bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of vinculin-binding proteins with an 125I-vinculin gel overlay technique.
Authors: Otto J J
Published: May 2004

Vinculin is an adhesion plaque component localized on the cytoplasmic side of the cell membrane where stress fibers end. To detect vinculin-binding proteins, we have developed an 125I-vinculin gel overlay method. SDS PAGE was used to separate different protein preparations. After fixing the proteins in the gel with methanol-acetic acid, the SDS was removed with ethanol and the proteins renatured in buffer. The gel was then incubated with 125I-vinculin. After extensive washing to remove nonspecifically associated label, the gel was dried and autoradiographed. Chick embryo fibroblasts, their Rous sarcoma virus transformants, and HeLa cells were found to contain two proteins (Mr 220,000 and 130,000) that bound 125I-vinculin strongly and another (Mr 42,000) that bound it moderately. The 130,000-mol-wt protein was identified as vinculin itself, which suggests that it may self-associate. The 42,000-mol-wt protein was identified as actin with which vinculin is known to interact. The identity of the 220,000-mol-wt protein is not known. It is not cellular fibronectin, myosin, or filamin. When fibroblast proteins were separated into Triton X-100 soluble and insoluble fractions, most of the vinculin and the 220,000-mol-wt protein was found to be in the soluble fraction. Chicken gizzard also contained these vinculin-binding proteins along with three others of Mr 190,000, 170,000, and 100,000.
Failure to detect immunocytochemically reactive endogenous lectin on the cell surface of Dictyostelium discoideum.
Authors: Erdos G W , Whitaker D
Published: May 2004

The endogenous lectins of Dictyostelium discoideum, called discoidins I and II, have been implicated in cell cohesion during the associative phase of this organism. In an effort to repeat and extend the studies of these putative cell-surface proteins, we attempted a variety of immunocytochemical techniques. Antibodies to a mixture of the purified discoidins were raised in rabbit. Both living and fixed cells were examined by indirect immunoferritin labeling using whole antiserum and by direct immunolabeling using purified specific IgG adsorbed to colloidal gold. Cells, at the appropriate stage, of strains A3, NC-4, and WS-582 were tested. In no instance were cell surface antigens detected despite meticulous efforts to duplicate the published techniques and to extend and refine them. Specific localization was found only in the cytosol and on the cytoplasmic face of certain endomembrane vesicles, and much less so on outer nuclear and mitochondrial membranes, in inadvertently disrupted cells. In no case was specific label found on either side of the plasma membrane or on food vacuoles. Exogenously supplied discoidins, bound to cells, were successfully localized by our technique. We conclude that the discoidins are not present on the cell surface, or are there in undetectable quantities, during the associative phase. We suggest that previous demonstrations of these proteins at the cell surface were artifacts resulting from the way in which the cells were handled, which caused the binding of externalized discoidins, possibly those released from lysed cells. We believe that the current notion that the discoidins play a direct role in cell cohesion by virtue of their carbohydrate-binding capacity should be reexamined. We suggest that the true role of the discoidins is solely intracellular.
Regionalization of transmembrane glycoproteins in the plasma membrane of boar sperm head is revealed by fracture-label.
Authors: Aguas A P , Pinto da Silva P
Published: May 2004

We used fracture-label and surface labeling techniques to characterize the distribution and topology of wheat germ agglutinin (WGA) receptors in the plasma membrane of boar sperm heads. We show that freeze-fracture results in preferential, but not exclusive, partition of WGA-binding sites with the outer (exoplasmic) half of the plasma membrane. Labeling of the inner (protoplasmic) half of the membrane is significant, and is denser over the areas that overlie the acrosome. Exoplasmic membrane halves are uniformly labeled. Analysis of freeze-fracture replicas revealed that the distribution of intramembrane particles over protoplasmic faces parallels that of WGA-binding sites as observed by fracture-label. Coating of intact spermatozoa with cationized ferritin results in drastic reduction of the labeling of both protoplasmic and exoplasmic membrane halves. Labeling of sperm cells lysed by short hypotonic shock fails to reveal the presence of WGA-binding sites at the inner surface of the plasma membrane. We conclude that: (a) all WGA-binding glycoconjugates are exposed at the outer surface of the membrane; (b) some of these glycoconjugates correspond to transmembrane glycoproteins that, on fracture, partition with the inner half of the membrane; (c) these transmembrane proteins are accumulated in the region of the plasma membrane that overlies the acrosome; and (d) parallel distribution of intramembrane particles and WGA-binding glycoproteins provides renewed support for the view of particles as the morphological counterpart of integral membrane proteins.
Association between the mammalian 110,000-dalton heat-shock protein and nucleoli.
Authors: Subjeck J R , Shyy T , Shen J , Johnson R J
Published: May 2004

A rabbit antiserum has been prepared using as antigen the 110,000-dalton mammalian heat-shock protein. This protein was purified for injection by two-dimensional PAGE of heat-shocked Chinese hamster ovary cells. Characterization by immunoautoradiography and immunoprecipitation reveals that the antiserum is specific for the 110,000-dalton protein. Both techniques also reveal that the protein against which the antiserum is directed is induced by heat shock. Indirect immunofluorescence shows that the antigen is primarily localized at or near the nucleolus in cultured cells and numerous murine tissues. Treatment of cultured cells with deoxyribonuclease destroys the organization of staining within the nucleus while ribonuclease appears to completely release the antigen from the nucleus. A binding of the antiserum to cytoplasmic structures is also observed by immunofluorescence. This association with nucleoli may have implications in the regulatory aspects of the heat-shock response.
Biochemical studies of the excitable membrane of paramecium tetraurelia. IX. Antibodies against ciliary membrane proteins.
Authors: Eisenbach L , Ramanathan R , Nelson D L
Published: May 2004

The excitable ciliary membrane of Paramecium regulates the direction of the ciliary beat, and thereby the swimming behavior of this organism. One approach to the problem of identifying the molecular components of the excitable membrane is to use antibodies as probes of function. We produced rabbit antisera against isolated ciliary membranes and against partially purified immobilization antigens derived from three serotypes (A, B, and H), and used these antisera as reagents to explore the role of specific membrane proteins in the immobilization reaction and in behavior. The immobilization characteristics and serotype cross-reactivities of the antisera were examined. We identified the antigens recognized by these sera using immunodiffusion and immunoprecipitation with 35S-labeled ciliary membranes. The major antigen recognized in homologous combinations of antigen-antiserum is the immobilization antigen (i-antigen), approximately 250,000 mol wt. Several secondary antigens, including a family of polypeptides of 42,000-45,000 mol wt, are common to the membranes of serotypes A, B, and H, and antibodies against these secondary antigens can apparently immobilize cells. This characterization of antiserum specificity has provided the basis for our studies on the effects of the antibodies on electrophysiological properties of cells and electron microscopic localization studies, which are reported in the accompanying paper. We have also used these antibodies to study the mechanism of cell immobilization by antibodies against the i-antigen. Monovalent fragments (Fab) against purified i-antigens bound to, but did not immobilize, living cells. Subsequent addition of goat anti-Fab antibodies caused immediate immobilization, presumably by cross-linking Fab fragments already bound to the surface. We conclude that antigen-antibody interaction per se is not sufficient for immobilization, and that antibody bivalency, which allows antigen cross-linking, is essential.
Mitogenic polypeptide of the mammalian seminiferous epithelium: biochemical characterization and partial purification.
Authors: Feig L A , Klagsbrun M , Bellvé A R
Published: May 2004

A mitogenic polypeptide, previously identified in Sertoli cells of the prepuberal mouse (Feig, L. A., A. R. Bellvé, N. Horbach-Erickson, and M. Klagsbrun, 1980, Proc. Natl. Acad. Sci. USA., 77:4774-4778), now has been shown to exist in Sertoli cells of the adult mouse and in the seminiferous epithelium of several other mammalian species, including the rat, guinea pig, and calf. The levels of this seminiferous growth factor (SGF) are not appreciably reduced in adult mouse testes following hypophysectomy. SGF purified from either the adult mouse or newborn calf seminiferous epithelium has a molecular weight (Mr) of 15,700 and a pl between 4.8 and 5.8, when exposed to denaturing conditions. Furthermore, SGF from these two mammalian species probably has few exposed hydrophobic domains and has a strong propensity to aggregate into multiple, high Mr species. A purification sequence based on these biochemical properties has enabled a greater than 350-fold enrichment of SGF activity from the calf seminiferous epithelium. The protocol involves a sequence of: (a) ammonium sulfate precipitation, (b) DEAE-cellulose ion exchange chromatography, (c) gel filtration chromatography on Bio-Gel P150 in 1.0 M ammonium acetate, (d) hydrophobic chromatography on dodecyl agarose, and (e) gel filtration chromatography in 6.0 M guanidine hydrochloride. Subsequent analysis of this purified preparation by SDS PAGE, followed by silver staining, reveals approximately 7 polypeptides with Mr between 14,000 and 20,000.
High-resolution three-dimensional views of membrane-associated clathrin and cytoskeleton in critical-point-dried macrophages.
Authors: Aggeler J , Takemura R , Werb Z
Published: May 2004

We obtained high-resolution topographical information about the distribution of clathrin and cytoskeletal filaments on cytoplasmic membrane surfaces of macrophages spreading onto glass coverslips by both critical-point drying of broken-open cells and preparation of rotary platinum replicas. Irregular patches of the adherent ventral surface of the plasma membrane were exposed in these cells, and large areas of these exposed membranes were covered with clathrin-coated patches, pits, and vesicles. Various amounts of cytoskeleton were attached to the plasma membranes of these spreading cells, either as distinct starlike foci, or as individual filaments and bundles radiating out from the cytoskeletal meshwork. In newly adherent cells a well developed Golgi-GERL complex, characterized by smooth, dish-like cisternae associated with rough endoplasmic reticulum, was observed. There were many coated vesicles budding off from the Golgi cisternae, and these were predominantly of the large type (150 nm) usually associated with the plasma membrane. In critical-point-dried samples, both cytoskeleton and membranes were preserved in detail comparable to that of quick-frozen samples, after appropriate fixation. Rotary replication of critical-point-dried cells provides a rapid, easily controlled, and generally easy to perform method for obtaining samples of exposed membrane large enough to permit quantification of membrane-associated clathrin and cytoskeleton under various experimental conditions.
Fibronectin-mediated binding and phagocytosis of polystyrene latex beads by baby hamster kidney cells.
Authors: McAbee D D , Grinnell F
Published: May 2004

The binding and phagocytosis of fibronectin (pFN)-coated latex beads by baby hamster kidney (BHK) cells was studied as a function of fibronectin concentration and bead diameter. Cells were incubated with radioactive pFN-coated beads, and total bead binding (cell surface or ingested) was measured as total radioactivity associated with the cells. Of the bound beads, those that also were phagocytosed were distinguished by their insensitivity to release from the cells by trypsin treatment. In continuous incubations, binding of pFN-coated beads to cells occurred at 4 degrees C or 37 degrees C, but phagocytosis was observed only at 37 degrees C. In addition, degradation of 3H-pFN from ingested beads occurred at 37 degrees C, as shown by the release of trichloroacetic acid-soluble radioactivity into the incubation medium. When the fibronectin density on the beads was varied, binding at 4 degrees C and ingestion at 37 degrees C were found to have the same dose-response dependencies, which indicated that pFN densities that permitted bead binding were sufficient for phagocytosis to occur. The fibronectin density for maximal binding of ingestion was approximately 250 ng pFN/cm2. When various sized beads (0.085-1.091 micron), coated with similar densities of pFN, were incubated with cells at 4 degrees C, no variation in binding as a function of bead size was observed. Under these conditions, the absolute amount of pFN ranged from less than 100 molecules on the 0.085-micron beads to greater than 15,000 molecules on the 1.091-micron beads. Based upon these results it can be concluded that the critical parameter controlling fibronectin-mediated binding of latex beads by BHK cells is the spacing of the pFN molecules on the beads. Correspondingly, it can be suggested that the spacing between pFN receptors on the cell surface that is optimal for multivalent interactions to occur is approximately 18 nM. When phagocytosis of various sized beads was compared, it was found that the largest beads were phagocytosed slightly better (two fold) than the smallest beads. This occurred both in continuous incubations of cells with beads and when the beads were prebound to the cells. Finally, the kinetic constants for the binding of 0.085 microM pFN-coated beads to the cells were analyzed. There appeared to be approximately 62,000 binding sites and the KD was 4.03 X 10(-9) M. Assuming a bivalent interaction, it was calculated that BHK cells have approximately 120,000 pFN receptors/cell and the binding affinity between pFN and its receptor is approximately 6 X 10(-5) M.
Reappearance of beta-adrenergic receptors after isoproterenol treatment in intact C6-cells.
Authors: Hertel C , Staehelin M
Published: May 2004

The reappearance of beta-adrenergic receptors in C6-glioma cells after desensitization with isoproterenol was studied using the antagonist [3H]CGP-12177. Reappearance had the following properties: (a) it occurred in intact cells only, (b) it was temperature dependent, (c) it required an Na+/H+ gradient, low intracellular Ca2+ activity, and (d) it required ATP, and (e) intact lysosomes. The results suggest endocytosis and recycling of the beta-adrenergic receptor after agonist treatment.
Intracellular and transcellular transport of secretory component and albumin in rat hepatocytes.
Authors: Sztul E S , Howell K E , Palade G E
Published: May 2004

The intra- and transcellular transports of hepatic secretory and membrane proteins were studied in rats in vivo using [3H]fucose and [35S]cysteine as metabolic precursors. Incorporated radioactivity in plasma, bile, and liver subcellular fractions was measured and the labeled proteins of the Golgi complex, bile, and plasma were separated by SDS PAGE and identified by fluorography. 3H-radioactivity in Golgi fractions peaked at 10 min postinjection (p.i.) and then declined concomitantly with the appearance of labeled glycoproteins in plasma. Maximal secretion of secretory fucoproteins from Golgi occurred between 10 and 20 min p.i. In contrast, the clearance of labeled proteins from Golgi membrane subfractions occurred past 30 min p.i., indicating that membrane proteins leave the Golgi complex at least 30 min later than the bulk of content proteins. A major 80,000-dalton form of secretory component (SC) was identified in the bile by co-precipitation with (IgA)2 by an anti-IgA antibody. An antibody (raised in rabbit) against the biliary 80,000-dalton peptide recognized two larger forms (116,000 and 94,000 dalton), presumably precursors, in Golgi membranes. A comparative study of kinetics of transport of 35S-SC and 35S-albumin showed that albumin peaked in bile at approximately 45 min p.i., whereas the SC peak occurred at 80 min p.i., suggesting that the transit time differs for plasma and membrane proteins that are delivered to the bile canaliculus.
An actin-depolymerizing protein (depactin) from starfish oocytes: properties and interaction with actin.
Authors: Mabuchi I
Published: May 2004

Physico-chemical properties and interaction with actin of an actin-depolymerizing protein from mature starfish oocytes were studied. This protein, which is called depactin, exists in a monomeric form under physiological conditions. Its molecular weight is approximately 20,000 for the native protein and approximately 17,000 for denatured protein. The Glu + Asp/Lys + Arg molar ratio of this protein is 1.55. The apparent pl of the denatured depactin is approximately 6. The extent of actin polymerization is reduced by the presence of depactin; however, the rate of polymerization seems to be accelerated as measured spectrophotometrically at 238nm. This effect is interpreted to indicate that depactin cut the newly formed filaments into small fragments, thereby increasing the number of the filament ends to which monomers are added. The apparent critical concentration of actin for polymerization, as determined by viscometry or flow birefringence measurement, is increased by the presence of depactin in a concentration-dependent manner. Raising the pH of the solution does not reverse the action of depactin. The molar ratio of actin and depactin, which interact with each other, is estimated to be 1:1 by means of a cross-linking experiment using a water-soluble carbodiimide. Depactin binds to a DNase I-Sepharose column via actin and is selectively eluted with 0.6 M KCl or 0.6 M Kl. The association constant between actin and depactin is estimated, using the column, to be 2-3 X 10(6) M-1. The content of depactin in the high-speed supernatant of the oocyte extract is determined to be 1%; this can act upon approximately 63% of the actin in the supernatant.
Newly synthesized G protein of vesicular stomatitis virus is not transported to the cell surface during mitosis.
Authors: Warren G , Featherstone C , Griffiths G , Burke B
Published: May 2004

Indirect immunofluorescence, immunoelectron microscopy, and digestion by protease were used to study intracellular transport of the G protein of vesicular stomatitis virus in mitotic and interphase cells. Quantitation showed that the appearance of G protein on the surface of mitotic cells was inhibited at least 10-fold when compared with that on interphase cells, even though similar amounts of viral protein were being synthesized. This dramatic inhibition, taken together with the simultaneous inhibition of endocytosis (Berlin, R. D., and J. M. Oliver, 1980, J. Cell Biol. 85: 660-671), points to a general cessation of membrane traffic in the mitotic cell.
Migration of rat RNA polymerase I into chick erythrocyte nuclei undergoing reactivation in chick-rat heterokaryons.
Authors: Scheer U , Lanfranchi G , Rose K M , Franke W W , Ringertz N R
Published: May 2004

Transcriptionally inactive chick erythrocyte nuclei were reactivated by Sendai virus-induced fusion of erythrocytes with rat L6J1 myoblasts. We used antibodies to trace the appearance of a specific protein engaged in transcription of a defined class of genes, those coding for rRNA, during reactivation. Using immunofluorescence microscopy, we found increasing amounts of rat RNA polymerase I to appear, during a certain period of time after fusion, in the reforming nucleoli of the chick nuclei. Amounts of rat RNA polymerase I sufficient to be detected by immunofluorescence microscopy had accumulated in the newly developed chick nucleoli 72-190 h after fusion was initiated. This time interval coincides with the time when chick rRNA synthesis can first be detected. The results raise the possibility that during these stages of the reactivation process chick rRNA genes are transcribed by heterologous RNA polymerase I molecules of rat origin.
Analysis of suborganellar fractions from spinach and pea chloroplasts for calmodulin-binding proteins.
Authors: Roberts D M , Zielinski R E , Schleicher M , Watterson D M
Published: May 2004

Purified chloroplasts from spinach and pea leaves were subfractionated into envelope, thylakoid, and stroma fractions and were analyzed for calmodulin-binding proteins using a 125I-calmodulin gel overlay assay. Calmodulin binding was primarily associated with a major polypeptide (Mr 33,000) in the envelope membrane fraction. In contrast, major calmodulin-binding proteins were not detected in the thylakoid or stroma fractions. Our results provide the first evidence of calmodulin-binding proteins in the chloroplast envelope, and raise the possibility that calmodulin may contribute to the regulation of chloroplast function through its interaction with calmodulin-binding proteins in the chloroplast envelope. In addition, our results combined with those of other investigators support the proposal that subcellular organelles may be a primary site of calmodulin action.
In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices.
Authors: Montesano R , Orci L , Vassalli P
Published: May 2004

We have studied the behavior of cloned capillary endothelial cells grown inside a three dimensional collagen matrix. Cell monolayers established on the surface of collagen gels were covered with a second layer of collagen. This induced the monolayers of endothelial cells to reorganize into a network of branching and anastomosing capillary-like tubes. As seen by electron microscopy, the tubes were formed by at least two cells (in transverse sections) delimiting a narrow lumen. In addition, distinct basal lamina material was present between the abluminal face of the endothelial cells and the collagen matrix. These results showed that capillary endothelial cells have the capacity to form vessel-like structures with well-oriented cell polarity in vitro. They also suggest that an appropriate topological relationship of endothelial cells with collagen matrices, similar to that occurring in vivo, has an inducive role on the expression of this potential. This culture system provides a simple in vitro model for studying the factors involved in the formation of new blood vessels (angiogenesis).
Studies on the motility of the foraminifera. II. The dynamic microtubular cytoskeleton of the reticulopodial network of Allogromia laticollaris.
Authors: Travis J L , Kenealy J F , Allen R D
Published: May 2004

Lamellipodia have been induced to form within the reticulopodial networks of Allogromia laticollaris by being plated on positively charged substrata. Video-enhanced, polarized light, and differential interference contrast microscopy have demonstrated the presence of positively birefringent fibrils within these lamellipodia. The fibrils correspond to the microtubules and bundles of microtubules observed in whole-mount transmission electron micrographs of lamellipodia. Microtubular fibrils exhibit two types of movements within the lamellipodia: lateral and axial translocations. Lateral movements are often accompanied by reversible lateral associations between adjacent fibrils within a lamellipodium. This lateral association-dissociation of adjacent fibrils has been termed 'zipping' and 'unzipping'. Axial translocations are bidirectional. The axial movements of the microtubular fibrils can result in the extension of filopodia by pushing against the plasma membrane of the lamellipodia. Shortening, or complete withdrawal, of such filopodia is accomplished by the reversal of the direction of the axial movement. The bidirectional streaming characteristic of the reticulopodial networks also occurs within the lamellipodia. In these flattened regions the streaming is clearly seen to occur exclusively in association with the intracellular fibrils. Transport of both organelles and bulk hyaline cytoplasm occurs bidirectionally along the fibrils.
Subcellular distribution of signal recognition particle and 7SL-RNA determined with polypeptide-specific antibodies and complementary DNA probe.
Authors: Walter P , Blobel G
Published: May 2004

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL-RNA. The particle was previously shown to function in protein translocation across and protein integration into the endoplasmic reticulum membrane. Polypeptide specific antibodies were raised in rabbits against the 72,000-, 68,000-, and 54,000-mol-wt polypeptide of SRP. All three antibodies are shown to neutralize SRP activity in vitro. A solid phase radioimmune assay is described and used to follow SRP in various cell fractions. The partitioning of SRP is shown to be dependent on the ionic conditions of the fractionation. Under conditions approximating physiological ionic strength, SRP is found to be about equally distributed between a membrane associated (38%) and a free (15%) or ribosome associated (47%) state. Furthermore, it is shown that greater than 75% of the total cellular 7SL-RNA is associated with SRP polypeptide in these fractions. Thus it is likely that the major--if not the only--cellular function of 7SL-RNA is as a part of SRP.
Mass distributions of coated vesicles isolated from liver and brain: analysis by scanning transmission electron microscopy.
Authors: Steven A C , Hainfeld J F , Wall J S , Steer C J
Published: May 2004

Populations of coated vesicles purified from bovine brain (BCV) and from rat liver (LCV) have been characterized with respect to the parameters of mass and diameter by analysis of scanning transmission electron micrographs of unstained specimens. Coated vesicles from both sources are heterogeneous, particularly in their masses. The respective distributions, compiled from mass measurements of many individual particles, are complex and markedly different. BCV range from 20 Mdaltons to approximately 100 Mdaltons with a weighted average of 35 Mdaltons: most BCV (80%) lie between 20 and 40 Mdaltons, including peaks at approximately 26 Mdaltons and at approximately 34 Mdaltons. In contrast, LCV masses tend to be substantially higher, ranging from 20 to 220 Mdaltons with a weighted average of 66 Mdaltons. There is a prominent subpopulation at approximately 35 Mdaltons, and 59% of all LCV belong to a broad peak between 50 and 120 Mdaltons. The Kolmogorov-Smirnov distribution-free test was used to affirm the statistical reproducibility of these isolates. BCV diameters vary from 50 to 90 nm, and those of LCV from 50 to 150 nm. Both protein compositions, determined by SDS PAGE, are dominated by clathrin and they are generally similar, except that corresponding secondary bands, notably the clathrin-associated light chains, appear to have lower molecular weights in the case of LCV. From consideration of the joint mass-diameter distribution, it is apparent that coated vesicles of a given diameter vary considerably in mass and that this variation is due primarily to widely differing amounts of material enclosed within the clathrin coat.
Immunological characterization of the major chick cartilage proteoglycan and its intracellular localization in cultured chondroblasts: a comparison with Type II procollagen.
Authors: Pacifici M , Soltesz R , Thal G , Shanley D J , Boettiger D , Holtzer H
Published: May 2004

Polyclonal antibodies were raised in a rabbit against the major proteoglycan of chick sternal cartilage. A total of six antisera was obtained, three after the first booster injection (A1, A2, and A3) and three after the second booster injection (A4, A5, and A6). The A1 antiserum, which was characterized in most detail, immunoprecipitated native as well as chondroitinase ABC-digested or chondroitinase ABC/keratanase-digested cartilage proteoglycan synthesized by cultured chick chondroblasts, but failed to immunoprecipitate the major proteoglycan synthesized by chick skin fibroblasts. This antiserum was also able to immunoprecipitate the cartilage proteoglycan core protein newly synthesized by cultured chondroblasts, but no other major cell protein. However, the late bleed antisera obtained from the same rabbit after a second booster injection reacted with a new chondroblast-specific polypeptide(s) of approximately 60,000 mol wt in addition to the cartilage proteoglycan. By immunofluorescence procedures, the A1 antiserum stained the extracellular proteoglycan matrix of cultured chondroblasts but not that of skin fibroblasts. Following enzymatic removal of the extracellular matrix and cell membrane permeabilization, this antiserum stained primarily a large, juxtanuclear structure. Additional radioautographic evidence suggests that this structure represents the Golgi complex. Similar immunofluorescent staining with antibodies to the cartilage-characteristic Type II collagen revealed that type II procollagen was localized in numerous cytoplasmic, vacuole-like structures which were scattered throughout most of the chondroblast cytoplasm but were notably scanty in the Golgi complex area. In conclusion, our data suggest the transit of the major cartilage proteoglycan through the Golgi complex of cultured chondroblasts and possible differences in the intracellular distribution of newly synthesized cartilage proteoglycan and Type II procollagen.
Interconversion of structural and contractile actin gels by insertion of myosin during assembly.
Authors: Kane R E
Published: May 2004

Extracts of the soluble cytoplasmic proteins of the sea urchin egg form gels of different composition and properties depending on the temperature used to induce actin polymerization. At temperatures that inactivate myosin, a gel composed of actin, fascin, and a 220,000-mol-wt protein is formed. Fascin binds actin into highly organized units with a characteristic banding pattern, and these actin-fascin units are the structural core of the sea urchin microvilli formed after fertilization and of the urchin coelomocyte filopods. Under milder conditions a more complex myosin-containing gel is formed, which contracts to a small fraction of its original volume within an hour after formation. What has been called "structural" gel can be assembled by combining actin, fascin, and the 220,000-mol-wt protein in 50-100 mM KCl; the aim of the experiments reported here was to determine whether myosin could be included during assembly, thereby interconverting structural and contractile gel. This approach is limited by the aggregation of sea urchin myosin at the low salt concentrations utilized in gel assembly. A method has been devised for the sequential combination of these components under controlled KCl and ATP concentrations that allows the formation of a gel containing dispersed myosin at a final concentration of 60-100 mM KCl. These gels are stable at low (approximately 10 micron) ATP concentrations, but contract to a small volume in the presence of higher (approximately 100 micron) ATP. Contraction can be controlled by forming a stable gel at low ATP and then overlaying it with a solution containing sufficient ATP to induce contraction. This system may provide a useful model for the study of the interrelations between cytoplasmic structure and motility.
Regulation of synthesis of the photosystem I reaction center.
Authors: Vierling E , Alberte R S
Published: May 2004

The in vivo biosynthesis of the P700 chlorophyll a-apoprotein was examined to determine whether this process is light regulated and to determine its relationship to chlorophyll accumulation during light-induced chloroplast development in barley (Hordeum vulgare L.). Rabbit antibodies to the 58,000-62,000-mol-wt apoprotein were used to measure relative synthesis rates by immunoprecipitation of in vivo labeled leaf proteins and to detect apoprotein accumulation on nitrocellulose protein blots. 5-d-old, dark-grown barley seedlings did not contain, or show net synthesis of, the 58,000-62,000-mol-wt polypeptide. When dark-grown barley seedlings were illuminated, net synthesis of the apoprotein was observed within the first 15 min of illumination and accumulated apoprotein was measurable after 1 h. After 4 h, P700 chlorophyll a-apoprotein biosynthesis accounted for up to 10% of the total cellular membrane protein synthesis. Changes in the rate of synthesis during chloroplast development suggest coordination between production of the 58,000-62,000-mol-wt polypeptide and the accumulation of chlorophyll. However, when plants were returned to darkness after a period of illumination (4 h) P700 chlorophyll a-apoprotein synthesis continued for a period of hours though at a reduced rate. Thus we found that neither illumination nor the rate of chlorophyll synthesis directly control the rate of apoprotein synthesis. The rapidity of the light-induced change in net synthesis of the apoprotein indicates that this response is tightly coupled to the primary events of light-induced chloroplast development. The data also demonstrate that de novo synthesis of the apoprotein is required for the onset of photosystem I activity in greening seedlings.
Cell killing by lysosomotropic detergents.
Authors: Miller D K , Griffiths E , Lenard J , Firestone R A
Published: May 2004

We have studied the mechanism by which lysosomotropic detergents kill baby hamster kidney cells. Lysosomotropic detergents are lysosomotropic amines (compounds with pK between 5 and 9, such as imidazole or morpholine) containing straight-chain hydrocarbon "tails" of 9-14 carbon atoms (Firestone, R. A., J. M. Pisano, and R. J. Bonney. 1979, J. Med. Chem., 22:1130-1133). Using lucifer yellow CH as a specific fluorescent label for lysosomes, it was shown by light microscopy that N-dodecyl (C12)-imidazole acted rapidly to damage lysosomes, causing leakage of dye into the cytoplasm. This was followed at later times by vacuolization, blebbing of the plasma membrane, cell rounding, and cell death. 3H-labeled C12-imidazole rapidly diffused into cells where much of it was trapped in lysosomes as shown by its co-migration with lysosomes in Percoll gradients. Cells preincubated with C12-imidazole released it slowly into C12-imidazole-free media, permitting the cells to be killed by the preincubation dose. Cell killing by the lysosomotropic detergents exhibited strongly sigmoidal dose-response curves. The sensitivity of baby hamster kidney cells to killing by C12-imidazole was density dependent, the cells being most sensitive at lowest cell densities, and relatively resistant at confluence. The amount of 3H-C12-imidazole taken up by the cells was also density dependent, with highest specific uptake occurring at the lowest cell density. A rise in lysosomal pH, measured in fluoresceinated dextran-labeled cells, commenced immediately upon addition of C12-imidazole to cells, and continued for over an hour. This was followed after a lag of 1-2 h by inhibition of protein and RNA synthesis and by lactate dehydrogenase release. Ionophores or lysosomotropic amines, such as methylamine, that raise intralysosomal pH provided substantial protection of the cells from killing by lysosomotropic detergents. These findings provide strong support for the idea that lysosomotropic detergents kill cells by disrupting lysosomes from within.
Laminin promotes neuritic regeneration from cultured peripheral and central neurons.
Authors: Manthorpe M , Engvall E , Ruoslahti E , Longo F M , Davis G E , Varon S
Published: May 2004

The ability of axons to grow through tissue in vivo during development or regeneration may be regulated by the availability of specific neurite-promoting macromolecules located within the extracellular matrix. We have used tissue culture methods to examine the relative ability of various extracellular matrix components to elicit neurite outgrowth from dissociated chick embryo parasympathetic (ciliary ganglion) neurons in serum-free monolayer culture. Purified laminin from both mouse and rat sources, as well as a partially purified polyornithine-binding neurite promoting factor (PNPF-1) from rat Schwannoma cells all stimulate neurite production from these neurons. Laminin and PNPF-1 are also potent stimulators of neurite growth from cultured neurons obtained from other peripheral as well as central neural tissues, specifically avian sympathetic and sensory ganglia and spinal cord, optic tectum, neural retina, and telencephalon, as well as from sensory ganglia of the neonatal mouse and hippocampal, septal, and striatal tissues of the fetal rat. A quantitative in vitro bioassay method using ciliary neurons was used to (a) measure and compare the specific neurite-promoting activities of these agents, (b) confirm that during the purification of laminin, the neurite-promoting activity co-purifies with the laminin protein, and (c) compare the influences of antilaminin antibodies on the neurite-promoting activity of laminin and PNPF-1. We conclude that laminin and PNPF-1 are distinct macromolecules capable of expressing their neurite-promoting activities even when presented in nanogram amounts. This neurite-promoting bioassay currently represents the most sensitive test for the biological activity of laminin.
Microinjection of Ca++-calmodulin causes a localized depolymerization of microtubules.
Authors: Keith C , DiPaola M , Maxfield F R , Shelanski M L
Published: May 2004

The microinjection of calcium-saturated calmodulin into living fibroblasts causes the rapid disruption of microtubules and stress fibers in a sharply delimited region concentric with the injection site. This effect is specific to the calcium-bearing form of calmodulin; neither calcium-free calmodulin nor calcium ion at similar levels affects the cytoskeleton. If cells have previously been microinjected with calcium-free calmodulin, elevation of their intracellular calcium levels to 25 mM potentiates the disruption of microtubules throughout the cytoplasm. Approximately 400 mM free calcium is required to cause an equivalent disruption in uninjected cells. The level of calmodulin necessary to disrupt the full complement of cellular microtubules is found to be approximately in 2:1 molar ratio to tubulin dimer. These results indicate that calmodulin can be localized within the cytoplasm in a calcium-dependent manner and that it can act to regulate the calcium lability of microtubules at molar ratios that could be achieved locally within the cell. Our results are consistent with the hypothesis that calmodulin may be controlling microtubule polymerization equilibria in areas of high local concentration such as the mitotic spindle.
Epidermal keratin filaments assembled in vitro have masses-per-unit-length that scale according to average subunit mass: structural basis for homologous packing of subunits in intermediate filaments.
Authors: Steven A C , Hainfeld J F , Trus B L , Wall J S , Steinert P M
Published: May 2004

We have used scanning transmission electron microscopy to elucidate the question of how intermediate filament (IF) subunits of widely differing mass can all form morphologically similar IF. From scanning transmission electron micrographs, the distributions of mass were determined for three types of epidermal keratin IF reassembled in vitro from mixtures of subunits with substantially different masses, viz., "light" and "heavy" human keratins with [Mr] = 50,000 and 56,000, respectively, and mouse keratins of [Mr] = 63,000. Their principal assembly products were found to average 22, 25, and 29 kdalton/nm, respectively. These densities, which correspond to immature "minimal form" IF (Steven, A. C., J. Wall, J. Hainfeld, and P. M. Steinert, 1982, Proc. Natl. Acad. Sci. USA., 79:3101-3105), are directly proportional to the average subunit masses. The human keratin IF (but not those of mouse) also contained minor amounts (15-20%) of more massive polymers averaging 33 and 35 kdalton/nm, respectively, which probably represent mature IF. Taken together with earlier results on IF of other subclasses, these results indicate that the average linear density of IF scales according to the average mass of their constituent subunits, both for "minimal form" and for mature IF. As underlying mechanism for this homology, we propose that the fundamental building-blocks of all these IF contain a common structural element whose packing within the various IF is likewise conserved and which specifies the overall structure. The variable amounts of mass in the nonconserved moieties account for the observed proportionality. This scheme fits with amino acid sequence data for several IF subunits that have revealed, as a likely candidate for the common element, an essentially conserved alpha-helical domain, contrasting with the highly variable sequences of their non-alpha-helical terminal domains.
Tropomyosin-enriched and alpha-actinin-enriched microfilaments isolated from chicken embryo fibroblasts by monoclonal antibodies.
Authors: Lin J J , Matsumura F , Yamashiro-Matsumura S
Published: May 2004

Antitropomyosin and anti-alpha-actinin monoclonal antibodies have been used to isolate two classes of microfilaments, i.e., tropomyosin-enriched and alpha-actinin-enriched microfilaments, respectively, from cultured chicken embryo fibroblasts. Electron microscopic studies of the isolated tropomyosin-enriched microfilaments showed periodic localization of tropomyosin along the microfilaments, with a 35-nm repeat. On the contrary, the isolated alpha-actinin-enriched microfilaments showed no obvious periodicity. Many individual alpha-actinin-enriched microfilaments with length greater than 1 micron (ranging from 1 to 10 microns) were aggregated by anti-alpha-actinin monoclonal antibodies. Both of the isolated microfilaments had the ability to activate the Mg2+-ATPase activity of skeletal muscle myosin, although different extents of activation were observed. These two classes of microfilaments also differed in their protein composition. Molar ratios of major identifiable proteins in the isolated microfilaments were alpha-actinin(dimer):actin(monomer):tropomyosin(dimer) = less than 0.02:8.06:1.00 for tropomyosin-enriched microfilaments and 0.44:13.91:1.00 for alpha-actinin-enriched microfilaments. By two-dimensional gel analysis of the isolated microfilaments, we have found seven spots which possess typical tropomyosin properties including pI 4.5, immunological cross-reaction, lack of proline and tryptophan, and heat stability. Pulse-chase experiments suggested that the assembly of microfilament-associated proteins, at least for alpha-actinin and tropomyosins, was coordinately regulated by the assembly of actin into microfilaments.
Membrane fusion mutants of Semliki Forest virus.
Authors: Kielian M C , Keränen S , Kääriäinen L , Helenius A
Published: May 2004

Previous reports have indicated that the entry of Semliki Forest virus (SFV) into cells depends on a membrane fusion reaction catalyzed by the viral spike glycoproteins and triggered by the low pH prevailing in the endosomal compartment. In this study the in vitro pH-dependent fusion of SFV with nuclease-filled liposomes has been used to select for a new class of virus mutants that have a pH-conditional defect. The mutants obtained had a threshold for fusion of pH 5.5 as compared with the wild-type threshold of 6.2, when assayed by polykaryon formation, fusion with liposomes, or fusion at the plasma membrane. They were fully capable of infecting cells under standard infection conditions but were more sensitive to lysosomotropic agents that increase the pH in acidic vacuoles of the endocytic pathway. The mutants were, moreover, able to penetrate and infect baby hamster kidney-21 cells at 20 degrees C, indicating that the endosomes have a pH below 5.5. The results confirm the involvement of pH-triggered fusion in SFV entry, emphasize the central role played by acidic endosomal vacuoles in this reaction, shed further light on the mechanism of SFV inhibition by lysosomotropic weak bases, and demonstrate the usefulness of mutant viruses as biological pH probes of the endocytic pathway.
Investigations of the role of the main light-harvesting chlorophyll-protein complex in thylakoid membranes. Reconstitution of depleted membranes from intermittent-light-grown plants with the isolated complex.
Authors: Day D A , Ryrie I J , Fuad N
Published: May 2004

The functions of the light-harvesting complex of photosystem II (LHC-II) have been studied using thylakoids from intermittent-light-grown (IML) plants, which are deficient in this complex. These chloroplasts have no grana stacks and only limited lamellar appression in situ. In vitro the thylakoids showed limited but significant Mg2+-induced membrane appression and a clear segregation of membrane particles into such regions. This observation, together with the immunological detection of small quantities of LHC-II apoproteins, suggests that the molecular mechanism of appression may be similar to the more extensive thylakoid stacking seen in normal chloroplasts and involve LHC-II polypeptides directly. To study LHC-II function directly, a sonication-freeze-thaw procedure was developed for controlled insertion of purified LHC-II into IML membranes. Incorporation was demonstrated by density gradient centrifugation, antibody agglutination tests, and freeze-fracture electron microscopy. The reconstituted membranes, unlike the parent IML membranes, exhibited both extensive membrane appression and increased room temperature fluorescence in the presence of cations, and a decreased photosystem I activity at low light intensity. These membranes thus mimic normal chloroplasts in this regard, suggesting that the incorporated LHC-II interacts with photosystem II centers in IML membranes and exerts a direct role in the regulation of excitation energy distribution between the two photosystems.
Mechanism of concanavalin A-induced anchorage of the major cell surface glycoproteins to the submembrane cytoskeleton in 13762 ascites mammary adenocarcinoma cells.
Authors: Jung G , Helm R M , Carraway C A , Carraway K L
Published: May 2004

Concanavalin A (Con A)-induced anchorage of the major cell surface sialoglycoprotein component complex (ASGP-1/ASGP-2) was studied in 13762 rat mammary adenocarcinoma sublines with mobile (MAT-B1 subline) and immobile (MAT-C1 subline) cell surface Con A receptors. Treatment of cells, isolated microvilli, or microvillar membranes with Con A resulted in marked retention of ASGP-1 and ASGP-2, a Con A-binding protein, in cytoskeletal residues of both sublines obtained by extraction with Triton X-100 in PBS. When Con A-treated microvillar membranes were extracted with a buffer containing Triton X-100, the sialoglycoprotein complex was found associated in the residues with a transmembrane complex composed of actin, a 58,000-dalton polypeptide, and a cytoskeleton-associated glycoprotein (CAG), also a Con A-binding protein, in MAT-C1 membranes, and of actin and CAG in MAT-B1 membranes. Untreated membrane Triton residues retained very little ASGP-1/ASGP-2 complex. Association of the sialoglycomembrane complex and the transmembrane complex was also demonstrated in Con A-treated, but not untreated, microvilli by their comigration on CsCl gradients. Association of both complexes with the cytoskeleton of microvilli was shown by sucrose density gradient centrifugation. A fraction of the polymerized actin comigrated with the transmembrane complex alone in the absence of Con A and with both the transmembrane complex and the sialoglycoprotein complex in the presence of Con A. From these results we propose that anchorage of the sialoglycoprotein complex to the cytoskeleton on Con A treatment occurs by cross-linking ASGP-2, the major cell surface Con A-binding component, to CAG of the transmembrane complex, which is natively linked to the cytoskeleton via its actin component. Since Con A-induced anchorage occurs in sublines with mobile and immobile receptors, the anchorage process cannot be responsible for the differences in receptor mobility between the sublines.