bluuebunny/crossref_metadata_2025 huggingface dataset:
Transmembrane biogenesis of the vesicular stomatitis virus glycoprotein.
Authors: Katz F N , Lodish H F
Published: May 2004

Previous work has shown that the mRNA encoding the vesicular stomatitis virus (VSV) glycoprotein (G) is bound to the rough endoplasmic reticulum (RER) and that newly made G protein is localized to the RER. In this paper, we have investigated the topology and processing of the newly synthesized G protein in microsomal vesicles. G was labeled with [35S]methionine ([35S]met), either by pulse-labeling infected cells or by allowing membrane-bound polysomes containing nascent G polipeptides to complete G synthesis in vitro. In either case, digestion of microsomal vesicles with any of several proteases removes approximately 5% (30 amino acids) from each G molecule. These proteases will digest the entire G protein if detergents are present during digestion. Using the method of Dintzis (1961, Proc. Natl. Acad. Sci. U. S. A. 47:247--261) to order tryptic peptides (8), we show that peptides lost from G protein by protease treatment of closed vesicles are derived from the carboxyterminus of the molecule. The newly made VSV G in microsomal membranes is glycosylated. If carbohydrate is removed by glycosidases, the resultant peptide migrates more rapidly on polyacrylamide gels than the unglycosylated, G0, form synthesized in cell-free systems in the absence of membranes. We infer that some proteolytic cleavage of the polypeptide backbone is associated with membrane insertion of G. Further, our findings demonstrate that, soon after synthesis, G is found in a transmembrane, asymmetric orientation in microsomal membranes, with its carboxyterminus exposed to the extracisternal, or cytoplasmic, face of the vesicles, and with most or all of its amino-terminal peptides and its carbohydrate sequestered within the bilayer and lumen of the microsomes.
Life history of mouse sperm protein. Intratesticular stages.
Authors: Rodman T C , Litwin S D , Romani M , Vidali G
Published: May 2004

A basic protein fraction, migrating as a single band in acetic acid-urea gel, distinct from histones, was isolated from mouse sperm collected from vasa deferentia and caudae epididymides and was used to immunize female rabbits. The presence of antibodies to the mouse sperm protein (MSP) in the rabbit antisera was demonstrated by a cytoimmunofluorescence procedure using the cells of origin of the antigenic protein, the mature mouse sperm. The specificity of the antisera was verified by fluid and gel precipitation tests and by crossed immunoelectrophoresis. The latter procedure demonstrated the presence of two antigen-antibody systems, consonant with earlier reports that the basic chromosomal protein of mouse sperm is heterogeneous. MSP antigen in situ was recognized by the specific antibodies of the rabbit antisera only after the smear of mature sperm was treated with either of two reducing agents: 2-mercaptoethanol or dithiothreitol. However, when the immunofluorescence procedure was applied to untreated smears of mouse testicular cells, spermatids of all stages from 1 to 14-15 were positive, while spermatocytes, stage 16 spermatids and spermatozoa were negative. After treatment of testes smears with reducing agent, only spermatocytes remained negative. Those observations indicate the following: (a) MSP is immunogenic in a heterologous species; (b) its antigenic sites are detectable in spermatozoa and spermatids of all stages, but not in primary spermatocytes; (c) those antigenic sites become masked at about stage 15 of spermiogenesis and may be unmasked by treatment with a reducing agent. The interpretation is made, therefore, that one or more components of MSP are assembled at the beginning of spermiogenesis and undergo an alteration in the final intratesticular stage of spermatid maturation. That alteration may be presumed to be the formation of disulfide linkages between the cysteine residues.
Active movements of the chromatoid body. A possible transport mechanism for haploid gene products.
Authors: Parvinen M , Parvinen L M
Published: May 2004

Recent data indicate that the chromatoid body typical of rat spermatogenesis may contain RNA synthesized in early spermatids by the haploid genome. Analyses of living step-1 and step-3 spermatids by time-lapse cinephotomicrography have shown that the chromatoid body moves in relation to the nuclear envelope in two different ways. Predominantly in step 1, the chromatoid body moves along the nuclear envelope on a wide area surrounding the Golgi complex and has frequent transient contacts with the latter organelle. In step 3, the chromatoid body was shown to move perpendicular to the nuclear envelope. It was seen located very transiently at the top of prominent outpocketings of the nuclear envelope with apparent material continuities through nuclear pore complexes to intranuclear particles. The rapid movements of the chromatoid body are suggested to play a role in the transport of haploid gene products in the early spermatids, including probably nucleocytoplasmic RNA transport.
Nerve fibers in culture and their interactions with non-neural cells visualized by immunofluorescence.
Authors: Jockusch H , Jockusch B M , Burger M M
Published: May 2004

Cultures of embryonic mouse spinal cord explants, alone or in combination with rat myotubes, were stained by indirect immunofluorescence using antibodies against three structural proteins to: (a) reveal the distribution of these proteins among different cell types, and (b) test the usefulness of antibody staining to reveal the gross morphology of the neurite network in complex cultures. Affinity column purified antibodies were used against chicken gizzard actin, porcine brain tubulin, and skeletal muscle alpha-actinin. Neurites were stained intensely by anti-actin as was the stress fiber pattern of underlying fibroblasts. With anti-tubulin, the staining of neurites was an order of magnitude more intense than the staining of the microtubule pattern of background fibroblasts. Neurite cell bodies and astrocyte-like glia cells were stained with anti-tubulin and their nuclei remained unstained. Anti-tubulin could thus be used to trace even the finest extensions of nerve processes in spinal cord and spinal cord-muscle cultures. Furthermore, it could be combined with the histochemical reaction for acetylcholinesterase (AChE, EC 3.1.1.7) to demonstrate AChE-positive neurons and specialized nerve-muscle contact sites. The staining of neural elements with anti-alpha-actinin was generally much weaker than with anti-actin and anti-tubulin. Neurites were stained only moderately in comparison to myotube Z lines in the same culture. However, a distinct staining of the periphery of dorsal root ganglion cells was observed. Thus, a protein immunologically related to muscle alpha-actinin is present in the nervous system. In myotubes, Z lines were stained intensely with anti-alpha-actinin while I bands were only faintly stained with anti-actin. In isolated myofibrils, both structures were stained intensely with the same antibody preparations.
A morphometrical study of the exocrine pancreatic cell in fasted and fed frogs.
Authors: Slot J W , Geuze J J
Published: May 2004

The influence of feeding on the ultrastruct of the frog exocrine pancreatic cell was studied by morphometrical procedures. Volume and surface of various cell structures were measured and expressed per unit cell volume. The average cellular size was not influenced by feeding. Though protein synthesis changes 5-to 10-fold (van Venrooij, W. J., and C. Poort. 1971. Biochim. Biophys. Acta. 247:468-470), no significant differences were observed in the amount of membrane that constitutes the rough endoplasmic reticulum (RER) and that represented the major part of total cellular membranes. The appearance of the RER changed. When fasted, most of its membrane was arranged in stacks of tightly packed, narrow cisternae. Within 4 h after feeding, these cisternae were separated and irregularly dilated, and ribosomes became ordered in typical rosettes on their surface. The total volume of the Golgi system increased twofold after feeding. The vesicular and tubular elements at the Golgi periphery did not change, but the volumes of the Golgi cisternae and the condensing vacuoles increased 2.5- and 6-fold, respectively. The increased in the amount of membrane present in these structures was only 1.6- and 3.5-fold, which reflects the more distended appearance of the cisternae and the rounded shape of the condensing vacuoles after feeding. Feeding halved the number of secretory granules per cell, and signs of exocytosis were more common than in fasted animals. These findings suggest that, in the frog pancreatic cell, fluctuations in the production of secretory proteins are not accompanied by an important breakdown and renewal of cellular membranes. This may favor a rapid and strong response of the cell to feeding.
Ultrastructure of microfilament bundles in baby hamster kidney (BHK-21) cells. The use of tannic acid.
Authors: Goldman R D , Chojnacki B , Yerna M J
Published: May 2004

After standard glutaraldehyde-osmium tetroxide fixation procedures, the majority of microfilament bundles in BHK-21 cells exhibit relatively uniform electron density along their long axes. The inclusion of tannic acid in the glutaraldehyde fixation solution results in obvious electron density shifts along the majority of microfilament bundles. Striated patterens are frequently observed which consist of regularly spaced electron dense (D) and electron lucid (L) bands. A striated pattern is also observed along many BHK-21 stress fibers after processing for indirect immunofluorescence utilizing BHK-21 myosin antiserum. A direct correlation of these periodicities seen by light and electron microscope techniques is impossible at the present time. However, comparative measurements indicate that the overall patterns seen in the immunofluorescence and electron microscope preparations are similar. The ultrastructural results provide an initial clue for the ultimate determination of the supramolecular organization of contracile proteins other than actin within the microfilament bundles of non-muscle cells.
Visualization of pores (export sites) correlated with cellulose production in the envelope of the gram-negative bacterium Acetobacter xylinum.
Authors: Zaar K
Published: May 2004

The Gram-negative bacterium Acetobacter xylinum assembles a cellulse ribbon composed of a number of microfibrils in the longitudinal axis of its envelope. The zone of ribbon assembly was investigated by freeze-etch electron microscopy. Freeze-etching revealed, beneath the cellulose ribbons, a linear array of pores on the lipopolysaccharide membrane. These pores have a rim diameter of 120--150 A and a central hole or deepening of approximately 35 A. The axes of pore arrays closely coincide with linear arrays of 100 A particles on the E- and P-faces of the fractured lipopolysaccharide membranes. Pores and particles in the lipopolysaccharide membrane are probably congruent. The pores are hypothesized to be the export sites (penetration sites) for cellulose.
Fibronectin from chicken embryo fibroblasts contains covalently bound phosphate.
Authors: Teng M H , Rifkin D B
Published: May 2004

Fibronectin isolated from cultures of chicken embryo fibroblasts (CEF) contains phosphorus linked to serine and threonine by monoester bonds. Normal and Rous sarcoma virus (RSV)-transformed cells were incubated with [32P]orthophosphate, and fibronectin was isolated from the cell surfaces and conditioned media. 32P was stably associated with fibronectin during immunoprecipitation, SDS-polyacrylamide gel electrophoresis, phospholipid solvent extraction, and hot acid but not alkaline treatment. After a limited acid hydrolysis of fibronectin, both phosphoserine and phosphothreonine were found. The specific radioactivity of the 32P-labeled fibronectin from the conditioned medium of normal CEF was higher than that from the cultures of transformed CEF.
On the mechanism of 5-bromodeoxyuridine induction of prolactin synthesis in rat pituitary tumor cells.
Authors: Biswas D K , Abdullah K T , Brennessel B A
Published: May 2004

GH12C1, a clonal strain of rat pituitary tumor cells in culture (GH cells), does not produce detectable amounts of prolactin. 5-Bromodeoxyuridine (BrdUrd), the thymidine analogue, at sublethal concentrations (3-5 microgram/ml) induces prolactin synthesis in these cells. BrdUrd also induces prolactin synthesis in F1BGH12C1 cells, a BrdUrd resistant (BrdUrdr) substrain isolated from GH12C1 cells. The F1BGH12C1 strain is not drug dependent, but its resistance to BrdUrd is a stable phenotype. The significant features of the induction of prolactin synthesis in the BrdUrdr strain are the increased net synthesis of prolactin and the shortening of the lag period of prolactin induction. As BrdUrd concentration in the growth medium is increased, the rise in prolactin synthesis parallels the increased incorporation of BrdUrd into DNA. Prolactin synthesis is first detected when BrdUrd replaces 20-25% of the thymidine in DNA. BrdUrd can replace up to 75-80% of the thymidine within 2 d of treatment. Partial starvation of these cells under specified growth conditions does not affect the general growth pattern of the cells, general protein synthesis, and thymidine uptake, but does affect DNA synthesis. When cells are cultured under conditions in which DNA synthesis is preferentially inhibited, BrdUrd does not induce prolactin synthesis, suggestive of a DNA-mediated mechanism of action for the drug.
Hormonal modulation of ovarian interstitial cells with particular reference to gap junctions.
Authors: Burghardt R C , Anderson E
Published: May 2004

Thin-section and freeze-fracture studies on the rat ovarian interstitial cells revealed reductions in the size and the number of gap junctions after pituitary ablation. Small gap junctions, however, persist as long as 90 days after hypophysectomy even though regressive cytoplasmic changes are completed 75 d earlier. Administration of exogenous human chorionic gonadotrophin (HCG) results in the restoration of the normal interstitial cell morphology which is accompanied by amplification of junctional membrane. Within 24 h of hormone application, gap junction growth is characterized by the appearance of formation plaques. These studies suggest that the effect of hormone on interstitial cell gap junctions is to modulate the junctional surface area.
Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. I. Effects of black widow spider venom and Ca2+-free solutions on the structure of the active zone.
Authors: Ceccarelli B , Grohovaz F , Hurlbut W P
Published: May 2004

Black widow spider venom (BWSV) was applied to frog nerve-muscle preparations bathed in Ca2+-containing, or Ca2+-free, solutions and the neuromuscular junctions were studied by the freeze-fracture technique. When BWSV was applied for short periods (10-15 min) in the presence of Ca2+, numerous dimples (P face) or protuberances (E face) appeared on the presynaptive membrane and approximately 86% were located immediately adjacent to the double rows of large intramembrane particles that line the active zones. When BWSV was applied for 1 h in the presence of Ca2+, the nerve terminals were depleted of vesicles, few dimples or protuberances were seen, and the active zones were almost completely disorganized. The P face of the presynaptic membrane still contained large intramembrane particles. When muscles were soaked for 2-3 h in Ca2+-free solutions, the active zones became disorganized, and isolated remnants of the double rows of particles were found scattered over the P face of the presynaptic membrane. When BWSV was applied to these preparations, dimples or protuberances occurred almost exclusively alongside disorganized active zones or alongside dispersed fragments of the active zones. The loss of synaptic vesicles from terminals treated with BWSV probably occurs because BWSV interferes with the endocytosis of vesicle membrane. Therefore, we assume that the dimples or protuberances seen on these terminals identify the sites of exocytosis, and we conclude that exocytosis can occur mostly in the immediate vicinity of the large intramembrane particles. Extracellular Ca2+ seems to be required to maintain the grouping of the large particles into double rows at the active zones, but is not required for these particles to specify the sites of exocytosis.
Immunological studies of the embryonic muscle cell surface. Antiserum to the prefusion myoblast.
Authors: Friedlander M , Fischman D A
Published: May 2004

Xenogeneic antisera raised in rabbits have been used to detect compositional changes at the cell surfaces of differentiating embryonic chick skeletal muscle. In this report, we present the serological characterization of antiserum (Anti-M-24) against muscle tissue and developmental stage-specific cell surface antigens of the prefusion myoblast. Cells from primary cultures of 12-d-old embryonic chick hindlimb muscle were injected into rabbits, and the resulting antisera were selectively absorbed to obtain immunological specificity. Cytotoxicity and immunohistochemical assays were used to test this antiserum. Absorption with embryonic or adult chick heart, brain, retina, liver, erythrocytes, or skeletal muscle fibroblasts failed to remove all reactivity of Anti-M-24 for myogenic cells at all stages of development. After absorption with embryonic myotubes, however, Anti-M-24 no longer reacted with differentiated myofibers, but did react with prefusion myoblasts. The myoblast surface antigens detected with Anti-M-24 are components of the muscle cell membrane: (a) these macromolecules are free to diffuse laterally within the myoblast membrane; (b) Anti-M-24, in the presence of complement, induced lysis of the muscle cell membrane; and (c) intact monolayers of viable myoblasts completely absorbed reactivity of Anti-M-24 for myoblasts. These antigens are not loosely adsorbed culture medium components or an artifact of tissue culture because: (a) absorption of Anti-M-24 with homogenized embryonic muscle removed all antibodies to cultured myoblasts; (b) Anti-M-24 reacted with myoblast surfaces in vivo; and (c) absorption of Anti-M-24 with culture media did not affect the titer of this antiserum for myoblasts. We conclude that myogenic cells at all stages of development possess externally exposed antigens which are undetected on other embryonic and adult chick tissues. In addition, myoblasts exhibit surface antigenic determinants that are either masked, absent, or present in very low concentrations on skeletal muscle fibroblasts, embryonic myotubes, or adult myofibers. These antigens are free to diffuse laterally within the myoblast membrane and may be modulated in response to appropriate environmental cues during myodifferentiation.
Stereo electron microscopy of the 25-nm chromatin fibers in isolated nuclei.
Authors: Olins A L , Olins D E
Published: May 2004

Thin sections (0.1-0.25 micron) of isolated chicken erythrocyte nuclei were examined at various tilt angles. Stereo pairs of electron micrographs document the parallel alignment of 25-nm chromatin fibers adjacent to the nuclear envelope, and demonstrate a fiber substructure consistent with close-packed arrays of nucleosomes.
DNase-I-dependent dissociation of erythrocyte cytoskeletons.
Authors: Sheetz M P
Published: May 2004

The human erythrocyte contains a complex of peripheral membrane proteins which forms an extensive network or cytoskeleton on the cytoplasmic membrane surface. When I treat erythrocyte cytoskeletons with deoxyribonuclease I (DNase I), the cytoskeletons dissociate and erythrocyte actin is solubilized. The dissociation of the cytoskeletons by DNase I parallels the disruption of actin filaments in vitro by DNase I and is blocked by the addition of action to the DNase I. Large protein complexes remain after DNase I disrupts the cytoskeletons, but these complexes are no longer visible in the light microscope nor sedimentable and are selectively depleted with respect to actin. From these studies, I suggest that DNase I binds to and solubilizes actin, which serves as a structural link between protein complexes in the erythrocyte cytoskeleton.
Andrenocorticotropin and β-lipotropin in the hypothalamus
Authors: Nivaler G , Zimmerman EA , Defendini R , Liotta AS , Kreiger DT , Brownstein MJ
Published: May 2004

Adrenocorticotropin and β-lipotropin (β-LPH) have been localized by immunoperoxidase methods in nerve cells and fibers of the hypothalamus and brain stem of the ewe. 6-μm sections were immunostained first for either ACTH or β-LPH. The reaction products and the antibody complexes were then eluted completely from the tissue, and the same section was immunostained for the second peptide. Absorption of the primary antisera with a variety of peptide fragments of ACTH and β-LPH demonstrated, immunocytochemically as well as by radioimmunoassay, that the ACTH and β-LPH antisera were directed to the COOH- and NH(2)-termini of the peptides, respectively. Neither antiserum recognized any portion of the heterologous peptide. In the sequential staining procedure on the same tissue section, preincubation of the antisera with the homologous peptide abolished the staining, whereas preincubation with the heterologous peptide did not affect it, regardless of the order followed.

Every nerve cell in the arcuate nucleus that contained ACTH also contained β-LPH, but β-LPH cells appeared, probably falsely, to be twice as numerous as ACTH cells. β-LPH-positive fibers in and beyond the hypothalamus were also more numerous and stained more intensively than ACTH fibers. The salient exception was fibers in the infundibular zona externa, where the opposite was true.

Our observations establish that ACTH and β-LPH are contained in the same nerve cells They stongly favor biosynthesis in brain, probably from a common precursor molecule, as has been demonstrated in the pituitary gland. The complexity of the cytologic distribution pattern described suggests that the two peptides are not processed in the same manner by the nerve cell.
Troponin in embryonic chick skeletal muscle cells in vitro. An immunoelectron microscope study.
Authors: Obinata T , Shimada Y , Matsuda R
Published: May 2004

The fine structurel distribution of troponin on thin filaments in developing myofibrils was investigated by the use of immunoelectron microscopy. Embryonic chick skeletal muscle cells grown in vitro were treated with antibodies against each of the troponin components (troponin T, I, and C) from adult chicken muscles. Each antibody was distributed along the thin filaments with a period of 38 nm. It is concluded that these newly synthesized regulatory proteins are assembled at their characteristic position from the initial phases of myofibrillogenesis.
Isolation of the pericellular matrix of human fibroblast cultures.
Authors: Hedman K , Kurkinen M , Alitalo K , Vaheri A , Johansson S , Höök M
Published: May 2004

The pericellular matrix of human fibroblast cultures was isolated, using sequential extraction with sodium deoxycholate and hypotonic buffer in the presence of protease inhibitor. The matrix attached to the growth substratum had a "sackcloth-like" structure as seen by phase contrast, immunofluorescence, and scanning electron microscopy, and it had a vaguely filamentous ultrastructure similar to that seen in intact cell layers. The matrix consisted of hyaluronic acid and heparan sulfate as the major glycosaminoglycan components and fibronectin and procollagen as major polypeptides as shown by metabolic labeling, gel electrophoresis, immunofluorescence, and collagenase digestion. This pericellular matrix can be regarded as an in vitro equivalent of the loose connective tissue matrix.
Sperm binding and fertilization envelope formation in a cell surface complex isolated from sea urchin eggs.
Authors: Decker G L , Lennarz W J
Published: May 2004

An isolated surface complex consisting of the vitelline layer, plasma membrane, and attached secretory vesicles has been examined for its ability to bind sperm and to form the fertilization envelope. Isolated surface complexes (or intact eggs) fixed in glutaraldehyde and then washed in artificial sea water are capable of binding sperm in a species-specific manner. Sperm which bind to the isolated surface complex exhibit the acrosomal process only when they are associated with the exterior surface (vitelline layer) of the complex. Upon resuspension of the unfixed surface complex in artificial sea water, a limiting envelope is formed which, based on examination of thin sections and negatively stained surface preparations, is structurally similar to the fertilization envelope formed by the fertilized egg. These results suggest that the isolated egg surface complex retains the sperm receptor, as well as integrated functions for the secretion of components involved in assembly of the fertilization envelope.
Specific fluorescent labeling of chicken myofibril Z-line proteins catalyzed by guinea pig liver transglutaminase.
Authors: Gard D L , Lazarides E
Published: May 2004

Guinea pig liver transglutaminase has been found to catalyze the covalent incorporation of dansylcadaverine into chicken skeletal muscle myofibril proteins. Epifluorescence microscopy reveals that the incorporated dansylcadaverine is specifically localized at or near the myofibril Z line. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) indicates that actin constitutes a major fraction of the labeled material; the Z-line proteins alpha-actinin and desmin also show significant labeling, as well as tropomyosin, several additional unidentified proteins, and material with an extremely high molecular weight. The Z-line-specific fluorescence can be removed by brief trypsinization, which releases fluorescent alpha-actinin into the supernate. The majority of the fluorescent protein species are resistant to extraction by either 0.6 M KCl or KI. These results, in conjunction with the microscopic localization, suggest that the dansyl-labeled proteins are constituents of the myofibril Z line. A significant amount of fluorescently labeled transglutaminase is also present in labeled myofibrils, which is resistant to extraction with either 0.6 M KCl or KI. This result indicates a strong, noncovalent interaction between the transglutaminase molecule and the myofibril Z line.
Cytochalasin B inhibition of endothelial proliferation at wound edges in vitro.
Authors: Selden S C , Schwartz S M
Published: May 2004

Cytochalasin B prevents both migration and wound-associated replication when applied to wounded monolayers of bovine endothelium in tissue culture. The normal low background rate of replication in undisturbed areas of the cultures is not inhibited by cytochalasin B. These results suggest that some form of movement may be required for initiation of wound-associated replication by endothelial cells.
Sodium uptake and membrane excitation in Paramecium.
Authors: Hansma H G
Published: May 2004

Although the phenotypes of many membrane-excitation mutants of Paramecium are best expressed in Na+-containing solutions, little is known about the role of Na+ in membrane excitation in Paramecium. By measuring 22Na fluxes, we have shown that: (a) The total cellular Na+ content is equivalent to a cytoplasmic concentration of 3--4 mM, if the Na+ concentration is uniform throughout the cell. (b) The kinetics of Na+ uptake can be divided into a saturable Na+ uptake with an apparent Km = 0.15 mM and a nonsaturable Na+ uptake seen at higher Na+ concentrations up to 20 mM. (c) The rate of Na+ uptake in high Na+ solutions is correlated with the duration of backward swimming and membrane excitation in wild type Paramecium and the mutants fast-2 and paranoiac. (d) Na+ uptake is inhibited at 4 degrees C. From these results, we postulate that Na+ uptake is faster when the membrane is depolarized than when it is at the resting potential level.
Cell cycle-dependent, in vitro assembly of microtubules onto pericentriolar material of HeLa cells.
Authors: Telzer B R , Rosenbaum J L
Published: May 2004

A centriolar complex comprising a pair of centrioles and a cloud of pericentriolar materials is located at the point of covergence of the microtubules of the mitotic apparatus. The in vitro assembly of microtubules was observed onto these complexes in the 1,400 g supernatant fraction of colcemid-blocked, mitotic HeLa cells lysed into solutions containing tubulin and Triton X-100. Dark-field microscopy provided a convenient means by which this process could be visualized directly. When this 1,400 g supernate was incubated at 30 degrees C and centrifuged into a discontinuous sucrose gradient, a band containing centriolar complexes and assembled microtubles was obtained at 50-60% sucrose interface. Ultrastructual analysis indicated that the majority of the microtubules assembled predominantly from the pericentriolar material but also onto the centrioles. When cells were synchronized by a double thymide block, the assembly of microtubules onto centriolar complexes was observed only in lysates of mitotic cells; no assembly was seen in lysed material of interphase cells. Microtubule assembly occured onto centriolar complexes in solutions of either 100,000 g brain supernate, 2 X cycled tubulin, or purified tubulin dimers. This study demonstrates that the pericentriolar material becomes competent as a microtubule-organizing center (MTOC) at the time of mitosis. With use of the techniques described, a method for the isolation of centriolar complexes may be developed.
Cellular interaction between fixed and living cells. Transfer of radioactive materials from living cells to fixed cells.
Authors: Sakiyama H , Otsu H , Kanegasaki S
Published: May 2004

Transfer of radioactive materials to fixed cells from an overlying layer of living cells has been examined to determine whether fixed cells can act as acceptors of glycosyltransferases of living cells. After the incubation of living cells were removed by EDTA treatment, and the radioactivity associated with the fixed cells was determined. Lipids, proteins and carbohydrates were found to be transfered from the living cells to the fixed cells. The amount of radioactivity transferred to the fixed cells was dependent on the number of both fixed and living cells and increased with the time of incubation. When fixed cells were treated with chloroform-methanol before the addition of living cells, the transfer of both lipids and proteins to the fixed cells decreased drastically, but only a slight decrease incarbohydrate transfer was observed. Most of the radioactive materials transferred from living cells labeled with glucosamine or fucose to chloroform-methanol-treated fixed cells were solubilized by trypsin but not by the detergents tested. Approximately 55% of the materials transferred from the cells labeled with glucosamine could be solubilized by hyaluronidase and chondroitinase, and the rest was solubilized by neuraminidase and a glycosidase mixture. The treatment of chloroform-methanol-extracted fixed cells with trypsin caused a significant decrease in the transfer from cells labeled with glucosamine. When nucleotide sugars were used as the radioactive precursor, no significant amount of radioactivity was transferred to the fixed cells.
Biosynthesis of cytochrome P-450 on membrane-bound ribosomes and its subsequent incorporation into rough and smooth microsomes in rat hepatocytes.
Authors: Fujii-Kuriyama Y , Negishi M , Mikawa R , Tashiro Y
Published: May 2004

Intracellular sites of synthesis of cytochrome P-450 and the subsequent incorporation of it into membrane structures of the endoplasmic reticulum (ER) in rat hepatocytes have been studied using an antibody monospecific for phenobarbital-inducible cytochrome P-450. The cytochrome is synthesized mainly on the "tightly bound" type of membrane-bound ribosomes whose release from the membrane requires treatment with puromycin in a high salt buffer (500 mM KCI, 5mM MgCl2, and 50 mM Tris-HCL [pH 7.5]). Subsequently the cytochrome is incorporated directly into the rough ER membranes with its major part exposed to the outer surface to the membrane and accessible to proteolytic enzymes added externally. The newly synthesized molecules, which appeared first in the rough membrane, are translocated to the smooth membrane, and are then distributed evenly between the two types of microsomeal membranes in approximately 1 h. Administration of cycloheximide, an inhibitor of protein biosynthesis, did not significantly inhibit the transfer of the enzyme from the rough to the smooth ER. It is suggested, therefore, that the translocation of the newly synthesized cythochrome P-450 between the rough and smooth microsomes is mainly due to the lateral movement of the molecules in the plane of the membranes rather than to the attachment and detachment of the ribosomes on the microsomal membranes after the ribosomal cycle for protein synthesis.
Calcium transport and exchange in mouse 3T3 and SV40-3T3 cells.
Authors: Hazelton B J , Tupper J T
Published: May 2004

The kinetics of Ca++ uptake have been evaluated in 3T3 and SV40-3T3 mouse cells. The data reveal at least two exchangeable cellular compartments in the 3T3 and SV40-3T3 cell over a 50-min exposure to 45Ca++. A rapidly exchanging compartment may represent surface-membrane-localized Ca++ whereas a more slowly exchanging compartment is presumably intracellular. The transition of the 3T3 cell from exponential growth (at 3 day's incubation) to quiescence (at 7 days) is characterized by a 7.5-fold increase in the size of the fast component. Quiescence of the 3T3 cell is also characterized by a 3.2-fold increase in the unidirectional Ca++ influx into the slowly exchanging compartment and a 3.6-fold increase in its size. The increase in size of the slow compartment at quiescence may result from a redistribution of intracellular Ca++ to a more readily exchangeable compartment, possibly reflecting a release of previously bound Ca++. In contrast, no significant change in any of these parameters is observed in the proliferatively active SV40-3T3 cells after corresponding period of incubation, even though these cells attained higher growth densities and underwent postconfluence.
Intermediate-sized filaments of human endothelial cells.
Authors: Franke W W , Schmid E , Osborn M , Weber K
Published: May 2004

Human endothelial cells prepared from unbilical cords are characterized in parallel by electron microscopy and indirect immunofluorescence microscopy using specific antibodies against different classes of intermediate-sized filaments. The strongly developed, loose bundles of intermediate-sized filaments typically found in these cells are not decorated by antibodies against prekeratin or antibodies against smooth muscle desmin. They are, however, strongly decorated by antibodies directed against murine "vimentin," i.e., the 57,000 mol wt polypeptide which is the major protein of the intermediate-sized filaments predominant in various cells of mesenchymal origin. Cytoskeletal preparations greatly enriched in intermediate-sized filaments show the enrichment of a polypeptide band comigrating with murine vimentin. This shows that the intermediate-sized filaments that are abundant in human endothelial cells are predominantly of the vimentin type and can be demonstrated by their cross-reaction with the vimentin of rodents. These data also strengthen the evidence for several subclasses of intermediate-sized filaments, which can be distinguished by immunological procedures.
Interaction of microtubule proteins with phospholipid vesicles.
Authors: Caron J M , Berlin R D
Published: May 2004

We have examined the interaction of unilamellar dimyristoyl phosphatidylcholine liposomes with the high-speed supernate of brain homogenate and with tubulin purified through one or two cycles of microtubule assembly-disassembly. Tubulin and certian high molecular weight proteins are selectively adsorbed from these mixtures onto liposomes. The composition of adsorbed proteins is similar to that obtained during corresponding cycles of microtubule assembly, suggesting the equivalency of these processes. Adsorption induces stacking and/or fusion of liposomes into multilamellar structures indicating strong protein-lipid interaction. In addition, liposome-adsorbed tubulin forms extensive intermolecular disulfide bridges that are inert to reducing agents in the aqueous medium. The observations form a basis for further study of the distribution, function, and properties of membrane-bound tubulin.
Induction of spreading during fibroblast movement.
Authors: Chen W T
Published: May 2004

This paper describes the phenomenon of retraction-induced spreading of embryonic chick heart fibroblasts moving in culture. Measurable criteria of cell spreading (increase in area of the spreading lamella, and total spread area of the cell) are found to change predictably with retraction of a portion of the cell margin. Ruffling activity was found to increase. The leading lamella of a spread fibroblast ordinarily advances slowly, with an average area increase of approximately 21 mu2m/min. A 10- to 30-fold increase in spreading occurs within 8 s after onset of retraction at the trailing edge and then decreases slightly so that by 1 min the increase in spreading is five to tenfold. During this period, there is a linear relationship between area increase at the leading edge and area decrease at the trailing edge. During the next 10--15 min, spreading gradually decreases to normal. Although the relationship between area spreading and area retracting of fibroblasts at different phases of movement is not significantly linear, it is highly correlated (Table II). These results suggest that the rate of fibroblast spreading may be inversely related to the degree of spreading of the cell as a whole.
Size classes of replication units in DNA from sea urchin embryos.
Authors: Kurek M P , Billig D , Stambrook P
Published: May 2004

Sea urchin DNA containing replication structures was isolated from two to four cell stage and blastula stage embryos, and examined by electron microscopy. In addition to the expected eye forms, we also observed molecules with large internal single-stranded gaps. Such structures were not present in DNA devoid of replicating molecules such as that isolated from sea urchin sperm. When the size of eye forms and interbubble distances between the two stages were compared, there was no detectable difference. In both stages, we observed two distinct size classes of bubbles and of interbubble distances. In the case of bubble sizes, the smaller size class was comprised of clustered microbubbles that ranged from 200 base pairs to 1 Kilobase (kb) with a mean of 432 base pairs. The large eye forms measured 1--35 kb with a mean of 6.8 kb. Interbubble distances also yielded two distinct populations, with the smaller class ranging from 400 base pairs to 2.3 kb (mean = 1.1 kb) and the larger population ranging from 2.8 to 36 kb (mean = 10.9 kb). Although other possibilities cannot be entirely excluded, the data support the contention that a substantial fraction of the larger eye-form population arises from the fusion of the clustered microbubbles.
Structural states of myelin observed by x-ray diffraction and freeze-fracture electron microscopy.
Authors: Kirschner D A , Hollingshead C J , Thaxton C , Caspar D L , Goodenough D A
Published: May 2004

Coordinated freeze-fracture electron microscopy and x-ray diffraction were used to visualize the morphological relation between compacted and native period membrane arrays in myelinated nerves treated with dimethylsulfoxide (DMSO). Comparison of x-ray diffraction at room temperature and at low temperature was used as a critical measure of the extent of structural preservation. Our x-ray diffraction patterns show that in the presence of cryoprotective agents, it is possible to preserve with only small changes the myelin structure which exists at room temperature. These changes include a slight increase in packing disorder of the membrane, a small, negative thermal expansion of the membrane unit, and some reorganization in the cytoplasmic half of the bilayer. The freeze-fracture electron microscopy clearly demonstrates continuity of compact and native period phases in DMSO-treated myelin. Finally, the use of freezing to trap the transient, intermediate structure during a structural transition in glycerol is demonstrated.
Mitochondrial ribosome assembly in Neurospora. Two-dimensional gel electrophoretic analysis of mitochondrial ribosomal proteins.
Authors: Lambowitz A M , LaPolla R J , Collins R A
Published: May 2004

Recent results with Neurospora crassa show that one protein (S-5, mol wt 52,000) associated with the mitochondrial (mit) small ribosomal subunit is translated within the mitochondria (Lambowitz et al. 1976. J. Mol. Biol. 107:223-253). In the present work, Neurospora mit ribosomal proteins were analyzed by two-dimensional gel electrophoresis using a modification of the gel system of Mets and Bogorad. The results show that S-5 is present in near stoichiometric concentrations in high salt (0.5 MKCl)-washed mit small subunits from wild-type strains. S-5 is among the most basic mit ribosomal proteins (pI greater than 10) and has a high affinity for RNA under the conditions of the urea-containing gel buffers. The role of S-5 in mit ribosome assembly was investigated by an indirect method, making use of chloramphenicol to specifically inhibit mit protein synthesis. Chloramphenicol was found to rapidly inhibit the assembly of mit small subunits leading to the formation of CAP-30S particles which sediment slightly behind mature small subunits (LaPolla and Lambowitz. 1977. J. Mol. 116: 189-205). Two-dimensional gel analysis shows that the more slowly sedimentaing CAP-30S particles are deficient in S-5 and in several other proteins, whereas these proteins are present in normal concentrations in mature small subunits from the same cells. Because S-5 is the only mit ribosomal protein whose synthesis is directly inhibited by chloramphenicol, the results tentatively suggest that S-5 plays a role in the assembly of mit small subunits. In addition, the results are consistent with the idea that S-5 stabilizes the binding of several other mit small subunit proteins. Two-dimensional gel electrophoresis was used to examine mit ribosomal proteins from [poky] and six additional extra-nuclear mutants with defects in the assembly of mit small subunits. The electrophoretic mobility of S-5 is not detectably altered in any of the mutants. However, [poky] mit small subunits are deficient in S-5 and also contain several other proteins in abnormally low or high concentrations. These and other results are consistent with a defect in a mit ribosomal constituent in [poky].
Role of tropomyosin in actin filament formation in embryonic salamander heart cells.
Authors: Lemanski L F
Published: May 2004

Recessive mutant gene c in Ambystoma mexicanum embryos causes a failure of the heart to function even though initial heart development appears normal. An analysis of the constituent proteins of normal and mutant hearts by SDS-poly-acrylamide gel electrophoresis shows that actin (43,000 daltons) is present in almost normal amounts, while myosin heavy chain (200,000 daltons) is somewhat reduced in mutants. Both SDS-polyacrylamide gel electrophoresis and immunofluorescence studies reveal that tropomyosin is abundant in normal hearts, but very much reduced in mutants. Electron microscope studies of normal hearts show numerous well-organized myofibrils. Although mutant cardiomyocytes contain a few 60- and 150-A filaments, organized sacromeres are absent. Instead, amorphous proteinaceous collections are prominent. Previously reported heavy meromyosin (HMM)-binding experiments on glycerinated hearts demonstrate that most of the actin is contained within the amorphous collections in a nonfilamentous state, and the addition of HMM causes polymerization into F actin (Lemanski et al., 1976, J. Cell. Biol. 68:375-388). In the present study, glycerol-extracted hearts are incubated with tropomyosin, purified from rabbit or chicken skeletal muscle. This treatment causes the amorphous collections to disappear, and large numbers of distinct thin actin (60- to 80-A) filaments are seen in their place. Negative staining experiments corroborate this observation. These results suggest that the nonfilamentous actin located in the amorphous collections of mutant heart cells is induced to form into filaments with the addition of tropomyosin.
Characterization of the molecular components in kinetoplast-mitochondrial DNA of Trypanosoma equiperdum. Comparative study of the dyskinetoplastic and wild strains.
Authors: Riou G F , Saucier J M
Published: May 2004

The structure of the kinetoplast DNA of Trypanosoma equiperdum has been studied and compared to the structure of the circular mitochondrial DNA extracted from a dyskinetoplastic strain of T. equiperdum. In T. equiperdum wild type, the kinetoplast DNA constitutes approximately 6% of the total cellular DNA and is composed of approximately 3,000 supercoiled minicircles of 6.4 x 10(5) daltons and approximately 50 circular supercoiled molecules of 15.4 x 10(6) daltons topologically interlocked; The buoyant density in CsCl of the minicircles is 1.691 g/cm 3. The large circles have a buoyant density of 1.684 g/cm 3, are homogeneous in size and are selectively cleaved by several restriction endonucleases which do not cleave the minicircles. The cleavage sites of six different restriction endonucleases have been mapped on the large circle. The minicircles are cleaved by two other restriction endonucleases, and their cleavage sites have been mapped. The mitochondrial DNA extracted from the dyskinetoplastic strain of T. equiperdum represents 7% of the total DNA of the cell and is composed of supercoiled circles, heterogeneous in size, and topologically associated in catenated oligomers. Its buoyant density in CsCl is 1.688 g/cm 3. These molecules are not cleaved by any of the eight restriction endonucleases tested. The reassociation kinetics of in vitro labeled kDNA minicircles and large circles has been studied. The results indicate that the minicircles as well as the large circles are homogeneous in sequence and that the circular DNA of the dyskinetoplastic strain has no sequence in common with the kDNA of the wild strain.
Study of the mechanism of vanadate inhibition of the dynein cross-bridge cycle in sea urchin sperm flagella
Authors: Sale WS , Gibbon IR
Published: May 2004

The effect of vanadate on the ATP-induced disruption of trypsin-treated axonemes and the ATP-induced straightening of rigor wave preparations of sea urchin sperm was investigated. Addition of ATP to a suspension of trypsin-treated axonemes results in a rapid decrease in turbidity (optical density measured at 350 nm) concomitant with the disruption of the axonemes by sliding between microtubules to form tangles of connected doublet microtubules (Summers and Gibbons, 1971; Sale and Satir, 1977). For axonemes digested to approximately 93 percent of their initial turbidity, 5 {muM} vanadate completely inhibits the ATP-induced decrease in turbidity and the axonemes maintain their structural integrity. However, with axonemes digested to approximately 80 percent of their initial turbidity, vanadate fails to inhibit the ATP-induced decrease in turbidity and the ATP-induced structural disruption of axonemes, even when the vanadate concentration is raised as high as 100 μm. For such axonemes digested to 80 percent of their initial turbidity, the form of ATP-induced structural changes, in the presence of 25 μM vanadate, was observed by dark-field light microscopy and revealed that the axonemes become disrupted into curved, isolated doublet microtubules, small groups of doublet microtubules, and "banana peel" structures in which tubules have peeled back from the axoneme. Addition of 5 μM ATP to rigor wave sperm, which were prepared by abrupt removal of ATP from reactivated sperm, causes straightening of the rigor waves within 1 min, and addition of more than 10 μM ATP causes resumption of flagellar beating. Addition of 40 μM vanadate to the rigor wave sperm does not inhibit straightening of the rigor waves of 2 μM-1 mM ATP, although oscillatory beating is completely inhibited. These results suggest that vanadate inhibits the mechanochemical cycle of dyein at a step subsequent to the MgATP(2-)-induced release of the bridged dynein arms.