bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of phosphorylation and cell surface redistribution of acetylcholine receptors by phorbol ester and carbamylcholine in cultured chick muscle cells.
Authors: Ross A , Rapuano M , Prives J
Published: May 2004

We have investigated the mechanisms regulating the clustering of nicotinic acetylcholine receptor (AChR) on the surface of cultured embryonic chick muscle cells. Treatment of these cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, was found to cause a rapid dispersal of AChR clusters, as monitored by fluorescence microscopy of cells labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin. The loss of AChR clusters was not accompanied by an appreciable change in the amount of AChR on the surface of these cells, as measured by the specific binding of [125I]Bgt. Analysis of the phosphorylation pattern of immunoprecipitable AChR subunits showed that the gamma- and delta-subunits are phosphorylated by endogenous protein kinase activity in the intact muscle cells, and that the delta-subunit displays increased phosphorylation in response to TPA. Structural analogues of TPA which do not stimulate protein kinase C have no effect on AChR surface topography or phosphorylation. Exposure of chick myotubes to the cholinergic agonist carbamylcholine was found to cause a dispersal of AChR clusters with a time course similar to that of TPA. Like TPA, carbamylcholine enhances the phosphorylation of the delta-subunit of AChR. The carbamylcholine-induced redistribution and phosphorylation of AChR is blocked by the nicotinic AChR antagonist d-tubocurarine. TPA and carbamylcholine have no effect on cell morphology during the time-course of these experiments. These findings indicate that cell surface topography of AChR may be regulated by phosphorylation of its subunits and suggest a mechanism for dispersal of AChR clusters by agonist activation.
Response of neutrophils to stimulus infusion: differential sensitivity of cytoskeletal activation and oxidant production.
Authors: Omann G M , Sklar L A
Published: May 2004

The response of human neutrophils to N-formyl peptides were studied under conditions where ligand binding was controlled by infusing a cell suspension with the peptide over a time period comparable to the normal half-time for binding. Receptor occupancy was measured in real time with a fluorescently labeled peptide using flow cytometry. This binding was approximated by a simple reversible model using typical on (7 X 10(8) M- min-1) and off (0.35/min) rate constants and the infusion rates (0.02-0.2 nM/min). Under conditions of stimulus infusion intracellular calcium elevation, superoxide generation, and right angle light scatter and F-actin formation were measured. As the infusion rate was decreased into the range of 10 pM/min, lowering the rate of increase of receptor occupancy to approximately 0.5% per min, the calcium and right angle light scatter responses elongated in time and decreased in magnitude. Superoxide generation decreased below infusion rates of approximately 100 pM/min (occupancy increasing at a rate in the range of 5% per min). This behavior could contribute to differences between chemotactic responses, which appear to require low rates of receptor occupancy over long periods, and bactericidal or inflammatory responses (free radical generation and degranulation), which require bursts of occupancy of several percent of the receptors.
N-myc proto-oncogene expression during organogenesis in the developing mouse as revealed by in situ hybridization.
Authors: Mugrauer G , Alt F W , Ekblom P
Published: May 2004

The N-myc proto-oncogene is expressed during embryogenesis, suggesting that it plays a role in normal development. Since the myc-family oncogenes have been implicated in the control of cell growth, the embryonic expression may reflect rapid proliferation known to occur in development. Alternatively, N-myc expression may be involved in specific differentiation stages. In many embryonic tissues, early and late differentiation events occur in different locations. By in situ hybridization of tissue sections, we now demonstrate a restricted expression of N-myc mRNA to a few tissues and to areas where the first differentiation stages occur. N-myc expression was most strongly expressed in the developing kidney, hair follicles, and in various parts of the central nervous system. In these tissues, expression was restricted to a few cell lineages. In all lineages, expression was confined to early differentiation stages, and, at onset of overt differentiation, the level of expression decreased dramatically. Several rapidly proliferating tissues showed very little, if any, N-myc expression. In the brain, post-mitotic but not yet differentiated cells expressed high levels of N-myc mRNA. Therefore, N-myc expression is not a simple marker for proliferation in the embryo. Rather, N-myc expression seems to be a feature of early differentiation stages of some cell lineages in kidney, brain, and hair follicles, regardless of the proliferative status of the cell. The results raise the possibility that N-myc may participate in the control of these early differentiation events.
Signal transduction by glycophorin A: role of extracellular and cytoplasmic domains in a modulatable process.
Authors: Chasis J A , Reid M E , Jensen R H , Mohandas N
Published: May 2004

Binding of ligands to the extracellular region of the erythrocyte transmembrane protein glycophorin A induces a decrease in membrane deformability. Since the property of membrane deformability is regulated by the skeletal proteins on the cytoplasmic side of the membrane, this suggests that ligand binding may initiate a transmembrane signal. To further study this process, we examined which domains of the extracellular region of glycophorin are involved in signal transduction and whether the cytoplasmic domain of the molecule is necessary for transmitting the signal. Using the ektacytometer, we compared the effect on deformability of four monoclonal antibodies that detect different epitopes on glycophorin A. We found that 9A3 (which recognized the amino terminus of glycophorin) caused a 5.8-fold increase in rigidity, R-10 and 10F7 (which recognized epitopes in the mid-region of the extracellular domain) caused a 10.8-fold increase in rigidity and B14 (which binds to glycophorin close to the membrane) caused a 18-fold increase in rigidity. Further, a direct relationship was observed between the degree of antibody-induced rigidity and the amount of glycophorin A that became associated with the skeletal proteins in a Triton shell assay. In Miltenberger V erythrocytes, which contain a hybrid sialoglycoprotein with no cytoplasmic domain, antibody binding did not induce an increase in rigidity. These results imply that glycophorin A is capable of a modulatable form of transmembrane signaling that is determined by the extracellular domain to which the ligand binds, and the cytoplasmic domain of glycophorin A is crucial for this process.
Functional alterations in beta'-actin from a KB cell mutant resistant to cytochalasin B.
Authors: Toyama S , Toyama S
Published: May 2004

We recently described the isolation of mutant KB cells (Cyt 1 cells) resistant to the cytotoxic effect of cytochalasin B (CB). This mutant carried an altered beta-actin; i.e., beta'-actin (Toyama, S., and S. Toyama. 1984. Cell. 37:609-614). In the present study, we have examined the functional properties of actin in Cyt 1 cells. Our results showed that increased resistance of Cyt 1 cells to CB was reflected in altered properties of beta'-actin itself. This was shown directly by two findings. First, the polymerization of beta'-actin was more resistant than that of beta- or gamma-actin to the multiple effects of CB. Second, beta'-actin bound less CB than beta- or gamma-actin. The functional alteration of beta'-actin in Cyt 1 cells was further supported by the observation that, although treatment of KB cells with CB increased the pool of unpolymerized actin, the same treatment did not affect the pool of unpolymerized actin in Cyt 1 cells, and that microfilaments of Cyt 1 cells were more resistant to the disrupting action of CB than those of KB cells. These results strongly suggest that the primary site of action of CB on cell motility processes is actin.
The association of prosomes with some of the intermediate filament networks of the animal cell.
Authors: Grossi de Sa M F , Martins de Sa C , Harper F , Olink-Coux M , Huesca M , Scherrer K
Published: May 2004

The small RNP complexes of defined morphology and biochemical composition termed prosomes, first isolated from the cytoplasm associated with repressed mRNA (Martins de Sa, C., M.-F. Grossi de Sa, O. Akhayat, F. Broders, and K. Scherrer. J. Mol. Biol. 1986. 187:47-493), were found also in the nucleus (Grossi de Sa, M.-F., C. Martins de Sa, F. Harper, O. Coux, O. Akhayat, P. Gounon, J. K. Pal, Y. Florentin, and K. Scherrer. 1988. J. Cell Sci. 89:151-165). Immunofluorescence, immunoelectron microscopy, and immunochemical studies using mAbs directed against some of the prosomal proteins of duck erythroblasts indicate that in the cytoplasm of HeLa and PtK cells, prosome antigens are associated with the intermediate filament network of the cytokeratin type.
Alcohol oxidase expressed under nonmethylotrophic conditions is imported, assembled, and enzymatically active in peroxisomes of Hansenula polymorpha.
Authors: Distel B , Van der Leÿ I , Veenhuis M , Tabak H F
Published: May 2004

We have introduced into Hansenula polymorpha an extra copy of its alcohol oxidase gene. This gene which is under the control of the Saccharomyces cerevisiae phosphoglycerate kinase promoter is integrated in a chromosome different from the one containing the endogenous gene. Cells with the extra alcohol oxidase gene, grown on glucose or ethanol as the sole carbon source, express enzymatically active alcohol oxidase. However, other enzymes characteristic for methylotrophic growth conditions are absent or present at low levels. Most of the alcohol oxidase occurs in the octameric state and immuno- and cytochemical evidence shows that it is located in a single enlarged peroxisome per cell. Such peroxisomes show crystalloid inclusions which are lacking in the peroxisomes present in glucose grown control cells. Our results suggest that import into peroxisomes of H. polymorpha, assembly and activation of alcohol oxidase is not conditionally dependent on adaptation to methylotrophic growth conditions and that proliferation of peroxisomes is a well-programmed process that is not triggered solely by overproduction of a peroxisomal protein.
Binding and transcytosis of glycoalbumin by the microvascular endothelium of the murine myocardium: evidence that glycoalbumin behaves as a bifunctional ligand.
Authors: Predescu D , Simionescu M , Simionescu N , Palade G E
Published: May 2004

The binding and transport of glycoalbumin (gA) by the endothelium of murine myocardial microvessels were studied by perfusing in situ 125I-gA or gA-gold complexes (gA-Au) and examining the specimens by radioassays and EM, respectively. After a 3-min perfusion, the uptake of radioiodinated gA is 2.2-fold higher than that of native albumin; it is partially (approximately 55%) competed by either albumin or D-glucose, and almost completely abolished by the concomitant administration of both competitors or by gA. D-mannose and D-galactose are not effective competitors. Unlike albumin-gold complexes that bind restrictively to plasmalemmal vesicles, gA-Au labels the plasma-lemma proper, plasmalemmal vesicles open on the lumen, and most coated pits. Competing albumin prevents gA-Au binding to the membrane of plasmalemmal vesicles, while glucose significantly reduces the ligand binding to plasmalemma proper. Competition with albumin and glucose gives additive effects. Transcytosis of gA-Au, already detected at 3 min, becomes substantial by 30 min. No tracer exit via intercellular junctions was detected. gA-Au progressively accumulates in multivesicular bodies. The results of the binding and competition experiments indicate that the gA behaves as a bifunctional ligand which is recognized by two distinct binding sites: one, located on the plasma membrane, binds as a lectin the glucose residues of gA; whereas the other, confined to plasmalemmal vesicles, recognizes presumably specific domains of the albumin molecule.
Structural and immunological characterization of the myosin-like 110-kD subunit of the intestinal microvillar 110K-calmodulin complex: evidence for discrete myosin head and calmodulin-binding domains.
Authors: Carboni J M , Conzelman K A , Adams R A , Kaiser D A , Pollard T D , Mooseker M S
Published: May 2004

The actin bundle within each microvillus of the intestinal brush border is tethered laterally to the membrane by spirally arranged bridges. These bridges are thought to be composed of a protein complex consisting of a 110-kD subunit and multiple molecules of bound calmodulin (CM). Recent studies indicate that this complex, termed 110K-CM, is myosin-like with respect to its actin binding and ATPase properties. In this study, possible structural similarity between the 110-kD subunit and myosin was examined using two sets of mAbs; one was generated against Acanthamoeba myosin II and the other against the 110-kD subunit of avian 110K-CM. The myosin II mAbs had been shown previously to be cross-reactive with skeletal muscle myosin, with the epitope(s) localized to the 50-kD tryptic fragment of the subfragment-1 (S1) domain. The 110K mAbs (CX 1-5) reacted with the 110-kD subunit as well as with the heavy chain of skeletal but not with that of smooth or brush border myosin. All five of these 110K mAbs reacted with the 25-kD, NH2-terminal tryptic fragment of chicken skeletal S1, which contains the ATP-binding site of myosin. Similar tryptic digestion of 110K-CM revealed that these five mAbs all reacted with a 36-kD fragment of 110K (as well as larger 90- and 54-kD fragments) which by photoaffinity labeling was shown to contain the ATP-binding site(s) of the 110K subunit. CM binding to these same tryptic digests of 110K-CM revealed that only the 90-kD fragment retained both ATP- and CM-binding domains. CM binding was observed to several tryptic fragments of 60, 40, 29, and 18 kD, none of which contain the myosin head epitopes. These results suggest structural similarity between the 110K and myosin S1, including those domains involved in ATP- and actin binding, and provide additional evidence that 110K-CM is a myosin. These studies also support the results of Coluccio and Bretscher (1988. J. Cell Biol. 106:367-373) that the calmodulin-binding site(s) and the myosin head region of the 110-kD subunit lie in discrete functional domains of the molecule.
Oligospecificity of the cellular adhesion receptor Mac-1 encompasses an inducible recognition specificity for fibrinogen.
Authors: Altieri D C , Bader R , Mannucci P M , Edgington T S
Published: May 2004

Mitogenesis, cellular aggregation, and motility follow upon the interaction of fibrinogen with certain defined cell surface receptors. In addition to circulating platelets and vascular endothelium, monocytes express what appears to be a receptor for fibrinogen. Evidence is presented here that the leukocyte adhesion receptor Mac-1 can be specifically induced to bind fibrinogen with characteristics immunochemically and functionally distinct from the established Arg-Gly-Asp-directed fibrinogen receptors. The competence of Mac-1 as a fibrinogen receptor is a general property of cells of monocyte and myeloid lineage acquired after maturational changes of some regions of the alpha subunit of Mac-1 during the process of cell differentiation. This ligand recognition specificity of Mac-1 is lacking for the resting cell. Rather, induction of fibrinogen binding capacity of Mac-1 is due to a cellular response to selected agonists characterized by inducing rapid transients of cytosolic Ca2+. Although different in activation pathways and recognition specificity, Mac-1 exhibits an oligospecific ligand versatility characteristic of other homologous Arg-Gly-Asp-directed adhesion receptors.
Monoclonal antibodies to a membrane glycoprotein induce the phosphorylation of histone H1 in sea urchin spermatozoa.
Authors: Vacquier V D , Moy G W , Trimmer J S , Ebina Y , Porter D C
Published: May 2004

Two groups of mAbs reacting with external domains of a major sea urchin sperm membrane glycoprotein of 210 kD were isolated. Previous studies have shown that group I mAbs inhibit the acrosome reaction induced by egg jelly and also cause large increases in intracellular Ca2+ [( Ca2+]i). Group II mAbs, at comparable levels of cell surface binding, neither inhibit the egg jelly-induced acrosome reaction nor cause increases in [Ca2+]i. In this paper, we investigate the ability of these mAbs to induce the cAMP-dependent phosphorylation of sperm histone H1. Group I mAbs induce H1 phosphorylation to the same level and on the same peptide, as occurs upon treatment of sperm with egg jelly. These mAbs also activate adenylate cyclase to the same extent as egg jelly. Group II mAbs do not induce H1 phosphorylation and are only poor activators of adenylate cyclase. Group I mAbs compete with each other, but not with group II mAbs, for binding to the cell surface. These data indicate that the activation of adenylate cyclase is an initial event in the pathway leading from the binding of mAbs to a specific domain of the 210-kD protein at the cell surface, to the discrete phosphorylation of histone H1 in highly condensed sperm chromatin. The domain on the 210-kD protein recognized by group I mAbs plays a critical role in signal transduction during the early events of fertilization.
A quantitative model of traffic between plasma membrane and secondary lysosomes: evaluation of inflow, lateral diffusion, and degradation.
Authors: Draye J P , Courtoy P J , Quintart J , Baudhuin P
Published: May 2004

We present here a mathematical model that accounts for the various proportions of plasma membrane constituents occurring in the lysosomal membrane of rat fibroblasts (Draye, J.-P., J. Quintart, P. J. Courtoy, and P. Baudhuin. 1987. Eur. J. Biochem. 170: 395-403; Draye, J.-P., P. J. Courtoy, J. Quintart, and P. Baudhuin. 1987. Eur. J. Biochem. 170:405-411). It is based on contents of plasma membrane markers in purified lysosomal preparations, evaluations of their half-life in lysosomes and measurements of areas of lysosomal and plasma membranes by morphometry. In rat fibroblasts, structures labeled by a 2-h uptake of horseradish peroxidase followed by a 16-h chase (i.e., lysosomes) occupy 3% of the cellular volume and their total membrane area corresponds to 30% of the pericellular membrane area. Based on the latter values, the model predicts the rate of inflow and outflow of plasma membrane constituents into lysosomal membrane, provided their rate of degradation is known. Of the bulk of polypeptides iodinated at the cell surface, only 4% reach the lysosomes every hour, where the major part (integral of 83%) is degraded with a half-life in lysosomes of integral to 0.8 h. For specific plasma membrane constituents, this model can further account for differences in the association to the lysosomal membrane by variations in the rate either of lysosomal degradation, of inflow along the pathway from the pericellular membrane to the lysosomes, or of lateral diffusion.
Secreted hepatitis B surface antigen polypeptides are derived from a transmembrane precursor.
Authors: Simon K , Lingappa V R , Ganem D
Published: May 2004

Hepatitis B surface antigen (HBsAg), the major coat protein of hepatitis B virus, is also independently secreted from infected cells as a lipoprotein particle. Secretion proceeds without signal sequence removal or cleavage of other segments of the polypeptide. We have examined the synthesis and transport of HBsAg in cultured cells expressing the cloned surface antigen gene. Our results show that HBsAg is initially synthesized as a integral membrane protein. This transmembrane form is slowly converted to a secreted lipoprotein complex in the lumen of the endoplasmic reticulum via a series of definable intermediates, after which it is secreted from the cell. This unusual export process shares many features with the assembly and budding reactions of conventional enveloped animal viruses. However, it differs importantly in its absence of a requirement for the participation of nucleocapsid or other viral proteins.
Glioblastoma infiltration into central nervous system tissue in vitro: involvement of a metalloprotease.
Authors: Paganetti P A , Caroni P , Schwab M E
Published: May 2004

Differentiated oligodendrocytes and central nervous system (CNS) myelin are nonpermissive substrates for neurite growth and for cell attachment and spreading. This property is due to the presence of membrane-bound inhibitory proteins of 35 and 250 kD and is specifically neutralized by monoclonal antibody IN-1 (Caroni, P., and M. E. Schwab. 1988. Neuron. 1:85-96). Using rat optic nerve explants, CNS frozen sections, cultured oligodendrocytes or CNS myelin, we show here that highly invasive CNS tumor line (C6 glioblastoma) was not inhibited by these myelin-associated inhibitory components. Lack of inhibition was due to a specific mechanism as the metalloenzyme blocker 1,10-phenanthroline and two synthetic dipeptides containing metalloprotease-blocking sequences (gly-phe, tyr-tyr) specifically impaired C6 cell spreading on CNS myelin. In the presence of these inhibitors, C6 cells were affected by the IN-1-sensitive inhibitors in the same manner as control cells, e.g., 3T3 fibroblasts or B16 melanomas. Specific blockers of the serine, cysteine, and aspartyl protease classes had no effect. C6 cell spreading on inhibitor-free substrates such as CNS gray matter, peripheral nervous system myelin, glass, or poly-D-lysine was not sensitive to 1,10-phenanthroline. The nonpermissive substrate properties of CNS myelin were strongly reduced by incubation with a plasma membrane fraction prepared from C6 cells. This reduction was sensitive to the same inhibitors of metalloproteases. In our in vitro model for CNS white matter invasion, cell infiltration of optic nerve explants, which occurred with C6 cells but not with 3T3 fibroblasts or B16 melanomas, was impaired by the presence of the metalloprotease blockers. These results suggest that C6 cell infiltrative behavior in CNS white matter in vitro occurs by means of a metalloproteolytic activity, which probably acts on the myelin-associated inhibitory substrates.
Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance.
Authors: Stevenson B R , Anderson J M , Goodenough D A , Mooseker M S
Published: May 2004

The relationship of tight junction permeability to junction structure and composition was examined using two strains of Madin-Darby canine kidney (MDCK) cells (I and II) which differ greater than 30-fold in transepithelial resistance. This parameter is largely determined by paracellular, and hence junctional, permeability under most conditions. When these two strains of cells were grown on permeable filter supports, they formed monolayers with equivalent linear amounts of junction/area of monolayer. Ultrastructural analysis of these monolayers by thin section EM revealed no differences in overall cellular morphology or in tight junction organization. Morphometric analysis of freeze-fractured preparations indicated that the tight junctions of these two cell strains were similar in both number and density of junctional fibrils. Prediction of transepithelial resistance for the two strains from this freeze-fracture data and a published structure-function formulation (Claude, P. 1978, J. Memb. Biol. 39:219-232) yielded values (I = 26.5 omega/cm2, II = 35.7 omega/cm2) that were significantly lower than those observed (I = 2,500-5,000 omega/cm2, II = 50-70 omega/cm2). Consistent with these structural studies, a comparison of the distribution and cellular content of ZO-1, a polypeptide localized exclusively to the tight junction, revealed no significant differences in either the localization of ZO-1 or the amount of ZO-1 per micron of junction (I = 1,415 +/- 101 molecules/micron, II = 1,514 +/- 215 molecules/micron).
Detection of a specific isoform of alpha-actinin with antisera directed against dystrophin.
Authors: Hoffman E P , Watkins S C , Slayter H S , Kunkel L M
Published: May 2004

We have characterized a protein immunologically related to dystrophin, the protein product of the Duchenne muscular dystrophy gene. We identify this related protein as a fast-twitch glycolytic isoform (mouse extensor digitorum longus-specific) of myofibrillar alpha-actinin. This specific isoform of alpha-actinin exhibits a more restricted pattern of expression in skeletal muscle than fast-twitch-specific isoforms of both myosin and Ca2+-ATPase. Our results provide evidence that dystrophin and myofibrillar alpha-actinin are related proteins, reinforcing the previous data concerning the sequence homologies noted between nonmuscle cytoskeletal alpha-actinin and dystrophin. In addition, we describe the first antisera directed against a specific myofibrillar skeletal muscle isoform of alpha-actinin.
Expression of cytotactin in the normal and regenerating neuromuscular system.
Authors: Daniloff J K , Crossin K L , Pinçon-Raymond M , Murawsky M , Rieger F , Edelman G M
Published: May 2004

Cytotactin is an extracellular glycoprotein found in a highly specialized distribution during embryonic development. In the brain, it is synthesized by glia, not neurons. It is involved in neuron-glia adhesion in vitro and affects neuronal migration in the developing cerebellum. In an attempt to extend these observations to the peripheral nervous system, we have examined the distribution and localization of cytotactin in different parts of the normal and regenerating neuromuscular system. In the normal neuromuscular system, cytotactin accumulated at critical sites of cell-cell interactions, specifically at the neuromuscular junction and the myotendinous junction, as well at the node of Ranvier (Rieger, F., J. K. Daniloff, M. Pinçon-Raymond, K. L. Crossin, M. Grumet, and G. M. Edelman. 1986. J. Cell Biol. 103:379-391). At the neuromuscular junction, cytotactin was located in terminal nonmyelinating Schwann cells. Cytotactin was also detected near the insertion points of the muscle fibers to tendinous structures in both the proximal and distal endomysial regions of the myotendinous junctions. This was in striking contrast to staining for the neural cell adhesion molecule, N-CAM, which was accumulated near the extreme ends of the muscle fiber. Peripheral nerve damage resulted in modulation of expression of cytotactin in both nerve and muscle, particularly among the interacting tissues during regeneration and reinnervation. In denervated muscle, cytotactin accumulated in interstitial spaces and near the previous synaptic sites. Cytotactin levels were elevated and remained high along the endoneurial tubes and in the perineurium as long as muscle remained denervated. Reinnervation led to a return to normal levels of cytotactin both in inner surfaces of the nerve fascicles and in the perineurium. In dorsal root ganglia, the processes surrounding ganglionic neurons became intensely stained by anticytotactin antibodies after the nerve was cut, and returned to normal by 30 d after injury. These data suggest that local signals between neurons, glia, and supporting cells may regulate cytotactin expression in the neuromuscular system in a fashion coordinate with other cell adhesion molecules. Moreover, innervation may regulate the relative amount and distribution of cytotactin both in muscle and in Schwann cells.
Thrombospondin is an osteoblast-derived component of mineralized extracellular matrix.
Authors: Robey P G , Young M F , Fisher L W , McClain T D
Published: May 2004

Thrombospondin, the most abundant protein of platelet alpha granules, is a biosynthetic product of a variety of connective tissue cells and a component of many extracellular matrices. In this study, thrombospondin distribution in bone was investigated using a monoclonal antibody specific for the human protein. Thrombospondin was localized in osteoid of undemineralized, frozen sections of fetal subperiosteal bone, and identified as a component of mineralized bone matrix of neonatal and/or young (growing) bone of many animal species by Western blot analysis. Adult human bone cells were demonstrated to contain mRNA for thrombospondin by hybridization of a cDNA thrombospondin probe to a 6.1 kb mRNA. Pulse-chase experiments indicated that the protein was synthesized and the majority was secreted from osteoblastic cells. Treatment of the cells with TGF-beta (0.01-10 ng/ml) slightly decreased total thrombospondin synthesis, but caused an increase in the retention on newly synthesized thrombospondin in the cell layer/matrix fraction. In cell attachment assays, thrombospondin mediated adhesion, but not spreading of adult human bone cells.
Localization of nicotinic acetylcholine receptor alpha-subunit transcripts during myogenesis and motor endplate development in the chick.
Authors: Fontaine B , Changeux J P
Published: May 2004

In 15-d-old chick latissimi dorsi muscles, the nicotinic acetylcholine receptor (AChR) alpha-subunit mRNA is densely accumulated at the level of subsynaptic nuclei of the motor endplate (Fontaine et al., 1988). In this paper, using in situ hybridization with genomic probes, we further show that the expression of the AChR alpha-subunit gene in the embryo, revealed by the accumulation of mature mRNAs, starts in myotomal cells and persists during the first stages of muscle development in a majority of muscle nuclei. Subsequently, the distribution of AChR alpha-subunit mRNAs becomes restricted to the newly formed motor endplates as neuromuscular junctions develop. To assess the transcriptional activity of individual nuclei in developing muscles, a strictly intronic fragment of the AChR alpha-subunit gene was used to probe in situ the level of unspliced transcripts. AChR alpha-subunit unspliced transcripts accumulate around a large number of sarcoplasmic nuclei at embryonic day 11, but can no longer be detected at their level after embryonic day 16 in the embryo. A similar decrease in the accumulation of AChR alpha-subunit transcripts is observed between day 4 and day 6 in primary cultures of muscle cells. On the other hand, in vivo denervation and in vitro blocking of muscle electrical activity by the sodium channel blocker tetrodotoxin results in an increase in the labeling of muscle nuclei. Yet, only 6% of the muscle nuclei appear labeled by the strictly intronic probes after denervation. The possible significance of such heterogeneity of muscle nuclei during motor endplate formation in AChR gene expression is discussed.
The N-myc proto-oncogene and IGF-II growth factor mRNAs are expressed by distinct cells in human fetal kidney and brain.
Authors: Hirvonen H , Sandberg M , Kalimo H , Hukkanen V , Vuorio E , Salmi T T , Alitalo K
Published: May 2004

We studied the expression of the N-myc proto-oncogene and the insulin-like growth factor-II (IGF-II) gene in human fetuses of 16-19 gestational wk. Both genes have specific roles in the growth and differentiation of embryonic tissues, such as the kidney and neural tissue. Since continued expression of N-myc and IGF-II mRNAs is also a characteristic feature of Wilms' tumor, a childhood neoplasm of probable fetal kidney origin, we were particularly interested in the possibility that their expression might be linked or coordinately regulated in the developing kidney. Expression of N-myc mRNA was observed in the brain and in the kidney by Northern hybridization analysis. In in situ hybridization of the kidney, N-myc autoradiographic grains were primarily located over epithelially differentiating mesenchyme while most of the mesenchymal stromal cells showed only a background signal with the N-myc probe. N-myc mRNA was detectable throughout the developing brain with a slight accentuation in the intermediate zone cells in between the subependymal and cortical layers. Thus, even postmitotic neuroepithelial cells of the fetal cerebrum expressed N-myc mRNA. In Northern hybridization, IGF-II mRNA signal was abundant in the kidney but much weaker, though definite, in the brain. The regional distribution of IGF-II mRNA in the kidney was largely complementary to that of N-myc. IGF-II autoradiographic grains were located predominantly over the stromal and blastemal cells with a relative lack of hybridization over the epithelial structures. In the brain, IGF-II mRNA was about two- to threefold more abundant in the subependymal and intermediate layers than in the cortical plate and ependymal zone, respectively. The fetal expression patterns of the N-myc and IGF-II mRNAs are reflected by the types of tumors known to express the corresponding genes during postnatal life such as Wilms' tumor. However, the apparent coexpression of the IGF-II and N-myc genes in immature kidneys occurs largely in distinct cell types.
Tenascin mediates cell attachment through an RGD-dependent receptor.
Authors: Bourdon M A , Ruoslahti E
Published: May 2004

Tenascin is an extracellular matrix glycoprotein expressed in association with mesenchymal-epithelial interactions during development and in the neovasculature and stroma of undifferentiated tumors. This selective expression of tenascin indicates a specific role in cell matrix interactions. We now show that tenascin can support the adhesion of a variety of cell types, including various human tumor cells, normal fibroblasts, and endothelial cells, all of which can attach to a substrate coated with tenascin. Detailed studies on the mechanism of the tenascin-promoted cell attachment were carried out with the human glioma cell line U251MG. The attachment of these cells and others to tenascin were inhibited specifically by peptides containing the RGD cell attachment signal. Affinity chromatography procedures similar to those that have been used to isolate other adhesion receptors yielded a heterodimeric cell surface protein which bound to a tenascin affinity matrix in an RGD-dependent fashion. One of the subunits of this putative tenascin receptor comigrates with the beta subunit of the fibronectin receptor in SDS-PAGE and cross reacts with antibodies prepared against the fibronectin receptor in immunoblotting. These results identify the tenascin receptor as a member of the fibronectin receptor family within the integrin superfamily of receptors. The cell attachment response on tenascin is distinctly different from that seen on fibronectin, suggesting that cell adhesion and motility may be modulated at those sites where tenascin is expressed in the extracellular matrix.
Formation of coated vesicles from coated pits in broken A431 cells.
Authors: Smythe E , Pypaert M , Lucocq J , Warren G
Published: May 2004

Biochemical and morphological techniques were used to demonstrate the early steps in the endocytosis of transferrin in broken A431 cells. After binding 125I-transferrin, the cells were broken by scraping and then warmed. 125I-transferrin became inaccessible to exogenous anti-transferrin antibody providing a measure of the internalization process. Parallel morphological experiments using transferrin coupled to horseradish peroxidase confirmed internalization in broken cells. The process was characterized and compared with endocytosis in intact cells and showed many similar features. The system was used to show that both the appearance of new coated pits and the scission of coated pits to form coated vesicles were dependent on the addition of cytosol and ATP whereas invagination of pits was dependent on neither.
Type I collagen gel induces Madin-Darby canine kidney cells to become fusiform in shape and lose apical-basal polarity.
Authors: Zuk A , Matlin K S , Hay E D
Published: May 2004

In the embryo, epithelia give rise to mesenchyme at specific times and places. Recently, it has been reported (Greenburg, G., and E. D. Hay. 1986. Dev. Biol. 115:363-379; Greenberg, G., and E. D. Hay. 1988. Development (Camb.). 102:605-622) that definitive epithelia can give rise to fibroblast-like cells when suspended within type I collagen gels. We wanted to know whether Madin-Darby canine kidney (MDCK) cells, an epithelial line, can form mesenchyme under similar conditions. Small explants of MDCK cells on basement membrane were suspended within or placed on top of extracellular matrix gels. MDCK cells on basement membrane gel are tall, columnar in shape, and ultrastructurally resemble epithelia transporting fluid and ions. MDCK explants cultured on type I collagen gel give rise to isolated fusiform-shaped cells that migrate over the gel surface. The fusiform cells extend pseudopodia and filopodia, lose cell membrane specializations, and develop an actin cortex around the entire cell. Unlike true mesenchymal cells, which express vimentin and type I collagen, fusiform cells produce both keratin and vimentin, continue to express laminin, and do not turn on type I collagen. Fusiform cells are not apically-basally polarized, but show mesenchymal cell polarity. Influenza hemagglutinin and virus budding localize to the front end or entire cell surface. Na,K-ATPase occurs intracellularly and also symmetrically distributes on the cell surface. Fodrin becomes diffusely distributed along the plasma membrane, ZO-1 cannot be detected, and desmoplakins distribute randomly in the cytoplasm. The loss of epithelial polarity and acquisition of mesenchymal cell polarity and shape by fusiform MDCK cells on type I collagen gel was previously unsuspected. The phenomenon may offer new opportunities for studying cytoplasmic and nuclear mechanisms regulating cell shape and polarity.
A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis.
Authors: André E , Brink M , Gerisch G , Isenberg G , Noegel A , Schleicher M , Segall J E , Wallraff E
Published: May 2004

A severin deficient mutant of Dictyostelium discoideum has been isolated by the use of colony immunoblotting after chemical mutagenesis. In homogenates of wild-type cells, severin is easily detected as a very active F-actin fragmenting protein. Tests for severin in the mutant, HG1132, included viscometry for the assay of F-actin fragmentation in fractions from DEAE-cellulose columns, labeling of blots with monoclonal and polyclonal antibodies, and immunofluorescent-labeling of cryosections. Severin could not be detected in the mutant using these methods. The mutation in HG1132 is recessive and has been mapped to linkage group VII. The mutant failed to produce the normal severin mRNA, but small amounts of a transcript that was approximately 100 bases larger than the wild-type mRNA were detected in the mutant throughout all stages of development. On the DNA level a new Mbo II restriction site was found in the mutant within the coding region of the severin gene. The severin deficient mutant cells grew at an approximately normal rate, aggregated and formed fruiting bodies with viable spores. By the use of an image processing system, speed of cell movement, turning rates, and precision of chemotactic orientation in a stable gradient of cyclic AMP were quantitated, and no significant differences between wild-type and mutant cells were found. Thus, under the culture conditions used, severin proved to be neither essential for growth of D. discoideum nor for any cell function that is important for aggregation or later development.
Concurrent collapse of keratin filaments, aggregation of organelles, and inhibition of protein synthesis during the heat shock response in mammary epithelial cells.
Authors: Shyy T T , Asch B B , Asch H L
Published: May 2004

The sequence of heat shock-induced perturbations in protein synthesis and cytoskeletal organization was investigated in primary cultures of mouse mammary epithelial cells (MMEC). Exposure of the cells to 45 degrees C for 15 min caused a marked inhibition of protein synthesis through 2 h after heart. Resumption of protein synthesis began by 4 h, was complete by 8 h, and was accompanied by induction of four major heat shock proteins (HSPs) of 68, 70, 89, and 110 kD. Fluorescent cytochemistry studies indicated that heat shock elicited a reversible change in the organization of keratin filaments (KFs) and actin filaments but had a negligible effect on microtubules. Changes in the organization of KFs progressed gradually with maximal retraction and collapse into the perinuclear zone occurring at 1-2 h after heat followed by restoration to the fully extended state at 8 h. In contrast, actin filaments disappeared immediately after heat treatment and then rapidly returned within 30-60 min to their original appearance. The translocation of many organelles first into and then away from the juxtanuclear area along with the disruption and reformation of polyribosomes were concurrent with the sequential changes in distribution of KFs. The recovery of the arrangement of KFs coincided with but was independent of the resumption of protein synthesis and induction of HSPs. Thermotolerance could be induced in protein synthesis and KFs, but not in actin filaments, by a conditioning heat treatment. Neither protein synthesis nor induction of HSPs was necessary for the acquisition of thermotolerance in the KFs. The results are compatible with the possibility that protein synthesis may depend on the integrity of the KF network in MMEC. Heat shock thus can efficiently disarrange the KF system in a large population of epithelial cells, thereby facilitating studies on the functions of this cytoskeletal component.
NH2-terminal substitutions of basic amino acids induce translocation across the microsomal membrane and glycosylation of rabbit cytochrome P450IIC2.
Authors: Szczesna-Skorupa E , Kemper B
Published: May 2004

Insertion of rabbit cytochrome P450IIC2 and its modified form, [2-lys,3-arg]P450IIC2, into microsomal membranes was studied in an in vitro transcription/translation/translocation system. Cytochrome P450IIC2, synthesized in the presence of chicken oviduct microsomal membranes, was resistant to extraction by alkaline solutions, but was sensitive to proteolytic digestion. In contrast, when [2-lys,3-arg]-P450IIC2 was synthesized in the presence of membranes, two new species migrating more slowly during gel electrophoresis were observed. After treatment with endoglycosidase H, the more slowly migrating species comigrated with [2-lys,3-arg]P450IIC2 synthesized in the absence of membranes, indicating that the proteins had been glycosylated. Both the glycosylated and nonglycosylated forms of [2-lys,3-arg]P450IIC2 were resistant to proteolytic digestion and to extraction from the membranes by alkaline solutions. Similar results were obtained for a truncated species, [2-lys,3-arg]P450IIC2(1-55), except that only a single glycosylated species was observed, consistent with the single remaining glycosylation site. In contrast to the proteolytic processing observed previously in a hybrid [2-lys,3-arg]P450IIC2/parathyroid hormone protein, little or no cleavage of the NH2-terminal peptide of [2-lys,3-arg]P450IIC2 was observed in the presence of membranes. Since cleavage in the hybrid protein occurred after glycine 25, which is derived from [2-lys,3-arg]P450IIC2, cytochrome P450 sequences COOH terminal to the cleavage site must decrease cleavage efficiency. These results demonstrate that cytochrome P450, which is normally localized on the cytoplasmic side of the membrane, can be entirely translocated to the luminal side when two basic amino acids precede the hydrophobic core of its NH2-terminal insertion/stop-transfer signal. None of the several internal hydrophobic regions of cytochrome P450, previously proposed as membrane spanning, function as a stop-transfer signal.
Reconstitution of the Golgi apparatus after microinjection of rat liver Golgi fragments into Xenopus oocytes.
Authors: Paiement J , Jolicoeur M , Fazel A , Bergeron J J
Published: May 2004

We have studied the reconstitution of the Golgi apparatus in vivo using an heterologous membrane transplant system. Endogenous glycopeptides of rat hepatic Golgi fragments were radiolabeled in vitro with [3H]sialic acid using detergent-free conditions. The Golgi fragments consisting of dispersed vesicles and tubules with intraluminal lipoprotein-like particles were then microinjected into Xenopus oocytes and their fate studied by light (LM) and electron microscope (EM) radioautography. 3 h after microinjection, radiolabel was observed by LM radioautography over yolk platelet-free cytoplasmic regions near the injection site. EM radioautography revealed label over Golgi stacked saccules containing the hepatic marker of intraluminal lipoprotein-like particles. At 14 h after injection, LM radioautographs revealed label in the superficial cortex of the oocytes between the yolk platelets and at the oocyte surface. EM radioautography identified the labeled structures as the stacked saccules of the Golgi apparatus, the oocyte cortical granules, and the plasmalemma, indicating that a proportion of microinjected material was transferred to the surface via the secretion pathway of the oocyte. The efficiency of transport was low, however, as biochemical studies failed to show extensive secretion of radiolabel into the extracellular medium by 14 h with approximately half the microinjected radiolabeled constituents degraded. Vinblastine (50 microM) administered to oocytes led to the formation of tubulin paracrystals. Although microinjected Golgi fragments were able to effect the formation of stacked saccules in vinblastine-treated oocytes, negligible transfer of heterologous material to the oocyte surface could be detected by radioautography. The data demonstrate that dispersed fragments of the rat liver Golgi complex (i.e., unstacked vesicles and tubules) reconstitute into stacked saccules when microinjected into Xenopus cytoplasm. After the formation of stacked saccules, reconstituted Golgi fragments transport constituents into a portion of the exocytic pathway of the host cell by a microtubule-regulated process.
Structure, biosynthesis, and localization of dipeptidyl aminopeptidase B, an integral membrane glycoprotein of the yeast vacuole.
Authors: Roberts C J , Pohlig G , Rothman J H , Stevens T H
Published: May 2004

We have characterized the structure, biogenesis, and localization of dipeptidyl aminopeptidase B (DPAP B), a membrane protein of the yeast vacuole. An antibody specific for DPAP B recognizes a 120-kD glycoprotein in yeast that behaves like an integral membrane protein in that it is not removed from membranes by high pH Na2CO3 treatment. Inspection of the deduced amino acid sequence of DPAP B reveals a hydrophobic domain near the NH2 terminus that could potentially span a lipid bilayer. The in vitro enzymatic activity and apparent molecular weight of DPAP B are unaffected by the allelic state of PEP4, a gene essential for the proteolytic activation of a number of soluble vacuolar hydrolases. DPAP B is synthesized as a glycosylated precursor that is converted to the mature 120-kD species by carbohydrate addition. The precursor form of DPAP B accumulates in sec mutants (Novick, P., C. Field, and R. Schekman. 1980. Cell. 21:205-215) that are blocked at the ER (sec18) or Golgi apparatus (sec7), but not at secretory vesicles (sec1). Immunolocalization of DPAP B in wild-type or sec1 mutant cells shows that the protein resides in the vacuolar membrane. However, it is present in non-vacuolar compartments in sec18 and sec7 cells, confirming that the delivery of DPAP B is blocked in these mutants. Interestingly, DPAP B appears to stain the nuclear envelope in a sec18 mutant, which is consistent with the accumulation of DPAP B in the ER membrane at the restrictive temperature. These results suggest that soluble and membrane-bound vacuolar proteins use the same stages of the secretory pathway for their transport.