bluuebunny/crossref_metadata_2025 huggingface dataset:
High mobility group proteins of amphibian oocytes: a large storage pool of a soluble high mobility group-1-like protein and involvement in transcriptional events.
Authors: Kleinschmidt J A , Scheer U , Dabauvalle M C , Bustin M , Franke W W
Published: May 2004

Oocytes of several amphibian species (Xenopus laevis, Rana temporaria, and Pleurodeles waltlii) contained a relatively large pool of nonchromatin-bound, soluble high mobility group (HMG) protein with properties similar to those of calf thymus proteins HMG-1 and HMG-2 (protein HMG-A; A, amphibian). About half of this soluble HMG-A was located in the nuclear sap, the other half was recovered in enucleated ooplasms. This protein was identified by its mobility on one- and two-dimensional gel electrophoresis, by binding of antibodies to calf thymus HMG-1 to polypeptides electrophoretically separated and blotted on nitrocellulose paper, and by tryptic peptide mapping of radioiodinated polypeptides. Most, if not all, of the HMG-A in the soluble nuclear protein fraction, preparatively defined as supernatant obtained after centrifugation at 100,000 g for 1 h, was in free monomeric form, apparently not bound to other proteins. On gel filtration it eluted with a mean peak corresponding to an apparent molecular weight of approximately 25,000; on sucrose gradient centrifugation it appeared with a very low S value (2-3 S), and on isoelectric focusing it appeared in fractions ranging from pH approximately 7 to 9. This soluble HMG-A was retained on DEAE-Sephacel but could be eluted already at moderate salt concentrations (0.2 M KCl). In oocytes of various stages of oogenesis HMG-A was accumulated in the nucleus up to concentrations of approximately 14 ng per nucleus (in Xenopus), corresponding to approximately 0.2 mg/ml, similar to those of the nucleosomal core histones. This nuclear concentration is also demonstrated using immunofluorescence microscopy. When antibodies to bovine HMG-1 were microinjected into nuclei of living oocytes of Pleurodeles the lateral loops of the lampbrush chromosomes gradually retracted and the whole chromosomes condensed. As shown using electron microscopy of spread chromatin from such injected oocyte nuclei, this process of loop retraction was accompanied by the appearance of variously-sized and irregularly-spaced gaps within transcriptional units of chromosomal loops but not of nucleoli, indicating that the transcription of non-nucleolar genes was specifically inhibited by this treatment and hence involved an HMG-1-like protein. These data show that proteins of the HMG-1 and -2 category, which are usually chromatin-bound components, can exist, at least in amphibian oocytes, in a free soluble monomeric form, apparently not bound to other molecules. The possible role of this large oocyte pool of soluble HMG-A in early embryogenesis is discussed as well as the possible existence of soluble HMG proteins in other cells.
Developmental acquisition of basement membrane heterogeneity: type IV collagen in the avian lens capsule.
Authors: Fitch J M , Mayne R , Linsenmayer T F
Published: May 2004

To investigate potential heterogeneity and developmental changes in basement membranes during embryogenesis, we performed immunohistochemical analyses on lens capsules in chicken embryos of different ages using domain-specific monoclonal antibodies against type IV collagen. We found that the capsule of the newly formed lens stained uniformly with antibodies against this component of basement membranes, but with increasing age and differentiation of the lens cells the anterior lens capsule remained brightly fluorescent while staining of the posterior capsule became relatively much less intense. This antero-posterior gradient of anti-type IV collagen antibody reactivity demonstrated that developmentally-regulated changes can occur within a single, continuous basement membrane.
Relationship between axenic growth of Dictyostelium discoideum strains and their track morphology on substrates coated with gold particles.
Authors: Kayman S C , Clarke M
Published: May 2004

Amoebae of Dictyostelium discoideum produce tracks with two distinct morphologies on gold-coated coverslips. The wild-type strain and other strains that feed only by phagocytosis produced indistinct, fuzzy tracks, whereas mutants capable of axenic growth produced clear, sharp tracks. The sharp track morphology was found to be a recessive phenotype that segregates with axenicity and probably requires a previously unidentified axenic mutation. Axenic and nonaxenic strains also differed in their ability to pinocytose. When the two types of cells were shifted from bacterial growth plates to nutrient media, within 24 h the axenic strain established a rapid rate of pinocytosis, approximately 100-fold higher than the low rate detectable for the nonaxenic strain. However, track formation did not appear to be directly related to endocytosis. Electron microscopic examination of cells during track formation showed that both axenic and nonaxenic strains accumulated gold particles on their surfaces, but neither strain internalized the gold to any significant degree. Observation of living cells revealed that axenic strains collected all particles that they contacted, whereas wild-type strains left many particles undisturbed. The size of the gold particle clusters discarded by the cells also contributed to track morphology.
Cytoplasmic organization in cerebellar dendritic spines.
Authors: Landis D M , Reese T S
Published: May 2004

Three sets of filamentous structures were found to be associated with synaptic junctions in slices of cerebellar tissue prepared by rapid-freezing and freeze-etch techniques. The electron-dense fuzz subjacent to postsynaptic membranes corresponds to a web of 4-6-nm-diam filaments that were clearly visualized in rapid-frozen, freeze-etched preparations. Purkinje cell dendritic spines are filled with a meshwork of 5-7-nm filaments that were found to contact the spine membrane everywhere except at the synaptic junction, and extend through the neck of the spine into the parent dendrite. In addition, 8-10-nm microfilaments, possibly actin, were seen to be associated with the postsynaptic web and to extend into the body and neck of the spine. The arrangements and attachments of the filamentous elements in the Purkinje cell dendritic spine may account for its shape.
Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis.
Authors: Celis J E , Larsen P M , Fey S J , Celis A
Published: May 2004

Analysis by means of two-dimensional gel electrophoresis (IEF) of [32P]orthophosphate-labeled proteins from mitotic and interphase transformed amnion cells (AMA) has shown that keratins IEF 31 (Mr = 50,000; Hela protein catalogue number), 36 (Mr = 48,500), 44 (Mr = 44,000), 46 (Mr = 43,500), as well as vimentin (IEF 26; Mr = 54,000) are phosphorylated above their interphase level during mitosis. Similar studies of normal human amnion epithelial cells (AF type) confirmed the above observations except in the case of keratin IEF 44 whose relative proportion was too low to be analyzed. Immunofluorescent staining of methanol/acetone-treated mitotic transformed amnion cells with a mouse polyclonal antibody elicited against human keratin IEF 31 showed a dotted staining (with a fibrillar background) in all of the cells in late anaphase/early telophase (characteristic "domino" pattern) and in a sizeable proportion of the cells in other stages of mitosis. Normal mitotic amnion cells on the other hand showed a fine fibrillar staining of keratins at all stages of mitosis. Similar immunofluorescent staining of normal and transformed mitotic cells with vimentin antibodies revealed a fibrillar distribution of vimentin in both cell types. Taken together the results indicate that the transformed amnion cells may contain a factor(s) that modulates the organization of keratin filaments during mitosis. This putative factor(s), however, is most likely not a protein kinase as transformed amnion cells and amnion keratins are modified to similar extents. It is suggested that in general the preferential phosphorylation of intermediate-sized filament proteins during mitosis may play a role in modulating the various proposed associations of these filaments with organelles and other cellular structures.
Immunocytochemical localization of lysosomal beta-galactosidase in rat liver.
Authors: Novikoff P M , La Russo N F , Novikoff A B , Stockert R J , Yam A , Le Sage G D
Published: May 2004

beta-galactosidase is a ubiquitous lysosomal hydrolase that specifically cleaves terminal beta-galactosyl residues from glycoproteins, glycosaminoglycans, oligosaccharides, and glycolipids. To study the intracellular distribution of this enzyme, we prepared a specific polyclonal antibody to lysosomal beta-galactosidase by immunizing rabbits with a highly purified preparation of beta-galactosidase from rat liver. Using this antibody we employed an immunocytochemical technique (protein A coupled to horseradish peroxidase and diaminobenzidine cytochemistry) and showed that beta-galactosidase is present in all hepatocytes of the rat liver. All types of lysosomes, the rough endoplasmic reticulum, and the specialized region of smooth endoplasmic reticulum known as GERL showed immunoreactivity. This in situ distribution suggests that these organelles are involved in the biosynthesis and intracellular sorting of this lysosomal enzyme.
Studies of the biosynthesis of the mannose 6-phosphate receptor in receptor-positive and -deficient cell lines.
Authors: Goldberg D E , Gabel C A , Kornfeld S
Published: May 2004

Phosphomannosyl residues present on lysosomal enzymes are specifically recognized by the mannose 6-phosphate receptor protein. This interaction results in the selective targeting of lysosomal enzymes to lysosomes. While this pathway is operative in many cell types, we have found four cultured cell lines that are deficient in the ability to bind lysosomal enzymes containing phosphomannosyl residues to their intracellular or surface membranes (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). These cells appear to segregate lysosomal enzymes by an alternate intracellular pathway. To determine the basis for the lack of mannose 6-phosphate receptor activity in these cell lines, we studied the biosynthesis of the receptor in receptor-positive (BW5147) and receptor-deficient (P388D1 and MOPC 315) cells. The cells were labeled with [2-3H]mannose or [35S]methionine and the receptor was immunoprecipitated with an antireceptor antiserum. BW5147 cells synthesize a receptor protein whose size increases after translation/glycosylation. MOPC 315 cells produce an apparently normal receptor and degrade it rapidly. P388D1 cells fail to synthesize any detectable receptor. The receptor from BW5147 and MOPC 315 cells is a glycoprotein with both high mannose and complex asparagine-linked oligosaccharides. The complex-type units become fully sialylated and remain so during long periods of chase.
Ion currents and membrane domains in the cleaving Xenopus egg.
Authors: Kline D , Robinson K R , Nuccitelli R
Published: May 2004

We used an extracellular vibrating probe to measure ion currents through the cleaving Xenopus laevis egg. Measurements indicate sharp membrane heterogeneities. Current leaves the first cleavage furrow after new, unpigmented membrane is inserted. This outward current may be carried by K+ efflux. No direct involvement of the Na+,K+-ATPase in the generation of this outward current is detected at first cleavage. Inward current enters the old, pigmented membrane; however, it does not enter uniformly. The inward current is largest at the old membrane bordering the new membrane. This suggests a heterogeneous ion channel distribution within the old membrane. Experiments suggest that the inward current may be carried by Na+ influx, Ca2+ influx, and Cl- efflux. No steady currents were detected during grey crescent formation, the surface contraction waves preceding cleavage, or with groove formation at the beginning of cleavage.
Establishment of plasma membrane domains in hepatocytes. I. Characterization and localization to the bile canaliculus of three antigens externally oriented in the plasma membrane.
Authors: Cook J , Hou E , Hou Y , Cairo A , Doyle D
Published: May 2004

A membrane fraction denoted N2 upper was isolated from homogenates of rat liver by sucrose gradient centrifugation. This fraction, which was enriched 65-fold over the homogenate in 5'-nucleotidase activity, was used as an immunogen in goats. The antisera obtained contained antibodies to three predominant polypeptides in the N2 upper membrane fraction, as shown by crossed immunoelectrophoresis. These polypeptides had molecular weights of 105,000, 110,000, and 160,000 after recovery from the crossed immunoelectrophoretic gels and are denoted PM105, PM110, and PM160. Each was a distinct polypeptide, as shown by the distinct peptide patterns resulting from limited proteolysis in the presence of detergents. The three polypeptides were synthesized by primary cultures of hepatocytes and were externally oriented at the surface of these cells, as shown by their accessibility in situ to iodination catalyzed by lactoperoxidase. They were not detectable in the serum by crossed immunoelectrophoresis. The three antigens were present at very low (PM110) or nondetectable (PM105, PM160) concentrations in intracellular membrane fractions derived from the Golgi and smooth and rough endoplasmic reticulum of liver. The antigens also were reduced in concentration in a plasma membrane fraction most likely derived from the sinusoidal surface of the hepatocyte. The three membrane antigens bind to concanavalin A; hence, they are probably glycoprotein constituents of a discrete domain of the hepatocyte plasma membrane. Immune complexes were isolated after crossed immunoelectrophoresis and injected into rabbits. Each of the antisera obtained was reactive to one of the membrane polypeptides. Sections of fixed rat livers were reacted with each of the antibodies and then the primary antibody was localized by indirect immunocytochemical methods using horseradish peroxidase or colloidal gold as labels. Each of the three antigens was localized by this method to the bile canalicular domain of the hepatocyte plasma membrane.
Erythropoietin causes down regulation of colony-stimulating factor (CSF-1) receptors on peritoneal exudate macrophages of the mouse.
Authors: Van Zant G , Chen B D
Published: May 2004

We have shown that erythropoietin (epo), the primary regulator of erythrocyte formation, diminished the binding to peritoneal exudate macrophages (PEM) of the principal macrophage growth regulator, colony-stimulating factor (CSF-1). The effect of epo on 125I-CSF-1 binding was dose-dependent; at a concentration of 1-2 U of epo/ml (10(-10) M), CSF-1 binding was almost completely suppressed. Erythropoietin did not compete with CSF-1 for occupancy of the latter's receptors. The effect of epo on CSF-1 binding occurred at 37 degrees C but not at 2 degrees C, and during the continuous exposure of PEM to epo at 37 degrees C we found that CSF-1 binding reached a nadir at 1 h and recovered to pre-exposure levels in 7 h. Our novel results are consistent with the notion that specific receptors for epo exist on the cell surface of PEM and that binding of epo sets in motion a series of cellular events resulting in the internalization of CSF-1 receptors. Thus epo causes down regulation of CSF-1 receptors on PEM. We have previously shown that epo causes suppression of CSF-induced granulocyte-macrophage colony formation by mouse bone marrow cells. The results we present here provide a possible mechanism for these results.
Two forms of cerebellar glial cells interact differently with neurons in vitro.
Authors: Hatten M E , Liem R K , Mason C A
Published: May 2004

Specific interactions between neurons and glia dissociated from early postnatal mouse cerebellar tissue were studied in vitro by indirect immunocytochemical staining with antisera raised against purified glial filament protein, galactocerebroside, and the NILE glycoprotein. Two forms of cells were stained with antisera raised against purified glial filament protein. The first, characterized by a cell body 9 microns diam and processes 130-150 microns long, usually had two to three neurons associated with them and resembled Bergmann glia. The second had a slightly larger cell body with markedly shorter arms among which were nestled several dozen neuronal cells, and resembled astrocytes of the granular layer. Staining with monoclonal antisera raised against purified galactocerebroside revealed the presence of immature oligodendroglia in the cultures. These glial cells constituted approximately 2% of the total cell population in the cultures and, in contrast to astroglia, did not form specific contacts with neurons. Staining with two neuronal markers, antisera raised against purified NILE glycoprotein and tetanus toxin, revealed that most cells associated with presumed astroglia were small neurons (5-8 microns). After 1-2 d in culture, some stained neurons had very fine, short processes. Nearly all of the processes greater than 10-20 micron long were glial in origin. Electron microscopy also demonstrated the presence of two forms of astroglia in the cultures, each with a different organizing influence on cerebellar neurons. Most neurons associated with astroglia were granule neurons, although a few larger neurons sometimes associated with them. Time-lapse video microscopy revealed extensive cell migration (approximately 10 microns/h) along the arms of Bergmann-like astroglia. In contrast, cells did not migrate along the arms of astrocyte-like astroglia, but remained stationary at or near branch points. Growth cone activity, pulsating movements of cell perikarya, and ruffling of the membranes of glial and neuronal processes were also seen.
Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. I. Chondrocyte ultrastructure--implications for the theories of mineralization and vascular invasion.
Authors: Hunziker E B , Herrmann W , Schenk R K , Mueller M , Moor H
Published: May 2004

Electron microscopic examination of epiphyseal cartilage tissue processed by high pressure freezing, freeze substitution, and low temperature embedding revealed a substantial improvement in the preservation quality of intracellular organelles by comparison with the results obtained under conventional chemical fixation conditions. Furthermore, all cells throughout the epiphyseal plate, including the terminal chondrocyte adjacent to the region of vascular invasion, were found to be structurally integral. A zone of degenerating cells consistently observed in cartilage tissue processed under conventional chemical fixation conditions was not apparent. Hence, it would appear that cell destruction in this region occurs during chemical processing and is not a feature of cartilage tissue in the native state. Since these cells are situated in a region where tissue calcification is taking place, the implication is that the onset and progression of cartilage calcification are, at least partially, controlled by the chondrocytes themselves. The observation that the terminal cell adjacent to the zone of vascular invasion is viable has important implications in relation to the theory of vascular invasion. This may now require reconceptualization to accommodate the possibility that active cell destruction may be a precondition for vascular invasion.
Small oligomers of immunoglobulin E (IgE) cause large-scale clustering of IgE receptors on the surface of rat basophilic leukemia cells.
Authors: Menon A K , Holowka D , Baird B
Published: May 2004

We examined the distribution of small oligomers of IgE bound to rat basophilic leukemia cells using fluorescence microscopy. The oligomers were seen to cluster into visible patches on the cell surface at 4 degrees C; at higher temperatures internalization also was observed. In contrast, cells labeled with IgE monomers remained predominantly ring-stained. Evidence is provided that the observed clustering of IgE oligomers is a cell-induced phenomenon, and the possible significance of this clustering is discussed in the context of the oligomer-triggered degranulation of rat basophilic leukemia cells.
Interactions of thrombospondin with extracellular matrix proteins: selective binding to type V collagen.
Authors: Mumby S M , Raugi G J , Bornstein P
Published: May 2004

Thrombospondin (TS), a protein first described in platelets, was recently shown to be synthesized and secreted by endothelial cells, fibroblasts, and smooth muscle cells. The presence of TS in the extracellular matrix of cultured cells has prompted us to examine the associations of this protein with matrix macromolecules. Interactions of TS with both matrix and serum proteins were tested using an enzyme-linked immunosorbent assay. With this assay we assessed the binding of TS in solution to proteins adsorbed to polystyrene microtiter plates. Among collagens, platelet TS bound to type V but not to types I, III, or IV. This selective interaction was confirmed in experiments using proteins linked to cyanogen bromide-activated Sepharose. TS released from platelets in response to thrombin activation, as well as that secreted by endothelial cells in culture, bound to type V but not to type I collagen-Sepharose. No binding was observed to denatured type V collagen-Sepharose. The binding region for type V collagen was located in a chymotrypsin-produced fragment of TS with chains of Mr = 70,000, after reduction. Interactions of TS with a number of other proteins, including fibronectin, fibrinogen, and laminin, could be demonstrated using the enzyme-linked immunosorbent assay technique but the interpretation of these findings is difficult since comparable binding to protein-Sepharose was not always observed. Our findings suggest that both the extravascular distribution and function of TS in vivo may involve an interaction with type V collagen.
Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin.
Authors: Thompson W C , Asai D J , Carney D H
Published: May 2004

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.
Bidirectional polymerization of G-actin on the human erythrocyte membrane.
Authors: Tsukita S , Tsukita S , Ishikawa H
Published: May 2004

The directional polymerization of actin on the erythrocyte membrane has been examined at various concentrations of G-actin by thin-section electron microscopy. For this purpose, a new experimental system using single-layered erythrocyte membranes with the cytoplasmic surfaces freely exposed was developed. The preformed actin filaments did not bind with the cytoplasmic surface of the erythrocyte membranes. When the erythrocyte membranes were incubated at low concentrations (0.3 and 0.5 microM) of G-actin, greater than 80% of polymerized actin filaments pointed toward the membranes mainly in an end-on fashion, as judged by arrowhead formation with heavy meromyosin. At higher concentrations (2 and 4 microM) of G-actin, about half of the polymerized actin filaments were directed with arrowheads pointing toward the membranes, while the rest of the filaments showed the opposite polarity pointing away from the membranes. The majority of polymerized actin filaments formed loops at the points of attachment to the membranes. In contrast, when G-actin (2 and 4 microM) in the presence of cytochalasin B was polymerized into filaments, approximately 70% showed the polarity pointing away from the membrane mainly in an end-on fashion. To check the treadmilling phenomena, the erythrocyte membranes with bidirectionally polymerized actin filaments were further incubated with G-actin at the overall critical concentration. In this case, almost all (90%) of actin filaments showed the polarity with arrowheads pointing toward the membranes. The results obtained are discussed with special reference to the mode of association of actin filaments with the plasma membrane in general.
Regulation of murine oocyte meiosis: evidence for a gonadotropin-induced, cAMP-dependent reduction in a maturation inhibitor.
Authors: Freter R R , Schultz R M
Published: May 2004

We have developed an assay that can detect relative changes in the amount of a non-cAMP inhibitor of maturation present in cumulus cells (Eppig et al., 1983, Dev. Biol., 100:39-49). Using this assay in which accelerated maturation of a group of treated cumulus cell-oocyte complexes relative to untreated complexes indicates a decrease in the amount of inhibitor, results of the experiments described here suggest a possible relationship between elevation of cAMP levels and subsequent decreased amounts of a non-cAMP inhibitor. Mouse oocytes obtained from cumulus cell-oocyte complexes treated with luteinizing hormone (LH) resumed meiosis prior to oocytes obtained from untreated complexes; the degree of acceleration of maturation was dependent on LH concentration. A similar result was obtained with follicle-stimulating hormone (FSH). Correlated with LH- or FSH-acceleration of maturation was an LH- or FSH-induced elevation of cumulus cell cAMP levels. Inhibiting LH-induced elevation of cumulus cell cAMP levels inhibited LH-induced acceleration of maturation. An initial incubation of complexes in medium containing dibutyryl cAMP (dbcAMP) also promoted acceleration of maturation. In contrast, maturation of denuded oocytes was not altered by treatment with either LH, FSH, or dbcAMP. Complexes initially incubated in dbcAMP-containing medium still demonstrated acceleration of maturation after a subsequent 2 h incubation in dbcAMP-free medium. Relative to untreated complexes, none of these treatments disrupted intercellular communication between cumulus cells and the oocyte. Elevating follicle cAMP levels with cholera toxin induced maturation of follicle-enclosed oocytes when cumulus cell-oocyte coupling was still fully maintained. These results are interpreted to indicate that gonadotropin-mediated acceleration of maturation is via a cAMP-dependent reduction in the level of a maturation inhibitor present in granulosa/cumulus cells.
Genetic and biochemical dissection of the eucaryotic flagellum.
Authors: Luck D J
Published: May 2004

The axoneme is the basic functional unit of the eucaryotic flagellum. Periodic structures appended to its 9+2 microtubule core are responsible for generation of flagellar bending. An account of biochemical and genetic studies of flagellar-defective mutants of Chlamydomonas reinhardtii is presented. These studies provide insights into the complex molecular composition of the appended structures, their mode of assembly, and the way in which they interact to modulate flagellar function.
Flagellar waveform and rotational orientation in a Chlamydomonas mutant lacking normal striated fibers.
Authors: Hoops H J , Wright R L , Jarvik J W , Witman G B
Published: May 2004

The Chlamydomonas mutant vfl-3 lacks normal striated fibers and microtubular rootlets. Although the flagella beat vigorously, the cells rarely display effective forward swimming. High speed cinephotomicrography reveals that flagellar waveform, frequency, and beat synchrony are similar to those of wild-type cells, indicating that neither striated fibers nor microtubular rootlets are required for initiation or synchronization of flagellar motion. However, in contrast to wild type, the effective strokes of the flagella of vfl-3 may occur in virtually any direction. Although the direction of beat varies between cells, it was not observed to vary for a given flagellum during periods of filming lasting up to several thousand beat cycles, indicating that the flagella are not free to rotate in the mature cell. Structural polarity markers in the proximal portion of each flagellum show that the flagella of the mutant have an altered rotational orientation consistent with their altered direction of beat. This implies that the variable direction of beat is not due to a defect in the intrinsic polarity of the axoneme, and that in wild-type cells the striated fibers and/or associated structures are important in establishing or maintaining the correct rotational orientation of the basal bodies to ensure that the inherent functional polarity of the flagellum results in effective cellular movement. As in wild type, the flagella of vfl-3 coordinately switch to a symmetrical, flagellar-type waveform during the shock response (induced by a sudden increase in illumination), indicating that the striated fibers are not directly involved in this process.
Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins.
Authors: Sanger J W , Mittal B , Sanger J M
Published: May 2004

To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alpha-actinin, and tropomyosin. In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin. Conditions known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands. When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands. Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i.e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the A-band which lacks cross-bridges. In the presence of 0.1-0.2 mM ATP, no actin binding occurred. When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added fluorescence occurred not in a doublet pattern but along the entire length of the myofibril. Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the l-band. Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band. These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha-actinin and, if actin is added subsequently, the exogenous alpha-actinin in the Z-band will bind the newly formed fluorescent actin filaments. Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin. These observations are discussed in terms of the structure and in vivo assembly of myofibrils.
Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs.
Authors: Lohka M J , Masui Y
Published: May 2004

A cell-free cytoplasmic preparation from activated Rana pipiens eggs could induce in demembranated Xenopus laevis sperm nuclei morphological changes similar to those seen during pronuclear formation in intact eggs. The condensed sperm chromatin underwent an initial rapid, but limited, dispersion. A nuclear envelope formed around the dispersed chromatin and the nuclei enlarged. The subcellular distribution of the components required for these changes was examined by separating the preparations into soluble (cytosol) and particulate fractions by centrifugation at 150,000 g for 2 h. Sperm chromatin was incubated with the cytosol or with the particulate material after it had been resuspended in either the cytosol, heat-treated (60 or 100 degrees C) cytosol or buffer. We found that the limited dispersion of chromatin occurred in each of these ooplasmic fractions, but not in the buffer alone. Nuclear envelope assembly required the presence of both untreated cytosol and particulate material. Ultrastructural examination of the sperm chromatin during incubation in the preparations showed that membrane vesicles of approximately 200 nm in diameter, found in the particulate fraction, flattened and fused together to contribute the membranous components of the nuclear envelope. The enlargement of the sperm nuclei occurred only after the nuclear envelope formed. The pronuclei formed in the cell-free preparations were able to incorporate [3H]dTTP into DNA. This incorporation was inhibited by aphidicolin, suggesting that the DNA synthesis by the pronuclei was dependent on DNA polymerase-alpha. When sperm chromatin was incubated greater than 3 h, the chromatin of the pronuclei often recondensed to form structures resembling mitotic chromosomes within the nuclear envelope. Therefore, it appeared that these ooplasmic preparations could induce, in vitro, nuclear changes resembling those seen during the first cell cycle in the zygote.
Isolation and characterization of keratin-like proteins from cultured cells with fibroblastic morphology.
Authors: Zackroff R V , Goldman A E , Jones J C , Steinert P M , Goldman R D
Published: May 2004

Intermediate filaments (IF) isolated from a variety of cultured cells, conventionally described as fibroblasts, are composed predominantely of proteins of molecular weights of 54,000 and/or 55,000. Less than 15% of the protein found in native IF preparations from these cells is composed of three to four polypeptides of molecular weights 60,000-70,000. We have investigated some biochemical and immunological properties of these proteins isolated from BHK-21 and mouse 3T3 cells. They are capable of forming paracrystals that exhibit a light/dark banding pattern when negatively stained with uranyl acetate. The dark bands are composed of longitudinally aligned approximately 2-nm-diam filaments. The center-to-center spacing between either dark or light bands is 37-40 nm. These dimensions are consistent with the secondary structure of IF polypeptides and suggest that the dark bands represent lateral alignment of alpha-helical coiled-coil domains. Immunoblotting, secondary structure, as well as amino acid composition data indicate that the 60,000-70,000-mol-wt paracrystal polypeptides are similar to keratin. Thus, polypeptides with biochemical and immunological properties of epidermal keratin are present in cells normally considered to be fibroblasts.
Packing of a specific gene into higher order structures following repression of RNA synthesis.
Authors: Andersson K , Björkroth B , Daneholt B
Published: May 2004

Transcription of the Balbiani ring (BR) genes of the dipteran Chironomus tentans was inhibited by teh nucleoside analogue DRB (5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole). The BR genes were emptied of RNA polymerases and the subsequent packing of the genes was monitored by transmission electron microscopy. The thin chromatin axis of the transcriptionally active genes condensed into a thick (20-25 nm) chromatin fiber, which was recorded as a linear structure, an open loop or a supercoiled loop. The compacted genes were finally packed into dense clumps of chromatin. It was proposed that upon repression of RNA synthesis the BR gene template attains the following consecutive stages with increasing compaction: transcription loop----linear thick fiber----open thick fiber loop----supercoiled thick fiber loop----dense chromatin. Within the chromatin blocks structures that resembled the supercoiled loops were discerned, suggesting that the final packing of the template might be accomplished by a close alignment of supercoiled loops.
Biochemical analysis of mutants of a macrophage cell line resistant to the growth-inhibitory activity of interferon.
Authors: Nagata Y , Rosen O M , Makman M H , Bloom B R
Published: May 2004

While a multiplicity of cellular and biochemical effects are mediated by interferons on cultured cells, the mechanisms involved in the direct growth-inhibitory activity of interferons remain problematic. We have previously found that variants in cAMP metabolism in a macrophage cell line, J774.2, were at least 50-fold less sensitive to the growth inhibitory activity of interferons (IFN) than the parental clone. To test the hypothesis that cAMP mediates the growth inhibition produced by IFN in these cells, interferon-resistant variants were selected and characterized with respect to cAMP synthesis and function. Approximately one-third of the IFN-resistant clones were found to be resistant to growth inhibition produced by cholera toxin, but not 8Br-cAMP. IFN was fully able to protect all of the interferon-resistant/choleratoxin-resistant (IFNr/CTr) clones against infection by vesicular stomatitis virus and markedly stimulated 2', 5'-oligodenylate synthetase activity. These IFNr/CTr variants were shown to have a defect in adenylate cyclase. The remaining IFN-resistant clones were fully susceptible to the growth-inhibitory effects of cholera toxin because their basal and stimulated adenylate cyclase activity is similar to that of the parental clone. IFN failed to protect these IFNr/choleratoxin sensitive clones against infection by vesicular stomatitis virus and failed to stimulate 2', 5-oligodenylate synthetase, suggesting that they have defective or deficient IFN receptors. In addition, IFN failed to increase intracellular cAMP levels in both IFNr/CTr and IFNr/choleratoxin sensitive clones. These results provide firm genetic and biochemical evidence that the growth inhibitory effects of IFN on this cell line are mediated by cAMP.
Presence of an extensive clathrin coat on the apical plasmalemma of the rat kidney proximal tubule cell.
Authors: Rodman J S , Kerjaschki D , Merisko E , Farquhar M G
Published: May 2004

The nature of the cytoplasmic coat present on the apical invaginations of the kidney proximal tubule cell was investigated by immuneoverlay and immunocytochemistry of renal brush borders with anticlathrin antibodies. When kidney cortex was prepared for electron microscopy using methods that enhance visualization of clathrin coats, the apical invaginations at the base of the brush border microvilli were seen to be backed by a nearly continuous coating which resembles but is more extensive than the lattice-like clathrin coats found around brain coated vesicles. When isolated brush border fractions were prepared under conditions that preserve the coats, separated by SDS PAGE, and transferred to nitrocellulose, the presence of clathrin heavy and light chains was detected by immuneoverlay using two different affinity-purified anticlathrin IgGs--one that we prepared, which detects only the clathrin light chains, and the other, prepared by Louvard et al. ( Louvard , D., C. Morris, G. Warren, K. Stanley, F. Winkler , and H. Reggio , 1983, EMBO [Eur. Mol. Biol. Organ.] J., 2:1655-1664), which detects both the heavy and light chains. As viewed by light microscopy (immunofluorescence or immunoperoxidase), staining with both anticlathrins was concentrated at the base of the proximal tubule microvilli. Immunoelectron microscopic localizations carried out on brush border fractions (using peroxidase and gold conjugates) demonstrated specific binding of anticlathrin IgGs to the lattice-like cytoplasmic coat. When brush border fractions were reacted with monoclonal antibodies prepared against gp330 and maltase, proteins that serve as markers for the membrane of the apical invaginations and microvilli, respectively ( Kerjaschki , D., L. Noronha - Blob , B. Sacktor , and M. G. Farquhar , 1984, J. Cell Biol., 98:1505-1513), the two proteins retained their restrictive distribution in the brush border. The findings demonstrate (a) that the cytoplasmic coat of the proximal tubule intermicrovillar apical invaginations is composed of clathrin heavy and light chains, and (b) that the differential distribution of proteins in these two brush border microdomains is maintained in appropriately prepared brush border fractions.
Collagenase is a major gene product of induced rabbit synovial fibroblasts.
Authors: Aggeler J , Frisch S M , Werb Z
Published: May 2004

We have investigated the effects of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), on rabbit synovial fibroblasts, and found that this agent induced a major switch in gene expression in these cells that was marked by the specific induction of the neutral proteinase, collagenase, and was always accompanied by alterations in cell morphology. Procollagenase synthesis and secretion was first observed 6-12 h after the addition of TPA. The rate of collagenase production (1-5 U, or approximately 0.2-1 micrograms secreted procollagenase protein per 10(5) cells per 24 h) depended on the TPA concentration (1-400 ng/ml) and time of exposure (1-72 h). Procollagenase was the most prominent protein visible by direct silver staining or by autoradiography after SDS PAGE of [35S]methionine-labeled proteins. The two procollagenase bands of Mr 53,000 and 57,000, which migrated as a family of spots on two-dimensional gels and were immunoprecipitated by antibodies to purified rabbit collagenase, accounted for 23% of the newly synthesized, secreted protein in TPA-treated cells. Cell-free translation of mRNA from TPA-treated cells in rabbit reticulocyte lysate produced a single band of immunoprecipitable preprocollagenase (Mr 55,000) as a major product (5% of total) that migrated as a single spot on two-dimensional gels. Secreted procollagenase, preprocollagenase , and active collagenase (purified to homogeneity; specific activity 1.2 X 10(4) U/mg protein) had related peptide maps. Two other major secreted proteins, a neutral metalloproteinase of Mr 51,000 and a polypeptide of Mr 47,000, were also induced by TPA. In contrast to the induction of these four polypeptides, TPA decreased synthesis and secretion of a number of proteins, including collagen and fibronectin. Thus, collagenase is a convenient marker for major alterations in the pattern of protein synthesis and secretion by rabbit synovial fibroblasts treated with TPA.
Changes in cell shape correlate with collagenase gene expression in rabbit synovial fibroblasts.
Authors: Aggeler J , Frisch S M , Werb Z
Published: May 2004

Induction of the neutral proteinase, collagenase, is a marker for a specific switch in gene expression observed in rabbit synovial fibroblasts. A variety of agents, including 12-O-tetradecanoylphorbol-13-acetate, cytochalasins B and D, trypsin, chymotrypsin, poly(2-hydroxyethylmethacrylate), and trifluoperazine induced this change in gene expression. Induction of collagenase by these agents was always correlated with a marked alteration in cell morphology, although the cells remained adherent to the culture dishes. The amount of collagenase induced was positively correlated with the degree of shape change produced by a given concentration and, to some extent, with the duration of treatment. Altered cell morphology was required only during the first few hours of treatment with inducing agents; after this time collagenase synthesis continued for up to 6 d even when agents were removed and normal flattened cell morphology was regained. All agents that altered cell morphology also produced a characteristic switch in protein secretion phenotype, characterized by the induction of procollagenase (Mr 53,000 and 57,000) and a neutral metalloproteinase (Mr 51,000), which accounted for approximately 25% and 15% of the protein secreted, respectively. Secretion of another neutral proteinase, plasminogen activator, did not correlate with increased collagenase secretion. In contrast, synthesis and secretion of a number of other polypeptides, including the extracellular matrix proteins, collagen and fibronectin, were concomitantly decreased. That changes in cell shape correlated with a program of gene expression manifested by both degradation and synthesis of extracellular macromolecules may have broad implications in development, repair, and pathologic conditions.
Immunocytochemical localization of opsin in the cell membrane of developing rat retinal photoreceptors.
Authors: Nir I , Cohen D , Papermaster D S
Published: May 2004

Mature retinal rod photoreceptors sequester opsin in the disk and plasma membranes of the rod outer segment (ROS). Opsin is synthesized in the inner segment and is transferred to the outer segment along the connecting cilium that joins the two compartments. We have investigated early stages of retinal development during which the polarized distribution of opsin is established in the rod photoreceptor cell. Retinas were isolated from newborn rats, 3-21 d old, and incubated with affinity purified biotinyl-sheep anti-bovine opsin followed by avidin-ferritin. At early postnatal ages prior to the development of the ROS, opsin is labeled by antiopsin on the inner segment plasma membrane. At the fifth postnatal day, as ROS formation begins opsin was detected on the connecting cilium plasma membrane. However, the labeling density of the ciliary plasma membrane was not uniform: the proximal cilium was relatively unlabeled in comparison with the distal cilium and the ROS plasma membrane. In nearly mature rat retinas, opsin was no longer detected on the inner segment plasma membrane. A similar polarized distribution of opsin was also observed in adult human rod photoreceptor cells labeled with the same antibodies. These results suggest that some component(s) of the connecting cilium and its plasma membrane may participate in establishing and maintaining the polarized distribution of opsin.
Chloroplast ribosomal proteins of Chlamydomonas synthesized in the cytoplasm are made as precursors.
Authors: Schmidt R J , Myers A M , Gillham N W , Boynton J E
Published: May 2004

Polyadenylated RNA from Chlamydomonas was translated in a cell-free rabbit reticulocyte system that employed [35S]methionine. Antibodies made to four chloroplast ribosomal proteins synthesized in the cytoplasm and imported into the organelle were used for indirect immunoprecipitation of the labeled translation products, which were subsequently visualized on fluorographs of SDS gels. The cytoplasmically synthesized chloroplast ribosomal proteins were first seen as precursors with apparent molecular weights of 1,000 to 6,000 greater than their respective mature forms. Processing of the ribosomal protein precursors to mature proteins was affected by adding a postribosomal supernatant that had been extracted from cells of Chlamydomonas. In contrast to the chloroplast ribosomal proteins synthesized in the cytoplasm, two such proteins made within the chloroplast were found to be synthesized in mature form in cell-free wheat germ translation systems programmed with nonpolyadenylated RNA.