bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of stress fiber-like structures in assembling nascent myofibrils in myosheets recovering from exposure to ethyl methanesulfonate.
Authors: Antin P B , Tokunaka S , Nachmias V T , Holtzer H
Published: May 2004

When day 1 cultures of chick myogenic cells were exposed to the mutagenic alkylating agent ethyl methanesulfonate (EMS) for 3 d, 80% of the replicating cells were killed, but postmitotic myoblasts survived. The myoblasts fused to form unusual multinucleated "myosheets": extraordinarily wide, flattened structures that were devoid of myofibrils but displayed extensive, submembranous stress fiber-like structures (SFLS). Immunoblots of the myosheets indicated that the carcinogen blocked the synthesis and accumulation of the myofibrillar myosin isoforms but not that of the cytoplasmic myosin isoform. When removed from EMS, widely spaced nascent myofibrils gradually emerged in the myosheets after 3 d. Striking co-localization of fluorescent reagents that stained SFLS and those that specifically stained myofibrils was observed for the next 2 d. By both immunofluorescence and electron microscopy, individual nascent myofibrils appeared to be part of, or juxtaposed to, preexisting individual SFLS. By day 6, all SFLS had disappeared, and the definitive myofibrils were displaced from their submembranous site into the interior of the myosheet. Immunoblots from recovering myosheets demonstrated a temporal correlation between the appearance of the myofibrillar myosin isoforms and the assembly of thick filaments. The assembly of definitive myofibrils did not appear to involve desmin intermediate filaments, but a striking aggregation of sarcoplasmic reticulum elements was seen at the level of each I-Z-band. Our findings suggest that SFLS in the EMS myosheets function as early, transitory assembly sites for nascent myofibrils.
Detection of a ventricular-specific myosin heavy chain in adult and developing chicken heart.
Authors: Zhang Y , Shafiq S A , Bader D
Published: May 2004

In the present study, a monoclonal antibody (McAb), ALD19, generated against myosin of slow tonic muscle, was shown to react with the heavy chain of ventricular myosin in the adult chicken heart. With this antibody, it was possible to detect a ventricular-specific myosin during myocardial differentiation and to show that the epitope recognized by ALD19 was present from the earliest stages of ventricular differentiation and maintained throughout development only in the ventricle. A second McAb, specific for atrial myosin heavy chain (MHC) (Gonzalez-Sanchez, A., and D. Bader, 1984, Dev. Biol., 103:151-158), was used as a control to detect an atrial-specific myosin in the caudal portion of the developing heart at Hamburger-Hamilton stage 15. It was found that the appearance of ventricular MHC predated the expression of atrial MHC by approximately 1 d in ovo and that specific MHCs were always differentially distributed. While a common primordial MHC may be present in the early heart, this study showed the tissue-specific expression of a ventricular MHC during the initial stages of heart development and its differential accumulation throughout development.
Sequential expression of chicken actin genes during myogenesis.
Authors: Hayward L J , Schwartz R J
Published: May 2004

Embryonic muscle development permits the study of contractile protein gene regulation during cellular differentiation. To distinguish the appearance of particular actin mRNAs during chicken myogenesis, we have constructed DNA probes from the transcribed 3' noncoding region of the single-copy alpha-skeletal, alpha-cardiac, and beta-cytoplasmic actin genes. Hybridization experiments showed that at day 10 in ovo (stage 36), embryonic hindlimbs contain low levels of actin mRNA, predominantly consisting of the alpha-cardiac and beta-actin isotypes. However, by day 17 in ovo (stage 43), the amount of alpha-skeletal actin mRNA/microgram total RNA increased more than 30-fold and represented approximately 90% of the assayed actin mRNA. Concomitantly, alpha-cardiac and beta-actin mRNAs decreased by 30% and 70%, respectively, from the levels observed at day 10. In primary myoblast cultures, beta-actin mRNA increased sharply during the proliferative phase before fusion and steadily declined thereafter. alpha-Cardiac actin mRNA increased to levels 15-fold greater than alpha-skeletal actin mRNA in prefusion myoblasts (36 h), and remained at elevated levels. In contrast, the alpha-skeletal actin mRNA remained low until fusion had begun (48 h), increased 25-fold over the prefusion level by the completion of fusion, and then decreased at later times in culture. Thus, the sequential accumulation of sarcomeric alpha-actin mRNAs in culture mimics some of the events observed in embryonic limb development. However, maintenance of high levels of alpha-cardiac actin mRNA as well as the transient accumulation of appreciable alpha-skeletal actin mRNA suggests that myoblast cultures lack one or more essential components for phenotypic maturation.
Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis.
Authors: Waters M G , Blobel G
Published: May 2004

We describe an in vitro system with all components derived from the yeast Saccharomyces cerevisiae that can translocate a yeast secretory protein across microsomal membranes. In vitro transcribed prepro-alpha-factor mRNA served to program a membrane-depleted yeast translation system. Translocation and core glycosylation of prepro-alpha-factor were observed when yeast microsomal membranes were added during or after translation. A membrane potential is not required for translocation. However, ATP is required for translocation and nonhydrolyzable analogues of ATP cannot serve as a substitute. These findings suggest that ATP hydrolysis may supply the energy required for translocation of proteins across the endoplasmic reticulum.
Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas.
Authors: Bole D G , Hendershot L M , Kearney J F
Published: May 2004

A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.
Unusual forms of low density lipoprotein receptors in hamster cell mutants with defects in the receptor structural gene.
Authors: Kozarsky K F , Brush H A , Krieger M
Published: May 2004

The structure and processing of low density lipoprotein (LDL) receptors in wild-type and LDL receptor-deficient mutant Chinese hamster ovary cells was examined using polyclonal anti-receptor antibodies. As previously reported for human LDL receptors, the LDL receptors in wild-type Chinese hamster ovary cells were synthesized as precursors which were extensively processed by glycosylation to a mature form. In the course of normal receptor turnover, an apparently unglycosylated portion of the cysteine-rich N-terminal LDL binding domain of the receptor is proteolytically removed. The LDL receptor-deficient mutants fall into four complementation groups, ldlA, ldlB, ldlC, and ldlD; results of the analysis of ldlB, ldlC, and ldlD mutants are described in the accompanying paper (Kingsley, D. M., K. F. Kozarsky, M. Segal, and M. Krieger, 1986, J. Cell. Biol, 102:1576-1585). Analysis of ldlA cells has identified three classes of mutant alleles at the ldlA locus: null alleles, alleles that code for normally processed receptors that cannot bind LDL, and alleles that code for abnormally processed receptors. The abnormally processed receptors were continually converted to novel unstable intracellular intermediates. We also identified a compound-heterozygous mutant and a heterozygous revertant which indicate that the ldlA locus is diploid. In conjunction with other genetic and biochemical data, the finding of multiple mutant forms of the LDL receptor in ldlA mutants, some of which appeared together in the same cell, confirm that the ldlA locus is the structural gene for the LDL receptor.
Three types of low density lipoprotein receptor-deficient mutant have pleiotropic defects in the synthesis of N-linked, O-linked, and lipid-linked carbohydrate chains.
Authors: Kingsley D M , Kozarsky K F , Segal M , Krieger M
Published: May 2004

Biochemical, immunological, and genetic techniques were used to investigate the genetic defects in three types of low density lipoprotein (LDL) receptor-deficient hamster cells. The previously isolated ldlB, ldlC, and ldlD mutants all synthesized essentially normal amounts of a 125,000-D precursor form of the LDL receptor, but were unable to process this receptor to the mature form of 155,000 D. Instead, these mutants produced abnormally small, heterogeneous receptors that reached the cell surface but were rapidly degraded thereafter. The abnormal sizes of the LDL receptors in these cells were due to defective processing of the LDL receptor's N- and O-linked carbohydrate chains. Processing defects in these cells appeared to be general since the ldlB, ldlC, and ldlD mutants also showed defective glycosylation of a viral glycoprotein, alterations in glycolipid synthesis, and changes in resistance to several toxic lectins. Preliminary structural studies suggested that these cells had defects in multiple stages of the Golgi-associated processing reactions responsible for synthesis of glycolipids and in the N-linked and O-linked carbohydrate chains of glycoproteins. Comparisons between the ldl mutants and a large number of previously isolated CHO glycosylation defective mutants showed that the genetic defects in ldlB, ldlC, and ldlD cells were unique and that only very specific types of carbohydrate alteration could dramatically affect LDL receptor function.
In situ measurements of external pH and optical density oscillations in Dictyostelium discoideum aggregates.
Authors: Gottmann K , Weijer C J
Published: May 2004

In situ measurements of extracellular pH by means of microelectrodes and in situ measurements of optical density were performed on aggregating cells of Dictyostelium discoideum. Early aggregation stage AX2 cells showed sinusoidal pH oscillations, which could be inhibited by the specific relay inhibitor caffeine, indicating that they were coupled to cAMP oscillations. Sometimes biphasic pH oscillations were found, which can be explained by the superposition of two harmonic pH oscillations. These harmonic oscillations might arise by gating of the cAMP signal; a part of the cells respond to every cAMP signal and another subpopulation to every second cAMP pulse. Late aggregation-stage cells showed complex changes of the extracellular pH, which could be inhibited by caffeine. Optical density measurements of wave propagation in aggregation streams of HG220 also revealed gating behavior. In addition to sinusoidal optical density oscillations, biphasic and still more complex oscillations were observed.
In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes.
Authors: Binder M , Tourmente S , Roth J , Renaud M , Gehring W J
Published: May 2004

A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick-translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: rapid signal detection: superior morphological preservation and spatial resolution; and signal-to-noise ratios equivalent to radiolabeling.
Subaxolemmal cytoskeleton in squid giant axon. I. Biochemical analysis of microtubules, microfilaments, and their associated high-molecular-weight proteins.
Authors: Kobayashi T , Tsukita S , Tsukita S , Yamamoto Y , Matsumoto G
Published: May 2004

Using the squid giant axon, we analyzed biochemically the molecular organization of the axonal cytoskeleton underlying the axolemma (subaxolemmal cytoskeleton). The preparation enriched in the subaxolemmal cytoskeleton was obtained by squeezing out the central part of the axoplasm using a roller. The electrophoretic banding pattern of the subaxolemmal cytoskeleton was characterized by large amounts of two high-molecular-weight (HMW) proteins (260 and 255 kD). The alpha, beta-tubulin, actin, and some other proteins were also its major constituents. The 260-kD protein is known to play an important role in maintaining the excitability of the axolemma (Matsumoto, G., M. Ichikawa, A. Tasaki, H. Murofushi, and H. Sakai, 1983, J. Membr. Biol., 77:77-91) and was recently designated "axolinin" (Sakai, H., G. Matsumoto, and H. Murofushi, 1985, Adv. Biophys., 19:43-89). We purified axolinin and the 255-kD protein in their native forms and further characterized their biochemical properties. The purified axolinin was soluble in 0.6 M NaCl solution but insoluble in 0.1 M NaCl solution. It co-sedimented with microtubules but not with actin filaments. In low-angle rotary-shadowing electron microscopy, the axolinin molecule in 0.6 M NaCl solution looked like a straight rod approximately 105 nm in length with a globular head at one end. On the other hand, the purified 255-kD protein was soluble in both 0.1 and 0.6 M NaCl solution and co-sedimented with actin filaments but not with microtubules. The 255-kD protein molecule appeared as a characteristic horseshoe-shaped structure approximately 35 nm in diameter. Furthermore, the 255-kD protein showed no cross-reactivity to the anti-axolinin antibody. Taken together, these characteristics lead us to conclude that the subaxolemmal cytoskeleton in the squid giant axon is highly specialized, and is mainly composed of microtubules and a microtubule-associated HMW protein (axolinin), and actin filaments and an actin filament-associated HMW protein (255-kD protein).
Myosin types and fiber types in cardiac muscle. III. Nodal conduction tissue.
Authors: Gorza L , Sartore S , Thornell L E , Schiaffino S
Published: May 2004

The sinoatrial (SA) and atrioventricular (AV) nodes are specialized centers of the heart conduction system and are composed of muscle cells with distinctive morphological and electrophysiological properties. We report here results of immunofluorescence and immunoperoxidase studies on the bovine heart showing that a large number of SA and AV nodal cells share a distinct type of myosin heavy chain (MHC) which is not found in other myocardial cells and can thus be used as a cell-type-specific marker. The antibody used in this study was raised against fetal skeletal myosin and reacted with fetal skeletal but not with adult skeletal MHCs. Both atrial and ventricular fibers, as well as fibers of the ventricular conduction tissue were unlabeled by this antibody. Specific reactivity was exclusively seen in most cells in the central portions of the SA and AV nodes and rare cells in perinodal areas. However, a number of nodal cells, particularly those located in the peripheral nodal regions, were unreactive with this antibody. The myosin composition of nodal tissues was also explored using two antibodies reacting specifically with alpha-MHC, the predominant atrial isoform, and beta-MHC, the predominant ventricular isoform. Most nodal cells were reactive for alpha-MHC and a number of them also for beta-MHC. Variation in reactivity with the two antibodies was also observed in perinodal areas: at these sites a population of large fibers reacted exclusively for beta-MHC. These findings point to the existence of muscle cell heterogeneity with respect to myosin composition both in nodal and perinodal tissues.
Membrane and cytoplasmic proteins are transported in the same organelle complex during nematode spermatogenesis.
Authors: Roberts T M , Pavalko F M , Ward S
Published: May 2004

During the development of pseudopodial spermatozoa of the nematode, Caenorhabditis elegans, protein synthesis stops before differentiation is completed. Colloidal gold conjugates of monoclonal antibody SP56, which binds to the surface of spermatozoa, and TR20, which recognizes the major sperm cytoplasmic protein (MSP), were used to label thin sections of testes embedded in Lowicryl K4M in order to follow polypeptides from their synthesis early in spermatogenesis to their segregation to specific compartments of the mature cell. Both antigens are synthesized in primary spermatocytes and are assembled into a unique double organelle, the fibrous body-membranous organelle (FB-MO) complex. However, the antigens are localized in different regions of this FB-MO complex. As described in detail, the assembly of proteins into the FB-MO complex allows both membrane and cytoplasmic components to be concentrated in the spermatids after meiosis. Then, the stepwise disassembly of this transient structure ensures delivery of each component to its final destination in the mature spermatozoan: MSP filaments in the fibrous body depolymerize, releasing MSP into the cytoplasm and the membranous organelles fuse with the plasma membrane, delivering SP56 antigen to the surface.
Changes in membrane-microfilament interaction in intercellular adherens junctions upon removal of extracellular Ca2+ ions.
Authors: Volberg T , Geiger B , Kartenbeck J , Franke W W
Published: May 2004

EGTA-induced depletion of Ca2+ ions from the culture medium of Madin-Darby bovine kidney epithelial cells results in rapid splitting of adherens-type junctions and the detachment of the vinculin- and actin-containing filament bundle from the cytoplasmic faces of the plasma membrane of the zonula adhaerens. This process was monitored by phase-contrast microscopy, combined with electron microscopy and immunofluorescent localization of the two proteins. It is shown that shortly after extracellular free Ca2+ concentration is lowered to the micromolar range, the actin-containing, junction-associated belt of microfilaments, together with the vinculin-rich junctional plaque material, is irreversibly detached as one structural unit from the plasma membrane, contracts, and is displaced towards the perinuclear cytoplasm where it gradually disintegrates. Other actin- and vinculin-containing structures present in the same cells, notably the focal contacts at the substratum, are not similarly affected by the Ca2+ depletion and retain both the adhesion to the external surface and the association with the plaque and microfilament components. Electron microscopic examination has shown that the membrane domain of the zonulae adhaerentes, unlike that of desmosomes, is not endocytosed after Ca2+ removal and that the displaced actin- and vinculin-containing plaque and filament belt are not associated with a particular membrane. It is further shown that upon restoration of normal Ca2+ levels in the culture medium, new intercellular contacts are established gradually by accretion of both vinculin and actin into new belt-like plaque- and microfilament-containing structures.
Identification of actin-, alpha-actinin-, and vinculin-containing plaques at the lateral membrane of epithelial cells.
Authors: Drenckhahn D , Franz H
Published: May 2004

In this paper, a new type of spot desmosome-like junction (type II plaque) is described that is scattered along the entire lateral plasma membrane of rat and human intestinal epithelium. Ultrastructurally type II plaques differed from the classical type of epithelial spot desmosome ("macula adherens", further denoted as type I desmosome) by weak electron density of the membrane-associated plaque material, association of the plaques with microfilaments rather than intermediate filaments, and poorly visible material across the intercellular space. Thus, type II plaques resemble cross-sections of the zonula adherens. Immunofluorescence-microscopic studies were done using antibodies to a main protein associated with the plaques of type I desmosomes (desmoplakin I) and to the three major proteins located at the plaques of the zonula adherens (actin, alpha-actinin, and vinculin). Two types of plaques were visualized along the lateral surface of intestinal and prostatic epithelium: (a) the type I desmosomes, which were labeled with anti-desmoplakin but did not bind antibodies to actin, alpha-actinin, and vinculin, and (b) a further set of similarly sized plaques, which bound antibodies to actin, alpha-actinin, and vinculin but were not stained with anti-desmoplakin. Three-dimensional computer reconstruction of serial sections double-labeled with anti-desmoplakin and anti-alpha-actinin further confirmed that both types of plaques are spatially completely separated from each other along the lateral plasma membrane. The computer graphs further revealed that the actin-, alpha-actinin-, and vinculin-containing plaques have the tendency to form clusters, a feature also typical of type II plaques. It is suggested that the type II plaques represent spot desmosome-like intercellular junctions, which, like the zonula adherens, appear to be linked to the actin filament system. As the type II plaques cover a considerable part of the lateral cell surface, they might play a particular role in controlling cellular shape and intercellular adhesion.
Microtubules and actin filaments are not critically involved in the biogenesis of epithelial cell surface polarity.
Authors: Salas P J , Misek D E , Vega-Salas D E , Gundersen D , Cereijido M , Rodriguez-Boulan E
Published: May 2004

We have studied the role of microtubules and actin filaments in the biogenesis of epithelial cell surface polarity, using influenza hemagglutinin and vesicular stomatitis G protein as model apical and basolateral proteins in infected Madin-Darby canine kidney cells. Addition of colchicine or nocodazole to confluent monolayers at concentrations sufficient to completely disassemble microtubules did not affect the asymmetric budding of influenza or vesicular stomatitis virus and only slightly reduced the typical asymmetric surface distribution of their envelope proteins, despite extensive cytoplasmic redistribution of the Golgi apparatus. Alteration of microtubular function by taxol or dissociation of actin filaments by cytochalasin D also failed to have a significant effect. Furthermore, neither colchicine nor cytochalasin D pretreatment blocked the ability of subconfluent Madin-Darby canine kidney cells to sustain polarized budding of influenza virus a few hours after attachment to the substrate. Our results indicate that domain-specific microtubule or actin filament "tracks" are not responsible for the vectorial delivery of apically or basolaterally directed transport vesicles. In conjunction with currently available evidence, they are compatible with a model in which receptors in the cytoplasmic aspect of apical or basolateral regions provide vectoriality to the transport of vesicles carrying plasma membrane proteins to their final surface localization.
Effect of human serum and some of its components on neutrophil adherence and migration across an epithelium.
Authors: Cramer E B , Milks L C , Brontoli M J , Ojakian G K , Wright S D , Showell H J
Published: May 2004

The effect of human serum and some of its components on the process of transepithelial migration of human neutrophils was investigated in an in vitro system. 10% autologous serum caused an increase in neutrophil adherence to and migration across canine kidney epithelial cells. This increase in neutrophil binding also occurred if the epithelium but not the neutrophils had been preincubated with serum. The binding was lost if the serum was either preabsorbed over the kidney epithelium before use or heat inactivated. Indirect immunofluorescence studies indicated that IgG, IgM, and a component of C3 bound to the epithelial surface, whereas IgA, IgE, or C5a were not detectable. The majority of epithelial cells were immunofluorescent, however epithelial cells with varying degrees of reactivity were also apparent and approximately 5% of the epithelial cells did not bind IgG, IgM, and C3. When epithelia were simultaneously tested for the presence of either IgG, IgM, or C3, and bound neutrophils the few epithelial cells which did not bind IgG or IgM also did not bind C3 or neutrophils. Studies with monoclonal antibodies against Fc and C3 receptors indicate that neutrophil adherence to the epithelial surface was mediated predominately by the receptors for C3b and C3bi. In response to a chemotactic gradient, bound neutrophils were able to detach and migrate across the epithelium. A separate heat-stable factor(s) in serum was able to increase neutrophil migration across the epithelial monolayer. This factor acted independently of the factors which caused the increase in neutrophil binding as the increase in neutrophil migration also occurred under conditions (preabsorption over the kidney epithelium or heat inactivation) that prevented the increase in neutrophil binding. The increase in neutrophil migration may be caused by the permeability-increasing properties of this factor as both serum and heat-inactivated serum lowered the transepithelial electrical resistance an average of 38 and 35%, respectively, in 40 min. Upon removal of serum or heat-inactivated serum, the resistance returned 100 and 81%, respectively, in 5 h.
Studies on the regulation of beta-nerve growth factor gene expression in the rat iris: the level of mRNA-encoding nerve growth factor is increased in irises placed in explant cultures in vitro, but not in irises deprived of sensory or sympathetic innervation in vivo.
Authors: Shelton D L , Reichardt L F
Published: May 2004

Beta-nerve growth factor (NGF) is a protein necessary for the survival and maintenance of sympathetic and sensory neurons that appears to be produced by the target tissues of these neurons in vivo. Both denervation and the culture of explants of one model target, the rat iris, leads to an increase in the NGF content, suggesting that innervating neurons may regulate a step in synthesis or turnover of NGF. To determine whether there is a change in synthesis controlled at the mRNA level, the rat iris has been assayed for its content of NGF mRNA after surgical and chemical denervation and after explant into culture. Using a sensitive blot hybridization assay, a large, rapid increase in the content of NGF mRNA was observed upon explant of the rat iris. The increase was readily detectable within 1 h, reached a maximum increase of 10- to 20-fold by 6 to 12 h, and was still evident after 3 d in culture. The distribution of NGF mRNA in different areas of the iris does not change during this time. This rapid increase in NGF mRNA is also seen in the fully innervated iris in vivo after trauma to the anterior chamber. In contrast, denervation to varying degrees in situ had no effect on NGF mRNA levels. Neither removal of sympathetic innervation by surgical or chemical methods nor combined surgical removal of sympathetic and sensory innervation detectably altered NGF mRNA content. Thus, denervation of the rat iris in situ does not cause the observed accumulation of NGF by increasing the level of NGF mRNA, and the increase in NGF content must be due to other factors.
Regulation of the terminal event in cellular differentiation: biological mechanisms of the loss of proliferative potential.
Authors: Wier M L , Scott R E
Published: May 2004

Human plasma has been demonstrated to contain factors that induce the sequential expression of nonterminal and terminal adipocyte differentiation in 3T3 T mesenchymal stem cells. We now report the development of methods for the isolation of purified populations of nonterminally differentiated cells and terminally differentiated cells, and we show that it is possible to experimentally induce transition from the nonterminal to the terminal state of differentiation. With this model system it is therefore now possible to examine the biological and molecular processes associated with the terminal event in differentiation, i.e., the irreversible loss of proliferative potential. In this regard, we demonstrate that transition from the nonterminal to terminal state of differentiation is a complex metabolic process that consists of at least two steps and that this process can be triggered by pulse exposure to an inducer for approximately 12 h but that approximately 24-48 h is required for the process to be completed. The data also establish that induction of the terminal event in differentiation requires protein synthesis but not RNA and DNA synthesis. These and additional results suggest that loss of proliferative potential associated with the terminal event in cellular differentiation is a distinct regulatory process, and we suggest that defects in this regulatory process may be of etiological significance in the pathogenesis of specific human diseases, especially cancer.
Structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells. II. Evidence for a pentasaccharide sequence that contains a 3-O-sulfate group.
Authors: Castellot J J , Choay J , Lormeau J C , Petitou M , Sache E , Karnovsky M J
Published: May 2004

Earlier work from our laboratory demonstrated that heparin inhibited the proliferation of vascular smooth muscle cells in vivo and in vitro. Both anticoagulant and non-anticoagulant heparin species were equally effective as antiproliferative agents. Previous structure-function studies indicated that hexasaccharide and larger fragments retained antiproliferative activity, whereas tetra- and disaccharides were inactive. These experiments also suggested that both N- and O-sulfates of heparin were necessary for growth inhibitory capacity. In this paper, we have further analyzed the structural determinants of the antiproliferative activity of heparin. These experiments were done using synthetically prepared and therefore chemically defined heparin oligosaccharides. We present evidence that a pentasaccharide fragment retains antiproliferative activity, and that the 3-O-sulfate on the internal glucosamine residue is critical for growth inhibitory capacity of the pentasaccharide. We also show that heparins obtained from different manufacturers differ significantly in their ability to suppress smooth muscle cell proliferation.
Determination of the intracellular state of soluble macromolecules by gel filtration in vivo in the cytoplasm of amphibian oocytes.
Authors: Dabauvalle M C , Franke W W
Published: May 2004

A method to examine the diffusible state and the sizes of major cytoplasmic proteins in a living cell is described. Small (40-300 microns) commercially available gel filtration beads of a broad range of Mr exclusion limits were microsurgically implanted into the cytoplasm of oocytes of the frog, Xenopus laevis, usually after metabolic labeling of oocyte proteins with [35S]methionine. After equilibration in vivo for several hours, the appearance of the implanted cells, notably the bead-cytoplasm boundary, was examined by light and electron microscopy of sections and found to be unaffected. After incubation the beads were isolated, briefly rinsed, and their protein contents examined by one- or two-dimensional gel electrophoresis. We show that diffusible proteins can be identified by their inclusion in the pores of the gel filtration beads used and that their approximate sizes can be estimated from the size exclusion values of the specific materials used. The application of this method to important cell biological questions is demonstrated by showing that several "karyophobic proteins," i.e., proteins of the cytosolic fraction which accumulate in the cytoplasm in vivo, are indeed diffusible in the living oocyte and appear with sizes similar to those determined in vitro. This indicates that the nucleo-cytoplasmic distribution of certain diffusible proteins is governed, in addition to size exclusion at nuclear pore complexes and karyophilic "signals," by other, as yet unknown forces. Some possible applications of this method of gel filtration in vivo are discussed.
Probing the structure of cytoplasm.
Authors: Luby-Phelps K , Taylor D L , Lanni F
Published: May 2004

We have used size-fractionated, fluorescent dextrans to probe the structure of the cytoplasmic ground substance of living Swiss 3T3 cells by fluorescence recovery after photobleaching and video image processing. The data indicate that the cytoplasm of living cells has a fluid phase viscosity four times greater than water and contains structural barriers that restrict free diffusion of dissolved macromolecules in a size-dependent manner. Assuming these structural barriers comprise a filamentous meshwork, the combined fluorescence recovery after photobleaching and imaging data suggest that the average pore size of the meshwork is in the range of 300 to 400 A, but may be as small as 200 A in some cytoplasmic domains.
Dynamics of microtubule depolymerization in monocytes.
Authors: Cassimeris L U , Wadsworth P , Salmon E D
Published: May 2004

Human monocytes, which contain few interphase microtubules (35.+/- 7.7), were used to study the dynamics of microtubule depolymerization. Steady-state microtubule assembly was abruptly blocked with either high concentrations of nocodazole (10 micrograms/ml) or exposure to cold temperature (3 degrees C). At various times after inhibition of assembly, cells were processed for anti-tubulin immunofluorescence microscopy. Stained cells were observed with an intensified video camera attached to the fluorescence microscope. A tracing of the entire length of each individual microtubule was made from the image on the television monitor by focusing up and down through the cell. The tracings were then digitized into a computer. All microtubules were seen to originate from the centrosome, with an average length in control cells of 7.1 +/- 2.7 microns (n = 957 microtubules). During depolymerization, the total microtubule polymer and the number of microtubules per cell decreased rapidly. In contrast, there was a slow decrease in the average length of the persisting microtubules. The half-time for both the loss of total microtubule polymer and microtubule number per cell was approximately 40 s for nocodazole-treated cells. The rate-limiting step in the depolymerization process was the rate of initiation of disassembly. Once initiated, depolymerization appeared catastrophic. Further kinetic analysis revealed two classes of microtubules: 70% of the microtubule population was very labile and initiated depolymerization at a rate approximately 23 times faster than a minor population of persistent microtubules. Cold treatment yielded qualitatively similar characteristics of depolymerization, but the initiation rates were slower. In both cases there was a significant asynchrony and heterogeneity in the initiation of depolymerization among the population of microtubules.
Intermediate filaments in muscle and epithelial cells of nematodes.
Authors: Bartnik E , Osborn M , Weber K
Published: May 2004

Current concepts of the developmentally controlled multigene family of intermediate filament (IF) proteins expect the origin of their complexity in evolutionary precursors preceding all vertebrate classes. Among invertebrates, however, firm ultrastructural as well as molecular documentation of IFs is restricted to some giant axons and to epithelia of a few molluscs and annelids. As Ascaris lumbricoides is easily dissected into clean tissues, IF expression in this large nematode was analyzed by electron microscopic and biochemical procedures and a monoclonal antibody reacting with all mammalian IF proteins. We document for the first time the presence of IFs in muscle cells of an invertebrate. They occur in three muscle types (irregular striated pharynx muscle, obliquely striated body muscle, uterus smooth muscle). IFs are also found in the epithelia studied (syncytial epidermis, intestine, ovary, testis). Immunoblots on muscles, pharynx, intestine, uterus, and epidermis identify a pair of polypeptides (with apparent molecular masses of 71 and 63 kD) as IF constituents. In vitro reconstitution of filaments was obtained with the proteins purified from body muscle. In the small nematode Caenorhabditis elegans IF proteins are so far found only in the massive desmosome-anchored tonofilament bundles which traverse a special epithelial cell type, the marginal cells of the pharynx. We speculate that IFs may occur in most but perhaps not all invertebrates and that they may not occur in all cells in large amounts. As electron micrographs of the epidermis of a planarian--a member of the Platyhelminthes--reveal IFs, the evolutionary origin of this cytoplasmic structure can be expected either among the lowest metazoa or already in some unicellular eukaryotes.
The axonemal axis and Ca2+-induced asymmetry of active microtubule sliding in sea urchin sperm tails.
Authors: Sale W S
Published: May 2004

Structural studies of stationary principal bends and of definitive patterns of spontaneous microtubule sliding disruption permitted description of the bending axis in sea urchin sperm tail axonemes. Lytechinus pictus sperm were demembranated in a buffer containing Triton X-100 and EGTA. Subsequent resuspension in a reactivation buffer containing 0.4 mM CaCl2 and 1.0 mM MgATP2- resulted in quiescent, rather than motile, cells and each sperm tail axoneme took on an extreme, basal principal bend of 5.2 rad. Thereafter, such flagellar axonemes began to disrupt spontaneously into two subsets of microtubules by active sliding requiring ATP. Darkfield light microscopy demonstrated that subset "1" is composed of microtubules from the inside edge of the principal bend. Subset "2" is composed of microtubules from the outside edge of the principal bend and always scatters less light in darkfield than subset 1. Subset 2, which always slides in the proximal direction, relative to subset 1, results in a basal loop of microtubules, and the subset 2 loop is restricted to the bend plane during sliding disruption. Electron microscopy revealed that doublets 8, 9, 1, 2, 3 and the central pair comprise subset 1, and doublets 4, 5, the bridge, 6, and 7 comprise subset 2. The microtubules of isolated subset 2 are maintained in a circular arc in the absence of spoke-central pair interaction. Longitudinal sections show that the bending plane bisects the central pair. We therefore conclude that the bend plane passes through doublet 1 and the 5-6 bridge and that doublet 1 is at the inside edge of the principal bend. Experimental definition of the axis permits explicit discussion of the location of active axonemal components which result in Ca2+-induced stationary basal bends and explicit description of components responsible for alternating basal principal and reverse bends.
Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin.
Authors: Sanger J M , Mittal B , Pochapin M B , Sanger J W
Published: May 2004

Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were approximately 1.9-2.4 microns long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 micron apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at approximately day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 microns sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 microns. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 microns sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.
Brain spectrin(240/235) and brain spectrin(240/235E): two distinct spectrin subtypes with different locations within mammalian neural cells.
Authors: Riederer B M , Zagon I S , Goodman S R
Published: May 2004

Adult mouse brain contains at least two distinct spectrin subtypes, both consisting of 240-kD and 235-kD subunits. Brain spectrin(240/235) is found in neuronal axons, but not dendrites, when immunohistochemistry is performed with antibody raised against brain spectrin isolated from enriched synaptic/axonal membranes. A second spectrin subtype, brain spectrin(240/235E), is exclusively recognized by red blood cell spectrin antibody. Brain spectrin(240/235E) is confined to neuronal cell bodies and dendrites, and some glial cells, but is not present in axons or presynaptic terminals.
A lymphoma plasma membrane-associated protein with ankyrin-like properties.
Authors: Bourguignon L Y , Walker G , Suchard S J , Balazovich K
Published: May 2004

In this study we have used several complementary techniques to isolate and characterize a 72-kD polypeptide that is tightly associated with a major mouse T-lymphoma membrane glycoprotein, gp 85 (a wheat germ agglutinin-binding protein), in a 16 S complex. These two proteins do not separate in the presence of high salt but can be dissociated by treatment with 2 M urea. Further analysis indicates that the 72-kD protein has ankyrin-like properties based on the following criteria: (a) it cross-reacts with specific antibodies raised against erythrocyte and brain ankyrin; (b) it displays a peptide mapping pattern and a pI (between 6.5 and 6.8) similar to that of the 72-kD proteolytic fragment of erythrocyte ankyrin; (c) it competes with erythrocyte ghost membranes (spectrin-depleted preparations) for spectrin binding; and (d) it binds to purified spectrin and fodrin molecules. Most importantly, in intact lymphoma cells this ankyrin-like protein is localized directly underneath the plasma membrane and is found to be preferentially accumulated beneath receptor cap structures as well as associated with a membrane-cytoskeleton complex preparation. It is proposed that the ankyrin-like 72-kD protein may play an important role in linking certain surface glycoprotein(s) to fodrin which, in turn, binds to actin filaments required for lymphocyte cap formation.
Immunolocalization of the oligosaccharide trimming enzyme glucosidase II.
Authors: Lucocq J M , Brada D , Roth J
Published: May 2004

We used immunoelectron microscopy to localize glucosidase II in pig hepatocytes. The enzyme trims the two inner alpha 1,3-linked glucoses from N-linked oligosaccharide precursor chains of glycoproteins. Immunoreactive enzyme was concentrated in rough (RER) and smooth (SER) endoplasmic reticulum but not detectable in Golgi apparatus cisternae. Transitional elements of RER and smooth membraned structures close to Golgi apparatus cisternae contained labeling for glucosidase II. Specific labeling was also found in autophagosomes. These results indicate strongly that glucosidase II acts on glycoproteins before their transport to, and processing in Golgi apparatus cisternae, and suggest that an important transitional region for glucosidase II exists between RER and Golgi apparatus cisternae. Degradation in autophagolysosomes could form a normal catabolic pathway for glucosidase II.
Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane.
Authors: Puddington L , Machamer C E , Rose J K
Published: May 2004

Oligonucleotide-directed mutagenesis was used to construct chimeric cDNAs that encode the extracellular and transmembrane domains of the vesicular stomatitis virus glycoprotein (G) linked to the cytoplasmic domain of either the immunoglobulin mu membrane heavy chain, the hemagglutinin glycoprotein of influenza virus, or the small glycoprotein (p23) of infectious bronchitis virus. Biochemical analyses and immunofluorescence microscopy demonstrated that these hybrid genes were correctly expressed in eukaryotic cells and that the hybrid proteins were transported to the plasma membrane. The rate of transport to the Golgi complex of G protein with an immunoglobulin mu membrane cytoplasmic domain was approximately sixfold slower than G protein with its normal cytoplasmic domain. However, this rate was virtually identical to the rate of transport of micron heavy chain molecules measured in the B cell line WEHI 231. The rate of transport of G protein with a hemagglutinin cytoplasmic domain was threefold slower than wild type G protein and G protein with a p23 cytoplasmic domain, which were transported at similar rates. The combined results underscore the importance of the amino acid sequence in the cytoplasmic domain for efficient transport of G protein to the cell surface. Also, normal cytoplasmic domains from other transmembrane glycoproteins can substitute for the G protein cytoplasmic domain in transport of G protein to the plasma membrane. The method of constructing precise hybrid proteins described here will be useful in defining functions of specific domains of viral and cellular integral membrane proteins.