bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of fusion of embryonic muscle cells in culture by tunicamycin is prevented by leupeptin.
Authors: Olden K , Law J , Hunter V A , Romain R , Parent J B
Published: May 2004

The carbohydrate requirement for alignment and fusion of embryonic quail muscle cells has been examined in tissue culture by use of tunicamycin (TM). The mononucleated, spindle-shaped proliferating myoblasts were treated with TM at various times before fusion and differentiation into multinucleated muscle fibers capable of spontaneous contraction. Tm blocked protein glycosylation and expression of glycoproteins on the cell surface, and strongly inhibited fusion when added to cultures of differentiating muscle cells before the fusion "burst," but had no apparent effect on cell alignment. The inhibition of fusion was partially prevented when TM was administered in the presence of protease inhibitors such as leupeptin and pepstatin, but the inhibition of glycosylation was not prevented. Both glycosylation and fusion were completely restored to normal by the removal of the antibiotic from the medium. These studies provide strong support for the idea that myoblast fusion is partially mediated by glycoproteins with asparagine-linked oligosaccharides. However, the requirement for the carbohydrate portion of the glycoprotein appears to be indirect in that it acts to stabilize the protein moiety against proteolytic degradation. Our findings do not rule out the possibility that oligosaccharide units of surface glycolipids have some role in myoblast fusion.
Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration
Authors: Griffin JW , Price DL , Drachman DB , Morris J
Published: May 2004

The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the "pulse- labeled" glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches.

The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons.

One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout.
Monoclonal antibodies against a specific nonhistone chromosomal protein of Drosophila associated with active genes.
Authors: Howard G C , Abmayr S M , Shinefeld L A , Sato V L , Elgin S C
Published: May 2004

Hybridomas secreting monoclonal antibodies have been produced by fusion of NS-1 mouse myeloma cells with the spleen cells of mice inoculated with a 60-65,000-mol wt fraction of proteins released from Drosophila embryo nuclei treated with DNase I. The antibodies secreted by the hybridomas were examined with polytene chromosomes of formaldehyde-fixed salivary gland squashes by an immunofluorescence assay. Most of the clonal antibodies obtained resulted in specific staining of the chromosomes relative to the cytoplasmic debris. In the case of clone 28, the antibodies showed a preferential association with sites of gene activity, both puffs and loci identified as puffing at some time during the third instar and prepupal period. In larvae that were heat shocked (exposed to 35 degrees C for 15 min before removal and fixation of the glands), the antibodies of clone 28 stained preferentially the induced heat-shock loci while continuing to stain most of the normal set of loci. The antigen for clone 28 was identified as a single protein of approximately 62,000 mol wt by using the antibodies followed by 125I-rabbit anti-mouse Ig to stain nitrocellulose replicas of SDS polyacrylamide gels of total chromosomal proteins. This study demonstrates that monoclonal antibodies can be used successfully in immunofluorescence staining of formaldehyde-fixed polytene chromosomes. The results verify the hypothesis that a specific nonhistone chromosomal protein is preferentially associated with the set of loci that includes both active sites and those scheduled to be active at some time in this developmental program. Such proteins may play a general role in the mechanisms of cell determination and gene activation.
Myosin types and fiber types in cardiac muscle. I. Ventricular myocardium.
Authors: Sartore S , Gorza L , Pierobon Bormioli S , Dalla Libera L , Schiaffino S
Published: May 2004

Antisera against bovine atrial myosin were raised in rabbits, purified by affinity chromatography, and absorbed with insolubilized ventricular myosin. Specific anti-bovine atrial myosin (anti-bAm) antibodies reacted selectively with atrial myosin heavy chains, as determined by enzyme immunoassay combined with SDS-gel electrophoresis. In direct and indirect immunofluorescence assay, anti-bAm was found to stain all atrial muscle fibers and a minor proportion of ventricular muscle fibers in the right ventricle of the bovine heart. In contrast, almost all muscle fibers in the left ventricle were unreactive. Purkinje fibers showed variable reactivity. In the rabbit heart, all atrial muscle fibers were stained by anti-bAm, whereas ventricular fibers showed a variable response in both the right and left ventricle, with a tendency for reactive fibers to be more numerous in the right ventricle and in subepicardial regions. Diversification of fiber types with respect to anti-bAm reactivity was found to occur during late stages of postnatal development in the rabbit heart and to be influenced by thyroid hormone. All ventricular muscle fibers became strongly reactive after thyroxine treatment, whereas they became unreactive or poorly reactive after propylthiouracil treatment. These findings are consistent with the existence of different ventricular isomyosins whose relative proportions can vary according to the thyroid state. Variations in ventricular isomyosin composition can account for the changes in myosin Ca2+-activated ATPase activity previously observed in cardiac muscle from hyper- and hypothyroid animals and may be responsible for the changes in the velocity of contraction of ventricular myocardium that occur under these conditions. The differential distribution of ventricular isomyosins in the normal heart suggests that fiber types with different contractile properties may coexist in the ventricular myocardium.
Direct visualization of fluorescein-labeled microtubules in vitro and in microinjected fibroblasts.
Authors: Keith C H , Feramisco J R , Shelanski M
Published: May 2004

Microtubule proteins and tubulin have been purified from brain and labeled with dichlorotriazinyl fluorescein (DTAF). This procedure compromises neither the polymerizability of the proteins nor their affinities for unlabeled proteins. Within 15 min after microinjection of either DTAF-microtubule proteins or DTAF-tubulin into cultured gerbil fibroma cells, there was an evolution of a fluorescent fibrillar pattern with a distribution similar to that of the microtubular network seen after staining with fluorescent antitubulin. These filaments were colchicine sensitive and could be seen to elongate with time. DTAF-labeled microtubule accessory proteins from brain were not incorporated into filaments and appeared to label autophagic vacuoles.
Cell-cycle analysis of insulin binding and internalization on mouse melanoma cells.
Authors: Shimizu N , Shimizu Y , Fuller B B
Published: May 2004

Binding of 125I-labeled insulin to the surface receptors of Cloudman S-91 mouse melanoma cells (CCL 53.1) was studied at various phases (M, G1, S, and G2) in the cell cycle. Insulin-binding activity was persistently present during the cell cycle but the highest activity was noted at the S-phase. The insulin once bound to the cell surface receptors at any phase of the cell cycle was internalized and degraded, presumably through a lysosomal pathway. Insulin-indexing activity of melanoma cells was not affected by melanocyte-stimulating hormone.
Membrane-bound ribosomes of myeloma cells. IV. mRNA complexity of free and membrane-bound polysomes.
Authors: Mechler B , Rabbitts T H
Published: May 2004

We have analyzed the sequence complexity, frequency distribution, and template activity of free (F) and membrane-bound (MB) polysomal mRNA populations of MOPC 21 (P3K) mouse myeloma cells. Using the technique of mRNA-cDNA hybridization, we find that F poly(A)+ RNA, which represent 60% of total polysomal mRNA, consists of approximately 8,000 different mRNA sequences distributed in three abundance classes, while MB poly(A)+ RNA (20% of total polysomal mRNA) includes only 230 mRNA species and almost completely lacks very infrequent mRNA species. Cross-hybridization indicates that MB mRNA sequences are also present in F mRNA, but in reduced concentrations. Translation of F and MB RNA fractions in a messenger-dependent reticulocyte lysate indicates that essentially all MB RNA contains poly(A), whereas 25% of F mRNA lacks poly(A). Furthermore, the use of a cDNA highly specific for the immunoglobulin light (Ig L) chain mRNA allows the determination of the subcellular content of this message. Ig L mRNA, representing approximately 5% of total polysomal poly(A)+ RNA, is one of the most abundant MB mRNAs. 90% of Ig L mRNA is found in MB polysomes and 10% in F polysomes.
Membrane-bound ribosomes of myeloma cells. V. Subcellular distribution of immunoglobulin mRNA molecules.
Authors: Mechler B
Published: May 2004

The subcellular distribution of the most abundant mRNA sequences, particularly those of the immunoglobulin heavy (Ig H) and light (IG L) chain mRNA sequences, of MOPC 21 (P3K) mouse myeloma cells has been examined by translating the mRNA of various subcellular fractions in a messenger-dependent reticulocyte lysate (MDL) and by identifying Ig products with the use of a specific antiserum. Analyses of the distribution of the mRNA template activity and the translation products by SDS polyacrylamide gel electrophoresis reveal that approximately 85% of the mRNA present in the free ribosomal fraction is incorporated into polysomes and that the remainder is present as mRNP particles. On the endoplasmic reticulum (ER) the mRNA is found entirely in polysomes. In general, the size class of free (F) and membrane-bound (MB) polysomes corresponds to the size of their translation products. Thus, mRNAs coding Ig H (5.0 x 10(5) daltons in size) and Ig L (2.5 x 10(5) daltons in size) are incorporated into polysomes formed of 12 and 6 ribosomes, respectively. About 10% of the Ig mRNAs are not bound to membranes. A third of these are associated with mRNPs and the remainder incorporated into F polysomes of the same size as the Ig-synthesizing MB polysomes.
Serum-free growth of normal and transformed fibroblasts in milk: differential requirements for fibronectin.
Authors: Steimer K S , Klagsbrun M
Published: May 2004

Bovine milk may be used as a supplement for the serum-free growth of certain fibroblastic cells in culture. The growth properties of three representative cell types in milk-supplemented medium were examined; fibroblastic cell strains, fibroblastic cell lines, and transformed fibroblasts. Transformed fibroblasts, which included RNA and DNA tumor virus-transformed cells and carcinogen-transformed cells, grew in milk. Instead of growing attached to the culture dishes, as they normally do in serum, transformed fibroblasts grew in milk as large clusters in suspension. In contrast, nontransformed fibroblastic cell strains and cell lines did not grow in milk-supplemented medium. Fibroblasts transformed by a temperature-sensitive transformation mutant of Rous sarcoma virus were temperature-sensitive for growth in milk. The failure of cells to adhere to the substratum in milk-supplemented medium suggested that milk might be deficient in attachment factors for fibroblasts. When the attachment of fibroblastic cells in milk-supplemented medium was facilitated by pretreating culture dishes with fibronectin, (a) transformed cells grew attached rather than in suspension, (b) normal cell lines attached and grew to confluence, and (c) normal cell strains adhered and survived but did not exhibit appreciable cell proliferation.
Effect of oxygen tension on human peripheral blood leukocytes: lysosomal enzyme release and metabolic responses during phagocytosis
Authors: Skosey JL , Chow DC , Nusinow S , May J , Gestautas V , Niwa Y
Published: May 2004

We found that nonlethal lysosomal enzyme release from human peripheral blood leukocytes during phagocytosis of opsonized zymosan in vitro was modified by the oxygen tension under which the cells were incubated; with decreasing Po(2), zymosan-induced release of lysosomal enzymes was potentiated. The effect on enzyme release could not be attributed secondarily to an effect on phagocytosis, because, as others have reported, Po(2) had little effect on that response. Metabolic responses that accompany phagocytosis were also modified by oxygen tension. Stimulation of oxidation by way of the pentose cycle was further enhanced by increasing Po(2). Conversely, anaerobic glycolysis was promoted by decreasing oxygen tension. ATP levels fell as a function of time and concentration of phagocytic stimulus, mirroring lysosomal enzyme release as modified by Po(2). Cyclic AMP levels fell during phagocytosis and lysosomal enzyme release, a change that could act to facilitate lysosomal enzyme release. However, the fall in nucleotide level was greatest with highest Po(2) (i.e., when lysosomal enzyme release was least). The inverse relationship between oxidative metabolism and enzyme release suggested that a product of oxidative metabolism might adversely influence enzyme release. Sulfhydryl antioxidants (Cysteine, glutathione) and scavengers of oxygen-derived reactants (superoxide dismutase, catalase, benzoate, hypoxanthine, xanthine, histidine, azide) all potentiated zymosan- stimulated enzyme release. These findings are consistent with the interpretation that one or more factors (e.g., superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen), generated in association with the burst of oxidative metabolism which accompanies phagocytosis, acts to inhibit lysosomal enzyme release.
Fluorescence studies on modes of cytochalasin B and phallotoxin action on cytoplasmic streaming in Chara.
Authors: Nothnagel E A , Barak L S , Sanger J W , Webb W W
Published: May 2004

Various investigations have suggested that cytoplasmic streaming in characean algae is driven by interaction between subcortical actin bundles and endoplasmic myosin. To further test this hypothesis, we have perfused cytotoxic actin-binding drugs and fluorescent actin labels into the cytoplasm of streaming Chara cells. Confirming earlier work, we find that cytochalasin B (CB) reversibly inhibits streaming. In direct contrast to earlier investigators, who have found phalloidin to be a potent inhibitor of movement in amoeba, slime mold, and fibroblastic cells, we find that phalloidin does not inhibit streaming in Chara but does modify the inhibitory effect of CB. Use of two fluorescent actin probes, fluorescein, isothiocyanate-heavy meromyosin (FITC-HMM) and nitrobenzoxadiazole-phallacidin (NBD-Ph), has permitted visualization of the effects of CB and phalloidin on the actin bundles. FITC-HMM labeling in perfused but nonstreaming cells has revealed a previously unobserved alteration of the actin bundles by CB. Phalloidin alone does not perceptibly alter the actin bundles but does block the alteration by CB if applied as a pretreatment, NBD-Ph perfused into the cytoplasm of streaming cells stains actin bundles without inhibiting streaming. NBD-Ph staining of actin bundles is not initially observed in cells inhibited by CB but does appear simultaneously with the recovery of streaming as CB leaks from the cells. The observations reported here are consistent with the established effects of phallotoxins and CB on actin in vitro and support the hypothesis that streaming is generated by actin-myosin interactions.
Fate of plasma membrane during endocytosis. III. Evidence for incomplete breakdown of immunoglobulins in lysosomes of cultured fibroblasts
Authors: Scheider Y , Duve C , Trouet A
Published: May 2004

Rat embryo fibroblasts, when cultured in the presence of control rabbit immunoglobulins (C IgG), doubly labeled by (3)H-acetylation (A) and then conjugated with flourescein (F), take up FAC IgG continuously for at least 72 h. They return the major part of their intake back to the medium in the form of breakdown products of very low molecular weight. Gel filtration and immunological analyses of cells and medium at various times indicate that essentially all the FAC IgG molecules taken up undergo digestion of their Fc part, but that the Fab part of only about three-fourths of the molecules is degraded. The rest remains stored intracellularly in the form of F(ab')2-type fragments that slowly dissociate into Fab'-type fragments.

When FAC IgG was incubated in vitro in the presence of a hepatic lysosomal extract, complete digestion of the Fc part likewise occurred, but the Fab' part of most if not all the molecules proved resistant to breakdown, and remained as Fab'-type fragments.

Cell fractionation experiments have demonstrated that the storage compartment of the FAC IgG and of its digestion residues: (a) shows a density distribution pattern in a sucrose gradient identical to that of the lysosomal marker N-acetyl-β-glucosaminidase and clearly dissociated from that of the Golgi marker galactosyltransferase, and (b) accompanies the lysosomal marker in its density shift induced by exposure of the cells to chloroquine.

It is concluded that storage and processing of FAC IgG by rat fibroblasts occur in a single, digestively active compartment of lysosomal nature, and that resistance to digestion of certain Fab'-type fragments accounts largely for the inability of the lysososmal enzymes to completely digest the FAC IgG taken up. This conclusion implies that the intracellular storage compartment through which, in earlier work, plasma membrane patches were found to transit after endocytosis and before recycling to the cell surface consists of authentic lysosomes.
Involvement of contractile proteins in the changes in consistency of oocyte nucleoplasm of the newt Pleurodeles waltlii.
Authors: Gounon P , Karsenti E
Published: May 2004

In the present work, we show that actin is present in considerable quantities in the oocyte nucleus of the newt Pleurodeles waltlii. The nuclear sap, extracted in saline buffer containing Ca++, is fluid. DNAase I inhibition assays have shown that 90% of actin is under a globular state in such conditions. Chelation of Ca++ by EGTA leads to the formation of a nuclear gel composed of individual microfilaments. This nuclear gel contains approximately 50% of total nuclear actin in a filamentous form. Phalloidin, a drug known to stabilize F-actin, induces the formation of a network of actin cables in the nuclei. This network contains nearly 100% of total nuclear actin in the filamentous form. The observation of the cables in the electron microscope shows that they are made of tightly associated microfilaments to which RNP-like particles are bound. The actin antibodies stain the cables and the particles by the indirect immunoperoxidase technique; myosin antibodies mainly stain the particles. The formation of the phalloïdin-induced network seems to require the presence of Ca++, Mg++, and ATP. We propose a scheme for the regulation of the supramolecular forms of actin in oocyte nuclei in which a delicate equilibrium seems to exist between globular actin, microfilaments, and actin cables. This equilibrium would be controlled by the concentration of Ca++, ATP, and various actin-associated proteins.
Rapid rounding of human epidermoid carcinoma cells A-431 induced by epidermal growth factor
Authors: Chinkers M , McKanna JA , Cohen S
Published: May 2004

Epidermal growth factor (EGF) induces rapid rounding of A-431 human epidermoid carcinoma cells in Ca(++)-free medium. Cell rounding is not induced by a variety of other polypeptide hormones, antiserum to cell membranes, local anesthetics, colchicine, cytochalasin B, or cyclic nucleotides. However, trypsin, like EGF, induces rounding of A- 431 cells in the absence of Ca(++). Both trypsin- and EGF-induced rounding are temperature dependent, appear to be energy dependent, and are inhibited by cytochalasins, suggesting that the active participation of microfilaments in cell rounding. However, a medium transfer experiment suggests that EGF-induced rounding is not attributable to secretion of a protease, and a number of serine protease inhibitors have no effect on the EGF-induced rounding process. Cell rounding is not attributable to the slight stimulation by EGF of the release of Ca(++) that is observed in the Ca(++)-free medium, as stimulation of such release by the ionophore A23187 neither induces cell rounding nor blocks EGF-induced rounding.

Cells that have rounded up after treatment with EGF or trypsin spread out upon addition of Ca(++) to the medium, even in the continuing presence of EGF or typsin. Like the cell-rounding process, the cell-spreading process is temperature dependent, appears to be energy dependent, and is inhibited by cytochalasin B. Thus, EGF does not destroy the ability of the cell to spread; rather, in the presence of the EGF (or trypsin), cell spreading and the maintenance of the flattened state become dependent on external Ca(++). Because untreated cells remain flattened in the absence of Ca(++), the data suggest that EGF may disrupt Ca(++)-independent mechanisms of adhesion normally present in A-431 cells.
Inefficient internalization of receptor-bound low density lipoprotein in human carcinoma A-431 cells.
Authors: Anderson R G , Brown M S , Goldstein J L
Published: May 2004

Human epithelioid carcinoma A-431 cells are known to express unusually large numbers of receptors for the polypeptide hormone epidermal growth factor. The current studies demonstrate that this cell line also expresses 5- to 10-fold more low density lipoprotein (LDL) receptors per cell than either human fibroblasts or Chinese hamster ovary (CHO) cells. As visualized with an LDL-ferritin conjugate, the LDL receptors in A-431 cells appeared in clusters that were distributed uniformly over the cell surface, occurring over flat regions of the membrane as well as over the abundant surface extensions. Only 4% of the LDL receptors were located in coated pits. The LDL receptors in A-431 cells showed the same affinity and specificity as the LDL receptors in human fibroblasts and other cell types. In addition, they were subject to feedback regulation by sterols in the same manner as the LDL receptors in other cells. However, in contrast to other cell types in which the receptor-bound LDL is internalized with high efficiency, in the A-431 cells only a small fraction of the receptor-bound LDL entered the cell. In CHO cells approximately 66% of the LDL receptors were located over coated regions of membrane, and the efficiency of LDL internalization was correspondingly 10-fold higher than in A-431 cells. These findings support the concept that the rate of LDL internalization is proportional to the number of LDL receptors in coated pits and that the inefficiency of internalization in the A-431 cells is caused by a limitation in the ability of these cells to incorporate their LDL receptors into coated pits.
A factor from neurons induces partial immobilization of nonclustered acetylcholine receptors on cultured muscle cells.
Authors: Axelrod D , Bauer H C , Stya M , Christian C N
Published: May 2004

A factor or factors released by cultured NG108-15 neuroblastoma X glioma hybrid cells and added to the medium of rat myotube primary cultures was found to immobilize some of the previously mobile acetylcholine receptors in the myotube membrane. Partial receptor immobilization occurred within 3 h after the beginning of treatment with the NG108-15-conditioned medium factor and persisted for at least 24 h of continuous treatment. A similarly derived conditioned medium concentrate from the non-neuronal parent glioma cell line did not immobilize receptors, relative to untreated controls. Acetylcholine receptors were visualized by fluorescent alpha-bungarotoxin and their lateral motion was observed by the technique of fluorescence photobleaching recovery.
Self-assembly of spectrin oligomers in vitro: a basis for a dynamic cytoskeleton.
Authors: Morrow J S , Marchesi V T
Published: May 2004

Purified human erythrocyte spectrin is able to form large oligomeric species without the collaboration of any other proteins. This reversible self-assembly process is both temperature and concentration dependent and seems to be mediated by the same kinds of low affinity noncovalent associations between spectrin monomers that promote tetramer formation. Low ionic strength extracts of erythrocyte membranes also contain these oligomeric species. These results support the idea that spectrin oligomers and the factors that regulate their formation may be responsible for both the stability and the versatility of the erythrocyte membrane cytoskeleton. It is postulated that the high concentrations of spectrin necessary for oligomerization are maintained in vivo by a high-affinity interaction with ankyrin. Such a coupling of high and low affinity interactions in multifunctional proteins may have significant implications for membrane structure and function.
Mechanism of action of cytochalasin: evidence that it binds to actin filament ends.
Authors: Brown S S , Spudich J A
Published: May 2004

To test the idea that cytochalasin retards actin assembly by binding to filament ends, we have designed a new assay for cytochalasin binding in which the number of filament ends can be varied independently of the total actin concentration. Actin is reacted with polylysine-coated polystyrene beads to make filament ends (Brown and Spudich, 1979, J. Cell Biol. 80:499-504) and then reacted with [3H]cytochalasin B. We have found that cytochalasin B binds to beads in the presence of actin, and that the number of cytochalasin B binding sites can be varied as a function of the number of filament ends independent of the total actin concentration by varying the bead concentration.
Regulation of the synthesis of lactate dehydrogenase-X during spermatogenesis in the mouse.
Authors: Wieben E D
Published: May 2004

Total mouse testis RNA directs the synthesis of the sperm-specific C subunit of lactate dehydrogenase-X (LDH-X) when translated in a cell-free system derived from rabbit reticulocytes. The newly synthesized C subunits were isolated by immunoprecipitation with antibody specific for this isozyme, and quantitated by electrophoresis on SDS polyacrylamide gels. The amount of radioactivity incorporated into the enzyme subunit was directly proportional to the amount of testis RNA added to the translational system, thereby providing a sensitive and reliable method for assessing relative LDH-X mRNA activity. A combination of sucrose gradient centrifugation and oligo(dT)-cellulose chromatography resulted in a 23-fold purification of LDH-X mRNA over total cytoplasmic testis RNA. Analysis of LDH-X mRNA activity in the developing testis indicated that the appearance of functional LDH-X mRNA activity coincides with the appearance of LDH-X catalytic activity at 14 d postpartum. Measurement of LDH-X mRNA levels in separated testis cell populations prepared by centrifugal elutriation demonstrated that LDH-X mRNA represents 0.17-0.18% of the total functional mRNA activity in fractions enriched in pachytene spermatocytes and round spermatids, but only 0.09-0.10% of the translation products of elongated spermatids.
Separation of dissociated thyroid follicular and parfollicular cells: association of serotonin binding protein with parafollicular cells
Authors: Bernd P , Gershon MD , Nunez EA , Tamir H
Published: May 2004

Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.
Glucocorticoid regulation of amino acid transport in anucleate rat hepatoma (HTC) cells.
Authors: McDonald R A , Gelehrter T D
Published: May 2004

The transport of alpha-aminoisobutyric acid (AIB) by rat hepatoma tissue culture (HTC) cells is rapidly and reversibly inhibited by dexamethasone and other glucocorticoids. To investigate the role of the nucleus in the regulation of transport and to determine whether steroid hormones or steroid-receptor complexes may have direct effects on cytoplasmic or membrane functions, we have examined the regulation of transport by dexamethasone in anucleate HTC cells. Cytoplasts prepared from suspension cultures of HTC cells fully retain active transport of AIB with the same kinetic properties as intact cells. However, the uptake of AIB is not inhibited by dexamethasone or other corticosteroids. Neither is the inhibited rate of transport, manifested by cytoplasts prepared from dexamethasone-treated cells, restored to normal upon removal of the hormone. Anucleate cells exhibit specific, saturable binding of [3H]dexamethasone; however, the binding is reduced compared with that of intact cells. The nucleus is thus required for the glucocorticoid regulation of amino acid transport in HTC cells.
hnRNA and its attachment to a nuclear protein matrix.
Authors: van Eekelen C A , van Venrooij W J
Published: May 2004

In this study, DNA-depleted nuclear protein matrices are isolated from HeLa S3 cells. These nuclear matrices consist of peripheral laminae, residual nucleoli, and internal fibrillar structures. High molecular weight, heterogeneous nuclear RNA (hnRNA) is quantitatively associated with these structures and can be released intact only by affecting the integrity of the matrices. It is, therefore, concluded that hnRNA is part of a highly organized nuclear structure. By irradiation of intact cells or isolated nuclear matrices with ultraviolet light, proteins tightly associated with hnRNA can be induced to cross-link with the RNA. Performing the cross-linking in vivo is an extra guarantee that only hnRNA-protein (hnRNP) complexes existing in the intact cell are covalently linked. Such hnRNP complexes were isolated and purified under conditions that completely dissociate nonspecific RNA-protein complexes. By comparison of the hnRNP found in nuclear matrices and the published data on the composition of hnRNP particles, it was found that the so-called hnRNP "packaging" proteins (32,000-38,000 mol wt) were not efficiently cross-linked to hnRNA by UV irradiation. They were, however, present in the matrix preparations, bound to hnRNA, because they were released from nuclear matrices after ribonuclease treatment of these structures. On the other hand, two major hnRNPs (41,500 and 43,000 mol wt) were efficiently cross-linked to hnRNA. These proteins were not released by ribonuclease treatment, which suggests that they are involved in the binding of hnRNA to the nuclear matrix.
Structural changes after transmitter release at the frog neuromuscular junction.
Authors: Heuser J E , Reese T S
Published: May 2004

The sequence of structural changes that occur during synaptic vesicle exocytosis was studied by quick-freezing muscles at different intervals after stimulating their nerves, in the presence of 4-aminopyridine to increase the number of transmitter quanta released by each stimulus. Vesicle openings began to appear at the active zones of the intramuscular nerves within 3-4 ms after a single stimulus. The concentration of these openings peaked at 5-6 ms, and then declined to zero 50-100 ms late. At the later times, vesicle openings tended to be larger. Left behind at the active zones, after the vesicle openings disappeared, were clusters of large intramembrane particles. The larger particles in these clusters were the same size as intramembrane particles in undischarged vesicles, and were slightly larger than the particles which form the rows delineating active zones. Because previous tracer work had shown that new vesicles do not pinch off from the plasma membrane at these early times, we concluded that the particle clusters originate from membranes of discharged vesicles which collapse into the plasmalemma after exocytosis. The rate of vesicle collapse appeared to be variable because different stages occurred simultaneously at most times after stimulation; this asynchrony was taken to indicate that the collapse of each exocytotic vesicle is slowed by previous nearby collapses. The ultimate fate of synaptic vesicle membrane after collapse appeared to be coalescence with the plasma membrane, as the clusters of particles gradually dispersed into surrounding areas during the first second after a stimulus. The membrane retrieval and recycling that reverse this exocytotic sequence have a slower onset, as has been described in previous reports.
Identification of a novel class of tandemly repeated genes transcribed on lampbrush chromosomes of Pleurodeles waltlii.
Authors: Scheer U
Published: May 2004

Electron microscope preparations of lampbrush chromosomes from oocytes of Pleurodeles waltlii have revealed a new class of tandemly repeated genes. These genes are highly active, as judged by the close spacing of nascent transcripts. They occur in clusters of greater than 100 copies and are transcribed in units containing roughly 940 base pairs of DNA that are separated by nontranscribed spacers of an estimated DNA content of 2,410 base pairs. The size and the pattern of arrangement of these transcription units can not be correlated with any of the repetitious genes so far described.
Appearance of fibronectin during the differentiation of cartilage, bone, and bone marrow.
Authors: Weiss R E , Reddi A H
Published: May 2004

Fibronectin has been localized by indirect immunofluorescence during the various phases of endochondral bone formation in response to subcutaneously implanted demineralized bone matrix. Its histologic appearance has been correlated with results of biosynthetic experiments. (a) The implanted collagenous bone matrix was coated with fibronectin before and during mesenchymal cell proliferation. (b) During proliferation of mesenchymal precursor cells, the newly synthesized extracellular matrix exhibited a fibrillar network of fibronectin. (c) During cartilage differentiation, the fibronectin in the extracellular matrix was apparently masked by proteoglycans, as judged by hyaluronidase treatment. (d) Differentiating chondrocytes exhibited a uniform distribution of fibronectin. (e) Fibronectin was present in a cottony array around osteoblasts during osteogenesis. (f) The developing hematopoietic colonies revealed fibronectin associated with them. Therefore, it appears that fibronectin is ubiquitous throughout the development of endochondral bone and bone marrow.
Interaction of ouabain with the Na(+) pump in intact epithelial cells
Authors: Mills JW , MacKnight ADC , Jarrell JA , Dayer JM , Ausiello DA
Published: May 2004

To determine the specificity and efficacy of [(3)H]ouabain binding as a quantitative measure of the Na(+) pump (Na(+), K(+)-ATPase) and as a marker for the localization of pumps involved in transepithelial Na(+)-transport, we analyzed the interaction of [(3)H]ouabain with its receptor in pig kidney epithelial (LLC-PK(1)) cells. When these epithelial cells are depleted of Na(+) and exposed to 2 muM [(3)H]ouabain in a Na(+)-free medium, binding is reduced by 90 percent. When depleted of K(+) and incubated in a K(+)- free medium, the ouabain binding rate is increase compared with that measured at 5 mM. This increase is only demonstable when Na(+) is present. The increased rate could be attributed to the predominance of the Na(+)-stimulated phosphorylated form of the pump, as K(+) is not readily available to stimulate dephosphorylation. However, some binding in the K(+)-free medium is attributable to pump turnover (and therefore, recycling of K(+)), because analysis of K(+)-washout kinetics demonstrated that addition of 2 muM ouabain to K(+)-depleted cells increased the rate of K(+) loss. These results indicate that in intact epithelial cells, unlike isolated membrane preparations, the most favorable condition for supporting ouabain binding occurs when the Na(+), K(+)-ATPase is operating in the Na(+)-pump mode or is phosphorylated in the presence of Na(+).

When LLC-PK(1) cells were exposed to ouabain at 4 degrees C, binding was reduced by 97 percent. Upon rewarming, the rate of binding was greater than that obtained on cells kept at a constant 37 degrees C. However, even at this accelerated rate, the time to reach equilibrium was beyond what is required for cells, swollen by exposure to cold, to recover normal volume. Thus, results from studies that have attempted to use ouabain to eliminate the contribution of the conventional Na(+) pump to volume recovery must be reevaluated if the exposure to ouabain was done in the cold or under conditions in which the Na(+) pump is not operating.
Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores.
Authors: Pollard T D , Mooseker M S
Published: May 2004

We used actin filament bundles isolated from intestinal brush-border microvilli to nucleate the polymerization of pure muscle actin monomers into filaments. Growth rates were determined by electron microscopy by measuring the change in the length of the filaments as a function of time. The linear dependence of the growth rates on the actin monomer concentration provided the rate constants for monomer association and dissociation at the two ends of the growing filament. The rapidly growing ("barbed") end has higher association and dissociation rate constants than the slowly growing ("pointed") end. The values of these rate constants differ in 20 mM KCl compared with 75 mM KCl, 5 mM MgSO4. 2 microM cytochalasin B blocks growth entirely at the barbed end, apparently by reducing both association and dissociation rate constants to near zero, but inhibits growth at the pointed end to only a small extent.
Sea urchin tube feet: unique structures that allow a cytological and molecular approach to the study of actin and its gene expression.
Authors: Kabat-Zinn J , Singer R H
Published: May 2004

Actin is the major extractable protein component from the tube feet of four different species of sea urchin: Arbacia punctulata, Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, and Diadema setosum. Actin made up as much as 60% of the total Coomassie Blue-staining material after SDS polyacrylamide gel electrophoresis and densitometer analysis. Two-dimensional gel electrophoresis resolved two, and possible three, species of actin for each sea urchin of which the dominant component was analogous to the beta form in vertebrates. In a cell-free system from rabbit reticulocytes, total RNA from tube feet stimulated the synthesis of one protein that represented 80% of the total methionine incorporation, migrated with the properties characteristic of actin in a two-dimensional gel system, and on proteolysis yielded fragments identical to purified rabbit actin. The mRNAs from the tube feet of two divergent species of sea urchin, Arbacia punctulata and Strongylocentrotus purpuratus, synthesized actins differing by less than 0.02 pH unit for each isospecies 90% of the DNA copied from tube foot RNA by reverse transcriptase represented a highly abundant sequence class judged by copy DNA(cDNA)-RNA excess hybridization. At least two-thirds of this class represented a low-complexity component, with a Rot1/2 about three times that expected for actin messenger RNA. The remarkable degree of conservation of the actin protein is reflected in concomitant conservation of the protein-coding nucleotide sequences of the messenger RNA, which has allowed the use of a cDNA probe to isolate actin sequences from a human phage library.
Cell-substrate contacts illuminated by total internal reflection fluorescence.
Authors: Axelrod D
Published: May 2004

A technique for exciting fluorescence exclusively from regions of contact between cultured cells and the substrate is presented. The technique utilizes the evanescent wave of a totally internally reflecting laser beam to excite only those fluorescent molecules within one light wavelength or less of the substrate surface. Demonstrations of this technique are given for two types of cell cultures: rat primary myotubes with acetylcholine receptors labeled by fluorescent alpha-bungarotoxin and human skin fibroblasts labeled by a fluorescent lipid probe. Total internal reflection fluorescence examination of cells appears to have promising applications, including visualization of the membrane and underlying cytoplasmic structures at cell-substrate contacts, dramatic reduction of autofluorescence from debris and thick cells, mapping of membranes topography, and visualization of reversible bound fluorescent ligands at membrane receptors.
Transport of newly synthesized vesicular stomatitis viral glycoprotein to purified Golgi membranes.
Authors: Rothman J E , Fries E
Published: May 2004

In a previous communication we reported that the newly synthesized membrane glycoprotein of vesicular stomatitis virus could be transported in crude extracts of CHO cells from endoplasmic reticulum-derived membranes to membranes of the Golgi complex. This conclusion was an indirect one, based on the terminal glycosylation of this glycoprotein, a reaction that was dependent upon a Golgi-specific enzyme, UDP-GlcNAc transferase I. We show here that the Golgi fraction of rat liver will substitute for members of CHO cells as a source of transferase I in this reaction. The use of highly purified fractions of liver Golgi membranes, coupled with the ability to recover these membranes from incubations, has now permitted a direct demonstration of net transport of G protein to these heterologous Golgi membranes. This transport reaction is specific, in that the smooth endoplasmic reticulum fraction will not substitute for the Golgi fraction, is quantitatively significant, involving at least 30% of the viral glycoprotein, and is sustained only in the presence of both ATP and a soluble, cytosol fraction of liver cells.
Polarity of dynein-microtubule interactions in vitro: cross-bridging between parallel and antiparallel microtubules.
Authors: Warner F D , Mitchell D R
Published: May 2004

Ciliary doublet microtubules produced by sliding disintegration in 20 muM MgATP2-reassociate in the presence of exogenous 30S dynein and 6 mM MgSO4. The doublets form overlapping arrays, held together by dynein cross-bridges. Dynein arms on both A and B subfibers serve as unambiguous markers of microtubule polarity within the arrays. Doublets reassociate via dynein cross-bridges in both parallel and antiparallel modes, although parallel interactions are favored 2:1. When 20 muM ATP is added to the arrays, the doublets undergo both vanadate-sensitive and insensitive forms of secondary disintegration to reproduce the original population of doublets. The results demonstrate that both parallel and antiparallel doublet cross-bridging is sensitive to dissociation by ATP even though normal ciliary motion depends strictly on dynein interactions between parallel microtubules.
Microtubule assembly and disassembly at alkaline pH
Authors: Regula CS , JR Pfeiffer , Berlin RD
Published: May 2004

Although it is now apparent that the intracellular pH may rise considerably above neutrality under physiological conditions, information on the effect of alkaline pH on microtubule assembly and disassembly is still quite fragmentay. We have studied the assembly/disassembly of bovine brain microtubule protein at alkaline pH in vitro. When microtubules are assembled to a new steady state at pH less than 7 and pH is then made more alkaline, they undergo a rapid disassembly to a new steady state. This disassembly is reversed by acidification. The degree of disassembly is determined largely by the pH- dependence of the critical concentration, which increases five to eight times, from pH 7 to 8. A fraction of assembly-incompetent tubulin is identified that increases with pH, but its incompetency is largely reversed with acidification. Measurements of microtubule lengths are used to indicate that disassembly occurs by uniform shortening of microtubules. A comparison of shortening by alkalinization with dilution suggests that the intrinsic rate of disassembly is accelerated by increasing pH.

The capacity for initiating assembly is progressively lost with incubation at alkaline pH (although some protection is afforded by sulfhydryl-reducing agents). However, direct assembly from depolymerized mixtures is possible at least up to pH 8.3, and the steady state achieved at these alkaline pH values is stable. Such preparations are readily disassembled by cold and podophyllotoxin (PLN). Disassembly induced by PLN is also markedly enhanced at alkaline pH, suggesting a corresponding enhancement of "treadmilling."

The implications of physiological events leading to alkaline shifts of pH for microtubule assembly/disassembly are discussed, particularly in the light of recent hypotheses regarding treadmilling and its role in controlling the distribution of microtubules in vivo.