bluuebunny/crossref_metadata_2025 huggingface dataset:
A nucleus-basal body connector in Chlamydomonas reinhardtii that may function in basal body localization or segregation.
Authors: Wright R L , Salisbury J , Jarvik J W
Published: May 2004

We have isolated a nucleus-basal body complex from Chlamydomonas reinhardtii. The complex is strongly immunoreactive to an antibody generated against a major protein constituent of isolated Tetraselmis striata flagellar roots (Salisbury, J. L., A. Baron, B. Surek, and M. Melkonian, J. Cell Biol., 99:962-970). Electrophoretic and immunoelectrophoretic analysis indicates that, like the Tetraselmis protein, the Chlamydomonas antigen consists of two acidic isoforms of approximately 20 kD. Indirect immunofluorescent staining of nucleus-basal body complexes reveals two major fibers in the connector region, one between each basal body and the nucleus. The nucleus is also strongly immunoreactive, with staining radiating around much of the nucleus from a region of greatest concentration at the connector pole. Calcium treatment causes shortening of the connector fibers and also movement of nuclear DNA towards the connector pole. Electron microscopic observation of negatively stained nucleus-basal body complexes reveals a cluster of approximately 6-nm filaments, suspected to represent the connector, between the basal bodies and nuclei. A mutant with a variable number of flagella, vfl-2-220, is defective with respect to the nucleus-basal body association. This observation encourages us to speculate that the nucleus-basal body union is important for accurate basal body localization within the cell and/or for accurate segregation of parental and daughter basal bodies at cell division. A physical association between nuclei and basal bodies or centrioles has been observed in a variety of algal, protozoan, and metazoan cells, although the nature of the association, in terms of both structure and function, has been obscure. We believe it likely that fibrous connectors homologous to those described here for Chlamydomonas are general features of centriole-bearing eucaryotic cells.
Selective expression of the 180-kD component of the neural cell adhesion molecule N-CAM during development.
Authors: Pollerberg E G , Sadoul R , Goridis C , Schachner M
Published: May 2004

The rodent neural cell adhesion molecule (N-CAM) consists of three glycoprotein chains of 180, 140, and 120 kD in their adult forms. Although the proportions of the three components are known to change during development and differ between brain regions, their individual distribution and function are unknown. Here we report studies carried out with a monoclonal antibody that specifically recognizes the 180-kD component of mouse N-CAM (N-CAM180) in its highly sialylated embryonic and less glycosylated adult forms. In primary cerebellar cell cultures, N-CAM180 antibody reacts intracellularly with all types of neural cells including astrocytes, oligodendrocytes, and neurons. During cerebellar, telencephalic, and retinal development N-CAM180 is detectable by indirect immunohistology in differentiated neural cells, but, in contrast to total N-CAM, not in their proliferating precursors in the ventricular zone and primordial and early postnatal external granular layer. In monolayer cultures of C1300 neuroblastoma cells, N-CAM180 appears by immunofluorescence more concentrated at contact points between adjacent cells, while N-CAM comprising the 180- and 140-kD component shows a more uniform distribution at the plasma membrane. Treatment of neuroblastoma cells with dimethylsulfoxide, which promotes differentiation, induces a shift toward the predominant expression of N-CAM180. These observations support the notion that N-CAM180 is expressed selectively in more differentiated neural cells and suggest a differential role of N-CAM180 in the stabilization of cell contacts.
Retention of autoregulatory control of tubulin synthesis in cytoplasts: demonstration of a cytoplasmic mechanism that regulates the level of tubulin expression.
Authors: Pittenger M F , Cleveland D W
Published: May 2004

Virtually all animal cells rapidly and specifically depress synthesis of new alpha- and beta-tubulin polypeptides in response to microtubule inhibitors that increase the pool of depolymerized subunits, or in response to direct elevation of the cellular tubulin subunit content through microinjection of exogenous tubulin subunits. Collectively, these previous findings have documented the presence of an apparent eucaryotic, autoregulatory control mechanism that specifies the level of expression of tubulin in cultured animal cells. Mechanistically, this regulation of tubulin synthesis is achieved through modulation of tubulin mRNA levels. To dissect further the molecular pathway that underlies this autoregulatory phenomenon, we have now investigated whether enucleated cells still retain the requisite regulatory machinery with which to alter tubulin synthetic levels in response to fluctuations in the pool size of unpolymerized tubulin subunits. Using two-dimensional gel electrophoresis to analyze the patterns of new polypeptide synthesis, we have determined that such cytoplasts can indeed respond to drug-induced microtubule depolymerization by specific repression of new beta-tubulin synthesis. Moreover, the response of cytoplasts is, if anything, greater in magnitude than that of whole cells. We conclude that autoregulatory control of beta-tubulin gene expression must derive principally, if not exclusively, from a cytoplasmic control mechanism that modulates beta-tubulin mRNA stability. For alpha-tubulin, although the response of cytoplasts after drug-induced microtubule depolymerization is quantitatively less dramatic than that of whole cells, at least part of the regulatory machinery must also be activated through a cytoplasmic regulatory event.
Nerve growth cones isolated from fetal rat brain. IV. Preparation of a membrane subfraction and identification of a membrane glycoprotein expressed on sprouting neurons.
Authors: Ellis L , Wallis I , Abreu E , Pfenninger K H
Published: May 2004

This study describes the preparation of a membrane subfraction from isolated nerve growth cone particles (GCPs) (see Pfenninger, K. H., L. Ellis, M. P. Johnson, L. B. Friedman, and S. Somlo, 1983, Cell, 35:573-584) and the identification in this fraction of a glycoprotein expressed during neurite growth. While approximately 40 major polypeptides are visible in Coomassie Blue-stained SDS polyacrylamide gels of pelleted (partially disrupted) GCPs, a salt-washed membrane fraction prepared from lysed, detergent-permeabilized GCPs contains only 14% of this protein and has an unusually simple polypeptide pattern of seven major bands. Monoclonal antibodies have been generated to GCP membranes isolated from fetal rat brain. These antibodies have been screened differentially with synaptosomes from adult rat brain in order to identify those which recognize antigens expressed selectively during neurite growth. One such antibody (termed 5B4) recognizes a developmentally regulated membrane glycoprotein that is enriched in GCP membranes and expressed in fetal neurons sprouting in vitro. The 5B4 antigen in fetal brain migrates in SDS polyacrylamide gels as a diffuse band of approximately 185-255 kD, is rich in sialic acid, and consists of a small family of isoelectric variants. Freezing-thawing and neuraminidase digestion result in the cleavage of the native antigen into two new species migrating diffusely around 200 and 160 kD. Prolonged neuraminidase digestion sharpens these bands at about 180 and 135 kD, respectively. In the mature brain, antibody 5B4 recognizes a sparse polypeptide migrating at approximately 140 kD. As shown in the following paper (Wallis, I., L. Ellis, K. Suh, and K. H. Pfenninger, 1985, J. Cell Biol., 101:1990-1998), the fetal antigen is specifically associated with regions of neuronal sprouting and, therefore, can be used as a molecular marker of neurite growth.
Immunolocalization of a neuronal growth-dependent membrane glycoprotein.
Authors: Wallis I , Ellis L , Suh K , Pfenninger K H
Published: May 2004

Monoclonal antibody (mAb) 5B4 recognizes in the rat a large, developmentally regulated membrane glycoprotein. The larger form of this antigen (185-255 kD) occurs in the developing nervous system and is present in membranes of nerve growth cones, as determined by analysis of a growth cone particle fraction. An immunochemical characterization of this antigen and of a smaller form (140 kD), sparsely present in the mature nervous system, has been described (Ellis, L., I. Wallis, E. Abreu, and K. H. Pfenninger, 1985, J. Cell. Biol., 101:1977-1989). The present paper reports on the localization by immunofluorescence of 5B4 antigen in cultured cortical neurons, developing spinal cord, and the mature olfactory system. In culture, mAb 5B4 stains only neurons; it is sparsely present in neurons at the onset of sprouting while, during sprouting, it appears to be concentrated at the growth cone and in regions of the perikaryon. In the developing spinal cord, 5B4 labeling is faintly detectable on embryonic day 11 but is intense on fetal day 13. At this stage, the fluorescence is observed in regions of the cord where axonal growth is occurring, while areas composed of dividing or migrating neural cells are nonfluorescent. With maturation of the spinal cord, this basic pattern of fluorescence persists initially, but the staining intensity decreases dramatically. In the adult, faint fluorescence is detectable only in gray matter, presumably indicating the presence of the 140 kD rather than the fetal antigen. The only known structure of the adult mammalian nervous system where axonal growth normally occurs is the olfactory nerve. mAb 5B4 intensely stains a variable proportion of olfactory axons in the mucosa as well as in the olfactory bulb. Based on both immunochemical and immunofluorescence data, the 5B4 antigen of 185-255 kD is associated specifically with growing neurons, i.e., neurons that are generating neurites.
Secretogranins I and II: two tyrosine-sulfated secretory proteins common to a variety of cells secreting peptides by the regulated pathway.
Authors: Rosa P , Hille A , Lee R W , Zanini A , De Camilli P , Huttner W B
Published: May 2004

We report on the biochemical and immunological properties as well as on the cellular and subcellular distribution of two proteins, called secretogranins I and II. These proteins specifically occur in a wide variety of endocrine and neuronal cells that package and sort regulatory peptides into secretory granules. Both secretogranins take the same intracellular route as the peptides and are also sorted into secretory granules. Secretogranins I and II are biochemically and immunologically distinct proteins and differ from chromogranin A. Yet, these three proteins are similar to each other in many respects and therefore constitute one class of proteins. A remarkable feature of this protein class is a very acidic pI, brought about by a high content of acidic amino acids as well as by phosphorylation on serine and sulfation on tyrosine and O-linked carbohydrate. As a result, this class of proteins has a high net negative charge even at the acidic pH of the trans Golgi cisternae. We discuss the possibility that this property of the proteins may point to a role in the packaging of regulatory peptides into secretory granules.
Transit of alpha-mannosidase during its maturation in Dictyostelium discoideum.
Authors: Wood L , Kaplan A
Published: May 2004

We proposed that Dictyostelium discoideum contains two linked pools of mature alpha-mannosidase (Wood, L., R. N. Pannell, and A. Kaplan, 1983, J. Biol. Chem., 258:9426-9430). To obtain physical evidence for these pools, cells were pulse-labeled with [35S]methionine, homogenized, and subjected to Percoll gradient centrifugation. After immune precipitation of alpha-mannosidase, its polypeptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and detected by fluorography. After a 30-min pulse with [35S]methionine, the precursor and small amounts of cleaved enzyme were detected in a low density fraction (1.04 g/ml). Subsequently, cleaved enzyme was transferred to higher density fractions (1.05 and 1.07 g/ml) that were enriched in lysosomal enzymes. The half time for formation of the 1.07 g/ml pool was approximately 45 min, whereas formation of the 1.05 g/ml pool was not detected until 1.5 h after the pulse. The transfer of mature forms out of the 1.04 g/ml pool was inhibited by monensin (3.5 microM). Thus, alpha-mannosidase precursor appears to be cleaved in a prelysosomal organelle. The data also indicate that starving cells secrete precursor directly from this organelle to the extracellular space, whereas cleaved forms are first transferred into lysosomes before they are secreted. Furthermore, 2 h after starvation, the secretion of mature forms ceases even though both transit of mature forms between the two pools and secretion of precursor continues. From this we inferred that the cessation of secretion of mature forms is due to a halt in fusion of lysosomes with the plasma membrane and that precursor follows a different route to the plasma membrane.
Desmocalmin: a calmodulin-binding high molecular weight protein isolated from desmosomes.
Authors: Tsukita S , Tsukita S
Published: May 2004

A unique high molecular weight protein (240,000 mol wt) has been purified from isolated desmosomes of bovine muzzle epidermis, using low-salt extraction at pH 9.5-10.5 and gel-filtration followed by calmodulin-affinity column chromatography. This protein was shown to bind to calmodulin in a Ca2+-dependent manner, so we called it desmocalmin here. Desmocalmin also bound to the reconstituted keratin filaments in vitro in the presence of Mg2+, but not to actin filaments. By use of the antibody raised against the purified desmocalmin, desmocalmin was shown by both immunoelectron and immunofluorescence microscopy to be localized at the desmosomal plaque just beneath the plasma membrane. Judging from its isoelectric point and antigenicity, desmocalmin was clearly distinct from desmoplakins I and II, which were identified in the desmosomal plaque by Mueller and Franke (1983, J. Mol. Biol., 163:647-671). In the low-angle, rotary-shadowing electron microscope, the desmocalmin molecules looked like flexible rods approximately 100-nm long consisting of two polypeptide chains lying side by side. The similar rodlike structures were clearly identified in the freeze-etch replica images of desmosomes. Taken together, these findings indicate that desmocalmin could function as a key protein responsible for the formation of desmosomes in a calmodulin-dependent manner (Trinkaus-Randall, V., and I.K. Gipson, 1984, J. Cell Biol., 98:1565-1571).
Alpha-tubulin acetylase activity in isolated Chlamydomonas flagella.
Authors: Greer K , Maruta H , L'Hernault S W , Rosenbaum J L
Published: May 2004

We have previously shown that the alpha-tubulin of Chlamydomonas flagella is synthesized as a precursor which is modified by acetylation in the flagellum during flagellar assembly. In this report, we show the presence of an alpha-tubulin acetylase activity in isolated Chlamydomonas flagella that is highly specific for alpha-tubulin of both mammalian brain and Chlamydomonas.
Microtubule-associated proteins and in vitro astrocyte differentiation.
Authors: Couchie D , Fages C , Bridoux A M , Rolland B , Tardy M , Nunez J
Published: May 2004

Primary cultures of mouse brain astrocytes have been used to identify the microtubule-associated proteins (MAPs) present in this cell type at different stages of in vitro differentiation. The MAPs of the astrocyte have been identified by polyacrylamide gel electrophoresis and immunological detection. Two antisera were raised against two brain MAPs, tau and MAP-2. These antisera were also used to label the microtubular network in the intact astrocytes at different stages of the culture. The mature astrocyte contains a variety of MAP-like proteins. Anti-MAP-2 serum detected several proteins of high molecular weight (380,000, 260,000, 205,000 and 165,000 mol wt) and one microheterogeneous peak of 83,000 mol wt. Anti-tau also detected high molecular weight components (380,000 to approximately 200,000 mol wt) but not the 165,000-mol-wt peak; in addition two microheterogeneous peaks of 83,000 and 62,000 mol wt were detected by the anti-tau serum. The 62,000-mol-wt peak was therefore detected only by the anti-tau serum whereas the 83,000-mol-wt component cross-reacted with both antisera. At early stages of the culture the immature cell contained about two times less immunoreactive material than at mature stages. Qualitative changes of the high molecular weight components were also observed. In the intact cell both antisera revealed a dense fibrous network. At early stages of the culture the astroblasts were stained by the antisera but the reaction was very diffuse in the cytoplasm; few fibrous cells were intensively stained. Morphological differentiation, which began after serum deprivation and which was accelerated by forskolin (a drug that induces cyclic AMP accumulation), led to high labeling of both the cell body and the cellular processes. In the presence of colchicine the staining regressed, the processes shortened, and the cell returned to a less-apparently differentiated state.
Transcellular transport of polymeric IgA in the rat hepatocyte: biochemical and morphological characterization of the transport pathway.
Authors: Hoppe C A , Connolly T P , Hubbard A L
Published: May 2004

Polymeric IgA (pIgA) is transported by liver parenchymal cells (hepatocytes) from blood to bile via a receptor-mediated process. We have studied the intracellular pathway taken by a TEPC15 mouse myeloma pIgA. When from 1 microgram to 1 mg 125I-pIgA was injected into the saphenous vein of a rat, 36% was transported as intact protein into the bile over a 3-h period. The concentration of transported 125I-pIgA was maximal in bile 30-60 min after injection, and approximately 80% of the total 125I-pIgA ultimately transported had been secreted into bile by 90 min. A horseradish peroxidase-pIgA conjugate (125I-pIgA-HRP) was transported to a similar extent and with kinetics similar to that of unconjugated 125I-pIgA and was therefore used to visualize the transport pathway. Peroxidase cytochemistry of livers fixed in situ 2.5 to 10 min after 125I-pIgA-HRP injection demonstrated a progressive redistribution of labeled structures from the sinusoidal area to intermediate and bile canalicular regions of the hepatocyte cytoplasm. Although conjugate-containing structures began accumulating in the bile canalicular region at these early times, no conjugate was present in bile until 20 min. From 7.5 to 45 min after injection approximately 30% of the labeled structures were in regions that contained Golgi complexes and lysosomes; however, we found no evidence that either organelle contained 125I-pIgA-HRP. At least 85% of all positive structures in the hepatocyte were vesicles of 110-160-nm median diameters, with the remaining structures accounted for by tubules and multivesicular bodies. Vesicles in the bile canalicular region tended to be larger than those in the sinusoidal region. Serial sectioning showed that the 125I-pIgA-HRP-containing structures were relatively simple (predominantly vesicular) and that extensive interconnections did not exist between structures in the sinusoidal and bile canalicular regions.
Occluding junction structure-function relationships in a cultured epithelial monolayer.
Authors: Madara J L , Dharmsathaphorn K
Published: May 2004

Electrical circuit analysis was used to study the structural development of occluding junctions (OJs) in cultured monolayers composed to T84 cells. The magnitude of the increments in transepithelial resistance predicted by such analysis was compared with the magnitude of the measured increments in resistance. Confluent sheets of epithelial cells were formed after cells were plated at high density on collagen-coated filters. Using Claude's OJ strand count-resistance hypothesis (1978, J. Membr. Biol. 39:219-232), electrical circuit analysis of histograms describing OJ strand count distribution at different time points after plating predicted that junctional resistance should rise in a proportion of 1:21:50 from 18 h to 2 d to 5 d. This reasonably paralleled the degree of rise in transepithelial resistance over this period, which was 1:29:59. The ability to predict the observed resistance rise was eliminated if only mean strand counts were analyzed or if electrical circuit analysis of OJ strand counts were performed using an OJ strand count-resistance relationship substantially different from that proposed by Claude. Measurements of unidirectional fluxes of inulin, mannitol, and sodium indicated that restriction of transjunctional permeability accounted for the observed resistance rise, and that T84 junctional strands have finite permeability to molecules with radii less than or equal to 3.6 A but are essentially impermeable to molecules with radii greater than or equal to 15 A. The results suggest that general correlates between OJ structure and OJ ability to resist passive ion flow do exist in T84 monolayers. The study also suggests that such correlates can be obtained only if OJ structural data are analyzed as an electrical circuit composed of parallel resistors.
Membrane and cytoskeletal changes associated with IgE-mediated serotonin release from rat basophilic leukemia cells.
Authors: Pfeiffer J R , Seagrave J C , Davis B H , Deanin G G , Oliver J M
Published: May 2004

Binding of antigen to IgE-receptor complexes on the surface of RBL-2H3 rat basophilic leukemia cells is the first event leading to the release of cellular serotonin, histamine, and other mediators of allergic, asthmatic, and inflammatory responses. We have used dinitrophenol-conjugated bovine serum albumin (DNP-BSA) as well as the fluorescent antigen, DNP-B-phycoerythrin, and the electron-dense antigen, DNP-BSA-gold, to investigate dynamic membrane and cytoskeletal events associated with the release of [3H]serotonin from anti-DNP-IgE-primed RBL-2H3 cells. These multivalent antigens bind rapidly to cell surface IgE-receptor complexes. Their distribution is initially uniform, but within 2 min DNP-BSA-gold is found in coated pits and is subsequently internalized. Antigen internalization occurs in the presence and absence of extracellular Ca2+. The F-actin content of the detergent-extracted cell matrices analyzed by SDS PAGE decreases during the first 10-30 s of antigen binding and then increases by 1 min to almost double the control levels. A rapid and sustained increase is also observed when total F-actin is quantified by flow cytometry after binding of rhodamine-phalloidin. The antigen-stimulated increase in F-actin coincides with (and may cause) the transformation of the cell surface from a finely microvillous to a highly folded or plicated topography. Other early membrane responses include increased cell spreading and a 2-3-fold increase in the uptake of fluorescein-dextran by fluid pinocytosis. The surface and F-actin changes show the same dependence on DNP-protein concentration as stimulated [3H]serotonin release; and both the membrane responses and the release of mediators are terminated by the addition of the non-cross-linking monovalent ligand, DNP-lysine. These data indicate that the same antigen-stimulated transduction pathway controls both the membrane/cytoskeletal and secretory events. However, the membrane and actin responses to IgE-receptor cross-linking are independent of extracellular Ca2+ and are mimicked by phorbol myristate acetate, whereas ligand-dependent mediator release depends on extracellular Ca2+ and is mimicked by the Ca2+ ionophore A23187.
Monoclonal antibodies as probes of epithelial membrane polarization.
Authors: Turner R J , Thompson J , Sariban-Sohraby S , Handler J S
Published: May 2004

Monoclonal antibodies directed against antigens in the apical plasma membrane of the toad kidney epithelial cell line A6 were produced to probe the phenomena that underlie the genesis and maintenance of epithelial polarity. Two of these antibodies, 17D7 and 18C3, were selected for detailed study here. 17D7 is directed against a 23-kD peptide found on both the apical and basolateral surfaces of the A6 epithelium whereas 18C3 recognizes a lipid localized to the apical membrane only. This novel observation of an apically localized epithelial lipid species indicates the existence of a specific sorting and insertion process for this, and perhaps other, epithelial plasma membrane lipids. The antibody-antigen complexes formed by both these monoclonal antibodies are rapidly internalized by the A6 cells, but only the 18C3-antigen complex is recycled to the plasma membrane. In contrast to the apical localization of the free antigen, however, the 18C3-antigen complex is recycled to both the apical and basolateral surface of the epithelium, which indicates that monoclonal antibody binding interferes in some way with the normal sorting process for this apical lipid antigen.
Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein.
Authors: Poruchynsky M S , Tyndall C , Both G W , Sato F , Bellamy A R , Atkinson P H
Published: May 2004

Rotavirus, a non-enveloped reovirus, buds into the rough endoplasmic reticulum and transiently acquires a membrane. The structural glycoprotein, VP7, a 38-kD integral membrane protein of the endoplasmic reticulum (ER), presumably transfers to virus in this process. The gene for VP7 potentially encodes a protein of 326 amino acids which has two tandem hydrophobic domains at the NH2-terminal, each preceded by an in-frame ATG codon. A series of deletion mutants constructed from a full-length cDNA clone of the Simian 11 rotavirus VP7 gene were expressed in COS 7 cells. Products from wild-type, and mutants which did not affect the second hydrophobic domain of VP7, were localized by immunofluorescence to elements of the ER only. However, deletions affecting the second hydrophobic domain (mutants 42-61, 43-61, 47-61) showed immunofluorescent localization of VP7 which coincided with that of wheat germ agglutinin, indicating transport to the Golgi apparatus. Immunoprecipitable wild-type protein, or an altered protein lacking the first hydrophobic sequence, remained intracellular and endo-beta-N-acetylglucosaminidase H sensitive. In contrast, products of mutants 42-61, 43-61, and 47-61 were transported from the ER, and secreted. Glycosylation of the secreted molecules was inhibited by tunicamycin, resistant to endo-beta-N-acetylglucosaminidase H digestion and therefore of the N-linked complex type. An unglycosylated version of VP7 was also secreted. We suggest that the second hydrophobic domain contributes to a positive signal for ER location and a membrane anchor function. Secretion of the mutant glycoprotein implies that transport can be constitutive with the destination being dictated by an overriding compartmentalization signal.
Cell-mediated extracellular acidification and bone resorption: evidence for a low pH in resorbing lacunae and localization of a 100-kD lysosomal membrane protein at the osteoclast ruffled border.
Authors: Baron R , Neff L , Louvard D , Courtoy P J
Published: May 2004

The extracellular compartment where bone resorption occurs, between the osteoclast and bone matrix, is shown in this report to be actively acidified. The weak base acridine orange accumulates within this compartment but dissipates after incubation with ammonium chloride. Upon removal of ammonium chloride, the cells are able to rapidly reacidify this compartment. The highly convoluted plasma membrane of the osteoclast facing this acidic compartment (ruffled border) is shown to contain a 100-kD integral membrane protein otherwise present in limiting membranes of lysosomes and other related acidified organelles (Reggio, H., D. Bainton, E. Harms, E. Coudrier, and D. Louvard, 1984, J. Cell Biol., 99:1511-1526; Tougard, C., D. Louvard, R. Picart, and A. Tixier-Vidal, 1985, J. Cell Biol. 100:786-793). Antibodies recognizing this 100-kD lysosomal membrane protein cross-react with a proton-pump ATPase from pig gastric mucosae (Reggio, H., D. Bainton, E. Harms, E. Coudrier, and D. Louvard, 1984, J. Cell Biol., 99:1511-1526), therefore raising the possibility that it plays a role in the acidification of both intracellular organelles and extracellular compartments. Lysosomal enzymes are also directionally secreted by the osteoclast into the acidified extracellular compartment which can therefore be considered as the functional equivalent of a secondary lysosome with a low pH, acid hydrolases, the substrate, and a limiting membrane containing the 100-kD antigen.
Mechanism of benzo(a)pyrene induction of alpha-human chorionic gonadotropin gene expression in human lung tumor cells.
Authors: Wong D T , Biswas D K
Published: May 2004

Human lung cells (ChaGo) derived from a bronchogenic carcinoma synthesize and secrete in the culture medium the alpha subunit of the glycoprotein hormone, human chorionic gonadotropin (alpha-hCG). The synthesis of alpha-hCG by ChaGo cells could be further stimulated by treatment with sublethal concentrations of the polycyclic aromatic hydrocarbons (PAHs), benzo(a)pyrene (BaP), or dimethylbenzanthracene. The production of alpha-hCG could be correlated to the levels of alpha-hCG-specific mRNA sequences in control and PAH-treated cells. Further analysis of the RNA species (Northern blot) revealed that the level of the mature (approximately 1.0 kb) and the high molecular weight alpha-hCG specific nuclear RNA sequences (approximately 2.2 and 5 kb) were all greater in PAH-treated cells. Addition of [3H]BaP (0.25 microgram/ml) in the culture medium of ChaGo cells led to immediate uptake of the radioactive compound apparently by simple diffusion. SDS PAGE and subsequent fluorography revealed that the radioactive compound interacted and formed covalent complexes with cytoplasmic and nuclear proteins. This covalent interaction of the [3H]BaP molecule with cellular proteins could be significantly inhibited by either inhibiting the activity of the enzyme aryl hydrocarbon hydroxylase with 7,8-benzoflavone or by reducing the cellular concentration of the enzyme by simultaneous incubation with cycloheximide. These results suggested that in ChaGo cells, the observed covalent complexes were formed by the interaction of the BaP metabolites with cellular proteins. The concentrations at which 7,8-benzoflavone or cycloheximide inhibited formation of metabolites from [3H]BaP and their covalent interaction with cell protein did not affect the BaP-induced stimulation of alpha-hCG gene expression. However, the cytotoxic effects of BaP in ChaGo cells seemed to be exerted by the metabolism of the compounds. Results presented in this report suggest that BaP metabolism and the interaction of the metabolites with cell proteins were not essential for the BaP-induced modulation of alpha-hCG gene expression.
Possible pathways for lysosomal enzyme delivery.
Authors: Geuze H J , Slot J W , Strous G J , Hasilik A , von Figura K
Published: May 2004

Immunogold double-labeling and ultrathin cryosections were used to compare the subcellular distribution of albumin, mannose 6-phosphate receptor (MPR), galactosyltransferase, and the lysosomal enzymes cathepsin D, beta-hexosaminidase, and alpha-glucosidase in Hep G2 cells. MPR and lysosomal enzymes were found throughout the stack of Golgi cisternae and in a trans-Golgi reticulum (TGR) of smooth-surfaced tubules with coated buds and vesicles. The trans-Golgi orientation of TGR was ascertained by the co-localization with galactosyltransferase. MPR was particularly abundant in TGR and CURL, the compartment of uncoupling receptors and ligands. Both TGR and CURL also contained lysosomal enzymes, but endogenous albumin was detected in TGR only. The coated buds on TGR tubules contained MPR, lysosomal enzymes, as well as albumin. MPR and lysosomal enzymes were also found in coated pits of the plasma membrane. CURL tubules seemed to give rise to smooth vesicles, often of the multivesicular body type. In CURL, the enzymes were found in the lumina of the smooth vesicles while MPR prevailed in the tubules. These observations suggest a role of CURL in transport of lysosomal enzymes to lysosomes. When the cells were treated with the lysosomotropic amine primaquine, binding of anti-MPR to the cells in culture was reduced by half. Immunocytochemistry showed that MPR accumulated in TGR, especially in coated buds. Since these buds contain endogenous albumin and lysosomal enzymes also, these data suggest that coated vesicles originating from TGR provide for a secretory route in Hep G2 cells and that this pathway is followed by the MPR system as well.
Cell anchorage determines whether mammary tumor virus glycoproteins are processed for plasma membranes or secretion.
Authors: Kabat D , Gliniak B , Rohrschneider L , Polonoff E
Published: May 2004

The subcellular localization of mouse mammary tumor virus (MMTV) glycoproteins was analyzed in infected and cloned rat hepatocarcinoma cells cultured with the MMTV transcriptional inducer dexamethasone. When reacted with protein A-coated erythrocytes in the presence of antisera specific for viral glycoproteins or with fluorescent antisera, only some of the cells acquired surface label. This diversity was dependent on cell anchorage to the substratum. In general, the more rounded, less adherent cells contained the MMTV glycoproteins on their surfaces, whereas the flatter, more adherent cells did not. After a change in adherence, a delay preceded complete remodeling of the plasma membranes. Fluorescent antibody studies of fixed cells and analyses of viral glycoprotein synthesis and shedding using L-[35S]methionine indicated that the different expression of MMTV glycoproteins in round versus flat cells is caused by a switch in posttranslational processing. In round cells, the MMTV-encoded precursor glycoprotein is proteolytically cleaved and then transported to plasma membranes as a complex of two subunits, the smaller being the membrane anchor. In flat adherent cells, the smaller subunit is rapidly degraded in an intracellular organelle and the larger is then secreted into the medium. As indicated by labeling of cells with 125I, the concentrations of several host-encoded plasma membrane components are also influenced by cell anchorage. We propose that this switch in cell surfaces and in secretions dependent upon cell-substratum attachments may be a common control mechanism important for embryogenesis, wound healing, and cancer.
pH-induced alterations in the fusogenic spike protein of Semliki Forest virus.
Authors: Kielian M , Helenius A
Published: May 2004

The spike glycoproteins of Semliki Forest virus mediate membrane fusion between the viral envelope and cholesterol-containing target membranes under conditions of mildly acidic pH (pH less than 6.2). The fusion reaction is critical for the infectious cycle, catalyzing virus penetration from the acidic endosome compartment. To define the role of the viral spike glycoproteins in the fusion reaction, conformational changes in the spikes at acid pH were studied using protease digestion and binding assays to liposomes and nonionic detergent. A method was also developed to prepare fragments of both transmembrane subunit glycopolypeptides of the spike, E1 and E2, which lacked the hydrophobic anchor peptides. Unlike the intact spikes the fragments were monomeric and therefore useful for obtaining information on conformational changes in individual subunits. The results showed that both E1 and E2 undergo irreversible conformational changes at the pH of fusion, that the conformational change of E1 depends, in addition to acidic pH, on the presence of cholesterol, and that no major changes in the solubility properties of the spikes takes place. On the basis of these findings it was concluded that fusion involves both subunits of the spike and that E1 confers the stereo-specific sterol requirement. The results indicated, moreover, that acid-induced fusion of Semliki Forest virus differs in important respects from that of influenza virus, another well-defined model system for protein-mediated membrane fusion.
A former amino terminal signal sequence engineered to an internal location directs translocation of both flanking protein domains.
Authors: Perara E , Lingappa V R
Published: May 2004

To determine whether a functional amino terminal signal sequence can be active at an internal position, a hybrid gene was constructed in which the entire coding region of bovine preprolactin cDNA was inserted into chimpanzee alpha-globin cDNA 109 codons downstream from the initiation codon of globin. When RNA synthesized in vitro from this plasmid (pSPGP1) was translated in the rabbit reticulocyte cell-free system, a 32-kD protein was produced that was both prolactin and globin immunoreactive. When microsomal membranes were present during translation (but not when added posttranslationally), a 26-kD and a 14-kD product were also observed. By immunoreactivity and electrophoretic mobility, the 26-kD protein was identical to mature prolactin, and the 14-kD protein appeared to be the globin domain with the prolactin signal sequence attached at its carboxy terminus. From (a) posttranslational proteolysis in the presence and absence of detergent, (b) sedimentation of vesicles in the presence and absence of sodium carbonate pH 11.5, and (c) N-linked glycosylation of the globin-immunoreactive fragment after insertion of an Asn-X-Ser N-linked glycosylation site into the globin coding region of pSPGP1, it appears that all of the 26-kD and some of the 14-kD products, but none of the 32-kD precursor, have been translocated to the lumen of the membrane vesicles. Thus, when engineered to an internal position, the prolactin signal sequence is able to translocate both flanking protein domains. These data have implications for the understanding of translocation of proteins across the membrane of the endoplasmic reticulum.
Biosynthesis of the neural cell adhesion molecule: characterization of polypeptide C.
Authors: Nybroe O , Albrechtsen M , Dahlin J , Linnemann D , Lyles J M , Møller C J , Bock E
Published: May 2004

The biosynthesis of the neural cell adhesion molecule (N-CAM) was studied in primary cultures of rat cerebral glial cells, cerebellar granule neurons, and skeletal muscle cells. The three cell types produced different N-CAM polypeptide patterns. Glial cells synthesized a 135,000 Mr polypeptide B and a 115,000 Mr polypeptide C, whereas neurons expressed a 200,000 Mr polypeptide A as well as polypeptide B. Skeletal muscle cells produced polypeptide B. The polypeptides synthesized by the three cell types were immunochemically identical. The membrane association of polypeptide C was investigated with methods that distinguish peripheral and integral membrane proteins. Polypeptide C was found to be a peripheral membrane protein, whereas polypeptides A and B were integral membrane proteins with cytoplasmic domains of approximately 50,000 and approximately 25,000 Mr, respectively. The affinity of the membrane binding of polypeptide C increased during postnatal development. The posttranslational modifications of polypeptide C were investigated in glial cell cultures, and it was found to be N-linked glycosylated and sulfated.
Cell surface tubulin in leukemic cells: molecular structure, surface binding, turnover, cell cycle expression, and origin.
Authors: Quillen M , Castello C , Krishan A , Rubin R W
Published: May 2004

We report here new characteristics of cell surface tubulin from a human leukemia cell line. These cells (CEM cells) possess tubulin that is readily iodinated on the surface of living cells, turns over at a rate identical to that of other surface proteins, and is present throughout the cell cycle. When removed with trypsin, it rapidly returns to the surface. Peptide mapping of iodinated surface tubulin indicates that it possesses a similar, but not identical, primary structure to total CEM and rat brain tubulin. Living CEM cells are able to bind specifically a subfraction of CEM tubulin from metabolically labeled high speed supernatants of lysed CEM cells. Surface tubulin is more basic than the total tubulin pool. The binding, which is saturable, is inhibited by unlabeled CEM high speed supernatants but not by excess thrice-cycled rat or bovine brain tubulin. Surface tubulin is also shown to bind to living nontransformed normal rat kidney cells but not to normal, circulating, mononuclear white cells. Activated lymphocytes produce a tubulin that binds to CEM cells. Since CEM tubulin was detected in the media of 6-h cultures of CEM cells, we must conclude that at least some of the surface tubulin comes from the media. We further conclude that these leukemic cells produce an unusual tubulin that may bind specifically to any membrane. The presence of iodinatable surface tubulin, however, appears to require both the production of a unique tubulin and the presence of a "receptor-like" surface binding component.
Characterization of a gene product (Sec53p) required for protein assembly in the yeast endoplasmic reticulum.
Authors: Bernstein M , Hoffmann W , Ammerer G , Schekman R
Published: May 2004

SEC53, a gene that is required for completion of assembly of proteins in the endoplasmic reticulum in yeast, has been cloned, sequenced, and the product localized by cell fractionation. Complementation of a sec53 mutation is achieved with unique plasmids from genomic or cDNA expression banks. These inserts contain the authentic gene, a cloned copy of which integrates at the sec53 locus. An open reading frame in the insert predicts a 29-kD protein with no significant hydrophobic character. This prediction is confirmed by detection of a 28-kD protein overproduced in cells that carry SEC53 on a multicopy plasmid. To follow Sec53p more directly, a LacZ-SEC53 gene fusion has been constructed which allows the isolation of a hybrid protein for use in production of antibody. With such an antibody, quantitative immune decoration has shown that the sec53-6 mutation decreases the level of Sec53p at 37 degrees C, while levels comparable to wild-type are seen at 24 degrees C. An eightfold overproduction of Sec53p accompanies transformation of cells with a multicopy plasmid containing SEC53. Cell fractionation, performed with conditions that preserve the lumenal content of the endoplasmic reticulum (ER), shows Sec53p highly enriched in the cytosol fraction. We suggest that Sec53p acts indirectly to facilitate assembly in the ER, possibly by interacting with a stable ER component, or by providing a small molecule, other than an oligosaccharide precursor, necessary for the assembly event.
Mitochondria and nuclei move by different mechanisms in Aspergillus nidulans.
Authors: Oakley B R , Rinehart J E
Published: May 2004

We have examined the effects of the antimicrotubule agent benomyl and several mutations on nuclear and mitochondrial movement in germlings of the filamentous fungus Aspergillus nidulans. While, as previously reported, benomyl inhibited nuclear division and movement, it did not inhibit mitochondrial movement. To test the effects of benomyl more rigorously, we germinated two benomyl super-sensitive, beta-tubulin mutants at a benomyl concentration 50-100 times greater than that required to inhibit colony formation completely. Again nuclear division and movement were inhibited, but mitochondrial movement was not. We also examined conditionally lethal beta-tubulin mutations that disrupt microtubule function under restrictive conditions. Nuclear division and movement were inhibited but, again, mitochondrial movement was not. Finally we examined the effects of five heat-sensitive mutations that inhibit nuclear movement but not nuclear division at restrictive temperatures. These mutations strongly inhibited nuclear movement at a restrictive temperature but did not inhibit mitochondrial movement. These data demonstrate that the mechanisms of nuclear and mitochondrial movement in Aspergillus nidulans are not identical and suggest that mitochondrial movement does not require functional microtubules.
Clonal variation in cell surface display of an H-2 protein lacking a cytoplasmic tail.
Authors: Zuniga M C , Hood L E
Published: May 2004

Truncated variants of the gene encoding H-2Ld, an integral membrane protein encoded by the major histocompatibility complex, were constructed by in vitro mutagenesis to elucidate the function of charged amino acids found on the cytoplasmic side of the transmembrane (TM) region. Analysis of cloned L cells transfected with these genes shows that the seven amino acids following the TM segment, four of which are basic, enhance the cell surface expression of H-2Ld protein but are not required for it. However, some clones do not express a tailless H-2Ld protein on the cell surface but express it intracellularly where it has a long half-life. Turnover measurements on cell surface H-2Ld proteins suggest that the basic residues following the TM segment are not a "stop transfer" sequence (Blobel, G., 1980, Proc. Natl. Acad. Sci. USA., 77:1496-1500) which anchors the H-2Ld protein in the membrane. Pulse-chase and endoglycosidase H sensitivity studies show that H-2Ld proteins lacking some or all of the basic residues and H-2Ld proteins which have a full-length cytoplasmic tail are processed with different kinetics. These results suggest an involvement of the membrane-proximal region of the cytoplasmic tail in the intracellular transport of H-2Ld. We further suggest that the L cell clones which do and do not express a tailless H-2Ld protein on the cell surface differ in the ability to transport a tailless integral membrane protein to the cell surface.
Heat shock response of the rat lens.
Authors: de Jong W W , Hoekman W A , Mulders J W , Bloemendal H
Published: May 2004

The sequence relationship between the small heat shock proteins and the eye lens protein alpha-crystallin (Ingolia, T. D., and E. E. Craig, 1982, Proc. Natl. Acad. Sci. USA, 79: 2360-2364) prompted us to subject rat lenses in organ culture to heat shock and other forms of stress. The effects on protein synthesis were followed by labeling with [35S]methionine and analysis by one- and two-dimensional gel electrophoresis and fluorography. Heat shock gave a pronounced induction of a protein that could be characterized as the stress protein SP71. This protein probably corresponds to the major mammalian heat shock protein hsp70. Also two minor proteins of 16 and 85 kD were induced, while the synthesis of a constitutive heat shock-related protein, P73, was considerably increased. The synthesis of SP71 started between 30 and 60 min after heat shock, reached its highest level after 3 h, and had stopped again after 8 h. In rat lenses that were preconditioned by an initial mild heat shock, a subsequent shock did not cause renewed synthesis of SP71. This effect resembles the thermotolerance phenomenon observed in cultured cells. The proline analogue azetidine-2-carboxylic acid, zinc chloride, ethanol, and calcium chloride did not, under the conditions used, induce stress proteins in the rat lens. Sodium arsenite, however, had very much the same effects as heat shock. Calcium ionophore A23187 specifically and effectively induced the synthesis of the glucose-regulated protein GRP78. No special response to stress on crystallin synthesis was noticed.
Expression of muscle genes in heterokaryons depends on gene dosage.
Authors: Pavlath G K , Blau H M
Published: May 2004

We report that gene dosage, or the ratio of nuclei from two cell types fused to form a heterokaryon, affects the time course of differentiation-specific gene expression. The rate of appearance of the human muscle antigen, 5.1H11, is significantly faster in heterokaryons with equal or near-equal numbers of mouse muscle and human fibroblast nuclei than in heterokaryons with increased numbers of nuclei from either cell type. By 4 d after fusion, a high frequency of gene expression is evident at all ratios and greater than 75% of heterokaryons express the antigen even when the nonmuscle nuclei greatly outnumber the muscle nuclei. The kinetic differences observed with different nuclear ratios suggest that the concentration of putative trans-acting factors significantly influences the rate of muscle gene expression: a threshold concentration is necessary, but an excess may be inhibitory.