyme cells (Solursh, M., T. F. Linsenmayer, and K. L. Jensen, 1982, Dev. Biol., 94: 259-264). To test whether specific cytoskeletal components mediate this influence of cell shape, we examined different cytoskeleton disrupting agents for their ability to affect chondrogenesis. Limb bud cells cultured at subconfluent densities on plastic substrata normally become flattened, contain numerous cytoplasmic microtubules and actin bundles, and do not undergo spontaneous chondrogenesis. If such cultures are treated with 2 micrograms/ml cytochalasin D during the initial 3-24 h in culture, the cells round up, lose their actin cables, and undergo chondrogenesis, as indicated by the production of immunologically detectable type II collagen and a pericellular Alcian blue staining matrix. Cytochalasin D also induces cartilage formation by high-density cultures of proximal limb bud cells, which normally become blocked in a protodifferentiated state. In addition, cytochalasin D was found to reverse the normal inhibition by fibronectin of chondrogenesis by proximal limb bud cells cultured in hydrated collagen gels. Agents that disrupt microtubules have no apparent effect on the shape or chondrogenic differentiation of limb bud mesenchymal cells. These results suggest an involvement of the actin cytoskeleton in controlling cell shape and chondrogenic differentiation of limb bud mesenchyme. Interactions of the actin cytoskeleton and extracellular matrix components may provide a regulatory mechanism for mesenchyme cell differentiation into cartilage or fibrous connective tissue in the developing limb.
Ferritin immunoelectron microscopic localization of 5'-nucleotidase on rat liver cell surface.
Authors: Matsuura S , Eto S , Kato K , Tashiro Y
Published: May 2004

Rat livers were prefixed by perfusion with 0.6% glutaraldehyde and briefly homogenized with a Teflon-glass homogenizer. The prefixed cells isolated by low-speed centrifugation in high yield effectively preserved the original polygonal shape and polarity. These cells were incubated with ferritin-antibody conjugates monospecific for rat liver 5'-nucleotidase, and the localization of the enzymes on the surface of hepatocytes and endothelial cells was quantitatively investigated. It was revealed that the surface density of 5'-nucleotidase is much higher on the bile canalicular surface than on the sinusoidal surface and only a few ferritin particles were detected on the lateral surface. On the bile canalicular surface ferritin particles were almost exclusively found on the microvilli in larger clusters. Similar distribution was also observed on the sinusoidal surface but the size of cluster was much smaller. On both surfaces many fewer ferritin particles were found on the intermicrovillar region, including the coated pits region, than on the microvillar region. Ferritin particles were also found on the endothelial cell surface.
Substituted benzyl acetates: a new class of compounds that reduce gap junctional conductance by cytoplasmic acidification.
Authors: Spray D C , Nerbonne J , Campos de Carvalho A , Harris A L , Bennett M V
Published: May 2004

Conductance of gap junctions in many preparations has been shown to be sensitive to cytoplasmic pH, decreasing as pH decreases below 7.5 in fish and amphibian embryos and below 7.1 in crayfish septate axon. We have found a new class of compounds, benzyl acetate derivatives, that reversibly decrease junctional conductance, gj, when applied in low concentration (approximately 1 mM). Simultaneous intracellular pH (pHi) measurements show that the ester effects are attributable to reduction in pHi. The sensitivity of gj to these compounds and the relative lack of side effects make these agents attractive for studies of the role played by gap junctions in normal tissue function. In addition, the finding of cytoplasmic acidification in response to cell exposure to esters suggests caution in interpretation of results obtained using esterified compounds for intracellular loading.
Durable synthesis of high molecular weight heat shock proteins in G0 cells of the yeast and other eucaryotes.
Authors: Iida H , Yahara I
Published: May 2004

We report that eucaryotic cells were induced to synthesize a specific class of heat shock proteins (hsps) when they entered the resting state, G0. This finding was originally made with Saccharomyces cerevisiae cells by taking advantage of the system in which we can distinguish between G1 arrests leading to G0 and those that do not result in G0 (Iida, H., and I. Yahara, 1984, J. Cell Biol. 98:1185-1193). Similar observations were subsequently made with higher eucaryotic cells including chick embryonic fibroblasts (CEF), mouse T lymphocytes, and Drosophila GM1 cells. The induction of hsps in G0 cells was distinct from that in heat-shocked cells in two respects. First, hsps with molecular weight around 25,000 were not induced in G0 cells, whereas most, if not all, high molecular weight (HMW) hsps were commonly induced both in G0 cells and in heat-shocked cells. Second, in contrast to the transient synthesis of hsps in heat-shocked cells, G0 cells continued to synthesize hsps at the stimulated rate for a relatively long period. These results suggest the possibility that high molecular weight hsps might function in a transition from the proliferating state to G0 or in maintaining G0 in the eucaryote.
Acanthamoeba castellanii capping protein: properties, mechanism of action, immunologic cross-reactivity, and localization.
Authors: Cooper J A , Blum J D , Pollard T D
Published: May 2004

We report further characterization of the physical and immunologic properties, mechanism of action, and intracellular localization of Acanthamoeba castellanii capping protein, an actin regulatory protein discovered by Isenberg (Isenberg, G., U. Aebi, and T. D. Pollard, 1980, Nature (Lond.) 288:455-459). The native molecular weight calculated from measurements of Stokes' radius (3.8 nm by gel filtration chromatography) and sedimentation coefficient (4.8 S by sucrose gradient velocity sedimentation) was 74,000 daltons. The subunit molecular weights were 31,000 and 28,000 daltons, so the native molecule is a heterodimer. The two subunits did not immunologically cross-react with each other or with any other proteins from Acanthamoeba or several other organisms. In studies of the mechanism of action, Isenberg (see above reference) found that capping protein blocked polymerization from the barbed end of actin filaments and sedimented with actin filaments. We confirmed that capping protein binds to actin filaments with a gel filtration assay. Capping protein decreased the length distribution and high shear viscosity of actin filaments. Capping protein did not bundle or cross-link actin filaments. Low concentrations of capping protein increased the critical concentration for muscle and ameba actin polymerization from 0.1 to 0.6 microM in Mg++ and EGTA. Increasing amounts of capping protein did not increase the critical concentration further. In Ca++ capping protein did not change the critical concentration for muscle actin, but did increase the critical concentration for ameba actin. Ca++ had no effect on the ability of capping protein to decrease the low or high shear viscosity of actin filaments. By indirect fluorescent antibody staining, capping protein was localized to the cell cortex, an area rich in actin filaments. During subcellular fractionation of homogenates, about 1/3 of cellular capping protein banded with a crude membrane fraction. The other 2/3 of cellular capping protein was soluble, with a Stokes' radius equal to that of the purified protein. The molar ratio of capping protein to actin in the cell was 1:150.
Interactions between glycolytic enzymes and components of the cytomatrix.
Authors: Masters C
Published: May 2004

Evidence is provided that enzymes absorb to cellular structures in a wide range of tissues. In particular, the interactions between glycolytic enzymes and the microfilaments of the cytoplasm are described. The relevance of these interactions to the compartmentation of carbohydrate metabolism is discussed. Examples are given of the variations in degree of binding during alteration of tissue metabolism and, for individual glycolytic enzymes, during fetal development and differentiation. Overall, these data support the concept that metabolic activities in the cytoplasm have an organized structure. Just as the structural elements of the cytosolic compartment have evolved with the capacity to assemble and disassemble in response to the changing requirements of the organism, so the metabolic elements appear to have evolved a parallel system that provides for the appropriate positioning of an energy-producing sequence in relation to the specific, dynamic requirements of the cytoskeleton.
A cytological approach to the ordering of events in gene activation using the Sgs-4 locus of Drosophila melanogaster.
Authors: Steiner E K , Eissenberg J C , Elgin S C
Published: May 2004

The polytene chromosomes of Drosophila strains that differ in the synthesis of the major salivary gland glue protein sgs-4 were examined by indirect immunofluorescence using antisera to several nonhistone chromosomal proteins. The Oregon-R X chromosome, which produces sgs-4 messenger RNA, showed a strong fluorescent band at locus 3C11-12 when stained with anti-RNA polymerase II, whereas the null mutant Berkeley 1 failed to exhibit fluorescence at that locus. The presence of another antigen (Band 2), normally associated with developmentally active loci, was clearly evident at locus 3C11-12 of both transcriptionally competent and null strains, indicating that the association of Band 2 antigen with the chromatin is an event independent of RNA polymerase II binding. Antibodies directed against Drosophila topoisomerase I stained 3C11-12 in the Sgs-4+ (wild-type) strain brightly, but gave significantly less staining in the null strain. This indicates that the high concentrations of topoisomerase I seen at active loci are closely associated with the transcriptional event. In some of these analyses, we have made use of flies heterozygous for the wild-type and null alleles in order to make internally controlled comparisons. The results suggest that this type of analysis will enable conclusions to be drawn concerning the interdependence and order of action of chromosomal proteins involved in developmental gene activation.
Denervation supersensitivity in skeletal muscle: analysis with a cloned cDNA probe.
Authors: Merlie J P , Isenberg K E , Russell S D , Sanes J R
Published: May 2004

Motor neurons regulate the acetylcholine sensitivity of the muscles they innervate: denervated muscle fiber become "supersensitive" to acetylcholine, due to insertion of newly synthesized acetylcholine receptors (AChRs) in the plasma membrane. We used hybridization analysis with a cloned cDNA specific for AChR alpha-subunit to compare the abundance of AChR mRNA in innervated and denervated adult mouse muscles. Within 3 d of denervation, levels of AChR mRNA increased 100-fold; levels of actin mRNA changed little. The increase in AChR mRNA level was sufficiently large and rapid to account for denervation supersensitivity.
The eucaryotic aminoacyl-tRNA synthetase complex: suggestions for its structure and function.
Authors: Deutscher M P
Published: May 2004

Aminoacyl-tRNA synthetases from eucaryotic cells generally are isolated as high molecular weight complexes comprised of multiple synthetase activities, and often containing other components as well. A model is proposed for the synthetase complex in which hydrophobic extensions on the proteins serve to maintain them in their high molecular weight form, but are not needed for catalytic activity. The structural similarity of these enzymes to certain membrane-bound proteins, and its implications for synthetase localization and function in vivo, are discussed.
Biosynthesis of heparan sulfate proteoglycan by human colon carcinoma cells and its localization at the cell surface.
Authors: Iozzo R V
Published: May 2004

After 24 h of continuous labeling with radioactive precursors, a high molecular weight heparan sulfate proteoglycan (HS-PG) was isolated from both the medium and cell layer of human colon carcinoma cells (WiDr) in culture. The medium HS-PG eluted from a diethylaminoethyl anion exchange column with 0.45-0.50 M NaCl, had an average density of 1.46-1.49 g/ml on dissociative CsCl density-gradient ultracentrifugation, and eluted from Sepharose CL-2B with a Kav = 0.57. This proteoglycan had an estimated Mr of congruent to 8.5 X 10(5), with glycosaminoglycan chains of Mr = 3 X 10(4) which were all susceptible to HNO2 deaminative cleavage. Deglycosylation of the HS-PG with polyhydrogen fluoride resulted in a 3H-core protein with Mr congruent to 2.4 X 10(5). The cell layer contained a population of HS-PG with characteristics almost identical to that released into the medium but with a larger Mr = 9.5 X 10(5). Furthermore, an intracellular pool contained smaller heparan sulfate chains (Mr congruent to 1 X 10(4)) which were mostly devoid of protein core. In pulse chase experiments, only the large cell-associated HS-PG was released (approximately 58%) into the medium as intact proteoglycan and/or internalized and degraded (approximately 42%), with a t1/2 = 6 h. However, the small intracellular component was never released into the medium and was degraded at a much slower rate. When the cells were subjected to mild proteolytic treatment, only the large cell-associated HS-PG, but none of the small component, was displaced. Addition of exogenous heparin did not displace any HS-PG into the medium. Both light and electron microscopic immunocytochemistry revealed that the cell surface reacted with antibody against an HS-PG isolated from a basement membrane-producing tumor. Electron microscopic histochemistry using ruthenium red and/or cuprolinic blue revealed numerous 10-50-nm diam granules and 70-220-nm-long electron-dense filaments, respectively, on the surface of the tumor cells. The results indicate that colon carcinoma cells synthesize HS-PGs with distinct structural and metabolic characteristics: a large secretory pool with high turnover, which appears to be synthesized as an integral membrane component and localized primarily at the cell surface, and a small nonsecretory pool with low turnover localized predominantly within the cell interior. This culture system offers an opportunity to investigate in detail the mechanisms involved in the regulation of proteoglycan metabolism, and in the establishment of the neoplastic phenotype.
Association of newly synthesized islet prohormones with intracellular membranes.
Authors: Noe B D , Moran M N
Published: May 2004

Results from recent studies have indicated that pancreatic islet prohormone converting enzymes are membrane-associated in islet microsomes and secretory granules. This observation, along with the demonstration that proglucagon is topologically segregated to the periphery within alpha cell secretory granules in several species, led us to investigate the possibility that newly synthesized islet prohormones might be associated with intracellular membranes. Anglerfish islets were incubated with [3H]tryptophan and [14C]isoleucine for 3 h, then fractionated by differential and density gradient centrifugation. Microsome (M) and secretory granule (SG) fractions were halved, sedimented, and resuspended in the presence or absence of dissociative reagents. After membrane lysis by repeated freezing and thawing, the membranous and soluble components were separated by centrifugation. Extracts of supernatants and pellets were chromatographed by gel filtration; fractions were collected and counted. A high proportion (77-79%) of the newly synthesized proinsulin and insulin was associated with both M and SG membranes. Most of the newly synthesized proglucagons and prosomatostatins (12,000-mol-wt precursors) were also membrane-associated (86-88%) in M and SG. In contrast, glucagon- and somatostatin-related peptides exhibited much less membrane-association in SG (24-31%). Bacitracin, bovine serum albumin EDTA, RNAse, alpha-methylmannoside, N-acetylglucosamine, and dithiodipyridine had no effect on prohormone association with membranes. However, high salt (1 M KCl) significantly reduced membrane-association of prohormones. Binding of labeled prohormones to SG membranes from unlabeled tissue increased with incubation time and was inhibited by unlabeled prohormones. The pH optimum for prohormone binding to both M and SG membranes was 5.2. It is suggested that association of newly synthesized prohormones with intracellular membranes could be related to the facilitation of proteolytic processing of prohormones and/or transport from their site of synthesis to the secretory granules.
Amino acid control of autophagic sequestration and protein degradation in isolated rat hepatocytes.
Authors: Seglen P O , Gordon P B
Published: May 2004

Sequestration of the inert cytosolic marker [14C]sucrose by sedimentable organelles was measured in isolated rat hepatocytes made transiently permeable to sucrose by means of electropermeabilization. Lysosomal integrity, protein degradation, autophagic sequestration, and other cellular functions were not significantly impaired by the electric treatment. Hepatocytes sequestered sucrose at an initial rate of approximately 10%/h, which is threefold higher than the estimated rate of autophagic-lysosomal protein degradation. Almost one-third would appear to represent mitochondrial fluid uptake; the rest was nearly completely and specifically inhibited by 3-methyladenine (3MA) and can be regarded as autophagic sequestration. A complete amino acid mixture was somewhat less inhibitory than 3MA, and partially antagonized the effect of the latter. This paradoxical effect, taken together with the high sequestration rate, may suggest heterogeneity as well as selectivity in autophagic sequestration. There was no detectable recycling of sequestered [14C]sucrose between organelles and cytosol. Studies of individual amino acids revealed histidine as the most effective sequestration inhibitor. Leucine may have a regulatory function, as indicated by its unique additive/synergistic effect, and a combination of Leu + His was as effective as the complete amino acid mixture. Asparagine inhibited sequestration only 20%, i.e., its very strong effect on overall (long-lived) protein degradation must partially be due to post-sequestrational inhibition. The lysosomal (amine-sensitive) degradation of short-lived protein was incompletely inhibited by 3MA, indicating a contribution from nonautophagic processes like crinophagy and endocytic membrane influx. The ability of an amino acid mixture to specifically antagonize the inhibition of short-lived protein degradation by AsN + GIN (but not by 3MA) may suggest complex amino acid interactions at the level of fusion between lysosomes and other vesicles in addition to the equally complex interactions at the level of autophagic sequestration.
Isolation and characterization of epinectin, a novel adhesion protein for epithelial cells.
Authors: Enenstein J , Furcht L T
Published: May 2004

A 70,000-mol-wt protein was isolated from A431 carcinoma cell extracellular matrix that promotes cell substratum adhesion of these epidermoid tumor cells. Extracellular matrix was isolated by a modification of a procedure described by Hedman et al. (Hedman, K., M. Kurkinen, K. Alitalo, A. Vaheri, S. Johansson, and M. Höök, 1979 J. Cell Biol., 81:83-91) and Yamada and Weston (Yamada, K., and J. A. Weston, 1974, Proc. Natl. Acad. Sci. USA, 71:3492-3496). Cells were solubilized with 0.5% deoxycholate, 10 mM Tris, 0.9% NaCl, and 1 mM phenylmethylsulfonyl fluoride, pH 8.0. The residual matrix was then removed from the plates with 6 M urea and 1 mM phenylmethylsulfonyl fluoride and phosphate-buffered saline. SDS PAGE gels of the 6 M urea extract showed one major band at 70,000-mol-wt by Coomassie Blue staining. A 70,000-mol-wt isotopically-labeled band could also be extracted from the matrix of cells incubated with [35S]methionine. Because of the presence of this protein on squamous-derived epithelial cells we have called the 70,000-mol-wt molecule epinectin. Indirect immunofluorescence with polyclonal rabbit antibodies against epinectin stained A431 cells pericellularly in dense punctate accumulations and along the plasma membrane. Enzyme-linked immunoassays and gel-transfer immunolocalization studies showed that the extract did not cross-react with antibodies to fibronectin, laminin, serum-spreading factor, epibolin, or keratin. Additionally, antibodies to epinectin did not cross-react with these proteins. Further studies showed that epinectin does not bind to gelatin. Cell-adhesion assay, using radiolabeled A431 carcinoma cells on various adhesion-promoting substrates, showed that epinectin has similar adhesion-promoting capacity as serum-spreading factor, was somewhat less active than fibronectin, but more effective than laminin or epibolin. Epinectin appears to be a unique protein isolated from epidermoid tumor cells that is distinct from other known adhesion proteins.
Identification and characterization of osteoclast-like cells and their progenitors in cultures of feline marrow mononuclear cells.
Authors: Ibbotson K J , Roodman G D , McManus L M , Mundy G R
Published: May 2004

The predominant cell responsible for bone resorption, the multinucleated osteoclast, has been difficult to study because of inaccessibility. When feline marrow-derived mononuclear cells are established in long-term culture, multinucleated cells form within 48 h, reaching maximum numbers at 16 d. We have observed that these cultured cells have many of the features of osteoclasts. Morphologically, they are multinucleated, contain large numbers of branched mitochondria, have a peripheral cytoplasm lacking organelles (a clear zone), and have extensive cell-surface processes. In addition to these ultrastructural features, the cells contain a tartrate-resistant acid phosphatase, the activity of which is increased by parathyroid hormone (PTH) and inhibited by calcitonin. PTH, prostaglandin E2, and 1,25(OH)2 vitamin D3 increased multinucleated cell formation, while calcitonin inhibited the stimulatory effects of PTH. Time-lapse cinemicrographic and autoradiographic studies indicated that the multinucleated cells formed by fusion of the mononuclear progenitors. The multinucleated cells were phagocytic and stained with nonspecific esterase, consistent with their being derived from immature monocytes. Further, cell populations enriched for multinucleated cells release 45Ca from devitalized bone. Density-gradient centrifugation on Percoll was used to enrich and characterize the mononuclear progenitors of these multinucleated cells. The progenitor cells were found predominantly in Percoll density layers of 1.065 to 1.08 g/ml and were enriched up to 30-fold as compared to unfractionated cells. The bone marrow mononuclear cells that formed the multinucleated cells were initially nonadherent to plastic, stained heavily with nonspecific esterase, and appeared to be immature monocytes histologically. These data indicate that the multinucleated osteoclast-like cells in our cultures are derived from nonadherent monocytic progenitor cells that are responsive to osteotropic hormones. The ability to grow and characterize these cells in vitro should facilitate studies to elucidate the role these cells play in normal and pathologic states of bone resorption.
Membrane protein damage and repair: removal and replacement of inactivated 32-kilodalton polypeptides in chloroplast membranes.
Authors: Ohad I , Kyle D J , Arntzen C J
Published: May 2004

Incubation of Chlamydomonas reinhardii cells at light levels that are several times more intense than those at which the cells were grown results in a loss of photosystem II function (termed photoinhibition). The loss of activity corresponded to the disappearance from the chloroplast membranes of a lysine-deficient, herbicide-binding protein of 32,000 daltons which is thought to be the apoprotein of the secondary quinone electron acceptor of photosystem II (the QB protein). In vivo recovery from the damage only occurred following de novo synthesis (replacement) of the chloroplast-encoded QB protein. We believe that the turnover of this protein is a normal consequence of its enzymatic function in vivo and is a physiological process that is necessary to maintain the photosynthetic integrity of the thylakoid membrane. Photoinhibition occurs when the rate of inactivation and subsequent removal exceeds the rate of resynthesis of the QB protein.
Transcellular ion currents in the water mold Achlya. Amino acid proton symport as a mechanism of current entry.
Authors: Kropf D L , Caldwell J H , Gow N A , Harold F M
Published: May 2004

Achlya, like other tip-growing organisms, generates an endogenous electrical current such that positive charge flows into the hyphal apex and exits from the trunk. The present study is concerned with the mechanism of current generation by hyphae growing in a defined, complete medium. The intensity of the current, measured in the extracellular medium with a vibrating probe, was unaffected by the removal of all the inorganic constituents of the growth medium. However, an increase in the external pH or the deletion of amino acids abolished the current. Removal of methionine alone diminished the current by two thirds. Hyphae also generated a longitudinal pH gradient in the extracellular medium; the region surrounding the tip was more alkaline than the bulk medium, whereas the region around the trunk was relatively acidic. These findings suggest that a flux of protons, dependent upon amino acids in the medium, carries current into the tip and creates the surrounding alkaline zone. The proton current appears to result from the transport of amino acids rather than their metabolism. Conditions that abolished the current also inhibited methionine uptake but had little effect on the respiratory rate. The findings imply a connection between the proton current and chemiosmotic energy transduction. We propose that protons flow into the hyphal tip through amino acid/proton symporters that are preferentially localized there. The proton flux energizes the uptake of amino acids into the growing zone and may also contribute to the polarization of hyphal growth.
A cell surface integral membrane glycoprotein of 85,000 mol wt (gp85) associated with triton X-100-insoluble cell skeleton.
Authors: Tarone G , Ferracini R , Galetto G , Comoglio P
Published: May 2004

The Triton X-100-insoluble skeleton of baby hamster kidney BHK cells consists of the nucleus, intermediate-size filaments, and actin fibers. By transmission electron microscopy, membrane fragments were found to be associated with these insoluble structures. When radioiodinated or [3H]glucosamine-labeled cells were extracted with 0.5% Triton, most plasma membrane glycoproteins were solubilized except for a glycoprotein with a molecular weight of 85,000 (gp85) that remained associated with the insoluble skeletons. Immunoprecipitation with a specific antiserum indicated that the gp85 is not a proteolytic degradation product of fibronectin, an extracellular matrix glycoprotein insoluble in detergent. A monoclonal antibody of BHK cells specific for gp85 was produced. Immunofluorescence analysis with this monoclonal antibody indicated that gp85 is not associated with the extracellular matrix, but is confined to the cell membrane. Both in fixed and unfixed intact cells, fluorescence was concentrated in dots preferentially aligned in streaks on the cell surface. Gp85 was found to behave as an integral membrane protein interacting with the hydrophobic core of the lipid bilayer since it was extracted from membrane preparations by ionic detergents such as SDS, but not by 0.1 N NaOH (pH 12) in the absence of detergents, a condition known to release peripheral molecules. Association of gp85 with the cell skeleton was unaffected by increasing the Triton concentration up to 5%, but it was affected when actin filaments were dissociated or when a protein-denaturing agent (6 M urea) was used in the presence of Triton, suggesting that protein-protein interactions are involved in the association of gp85 with the cell skeleton. We conclude that gp85 is an integral plasma membrane glycoprotein that might have a role in cell surface-cytoskeleton interaction.
Glycosyltransferase activities in Golgi complex and endoplasmic reticulum fractions isolated from African trypanosomes.
Authors: Grab D J , Ito S , Kara U A , Rovis L
Published: May 2004

Highly enriched Golgi complex and endoplasmic reticulum fractions were isolated from total microsomes obtained from Trypanosoma brucei, Trypanosoma congolense, and Trypanosoma vivax, and tested for glycosyltransferase activity. Purity of the fractions was assessed by electron microscopy as well as by biochemical analysis. The relative distribution of all the glycosyltransferases was remarkably similar for the three species of African trypanosomes studied. The Golgi complex fraction contained most of the galactosyltransferase activity followed by the smooth and rough endoplasmic reticulum fractions. The dolichol-dependent mannosyltransferase activities were highest for the rough endoplasmic reticulum, lower for the smooth endoplasmic reticulum, and lowest for the Golgi complex. Although the dolichol-independent form of N-acetylglucosaminyltransferase was essentially similar in all the fractions, the dolichol-dependent form of this enzyme was much higher in the endoplasmic reticulum fractions than in the Golgi complex fraction. Inhibition of this latter activity in the smooth endoplasmic reticulum fraction by tunicamycin A1 suggests that core glycosylation of the variable surface glycoprotein may occur in this organelle and not in the rough endoplasmic reticulum as previously assumed.
Estrogen stimulates the transient association of calmodulin and myosin light chain kinase with the chicken liver nuclear matrix.
Authors: Simmen R C , Dunbar B S , Guerriero V , Chafouleas J G , Clark J H , Means A R
Published: May 2004

Previous work has demonstrated that estrogen administration to immature chickens results in a rapid but transient increase in nuclear estrogen receptor content, a large portion of which is associated with the nuclear matrix. The present studies were undertaken to determine whether estrogen produced a more generalized change in the protein composition of the nuclear matrix. High-resolution two-dimensional gel analysis of the matrix revealed a very complex protein pattern, but several major qualitative differences were observed after estrogen treatment. To simplify the number of proteins evaluated, we examined the effects of estrogen on a subset of matrix proteins, namely, calmodulin and its binding proteins. Calmodulin was measured by radioimmunoassay and the binding proteins were detected by interaction of 125I-calmodulin with matrix proteins distributed on one-dimensional polyacrylamide gels. Calmodulin and two specific Ca2+-dependent calmodulin-binding proteins were found to be associated with matrix preparations. The two binding proteins exhibited apparent Mr of 200,000 and 130,000. The Mr 130,000 protein was identified as myosin light chain kinase on the basis of enzymatic activity and immunoreactivity with a specific antibody to this enzyme. Estrogen treatment of immature chickens did not alter the hepatic content of calmodulin. However, the steroid did result in an enrichment of the proportion of calmodulin and its two binding proteins associated with the nuclear matrix within 4 h after injection. The time course of these changes paralleled those previously documented for estrogen receptor. Taken together, these data are compatible with a role for calmodulin and myosin light chain kinase in the response of chicken liver cells to steroid hormones.
Fetal bovine bone cells synthesize bone-specific matrix proteins.
Authors: Whitson S W , Harrison W , Dunlap M K , Bowers D E , Fisher L W , Robey P G , Termine J D
Published: May 2004

We isolated cells from both calvaria and the outer cortices of long bones from 3- to 5-mo bovine fetuses. The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type 1 collagen. In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by immunoprecipitation and fluorography of SDS polyacrylamide gels. Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (approximately 94%) type I collagen, with small amounts of types III and V collagens. In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and beta-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen. These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro.