bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminal plasma membranes.
Authors: Morel N , Thieffry M , Manaranche R
Published: May 2004

The crude extract of venom glands of the polychaete annelid Glycera convoluta triggers a large Ca2+-dependent acetylcholine release from both frog motor nerve terminals and Torpedo electric organ synaptosomes. This extract was partially purified by Concanavalin A affinity chromatography. The biological activity was correlated in both preparations to a 300,000-dalton band, as shown by gel electrophoresis. This confirmed previous determinations obtained with chromatographic methods. This glycoprotein binds to presynaptic but not postsynaptic plasma membranes isolated from Torpedo electric organ. Pretreatment of intact synaptosomes by pronase abolished both the binding and the venom-induced acetylcholine release without impairing the high K+-induced acetylcholine release. Pretreatment of nerve terminal membranes by Concanavalin A similarly prevented the binding and the biological response. Binding to Torpedo membranes was still observed in the presence of EGTA. An antiserum directed to venom glycoproteins inhibited the neurotoxin so we could directly follow its binding to the presynaptic membrane. Glycera convoluta neurotoxin has to bind to a ectocellularly oriented protein of the presynaptic terminal to induce transmitter release.
Rapid acidification of endocytic vesicles containing asialoglycoprotein in cells of a human hepatoma line.
Authors: Tycko B , Keith C H , Maxfield F R
Published: May 2004

Acidification of endocytic vesicles has been implicated as a necessary step in various processes including receptor recycling, virus penetration, and the entry of diphtheria toxin into cells. However, there have been few accurate pH measurements in morphologically and biochemically defined endocytic compartments. In this paper, we show that prelysosomal endocytic vesicles in HepG2 human hepatoma cells have an internal pH of approximately 5.4. (We previously reported that similar vesicles in mouse fibroblasts have a pH of 5.0.) The pH values were obtained from the fluorescence excitation profile after internalization of fluorescein labeled asialo-orosomucoid (ASOR). To make fluorescence measurements against the high autofluorescence background, we developed digital image analysis methods for estimating the pH within individual endocytic vesicles or lysosomes. Ultrastructural localization with colloidal gold ASOR demonstrated that the pH measurements were made when ligand was in tubulovesicular structures lacking acid phosphatase activity. Biochemical studies with 125I-ASOR demonstrated that acidification precedes degradation by more than 30 min at 37 degrees C. At 23 degrees C ligand degradation ceases almost entirely, but endocytic vesicle acidification and receptor recycling continue. These results demonstrate that acidification of endocytic vesicles, which causes ligand dissociation, occurs without fusion of endocytic vesicles with lysosomes. Methylamine and monensin raise the pH of endocytic vesicles and cause a ligand-independent loss of receptors. The effects on endocytic vesicle pH are rapidly reversible upon removal of the perturbant, but the effects on cell surface receptors are slowly reversible with methylamine and essentially irreversible with monensin. This suggests that monensin can block receptor recycling at a highly sensitive step beyond the acidification of endocytic vesicles. Taken together with other direct and indirect estimates of endocytic vesicle pH, these studies indicate that endocytic vesicles in many cell types rapidly acidify below pH 5.5, a pH sufficiently acidic to allow receptor-ligand dissociation and the penetration of some toxin chains and enveloped virus nucleocapsids into the cytoplasm.
Immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (G) of vesicular stomatitis virus in infected Chinese hamster ovary cells.
Authors: Bergmann J E , Singer S J
Published: May 2004

An immunoelectron microscopic study was undertaken to survey the intracellular pathway taken by the integral membrane protein (G-protein) of vesicular stomatitis virus from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane of virus-infected Chinese hamster ovary cells. Intracellular transport of the G-protein was synchronized by using a temperature-sensitive mutant of the virus (0-45). At the nonpermissive temperature (39.8 degrees C), the G-protein is synthesized in the cell infected with 0-45, but does not leave the rough endoplasmic reticulum. Upon shifting the temperature to 32 degrees C, the G-protein moves by stages to the plasma membrane. Ultrathin frozen sections of 0-45-infected cells were prepared and indirectly immunolabeled for the G-protein at different times after the temperature shift. By 3 min, the G-protein was seen at high density in saccules at one face of the Golgi apparatus. No large accumulation of G-protein-containing vesicles were observed near this entry face, but a few 50-70-mm electron-dense vesicular structures labeled for G-protein were observed that might be transfer vesicles between the rough endoplasmic reticulum and the Golgi complex. At blebbed sites on the nuclear envelope at these early times there was a suggestion that the G-protein was concentrated, these sites perhaps serving as some of the transitional elements for subsequent transfer of the G-protein from the rough endoplasmic reticulum to the Golgi complex. By 3 min after its initial asymmetric entry into the Golgi complex, the G-protein was uniformly distributed throughout all the saccules of the complex. At later times, after the G-protein left the Golgi complex and was on its way to the plasma membrane, a new class of G-protein-containing vesicles of approximately 200-nm diameter was observed that are probably involved in this stage of the transport process. These data are discussed, and the further prospects of this experimental approach are assessed.
Rearrangement of the keratin cytoskeleton after combined treatment with microtubule and microfilament inhibitors.
Authors: Knapp L W , O'Guin W M , Sawyer R H
Published: May 2004

In addition to containing microtubule and microfilament systems, vertebrate epithelial cells contain an elaborate keratin intermediate-filament cytoskeleton. Little is known about its structural organization or function. Using indirect immunofluorescence microscopy with an antikeratin antiserum probe, we found that destabilization of microtubules and microfilaments with cytostatic drugs induces significant alterations in the cytoskeletal organization of keratin filaments in HeLa and fetal mouse epidermal cells. Keratin filament organization was observed to undergo a rapid (1-2 h) transition from a uniform distribution to an open lattice of keratin fibers stabilized by membrane-associated focal centers. Since addition of any one drug alone did not elicit significant organizational change in the keratin cytoskeleton, we suggest that microfilaments and microtubules have a combined role in maintaining the arrangement of keratin in these cells. Vimentin filaments, the only other intermediate-sized filaments found in HeLa cells, did not co-distribute with keratin in untreated or drug-treated cells. These findings offer a new way to approach the study of the dynamics and functional roles of the keratin cytoskeleton in epithelial cells.
The changes in structural organization of actin in the sea urchin egg cortex in response to hydrostatic pressure.
Authors: Begg D A , Salmon E D , Hyatt H A
Published: May 2004

We have used hydrostatic pressure to study the structural organization of actin in the sea urchin egg cortex and the role of cortical actin in early development. Pressurization of Arbacia punctulata eggs to 6,000 psi at the first cleavage division caused the regression of the cleavage furrow and the disappearance of actin filament bundles from the microvilli. Within 30 s to 1 min of decompression these bundles reformed and furrowing resumed. Pressurization of dividing eggs to 7,500 psi caused both the regression of the cleavage furrow and the complete loss of microvilli from the egg surface. Following release from this higher pressure, the eggs underwent extensive, uncoordinated surface contractions, but failed to cleave. The eggs gradually regained their spherical shape and cleaved directly into four cells at the second cleavage division. Microvilli reformed on the egg surface over a period of time corresponding to that required for the recovery of normal egg shape and stability. During the initial stages of their regrowth the microvilli contained a network of actin filaments that began to transform into bundles when the microvilli had reached approximately 2/3 of their final length. These results demonstrate that moderate levels of hydrostatic pressure cause the reversible disruption of cortical actin organization, and suggest that this network of actin stabilizes the egg surface and participates in the formation of the contractile ring during cytokinesis. The results also demonstrate that actin filament bundles are not required for the regrowth of microvilli after their removal by pressurization. Preliminary experiments demonstrate that F-actin is not depolymerized in vitro by pressures up to 10,000 psi and suggest that pressure may act indirectly in vivo, either by changing the intracellular ionic environment or by altering the interaction of actin binding proteins with actin.
Vesicular stomatitis virus glycoprotein, albumin, and transferrin are transported to the cell surface via the same Golgi vesicles.
Authors: Strous G J , Willemsen R , van Kerkhof P , Slot J W , Geuze H J , Lodish H F
Published: May 2004

Human hepatoma cells, infected by vesicular stomatitis virus, offer a good system to study simultaneously the intracellular localization of a well defined transmembrane glycoprotein (VSV-G), a secretory glycoprotein (transferrin), and a nonglycosylated secretory protein (albumin). We used monospecific antibodies in combination with 5- and 8-nm colloidal gold particles complexed with protein A to immunolabel these proteins simultaneously in thin frozen sections of hepatoma cells. VSV-G, transferrin, and albumin are present in the same rough endoplasmic reticulum cisternae, the same Golgi compartments, and the same secretory vesicles. In the presence of the ionophore monensin intracellular transport is blocked at the trans cisternae of the Golgi complex, and VSV-G, transferrin, and albumin accumulate in dilated cisternae, which are apparently derived from the trans-Golgi elements. Glycoproteins, synthesized and secreted in the presence of monensin, are less acidic than those in control cultures. This is probably caused by a less efficient contact between the soluble secretory proteins and the membrane-bound glycosyltransferases that are present in the most monensin-affected (trans) Golgi cisternae.
Molecular morphology of the tetrodotoxin-binding sodium channel protein from Electrophorus electricus in solubilized and reconstituted preparations.
Authors: Ellisman M H , Miller J A , Agnew W S
Published: May 2004

The appearance of detergent-solubilized voltage-regulated sodium channel protein was recently characterized by this laboratory. Negative-staining revealed rod-shaped particles measuring 40 X 170 A. Further studies have suggested that the actual configuration of this protein may be quite different from the rod-shaped structures. Freeze-fracture and freeze-etch images of the protein in reconstituted membranes indicated that the channel is cylindrical with a diameter of 100 A and a minimum length of 80 A. Experiments with two detergent systems (Lubrol-PX and sodium cholate) enabled us to explain the discrepancy between this structure and the rod-shaped particles visualized earlier. Negative staining in either detergent at low pH (4.5) produced rod-shaped structures. As the pH was increased, doughnut-shaped particles, consistent with the structure of the protein in freeze-etch, appeared in negative stain. The tendency of the protein to change shape under different pH conditions appears to be a peculiar property of this protein.
Cell type-dependent expression of tubulins in Physarum.
Authors: Burland T G , Gull K , Schedl T , Boston R S , Dove W F
Published: May 2004

Three alpha-tubulins and two beta-tubulins have been resolved by two-dimensional gel electrophoresis of whole cell lysates of Physarum myxamoebae or plasmodia. Criteria used to identify the tubulins included migration on two-dimensional gels with myxamoebal tubulins purified by self-assembly into microtubules in vitro, peptide mapping with Staphylococcus V8 protease and with chymotrypsin, immunoprecipitation with a monoclonal antibody specific for beta-tubulin, and, finally, hybrid selection of specific mRNA by cloned tubulin DNA sequences, followed by translation in vitro. Differential expression of the Physarum tubulins was observed. The alpha 1- and beta 1-tubulins were detected in both myxamoebae and plasmodia; alpha 2 and beta 2 were detected only in plasmodia, alpha 3 was detected only in the myxamoebal phase, and may be specific to the flagellate. Observation of more tubulin species in plasmodia than in myxamoebae was remarkable; the only microtubules detected in plasmodia are those of the mitotoic spindle, whereas myxamoebae display cytoplasmic, centriolar, flagellar, and mitotic-spindle microtubules. In vitro translation of myxamoebal and plasmodial RNAs indicated that there are distinct mRNAs, and therefore probably separate genes, for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulins. Thus, the different patterns of tubulin expression in myxamoebae and plasmodia reflect differential expression of tubulin genes.
Epinemin: a new protein associated with vimentin filaments in non-neural cells.
Authors: Lawson D
Published: May 2004

In this report I describe a new protein, defined by a monoclonal antibody, which is associated with vimentin filaments in a variety of cultured cells and in skeletal muscle. By immunofluorescence it is absent in smooth muscle, in cells without vimentin, and in neural vimentin containing cells. This protein has a molecular weight of 44,500, a pl of 5, a two-dimensional tryptic peptide fingerprint pattern different from vimentin, is unrelated to actin by Cleveland peptide analysis and by light and electron microscopy, and is not recognized by either a polyclonal antivimentin antibody (Frank, E.D., and L. Warren, 1981, Proc. Natl. Acad. Sci. USA, 78:3020-3024) or a monoclonal antibody against all classes of intermediate filaments (Pruss, R.M., R. Mirsky, M.C. Raff, R. Thorpe, A.J. Dowding, and B.H. Anderton, 1981, Cell, 27:419-428). The protein is resistant to nonionic detergent extraction, is soluble in high salt and can thus be removed from vimentin filaments, but fragments with vimentin in either low salt or anionic detergent and collapses with vimentin in colchicine-treated cells. By light microscopy, the distribution of the protein is indistinguishable from vimentin filaments and appears uniform along them. In contrast, immunoferritin electron microscopy reveals that the molecule is distributed in an intermittent pattern on vimentin filaments. Adopting the terminology of Granger and Lazarides (1980, Cell, 30:263-275), the molecule is called epinemin, meaning "upon filaments."
Role of fibronectin as a growth factor for fibroblasts.
Authors: Bitterman P B , Rennard S I , Adelberg S , Crystal R G
Published: May 2004

Fibroblast replication is regulated by exogenous signals provided by growth factors, mediators that interact with the target cell surface and signal the cell to proliferate. A useful model of growth regulation, the "dual control model," suggests that growth factors can be grouped either as competence factors or as progression factors, and that optimal replication of fibroblasts requires the presence of both types of growth factors. Although most growth factors are soluble mediators, recent studies have demonstrated that, for some cell types, the extracellular matrix can replace the requirement for a competence factor. Since fibronectin is an important constituent of the extracellular matrix that interacts with specific domains on the fibroblast surface, we examined the ability of fibronectin to act as a competence factor to promote the growth of human diploid fibroblasts. To accomplish this, fibronectins purified from two sources, human plasma and human alveolar macrophages, were tested for their ability to (a) stimulate fibroblast replication in serum-free medium containing characterized progression factors (insulin or alveolar macrophage-derived growth factor); (b) provide a growth-promoting signal early in G1. Fibronectin stimulated fibroblast replication in a dose-dependent manner in the presence of a fixed dose of a progression factor. Conversely, fibronectin conferred on previously unresponsive fibroblasts the ability to replicate in a dose-dependent manner when cultured with increasing amounts of a progression factor. Moreover, fibronectin signaled growth-arrested fibroblasts to traverse G1 approximately 4 h closer to S phase. No differences were observed in the ability of plasma or macrophage fibronectins to provide a competence signal for fibroblast replication. Since fibronectin is a major component of the extracellular matrix, these observations suggest that it may provide at least one of the signals by which the matrix conveys the "competence" that permits fibroblasts to replicate in the presence of an appropriate progression signal.
Fetal calf ligament fibroblasts in culture secrete a low molecular weight collagen with a unique resistance to proteolytic degradation.
Authors: Sage H , Mecham R , Johnson C , Bornstein P
Published: May 2004

A highly unusual collagen was secreted by fibroblasts cultured from 150- and 270-d-old fetal calf nuchal ligaments. Purification revealed that this protein (which may be synthesized in a higher molecular weight form) was precipitated at unusually high concentrations of ammonium sulfate and was also eluted from DEAE-cellulose at greater salt concentrations than were types I and III procollagens. On SDS PAGE, the collagenous protein exhibited an Mr of approximately 12,750 that was not altered in the presence of reducing agent. The low molecular weight collagen (FCL-1) was sensitive to bacterial collagenase and had a [3H]glycine content comparable to that found in type I procollagen, although the [3H]Hyp to [3H]Pro ratio was 0.43. FCL-1 was not cleaved by human skin collagenase, mast cell protease, trypsin, Staphylococcal V8 protease, or proteinase K at 37 degrees C. The collagen was susceptible to trypsin, but not to V8 protease, only after heating at 80 degrees C for 30 min. Preliminary structural studies indicate that FCL-1 was resistant to cleavage by CNBr but exhibited limited proteolysis with pepsin. Both 150- and 270-d-old fibroblasts produced comparable levels of interstitial (types I and III) procollagens, which comprised approximately 70% of the total protein secreted into the culture medium. However, 270-d-old (term) fibroblasts secreted approximately 50% more FCL-1, as percent of total culture medium protein, in comparison to the cells from the earlier gestational stage. This collagen may therefore play a role in the development of the nuchal ligament.
In vitro induction of cartilage-specific macromolecules by a bone extract.
Authors: Seyedin S M , Thompson A Y , Rosen D M , Piez K A
Published: May 2004

An in vitro system has been developed to study the onset of chondrogenesis. Embryonic rat muscle mesenchymal cells, when treated in suspension culture with an extract of bovine bone matrix, synthesized cartilage-specific proteoglycan and type II collagen. The synthesis of these two macromolecules was assayed by the enzyme-linked immunosorbent assay inhibition technique. Further evidence of chondrogenesis was demonstrated by morphological changes of treated cells when cultured in firm agarose and stained for metachromatic matrix. Even with crude bone matrix extracts, the assay was sensitive at the microgram level and significant differences in cartilage macromolecules compared with controls were observed in 2-3 d. In vivo the same extract induced first cartilage and then bone.
Correlation between changes in light energy distribution and changes in thylakoid membrane polypeptide phosphorylation in Chlamydomonas reinhardtii.
Authors: Wollman F A , Delepelaire P
Published: May 2004

We have used a new method to extensively modify the redox state of the plastoquinone pool in Chlamydomonas reinhardtii intact cells. This was achieved by an anaerobic treatment that inhibits the chlororespiratory pathway recently described by P. Bennoun (Proc. Natl. Acad. Sci. USA, 1982, 79:4352-4356). A state I (plus 3,4-dichlorophenyl-1,1-dimethylurea) leads to anaerobic state transition induced a decrease in the maximal fluorescence yield at room temperature and in the FPSII/FPSI ratio at 77 degrees K, which was three times larger than in a classical state I leads to state II transition. The fluorescence changes observed in vivo were similar in amplitude to those observed in vitro upon transfer to the light of dark-adapted, broken chloroplasts incubated in the presence of ATP. We then compared the phosphorylation pattern of thylakoid polypeptides in C. reinhardtii in vitro and in vivo using gamma-[32P]ATP and [32P]orthophosphate labeling, respectively. The same set of polypeptides, mainly light-harvesting complex polypeptides, was phosphorylated in both cases. We observed that this phosphorylation process is reversible and is mediated by the redox state of the plastoquinone pool in vivo as well as in vitro. Similar changes of even larger amplitude were observed with the F34 mutant intact cells lacking in photosystem II centers. The presence of the photosystem II centers is then not required for the occurrence of the plastoquinone-mediated phosphorylation of light-harvesting complex polypeptides.
Modulation of secreted proteins of mouse mammary epithelial cells by the collagenous substrata.
Authors: Lee E Y , Parry G , Bissell M J
Published: May 2004

It has been shown previously that cultures of mouse mammary epithelial cells retain their characteristic morphology and their ability to produce gamma-casein, a member of the casein gene family, only if they are maintained on floating collagen gels (Emerman, J.T., and D.R. Pitelka, 1977, In Vitro, 13:316-328). In this paper we show: (a) Cells on floating collagen gels secrete not only gamma-casein but also alpha 1-, alpha 2-, and beta-caseins. These are not secreted by cells on plastic and are secreted to only a very limited extent by cells on attached collagen gels. (b) The floating collagen gel regulates at the level of synthesis and/or stabilization of the caseins rather than at the level of secretion alone. Contraction of the floating gel is important in that cells cultured on floating glutaraldehyde cross-linked gels do not secrete any of the caseins. (c) The secretion of an 80,000-mol-wt protein, most probably transferrin, and a 67,000-mol-wt protein, probably butyrophilin, a major protein of the milk fat globule membrane are partially modulated by substrata. However, in contrast to the caseins, these are always detectable in media from cells cultured on plastic and attached gels. (d) Whey acidic protein, a major whey protein, is actively secreted by freshly isolated cells but is secreted in extremely limited quantities in cultured cells regardless of the nature of the substratum used. alpha-Lactalbumin secretion is also decreased significantly in cultured cells. (e) A previously unreported set of proteins, which may be minor milk proteins, are prominently secreted by the mammary cells on all substrata tested. We conclude that while the substratum profoundly influences the secretion of the caseins, it does not regulate the expression of every milk-specific protein in the same way. The mechanistic implications of these findings are discussed.
Distribution of F-actin during cleavage of the Drosophila syncytial blastoderm.
Authors: Warn R M , Magrath R , Webb S
Published: May 2004

The process of cleavage during the syncytial blastoderm stage of the Drosophila embryo was studied in fixed whole-mounts using a triple-staining technique. Plasmalemma was stained with Concanavalin A conjugated to tetramethylrhodamine isothiocyanate, the underlying cortical F-actin with a fluorescein derivative of phalloidin, and nuclei with 4',-6 diamidine-2-phenylindole dihydrochloride. The surface caps, which overlie the superficial nuclei at this stage, were found to be rich in F-actin as compared with the rest of the cortex. After the caps formed, they extended over the surface and flattened. Whilst this was occurring the F-actin network within the caps became more diffuse. By the end of the expansion process F-actin had become concentrated at both poles of the caps. The caps then split in two. The cleavage was not accompanied by the formation of any apparent contractile ring of microfilaments across the cap, rather the break region was depleted in F-actin. The cortical actin associated with each half of the old cap then became reorganized around a nucleus to form a new daughter cap, and the cycle began again.
Attractant-induced changes and oscillations of the extracellular Ca++ concentration in suspensions of differentiating Dictyostelium cells.
Authors: Bumann J , Wurster B , Malchow D
Published: May 2004

We used a Ca++-sensitive electrode to measure changes in extracellular Ca++ concentration in cell suspensions of Dictyostelium discoideum during differentiation and attractant stimulation. The cells maintained an external level of 3-8 microM Ca++ until the beginning of aggregation and then started to take up Ca++. The attractants, folic acid, cyclic AMP, and cyclic GMP, induced a transient uptake of Ca++ by the cells. The response was detectable within 6 s and peaked at 30 s. Half-maximal uptake occurred at 5 nM cyclic AMP or 0.2 microM folic acid, respectively. The apparent rate of uptake amounted to 2 X 10(7) Ca++ per cell per min. Following uptake, Ca++ was released by the cells with a rate of 5 X 10(6) ions per cell per min. Specificity studies indicated that the induced uptake of Ca++ was mediated by cell surface receptors. The amount of accumulated Ca++ remained constant as long as a constant stimulus was provided. No apparent adaptation occurred. The cyclic AMP-induced uptake of Ca++ increased during differentiation and was dependent on the external Ca++ concentration. Saturation was found above 10 microM external Ca++. The time course and magnitude of the attractant-induced uptake of external Ca++ agree with a role of Ca++ during contraction. During development the extracellular Ca++ level oscillated with a period of 6-11 min. The change of the extracellular Ca++ concentration during one cycle would correspond to a 30-fold change of the cellular free Ca++ concentration.
Intracellular site of U1 small nuclear RNA processing and ribonucleoprotein assembly.
Authors: Madore S J , Wieben E D , Pederson T
Published: May 2004

We have investigated the intracellular site and posttranscriptional immediacy of U1 small nuclear RNA processing and ribonucleoprotein (RNP) assembly in HeLa cells. After 30 or 45 min of labeling with [3H]uridine, a large amount of U1-related RNA radioactivity in the cytoplasm was found by using either hypotonic or isotonic homogenization buffers. The pulse-labeled cytoplasmic U1 RNA was resolved as a ladder of closely spaced bands running just behind mature-size U1 (165 nucleotides) on RNA sequencing gels, corresponding to a series of molecules between one and at least eight nucleotides longer than mature U1. They were further identified as U1 RNA sequences by gel blot hybridization with cloned U1 DNA. The ladder of cytoplasmic U1 RNA bands reacted with both RNP and Sm autoimmune sera and with a monoclonal Sm antibody, indicating a cytoplasmic assembly of these U1 RNA-related molecules into complexes containing the same antigens as nuclear U1 RNP particles. The cytoplasmic molecules behave as precursors to mature nuclear U1 RNA in both pulse-chase and continuous labeling experiments. While not excluding earlier or subsequent nuclear stages, these results suggest that the cytoplasm is a site of significant U1 RNA processing and RNP assembly. This raises the possibility that nuclear-transcribed eucaryotic RNAs are always processed in the cell compartment other than that in which they ultimately function, which suggests a set of precise signals regulating RNA and ribonucleoprotein traffic between nucleus and cytoplasm.
Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. II. Intercellular matrix ultrastructure - preservation of proteoglycans in their native state.
Authors: Hunziker E B , Schenk R K
Published: May 2004

The extracellular matrix of epiphyseal cartilage tissue was preserved in a state believed to resemble closely that of native tissue following processing by high pressure freezing, freeze substitution, and low temperature embedding (HPF/FS). Proteoglycans (PG) were preserved in an extended state and were apparent as a reticulum of fine filamentous threads throughout the matrix. Within this network, two morphologically discrete components were discernible and identified with the carbohydrate and protein components of PG molecules. Numerous points of contact were clearly visible between components of the PG network and cross-sectioned collagen fibrils and also between PG components and chondrocytic plasmalemmata. These observations provide direct morphological indication that such relationships may exist in native epiphyseal cartilage tissue.
Compartmentalization of intracellular membrane glycocomponents is revealed by fracture-label.
Authors: Torrisi M R , Pinto da Silva P
Published: May 2004

We used thin-section fracture-label to determine the distribution of wheat-germ agglutinin binding sites in intracellular membranes of secretory and nonsecretory rat tissues as well as in human leukocytes. In all cases, analysis of the distribution of wheat germ agglutinin led to the definition of two endomembrane compartments: one, characterized by absence of the label, includes the membranes of mitochondria and peroxisomes as well as those of the endoplasmic reticulum and nuclear envelope; the other, strongly labeled, comprises the membrane of lysosomes, phagocytic vacuoles, and secretory granules, as well as the plasma membrane. The Golgi apparatus was weakly labeled in all studied tissues.
Embryonic fibroblast motility and orientation can be influenced by physiological electric fields.
Authors: Erickson C A , Nuccitelli R
Published: May 2004

Epithelial layers in developing embryos are known to drive ion currents through themselves that will, in turn, generate small electric fields within the embryo. We hypothesized that the movement of migratory embryonic cells might be guided by such fields, and report here that embryonic quail somite fibroblast motility can be strongly influenced by small DC electric fields. These cells responded to such fields in three ways: (a) The cells migrated towards the cathodal end of the field by extending lamellipodia in that direction. The threshold field strength for this galvanotaxis was between 1 and 10 mV/mm when the cells were cultured in plasma. (b) The cells oriented their long axes perpendicular to the field lines. The threshold field strength for this response for a 90-min interval in the field was 150 mV/mm in F12 medium and between 50 and 100 mV/mm in plasma. (c) The cells elongated under the influence of field strengths of 400 mV/mm and greater. These fibroblasts were therefore able to detect a voltage gradient at least as low as 0.2 mV across their width. Electric fields of at least 10-fold larger in magnitude than this threshold field have been detected in vivo in at least one vertebrate thus far, so we believe that these field effects encompass a physiological range.
Widespread cellular distribution of MAP-1A (microtubule-associated protein 1A) in the mitotic spindle and on interphase microtubules.
Authors: Bloom G S , Luca F C , Vallee R B
Published: May 2004

In the accompanying paper (Bloom, G.S., T.A. Schoenfeld, and R.B. Vallee, 1983, J. Cell Biol. 98:320-330), we reported that microtubule-associated protein 1 (MAP 1) from brain comprises multiple protein species, and that the principal component, MAP 1A, can be detected in both neuronal and glial cells by immunofluorescence microscopy using a monoclonal antibody. In the present study, we sought to determine the cellular and subcellular distribution of MAP 1A in commonly used cultured cell systems. For this purpose we used immunofluorescence microscopy and immunoblot analysis with anti-MAP 1A to examine 18 types of mammalian cell cultures. MAP 1A was detected in every culture system examined. Included among these were cells of mouse, rat, Chinese hamster, Syrian hamster, Potoroo (marsupial), and human origin derived from a broad variety of tissues and organs. Anti-MAP 1A consistently labeled mitotic spindles and stained cytoplasmic fibers during interphase in most of the cultures. These fibers were identified as microtubules by co-localization with tubulin in double-labeling experiments, by their disappearance in response to colchicine or vinblastine, and by their reorganization in response to taxol. The anti-MAP 1A stained microtubules in a punctate manner, raising the possibility that MAP 1A is located along microtubules at discrete foci that might represent sites of interaction between microtubules and other organelles. Verification that MAP 1A was, indeed, the reactive material in immunofluorescence microscopy was obtained from immunoblots. Anti-MAP 1A stained a band at the position of MAP 1A in all cultures examined. These results establish that MAP 1A, a major MAP from brain, is widely distributed among cultured mammalian cells both within and outside of the nervous system.
Identification of coated vesicles in Saccharomyces cerevisiae.
Authors: Mueller S C , Branton D
Published: May 2004

Clathrin-coated vesicles were found in yeast, Saccharomyces cerevisiae, and enriched from spheroplasts by a rapid procedure utilizing gel filtration on Sephacryl S-1000. The coated vesicles (62-nm diam) were visualized by negative stain electron microscopy and clathrin triskelions were observed by rotary shadowing. The contour length of a triskelion leg was 490 nm. Coated vesicle fractions contain a prominent band with molecular weight of approximately 185,000 when analyzed by SDS PAGE. The presence of coated vesicles in yeast cells suggests that this organism will be useful for studying the function of clathrin-coated vesicles.
Studies on the spectrin-like protein from the intestinal brush border, TW 260/240, and characterization of its interaction with the cytoskeleton and actin.
Authors: Pearl M , Fishkind D , Mooseker M , Keene D , Keller T
Published: May 2004

The terminal web of the intestinal brush border contains a spectrin-like protein, TW 260/240 (Glenney, J. R., Jr., P. Glenney, M. Osborne, and K. Weber, 1982, Cell, 28:843-854.) that interconnects the "rootlet" ends of microvillar filament bundles in the terminal web (Hirokawa, N., R. E. Cheng, and M. Willard, 1983, Cell, 32:953-965; Glenney J. R., P. Glenney, and K. Weber, 1983, J. Cell Biol., 96:1491-1496). We have investigated further the structural properties of TW 260/240 and the interaction of this protein with actin. Salt extraction of TW 260/240 from isolated brush borders results in a loss of terminal web cross-linkers primarily from the apical zone directly beneath the plasma membrane. Morphological studies on purified TW 260/240 using the rotary shadowing technique confirm earlier results that this protein is spectrin-like and is in the tetrameric state in buffers of low ionic strength. However, examination of TW 260/240 tetramers by negative staining revealed a molecule much straighter and more uniform in diameter than rotary-shadowed molecules. At salt concentrations at (150 mM KCl) and above (300 mM KCl) the physiological range, we observed a partial dissociation of tetramers into dimers that occurred at both 0 degree and 37 degrees C. We also observed (in the presence of 75 mM KCl) a concentration-dependent self-association of TW 260/240 into sedimentable aggregates. We have studied the interaction of TW 260/240 with actin using techniques of co-sedimentation, viscometry, and both light and electron microscopy. We observed that TW 260/240 can bind and cross-link actin filaments and that this interaction is salt- and pH-dependent. Under optimum conditions (25-75 mM KCl, at pH 7.0) TW 260/240 cross-linked F-actin into long, large-diameter bundles. The filaments within these bundles were tightly packed but loosely ordered. At higher pH (7.5) such bundles were not observed, although binding and cross-linking were detectable by co-sedimentation and viscometry. At higher salt (greater than 150 mM KCl), the binding of TW 260/240 to actin was inhibited. The presence of skeletal muscle tropomyosin had no significant effect on the salt-dependent binding of TW 260/240 to F-actin.
Recycling of platelet phosphorylation and cytoskeletal assembly.
Authors: Cox A C , Carroll R C , White J G , Rao G H
Published: May 2004

The shape change and aggregation of washed platelets induced by 10 microM arachidonic acid (AA) can be reversed by 20 ng/ml prostacyclin (PGI2), but these platelets can be reactivated by treatment with 30 microM epinephrine and subsequent addition of 10 microM AA mixture. These events may be modulated by cAMP since 2 mM dibutyryl cAMP also reversed activation without reactivation by epinephrine and AA. We examined protein phosphorylation and formation of cytoskeletal cores resistant to 1% Triton X-100 extraction of these platelets and correlated these processes with aggregation, fibrinogen binding, and changes in ultrastructure. Unactivated platelet cores contained less than 15% of the total actin and no detectable myosin or actin-binding protein. AA-induced cytoskeletal cores, which contained 60-80% of the total actin, myosin, and actin-binding protein as the major components, were disassembled back to unactivated levels by PGI2 and then fully reassembled by epinephrine and AA. Phosphorylation of myosin light chain and a 40,000-dalton protein triggered by AA (two- to fivefold) was reversed to basal levels by PGI2 but was completely restored to peak levels upon addition of the epinephrine and AA mixture. The reversibility of actin-binding protein phosphorylation could not be established clearly because both PGI2 and dibutyryl cAMP caused its phosphorylation independent of activation. With this possible exception, cytoskeletal assembly with associated protein phosphorylation, aggregation, fibrinogen binding, and changes in ultrastructure triggered by activation are readily and concertedly recyclable.
Regulation of connective tissue collagenase production: stimulators from adult and fetal epidermal cells.
Authors: Johnson-Wint B , Gross J
Published: May 2004

We have examined the ability of primary adult rabbit skin cells to regulate collagenase production in vitro. Dermal cells constitutively produce collagenase in culture, and enzyme production by these cells can be influenced by epithelial cells. Co-culture with skin epidermal cells resulted in more enzyme production by dermal cells, whereas co-culture with corneal epithelial cells yielded less enzyme activity. Connective tissue cells from a different source, cornea, also produced collagenase when co-cultured with skin epidermal cells, although the stromal cells alone made no enzyme. The drug cytochalasin B had very little influence on collagenase production by dermal cells, either alone or in co-culture with epidermal cells, but did significantly potentiate enzyme production by corneal stromal cells responding to epidermal effector molecules. Epidermal-cell-conditioned medium from both fetal and adult rabbit skin was a potent source of stimulators (apparent mol wt 20,500 and 55,000) of connective-tissue-cell collagenase production. Stimulator production by epidermal cultures was cell density dependent. Optimal production of stimulators occurred in adult cultures containing 10(6) epidermal cells/ml of medium, and in fetal cultures containing 10(5) cells/ml. Inhibitors of connective tissue cell enzyme production were not detected in conditioned medium from either adult or fetal epidermal cells.
Cytochemical localization of terminal N-acetyl-D-galactosamine residues in cellular compartments of intestinal goblet cells: implications for the topology of O-glycosylation.
Authors: Roth J
Published: May 2004

The O-linked oligosaccharides of mucin-type glycoproteins contain N-acetyl-D-galactosamine (GalNAc) that is not found in N-linked glycoproteins. Because Helix pomatia lectin interacts with terminal GalNAc, we used this lectin, bound to particles of colloidal gold, to localize such sugar residues in subcellular compartments of intestinal goblet cells. When thin sections of low temperature Lowicryl K4M embedded duodenum or colon were incubated with Helix pomatia lectin-gold complexes, no labeling could be detected over the cisternal space of the nuclear envelope and the rough endoplasmic reticulum. A uniform labeling was observed over the first and several subsequent cis Golgi cisternae and over the last (duodenal goblet cells) or the two last (colonic goblet cells) trans Golgi cisternae as well as forming and mature mucin droplets. However, essentially no labeling was detected over several cisternae in the central (medial) region of the Golgi apparatus. The results strongly suggest that core O-glycosylation takes place in cis Golgi cisternae but not in the rough endoplasmic reticulum. The heterogenous labeling for GalNAc residues in the Golgi apparatus is taken as evidence that termination of certain O-oligosaccharide chains by GalNAc occurs in trans Golgi cisternae.
Processing of MOPC 315 immunoglobulin A oligosaccharides: evidence for endoplasmic reticulum and trans Golgi alpha 1,2-mannosidase activity.
Authors: Hickman S , Theodorakis J L , Greco J M , Brown P H
Published: May 2004

The processing of asparagine-linked oligosaccharides on the alpha-chains of an immunoglobulin A (IgA) has been investigated using MOPC 315 murine plasmacytoma cells. These cells secrete IgA containing complex-type oligosaccharides that were not sensitive to endo-beta-N-acetylglucosaminidase H. In contrast, oligosaccharides present on the intracellular alpha-chain precursor were of the high mannose-type, remaining sensitive to endo-beta-N-acetylglucosaminidase H despite a long intracellular half-life of 2-3 h. The major [3H]mannose-labeled alpha-chain oligosaccharides identified after a 20-min pulse were Man8GlcNAc2 and Man9GlcNAc2. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man6GlcNAc2, which was shown to contain a single alpha 1,2-linked mannose residue. Conversion of Man6GlcNAc2 to complex-type oligosaccharides occurred at the time of secretion since appreciable amounts of Man5GlcNAc2 or further processed structures could not be detected intracellularly. The subcellular locations of the alpha 1,2-mannosidase activities were studied using carbonyl cyanide m-chlorophenylhydrazone and monensin. Despite inhibiting the secretion of IgA, these inhibitors of protein migration did not effect the initial processing of Man9GlcNAc2 to Man6GlcNAc2. Furthermore, no large accumulation of Man5GlcNAc2 occurred, indicating the presence of two subcellular locations of alpha 1,2-mannosidase activity involved in oligosaccharide processing in MOPC 315 cells. Thus, the first three alpha 1,2-linked mannose residues were removed shortly after the alpha-chain was glycosylated, most likely in rough endoplasmic reticulum, since this processing occurred in the presence of carbonyl cyanide m-chlorophenylhydrazone. However, the removal of the final alpha 1,2-linked mannose residue as well as subsequent carbohydrate processing occurred just before IgA secretion, most likely in the trans Golgi complex since processing of Man6GlcNAc2 to Man5GlcNAc2 was greatly inhibited in the presence of monensin.
Induction of muscle genes in neural cells.
Authors: Wright W E
Published: May 2004

The regulation of skeletal muscle genes was examined in heterokaryons formed by fusing differentiated chick skeletal myocytes to four different rat neural cell lines. Highly enriched populations of heterokaryons isolated using irreversible biochemical inhibitors were labeled with [35S]methionine and analyzed on two-dimensional gels. Rat skeletal myosin light chains were induced in three of the four cell combinations. The one exception, the S-20 cholinergic cell line, not only failed to synthesize rat muscle proteins but also suppressed chick myogenic functions. Experiments with heterokaryons between chick myocytes and cells from whole embryonic rat brain cultures demonstrated that rat skeletal myosin light chains are inducible in normal diploid neural cells as well as in established neural cell lines. In contrast, dividing cell hybrids between rat myoblasts and rat glial cells were nonmyogenic. These results demonstrate that although neural cells may contain factors that prevent the decision to differentiate along myogenic lines in cell hybrids, most neural cell lines do not dominantly suppress the expression of muscle structural genes in heterokaryons. Furthermore, the skeletal myosin light chain genes in most neural cell lines are regulated by a mechanism that permits them to respond to putative chick skeletal myocyte-inducing factors. The "open" state of these myogenic genes may explain many of the reports of apparent "transdifferentiation" to muscle in neural cultures and neural tumors.
Comparison of rapid changes in surface morphology and coated pit formation of PC12 cells in response to nerve growth factor, epidermal growth factor, and dibutyryl cyclic AMP.
Authors: Connolly J L , Green S A , Greene L A
Published: May 2004

Scanning and transmission electron microscopic studies were carried out on the rapid cell surface response of PC12 pheochromocytoma cells to treatment with nerve growth factor (NGF), epidermal growth factor (EGF), and dibutyryl cyclic AMP. EGF induced a rapidly initiated series of surface changes identical to those previously observed with NGF. Ruffles appear over the dorsal surface of the cells by 30 s, are prominent at 3 min, and are absent by 7 min. Microvilli disappear as dorsal ruffles become prominent. Peripheral ruffles are seen by 3 min, are prominent on most of the cells by 7 min, and are virtually absent by 15 min. Large blebs are present on 50% of the cells by 2 h and are markedly decreased by 4 h. Within 30 s after NGF or EGF addition, an increase in the density of 60-130-nm coated pits per unit membrane is detectable. This reaches a maximum of two- to threefold in from 1 to 3 min and gradually decreases. Combined treatment with NGF and EGF increases surface ruffling and, after an early peak in coated pits which at 3 min is similar in magnitude to that observed for the separately administered factors, maintains a greater number of pits per unit area than either treatment alone. 3-d pretreatment with NGF greatly reduces the response of the cells to EGF both with respect to surface ruffling and coated pit formation while 4-h NGF pretreatment has no effect on the EGF response. Dibutyryl cyclic AMP induced none of the rapidly onsetting changes caused by NGF or EGF, and therefore it seems unlikely that cyclic AMP mediates these surface changes. Changes in cell surface architecture induced by NGF and EGF on PC12 cells and by NGF in normal sympathetic neurons (as previously described) indicates that such responses may be a widespread phenomenon associated with the interaction of at least some peptide growth factors/hormones with their receptors. These responses may represent or reflect primary events in the mechanism by which these factors act.