bluuebunny/crossref_metadata_2025 huggingface dataset:
K-252a, a potent protein kinase inhibitor, blocks nerve growth factor-induced neurite outgrowth and changes in the phosphorylation of proteins in PC12h cells.
Authors: Hashimoto S
Published: May 2004

Nerve growth factor (NGF) promotes neuronal differentiation of PC12 pheochromocytoma cells. One of the most prominent and distinguishing features of neuronal differentiation is neurite outgrowth. The mechanism by which NGF causes the cells to elaborate neurites is unknown. This study shows that K-252a, a potent protein kinase inhibitor, blocks NGF-induced neurite outgrowth and the changes in protein phosphorylation elicited by NGF. In the experiment with intact cells phosphorylated with 32P-orthophosphoric acid, an exposure of PC12h cells to NGF (50 ng/ml) caused an increase in the phosphorylation of tyrosine hydroxylase and a 35,000-D protein and a decrease in a 36,500-D protein. Pretreatment of PC12h cells with K-252a (100 nM) inhibited the effects of NGF on the phosphorylation of these three proteins. In the phosphorylation of cell-free extracts with [gamma-32P] ATP, treatment of PC12h cells with NGF (50 ng/ml) caused a decrease in the phosphorylation of Nsp100. Pretreatment of the cells with K-252a (30 nM) almost completely blocked the NGF effect on the phosphorylation of Nsp100 elicited by subsequent treatment of the cells with NGF. Treatment of PC12h cells with NGF promoted outgrowth of neurites. The addition of K-252a (100 nM) into the culture almost completely blocked the generation of neurites elicited by NGF. Earlier studies demonstrated that NGF-induced neurite outgrowth in PC12 cells involves at least two components: the first of these is transcription-dependent and the second is transcription-independent. To determine the component on which K-252a acts, experiments were carried out on NGF-induced priming or regeneration of neurites. When K-252a was present in the priming step, NGF induced only actinomycin D-sensitive neurites, showing that K-252a interferes with the transcription-dependent actions of NGF. When already primed cells were treated with NGF, actinomycin D-resistant neurites were formed and these were blocked by K-252a, showing that the inhibitor interferes with the transcription-independent actions of NGF as well. Although the exact mechanism of inhibition of NGF-promoted neurite formation by K-252a is unknown, the most probable explanation is that both transcription-dependent and -independent components are involved in at least one step of the activation of some specific protein kinase(s) that can be suppressed by K-252a.
RGD-containing peptides inhibit the synthesis of myelin-like membrane by cultured oligodendrocytes.
Authors: Cardwell M C , Rome L H
Published: May 2004

A synthetic peptide derived from the fibronectin cell-binding domain, GRGDSP, inhibits the adhesion of rat oligodendrocytes to a number of substrates. However, while GRGDSP inhibited the adhesion of cells in a short term adhesion assay, the presence of the peptide did not prevent cells from adhering and thriving in longer term culture. The morphological characteristics of individual cells cultured with 0.1 mg/ml GRGDSP were similar to untreated cultures; small rounded cell bodies radiating numerous fine processes. Peptide-treated cultures were inhibited in their ability to produce myelin specific components. The characteristic developmental peak in sulfolipid synthesis which occurs both in vivo and in vitro was completely inhibited when cells were cultured with GRGDSP. In addition, the synthesis of myelin basic protein was inhibited. Ultrastructurally, cells treated with GRGDSP showed a greatly reduced number of multilamellar myelin-like membrane figures than cells grown without peptide or those grown with GRADSP. Cultured oligodendrocytes did not become sensitive to inhibition of sulfolipid synthesis by GRGDSP until a period immediately preceding the peak in sulfolipid biosynthesis. The effects of pretreatment with peptide for 5 d before this time were completely reversible. Pretreatment which extended into the time of peak myelin synthesis resulted in permanent impairment in the cell's ability to synthesize sulfolipid. The oligodendrocyte's ability to synthesize a myelin-like membrane in culture is, in part, inherent since it occurs in the absence of neurons. The present results indicate that myelin membrane production is also subject to external control since it appears that occupancy of an RGD-dependent cell surface receptor during a critical period of in vitro development is required for the oligodendrocyte to produce myelin-like membrane.
Changes in cyclin/proliferating cell nuclear antigen distribution during DNA repair synthesis.
Authors: Toschi L , Bravo R
Published: May 2004

UV irradiation of quiescent human fibroblasts immediately triggers the appearance of the nuclear protein cyclin/proliferating cell nuclear antigen (PCNA) as detected by indirect immunofluorescent staining after methanol fixation. This was found to be independent of new synthesis of cyclin/PCNA by two-dimensional gel analysis and cycloheximide treatment. The intensity of the immunofluorescent staining of cyclin/PCNA observed in UV-irradiated cells corresponded with the UV dose used and with the DNA repair synthesis detected by autoradiography. The nuclear staining remains as long as DNA repair activity is detected in the cells. By extracting the UV-irradiated quiescent cells with Triton X-100 and fixing with formaldehyde, it was possible to demonstrate by indirect immunofluorescence rapid changes in the cyclin/PCNA population after irradiation, a small proportion (5-10%) of which is tightly associated to the nucleus as determined by high salt extraction. By incubating at low temperature and depleting the ATP pools of the cells before UV irradiation, we have demonstrated that the changes in cyclin/PCNA distribution observed involve at least two different nuclear associations.
Topogenic analysis of the human immunodeficiency virus type 1 envelope glycoprotein, gp160, in microsomal membranes.
Authors: Haffar O K , Dowbenko D J , Berman P W
Published: May 2004

The orientation in cellular membranes of the 856 amino acid envelope glycoprotein precursor, gp160, of human immunodeficiency virus type 1 was investigated in vitro. Variants of the env gene were transcribed using the bacteriophage SP6 promoter, translated using a rabbit reticulocyte lysate, and translocated into canine pancreatic microsomal membranes. Immunoprecipitation studies of gp160 variants using antibodies specific for various gp160-derived polypeptides provided evidence that the external (cell surface) domain of gp160 begins at the mature amino terminus of the protein and continues through amino acid 665. A stop-transfer sequence (transmembrane domain) was identified in a hydrophobic region COOH-terminal to amino acid 665 and NH2-terminal to amino acid 732. Protease protection experiments demonstrated that gp160 possesses a single cytoplasmic domain COOH-terminal to residue 707. Membrane extraction studies using carbonate buffer provided evidence that the 29 amino acid hydrophobic domain (residues 512-541) of gp160 was unable to serve as a stop-transfer sequence. Finally, we propose that the cytoplasmic tail of gp160 forms a secondary association with the microsomal membranes.
Primary sequence domains required for the retention of rotavirus VP7 in the endoplasmic reticulum.
Authors: Poruchynsky M S , Atkinson P H
Published: May 2004

Rotavirus VP7 is a membrane-associated protein of the endoplasmic reticulum (ER). It is the product of rotavirus gene 9 which potentially encodes a protein of 326 amino acids that contains two amino terminal hydrophobic domains, h1 and h2, each preceded by an initiation codon. Comparison of the size of products derived from altered genes containing coding sequences for both h1 and h2 with those lacking the h1 sequence ('dhl' mutants), indicates that initiation takes place at M30 immediately preceding h2 (residues F32 to L48) and that h2 is cleaved, confirming the studies of others (Stirzaker, S.C., P.L. Whitfeld, D.L. Christie, A.R. Bellamy, and G.W. Both. 1987. J. Cell Biol. 105:2897-2903). Our previous work had shown that deletions in the carboxy end of h2, extending to amino acid 61 in the open reading frame, resulted in secretion of VP7. The region from amino acid number 51-61, present in wild-type VP7 but missing in the secreted mutant delta 47-61, was thus implicated to have a role in ER retention. To test this, a series of chimeric genes were constructed by fusing the first 63 codons of wild-type VP7, delta 1-14 or delta 51-61/dhl, to the mouse salivary alpha-amylase gene, a secretory protein, such that the fusion junction was located at the exact mature terminus of amylase. The chimeric proteins VP7(63)/amylase, delta 1-14(63)/amylase and delta 51-61(63)/dhl/amylase were secreted when expressed in cells and the h2 domain was cleaved when mRNA was translated in vitro. These results imply that the sequence 51-61 is necessary but not sufficient for ER retention. When a second series of VP7/amylase chimera were constructed extending the VP7 contribution to amino acid 111, the product expressed by delta 1-14(111)/amylase was not secreted whereas that of delta 47-61(111)/amylase was. Significantly, the intracellular delta 1-14(111)/amylase product exhibited an amylase enzymatic specific activity that was similar to that of the wild-type amylase product. We conclude that two regions of VP7 mediate its retention in the ER, the first lies within the sequence 51-61 and the second within the sequence 62-111, which contains the glycosylation site for VP7. Both regions are necessary for retention, though neither is sufficient alone.
Exocytosis of vacuolar apical compartment (VAC): a cell-cell contact controlled mechanism for the establishment of the apical plasma membrane domain in epithelial cells.
Authors: Vega-Salas D E , Salas P J , Rodriguez-Boulan E
Published: May 2004

The vacuolar apical compartment (VAC) is an organelle found in Madin-Darby canine kidney (MDCK) cells with incomplete intercellular contacts by incubation in 5 microM Ca++ and in cells without contacts (single cells in subconfluent culture); characteristically, it displays apical biochemical markers and microvilli and excludes basolateral markers (Vega-Salas, D. E., P. J. I. Salas, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:1249-1259). The apical surface of cells kept under these culture conditions is immature, with reduced numbers of microvilli and decreased levels of an apical biochemical marker (184 kD), which is, however, still highly polarized (Vega-Salas, D. E., P. J. I. Salas, D. Gundersen, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:905-916). We describe here the morphological stages of VAC exocytosis which ultimately lead to the establishment of a differentiated apical domain. Addition of 1.8 mM Ca++ to monolayers developed in 5 microM Ca++ causes the rapid (20-40 min) fusion of VACs with the plasma membrane and their accessibility to external antibodies, as demonstrated by immunofluorescence, immunoperoxidase EM, and RIA with antibodies against the 184-kD apical plasma membrane marker. Exocytosis occurs towards areas of cell-cell contact in the developing lateral surface where they form intercellular pockets; fusion images are always observed immediately adjacent to the incomplete junctional bands detected by the ZO-1 antibody (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). Blocks of newly incorporated VAC microvilli and 184-kD protein progressively move from intercellular ("primitive" lateral) spaces towards the microvilli-poor free cell surface. The definitive lateral domain is sealed behind these blocks by the growing tight junctional fence. These results demonstrate a fundamental role of cell-cell contact-mediated VAC exocytosis in the establishment of epithelial surface polarity. Because isolated stages (intercellular pockets) of the stereotyped sequence of events triggered by the establishment of intercellular contacts in MDCK cells have been reported during normal differentiation of intestine epithelium (Colony, P. C., and M. R. Neutra. 1983. Dev. Biol. 97:349-363), we speculate that the mechanism we describe here plays an important role in the establishment of epithelial cell polarity in vivo.
Arrangement of myosin heads on Limulus thick filaments.
Authors: Levine R J , Chantler P D , Kensler R W
Published: May 2004

The two myosin heads with a single surface subunit on thick filaments from chelicerate arthropod muscle may originate from the same, or from axially sequential molecules, as suggested by three-dimensional reconstructions. The resolution attained in the reconstructions, however, does not permit one to distinguish unequivocally between these two possible arrangements. We examined the effect of 0.6 M KCl on relaxed thick filaments separated from Limulus muscle and filaments in which nearest myosin heads were cross-linked by the bifunctional agent, 3,3'-dithio-bis[3'(2')-O-[6-propionylamino)hexanoyl]adenosine 5'-triphosphate (bis22ATP), in the presence of vanadate (Vi). In high salt, surface myosin dissolved from both native, relaxed filaments and those exposed to 1-2 mM dithiothreitol after cross-linking, but was retained on filaments with cross-linked heads. Since bis22ATP must form intermolecular bonds between myosin heads within each subunit to prevent myosin solubilization in high salt, we conclude that each of these heads originates from a different myosin molecule, as was previously predicted by the reconstructions.
Sertoli cell processes have axoplasmic features: an ordered microtubule distribution and an abundant high molecular weight microtubule-associated protein (cytoplasmic dynein).
Authors: Neely M D , Boekelheide K
Published: May 2004

Microtubules in the cytoplasm of rat Sertoli cell stage VI-VIII testicular seminiferous epithelium were studied morphometrically by electron microscopy. The Sertoli cell microtubules demonstrated axonal features, being largely parallel in orientation and predominantly spaced one to two microtubule diameters apart, suggesting the presence of microtubule-bound spacer molecules. Testis microtubule-associated proteins (MAPs) were isolated by a taxol, salt elution procedure. Testis MAPs promoted microtubule assembly, but to a lesser degree than brain MAPs. High molecular weight MAPs, similar in electrophoretic mobilities to brain MAP-1 and MAP-2, were prominent components of total testis MAPs, though no shared immunoreactivity was detected between testis and brain high molecular weight MAPs using both polyclonal and monoclonal antibodies. Unlike brain high molecular weight MAPs, testis high molecular weight MAPs were not heat stable. Testis MAP composition, studied on postnatal days 5, 10, 15, and 24 and in the adult, changed dramatically during ontogeny. However, the expression of the major testis high molecular weight MAP, called HMW-2, was constitutive and independent of the development of mature germ cells. The Sertoli cell origin of HMW-2 was confirmed by identifying this protein as the major MAP found in an enriched Sertoli cell preparation and in two rat models of testicular injury characterized by germ cell depletion. HMW-2 was selectively released from testis microtubules by ATP and co-purified by sucrose density gradient centrifugation with MAP-1C, a neuronal cytoplasmic dynein. The inhibition of the microtubule-activated ATPase activity of HMW-2 by vanadate and erythro-(2-hydroxy-3-nonyl)adenine and its proteolytic breakdown by vanadate-dependent UV photocleavage confirmed the dynein-like nature of HMW-2. As demonstrated by this study, the neuronal and Sertoli cell cytoskeletons share morphological, structural and functional properties.
Isolated beta-heavy chain subunit of dynein translocates microtubules in vitro.
Authors: Sale W S , Fox L A
Published: May 2004

Our goal was to assess the microtubule translocating ability of individual ATPase subunits of outer arm dynein. Solubilized outer arm dynein from sea urchin sperm (Stronglocentrotus purpuratus) was dissociated into subunits by low ionic strength buffer and fractionated by zonal centrifugation. Fractions were assessed by an in vitro functional assay wherein microtubules move across a glass surface to which isolated dynein fractions had been absorbed. Microtubule gliding activity was coincident with the 12-S beta-heavy chain-intermediate chain 1 ATPase fractions (beta/IC1). Neither the alpha-heavy chain nor the intermediate chains 2 and 3 fractions coincided with microtubule gliding activity. The beta/IC1 ATPase induced very rapid gliding velocities (9.7 +/- 0.88 micron/s, range 7-11.5 micron/s) in 1 mM ATP-containing motility buffers. In direct comparison, isolated intact 21-S outer arm dynein, from which the beta/IC1 fraction was derived, induced slower microtubule gliding rates (21-S dynein, 5.6 +/- 0.7 micron/s; beta/IC1, 8.7 +/- 1.2 micron/s). These results demonstrate that a single subdomain in dynein, the beta/IC1 ATPase, is sufficient for microtubule sliding activity.
Immunogold localization of the regulatory subunit of a type II cAMP-dependent protein kinase tightly associated with mammalian sperm flagella.
Authors: Lieberman S J , Wasco W , MacLeod J , Satir P , Orr G A
Published: May 2004

We have shown previously that the regulatory subunit (RII) of a type II cAMP-dependent protein kinase is an integral component of the mammalian sperm flagellum (Horowitz, J.A., H. Toeg, and G.A. Orr. 1984. J. Biol. Chem. 259:832-838; Horowitz, J.A., W. Wasco, M. Leiser, and G.A. Orr. 1988. J. Biol. Chem. 263:2098-2104). The subcellular localization of this flagellum-associated RII in bovine caudal epididymal sperm was analyzed at electron microscope resolution with gold-conjugated secondary antibody labeling techniques using anti-RII monoclonal antibodies. By immunoblot analysis, the flagellum-associated RII was shown to interact with mAb 622 which cross reacts with both neural and nonneural isoforms of RII. In contrast, a neural specific monoclonal antibody (mAb 526) failed to interact with flagellar RII. In the midpiece of the demembranated sperm tail, gold label after mAb 622 incubation was primarily associated with the outer mitochondrial membrane. Although almost all specific labeling in the midpiece can be assigned to the mitochondria, in the principal piece, there is some labeling of the fibrous sheath. Labeling of the outer dense fibers and the axoneme was sparse. Specific labeling was virtually absent in the sperm head. Sections of sperm tails incubated in the absence of primary antisera or with mAb 526 showed little labeling. A beta-tubulin monoclonal antibody localized only to the 9 + 2 axoneme. These results raise the possibility that a type II cAMP-dependent protein kinase located at the outer mitochondrial membrane plays a role in the direct cAMP stimulation of mitochondrial respiration during sperm activation.
Topological distribution of two connexin32 antigenic sites in intact and split rodent hepatocyte gap junctions.
Authors: Goodenough D A , Paul D L , Jesaitis L
Published: May 2004

The membrane topology of connexin32, a principal polypeptide of gap junctions in diverse cell types, has been studied in rat and mouse hepatocyte gap junctions using site-specific antisera raised against synthetic oligopeptides corresponding to amino acid sequences deduced from cDNA clones. Based on published hydropathicity maps and identified protease-sensitive cleavage sites, oligopeptides were synthesized corresponding to two hydrophilic domains of connexin32, one predicted to face the cytoplasm, the other predicted to be directed extracellularly. Antisera were raised to keyhole limpet hemocyanin conjugates of the oligopeptides and used to map the distribution of their antigens using indirect immunocytochemistry on isolated gap junctions. The results directly demonstrated the cytoplasmic orientation of an antigen contained within amino acids 98-124 of the connexin32 sequence. The extracellular space in intact, isolated gap junctions is too small to permit binding of antibody molecules, necessitating the experimental separation of the junctional membranes to expose their extracellular surfaces using a urea/alkali procedure. While an antigen contained within amino acids 164-189 was visualized on the extracellular surfaces of some of the separated junctional membranes, variability in the observations and in the splitting procedure left ambiguities concerning the biological relevance of the observations after the denaturing conditions necessary to separate the junctional membranes. Using a different approach, however, the antigen could be exposed in intact liver using a hypertonic disaccharide junction-splitting procedure. The period of time of antigen exposure at the cell surface appears to peak at 30 s and disappear by 2-4 min. Taken together, these data demonstrate the extracellular orientation of an antigen contained within amino acids 164-189, which may be involved in cell-cell interaction within the gap junction.
Motor neurons contain agrin-like molecules.
Authors: Magill-Solc C , McMahan U J
Published: May 2004

Molecules antigenically similar to agrin, a protein extracted from the electric organ of Torpedo californica, are highly concentrated in the synaptic basal lamina of neuromuscular junctions in vertebrate skeletal muscle. On the basis of several lines of evidence it has been proposed that agrin-like molecules mediate the nerve-induced formation of acetylcholine receptor (AChR) and acetylcholinesterase (AChE) aggregates on the surface of muscle fibers at developing and regenerating neuromuscular junctions and that they help maintain these postsynaptic specializations in the adult. Here we show that anti-agrin monoclonal antibodies selectively stain the cell bodies of motor neurons in embryos and adults, and that the stain is concentrated in the Golgi apparatus. We also present evidence that motor neurons in both embryos and adults contain molecules that cause the formation of AChR and AChE aggregates on cultured myotubes and that these AChR/AChE-aggregating molecules are antigenically similar to agrin. These findings are consistent with the hypothesis that agrin-like molecules are synthesized by motor neurons, and are released from their axon terminals to become incorporated into the synaptic basal lamina where they direct the formation of synapses during development and regeneration.
Mapping of a cell-binding domain in the cell adhesion molecule gp80 of Dictyostelium discoideum.
Authors: Kamboj R K , Wong L M , Lam T Y , Siu C H
Published: May 2004

At the aggregation stage of Dictyostelium discoideum development, a cell surface glycoprotein of Mr 80,000 (gp80) has been found to mediate the EDTA-resistant type of cell-cell adhesion via homophilic interaction (Siu, C.-H., A. Cho, and A. H. C. Choi. 1987. J. Cell Biol. 105:2523-2533). To investigate the structure-function relationships of gp80, we have isolated full length cDNA clones for gp80 and determined the DNA sequence. The deduced structure of gp80 showed three major domains. An amino-terminal globular domain composed of the bulk of the protein is supported by a short stalk region, which is followed by a membrane anchor at the carboxy terminus. Structural analysis suggested that the cell-binding domain of gp80 resides within the globular domain near the amino terminus. To investigate the relationship of the cell-binding activity to this region of the polypeptide, three protein A/gp80 (PA80) gene fusions were constructed using the expression vector pRIT2T. These PA80 fusion proteins were assayed for their ability to bind to aggregation stage cells. Binding of 125I-labeled fusion proteins PA80I (containing the Val123 to Ile514 fragment of gp80) and PA80II (Val123 to Ala258) was dosage dependent and could be inhibited by precoating cells with the cell cohesion-blocking mAb 80L5C4. On the other hand, there was no appreciable binding of PA80III (Ile174 to Ile514) to cells. Reassociation of cells was significantly inhibited in the presence of PA80I or PA80II. In addition, 125I-labeled PA80II exhibited homophilic interaction with immobilized PA80I, PA80II, or gp80. The results of these studies lead to the mapping of a cell-binding domain in the region between Val123 and Leu173 of gp80 and provide direct evidence that the cell-binding activity of gp80 resides in the protein moiety.
Functionally distinct laminin receptors mediate cell adhesion and spreading: the requirement for surface galactosyltransferase in cell spreading.
Authors: Runyan R B , Versalovic J , Shur B D
Published: May 2004

The molecular mechanisms underlying cell attachment and subsequent cell spreading on laminin are shown to be distinct form one another. Cell spreading is dependent upon the binding of cell surface galactosyltransferase (GalTase) to laminin oligosaccharides, while initial cell attachment to laminin occurs independent of GalTase activity. Anti-GalTase IgG, as well as the GalTase modifier protein, alpha-lactalbumin, both block GalTase activity and inhibited B16-F10 melanoma cell spreading on laminin, but not initial attachment. On the other hand, the addition of UDP galactose, which increases the catalytic turnover of GalTase, slightly increased cell spreading. None of these reagents had any effect on cell spreading on fibronectin. When GalTase substrates within laminin were either blocked by affinity-purified GalTase or eliminated by prior galactosylation, cell attachment appeared normal, but subsequent cell spreading was totally inhibited. The laminin substrate for GalTase was identified as N-linked oligosaccharides primarily on the A chain, and to a lesser extent on B chains. That N-linked oligosaccharides are necessary for cell spreading was shown by the inability of cells to spread on laminin surfaces pretreated with N-glycanase, even though cell attachment was normal. Cell surface GalTase was distinguished from other reported laminin binding proteins, most notably the 68-kD receptor, since they were differentially eluted from laminin affinity columns. These data show that surface GalTase does not participate during initial cell adhesion to laminin, but mediates subsequent cell spreading by binding to its appropriate N-linked oligosaccharide substrate. These results also emphasize that some of laminin's biological properties can be attributed to its oligosaccharide residues.
Macrophage interactions with laminin: PMA selectively induces the adherence and spreading of mouse macrophages on a laminin substratum.
Authors: Mercurio A M , Shaw L M
Published: May 2004

The ability of thioglycollate (TG)-elicited mouse peritoneal macrophages to adhere to a laminin substratum has been studied. These cells do not adhere to laminin-coated (20 micrograms/ml) surfaces, but the addition of phorbol myristate acetate (PMA; 50 ng/ml) results in their rapid adherence and spreading on this substratum. TG-elicited and PMA-activated macrophages, however, can bind soluble laminin. Macrophages adhere to fibronectin-coated surfaces and tissue culture plastic without PMA stimulation, and PMA does not increase the number of cells that adhere to these surfaces. The predominant surface proteins that bind specifically to laminin-Sepharose exhibit an Mr of 67 and 36 kD, but the expression of these proteins does not increase after PMA stimulation. Laminin receptor antibodies immunoprecipitate the 67-kD protein from radiolabled surface lysates and are capable of blocking macrophage adherence to a laminin substratum. Indirect immunofluorescence microscopy indicates that PMA stimulation does not increase receptor expression, but that it may induce the aggregation of the receptor on the cell surface. PMA stimulation also promotes macrophage spreading and induces a reorganization of the actin cytoskeleton. Taken together, these data indicate the mechanism by which PMA promotes macrophage adherence to laminin does not involve increased 67-kD receptor surface expression, but that it is related to the changes in cytoskeletal and receptor surface organization that occur in response to PMA stimulation.
Differentiation of cardiac myocytes after mitogen withdrawal exhibits three sequential states of the ventricular growth response.
Authors: Ueno H , Perryman M B , Roberts R , Schneider M D
Published: May 2004

During cardiac myogenesis, ventricular muscle cells lose the capacity to proliferate soon after birth. It is unknown whether this developmental block to mitotic division and DNA replication might involve irreversible repression of the cellular oncogene c-myc. Ventricular myocytes from 2 d-old rats continued to differentiate in vitro during 15 d of mitogen withdrawal, as shown by the formation of cross-striations, increased proportion of the muscle isoenzyme of creatine kinase, stable expression of alpha-cardiac actin and myosin heavy chain mRNAs, and appropriate down-regulation of alpha-skeletal actin mRNA. After mitogen withdrawal for 2 d, serum evoked both DNA synthesis and mitotic division; after 7 d, DNA replication was uncoupled from cell division; after 15 d, DNA synthesis itself was markedly attentuated. These three distinct phenotypic states resemble the sequential properties of growth found in the neonatal rat heart in vivo. Despite failure to induce DNA replication or division after 15 d of mitogen withdrawal, serum elicited both c-myc and alpha-skeletal actin as found during hypertrophy of the intact heart. The results agree with previous evidence that one or more functional pathways that transduce the effects of serum factors may persist in older cardiac muscle cells, and indicate that irreversible down-regulation of c-myc cannot be the basis for the loss of growth responses.
Accumulation, localization, and compartmentation of transforming growth factor beta during endochondral bone development.
Authors: Carrington J L , Roberts A B , Flanders K C , Roche N S , Reddi A H
Published: May 2004

Endochondral bone formation was induced in postnatal rats by implantation of demineralized rat bone matrix. Corresponding control tissue was generated by implanting inactive extracted bone matrix, which did not induce bone formation. At various times, implants were removed and sequentially extracted with guanidine hydrochloride, and then EDTA and guanidine hydrochloride. Transforming growth factor beta (TGF beta) in the extracts was quantitated by a radioreceptor assay. TGF beta was present in demineralized bone matrix before implantation, and the concentration had decreased by 1 d after implantation. Thereafter, TGF beta was undetectable by radioreceptor assay until day 9. From day 9-21 the TGF beta was extracted only after EDTA demineralization, indicating tight association with the mineralized matrix. During this time, the content of TGF beta per milligram soluble protein rose steadily and remained high through day 21. This increased concentration correlated with the onset of vascularization and calcification of cartilage. TGF beta was detected only between days 3-9 in the controls; i.e., non-bone-forming implants. Immunolocalization of TGF beta in bone-forming implants revealed staining of inflammatory cells at early times, followed later by staining of chondrocytes in calcifying cartilage and staining of osteoblasts. The most intense staining of TGF beta was found in calcified cartilage and mineralized bone matrix, again indicating preferential compartmentalization of TGF beta in the mineral phase. In contrast to the delayed expression of TGF beta protein, northern blot analysis showed TGF beta mRNA in implants throughout the sequence of bone formation. The time-dependent accumulation of TGF beta when cartilage is being replaced by bone in this in vivo model of bone formation suggests that TGF beta may play a role in the regulation of ossification during endochondral bone development.
Antigenic and functional characterization of a rat central nervous system-derived cell line immortalized by a retroviral vector.
Authors: Geller H M , Dubois-Dalcq M
Published: May 2004

We have immortalized rat central nervous system (CNS) cells of primary cultures of rat optic nerve with murine leukemia virus psi-2,SV-40-6, which is defective in assembly and contains the SV-40 large T antigen and neomycin resistance genes, to produce a cell line that we named A7. After drug selection, greater than 90% of the growing cells expressed nuclear SV-40 large T cells and a fraction of these contained the astrocyte-specific marker, glial fibrillary acidic protein. The majority of these cells also expressed surface marker A4 (specific for neural tube derivatives), Ran 2, p185 (the 185-kD phosphoprotein product of the neu oncogene), and fibronectin, but did not express the astrocyte enzymes glutamine synthetase and monoamine oxidase B. Surface markers characteristic of glial progenitors (A2B5) and oligodendrocytes (galactocerebroside) were not detected. After two rounds of cell cloning, subclone A7.6-3 expressed Ran 2, fibronectin, and the neural cell adhesion molecule (N-CAM) but not glial fibrillary acidic protein and A4. The A7 cell line and subclones also displayed certain functions of type 1 astrocytes: the conditioned medium of these cells had a potent mitogenic activity for glial progenitor cells which could be neutralized by anti-platelet-derived growth factor antibodies and monolayers of these cells supported the growth of embryonic hypothalamic neurons. We conclude that a retrovirus containing SV-40 large T antigen can immortalize rat CNS cells and that such immortalized glial cells retain at least two important functions of type 1 astrocytes: the ability to secrete platelet-derived growth factor and to support the growth of embryonic CNS neurons. Moreover, such stable immortalized clonal cell lines can be used to study gene regulation in glial cells.
Integral membrane proteins specific to the inner nuclear membrane and associated with the nuclear lamina.
Authors: Senior A , Gerace L
Published: May 2004

We obtained a monoclonal antibody (RL13) that identifies three integral membrane proteins specific to the nuclear envelope of rat liver, a major 75-kD polypeptide and two more minor components of 68 and 55 kD. Immunogold labeling of isolated nuclear envelopes demonstrates that these antigens are localized specifically to the inner nuclear membrane, and that the RL13 epitope occurs on the inner membrane's nucleoplasmic surface where the nuclear lamina is found. When nuclear envelopes are extracted with solutions containing nonionic detergent and high salt to solubilize nuclear membranes and pore complexes, most of these integral proteins remain associated with the insoluble lamina. Since the polypeptides recognized by RL13 are relatively abundant, they may function as lamina attachment sites in the inner nuclear membrane. Major cross-reacting antigens are found by immunoblotting and immunofluorescence microscopy in all rat cells examined. Therefore, these integral proteins are biochemical markers for the inner nuclear membrane and will be useful models for studying nuclear membrane biogenesis.
A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites.
Authors: Vestweber D , Schatz G
Published: May 2004

Bovine pancreatic trypsin inhibitor (which contains three intramolecular disulfide bridges) was chemically coupled to the COOH terminus of a purified artificial mitochondrial precursor protein. When the resulting chimeric precursor was presented to energized isolated yeast mitochondria, its trypsin inhibitor moiety prevented the protein from completely entering the organelle; the protein remained stuck across both mitochondrial membranes, with its NH2 terminus in the matrix and its trypsin inhibitor moiety still exposed on the mitochondrial surface. The incompletely imported protein appeared to "jam" mitochondrial protein import sites since it blocked import of three authentic mitochondrial precursor proteins; it did not collapse the potential across the mitochondrial inner membrane. Quantification of the inhibition indicated that each isolated mitochondrial particle contains between 10(2) and 10(3) protein import sites.
Retrovirus-mediated expression of preprosomatostatin: posttranslational processing, intracellular storage, and secretion in GH3 pituitary cells.
Authors: Stoller T J , Shields D
Published: May 2004

Somatostatin (SRIF) is a 14-amino acid peptide hormone that is synthesized as part of a larger precursor, preproSRIF, consisting of a signal peptide and a proregion of 80-90 amino acids. The mature hormone, which is located at the carboxyl terminus of the precursor, is preceded by a single pair of basic amino acids. We are studying preproSRIF to investigate intracellular sorting, proteolytic processing, and storage of peptide hormone precursors in the secretory pathway. We used a retroviral expression vector to achieve the high levels of precursor synthesis which are necessary for detailed characterization of processing intermediates and mature somatostatin. Recombinant retroviruses containing RNA transcripts encoding anglerfish preproSRIF I were used to infect rat pituitary GH3 cells which secrete growth hormone and prolactin, neither of which are substrates for endoproteolytic cleavage. In these cells preproSRIF was accurately processed to the mature hormone with an efficiency of approximately 75%. Of the newly synthesized mature SRIF, 55% was sorted into the regulated secretory pathway and released in response to the secretagogue 8-Br-cAMP. The remaining 45% of mature SRIF and residual unprocessed precursor was rapidly secreted. In contrast to SRIF, only 5% of newly synthesized endogenous growth hormone was stored intracellularly, whereas 95% was sorted to the constitutive pathway and secreted rapidly with kinetics identical to proSRIF. Our results show that proSRIF processing is not necessarily dependent on a specific protease found only in SRIF-producing cells and suggest that proteolytic cleavage is not restricted to cells that process endogenous hormones. Moreover, these results demonstrate that GH3 cells have the capacity to discriminate between endogenous and foreign hormones and target the foreign molecule significantly more efficiently to the regulated secretory pathway.
Inhibition of early but not late proteolytic processing events leads to the missorting and oversecretion of precursor forms of lysosomal enzymes in Dictyostelium discoideum.
Authors: Richardson J M , Woychik N A , Ebert D L , Dimond R L , Cardelli J A
Published: May 2004

Lysosomal enzymes are initially synthesized as precursor polypeptides which are proteolytically cleaved to generate mature forms of the enzymatically active protein. The identification of the proteinases involved in this process and their intracellular location will be important initial steps in determining the role of proteolysis in the function and targeting of lysosomal enzymes. Toward this end, axenically growing Dictyostelium discoideum cells were pulse radiolabeled with [35S]methionine and chased in fresh growth medium containing inhibitors of aspartic, metallo, serine, or cysteine proteinases. Cells exposed to the serine/cysteine proteinase inhibitors leupeptin and antipain and the cysteine proteinase inhibitor benzyloxycarbonyl-L-phenylalanyl-L-alanine-diazomethyl ketone (Z-Phe-AlaCHN2) were unable to complete proteolytic processing of the newly synthesized lysosomal enzymes, alpha-mannosidase and beta-glucosidase. Antipain and leupeptin treatment resulted in both a dramatic decrease in the efficiency of proteolytic processing, as well as a sevenfold increase in the secretion of alpha-mannosidase and beta-glucosidase precursors. However, leupeptin and antipain did not stimulate secretion of lysosomally localized mature forms of the enzymes suggesting that these inhibitors prevent the normal sorting of lysosomal enzyme precursors to lysosomes. In contrast to the results observed for cells treated with leupeptin or antipain, Z-Phe-AlaCHN2 did not prevent the cleavage of precursor polypeptides to intermediate forms of the enzymes, but greatly inhibited the production of the mature enzymes. The accumulated intermediate forms of the enzymes, however, were localized to lysosomes. Finally, fractionation of cell extracts on Percoll gradients indicated that the processing of radiolabeled precursor forms of alpha-mannosidase and beta-glucosidase to intermediate products began in cellular compartments intermediate in density between the Golgi complex and mature lysosomes. The generation of the mature forms, in contrast, was completed immediately upon or soon after arrival in lysosomes. Together these results suggest that different proteinases residing in separate intracellular compartments may be involved in generating intermediate and mature forms of lysosomal enzymes in Dictyostelium discoideum, and that the initial cleavage of the precursors may be critical for the proper localization of lysosomal enzymes.
The dynamics of exocytosis in human neutrophils.
Authors: Nüsse O , Lindau M
Published: May 2004

We have investigated the dynamics of exocytosis in single human neutrophils. The increase of membrane area associated with granule fusion was followed by time-resolved patch-clamp capacitance measurements. Intracellular application of 20 microM guanosine-5'-O(3-thiotriphosphate) (GTP gamma S) in the presence of 2.5 mM ATP stimulated exocytosis and led to an increase of membrane capacitance from 3.0 to integral of 8.4 pF corresponding to a 540 micron 2 increase of membrane area. This capacitance change is very close to the value expected from morphological data if all primary and secondary granules fuse with the plasma membrane. High resolution measurements revealed stepwise capacitance changes corresponding to the fusion of individual granules. GTP gamma S-stimulated exocytosis did not require pretreatment with cytochalasin B and the amplitude was independent of the intracellular-free calcium concentration between 10 nM and integral of 2.5 microM. In the absence of GTP gamma S elevation of intracellular-free calcium concentration to the micromolar range led to the fusion of only a limited number of granules. Degranulation stimulated with GTP gamma S started after a lag phase of 2-7 min and was usually complete within 5-20 min. The time course was affected by the intracellular ATP and calcium concentration. Exocytosis was markedly accelerated by pretreatment with cytochalasin B. Our results demonstrate that the final steps leading to primary and secondary granule fusion are controlled by a guanine nucleotide-binding protein and do not require an elevation of intracellular calcium. Calcium and other factors are, however, involved in the regulation having pronounced effects on the dynamics of exocytosis.
p60c-src is complexed with a cellular protein in subcellular compartments involved in exocytosis.
Authors: Grandori C , Hanafusa H
Published: May 2004

We found high levels of the c-src gene product in neuroendocrine tissues from adult animals. To understand the role of this proto-oncogene product, the subcellular localization of p60c-src was studied in neuroendocrine tissue from adrenal medulla. The results indicate that p60c-src was highly enriched in chromaffin granule membranes, in stable association with a protein of 38 kD. The complex with the 38-kD protein was also detected in brain, a tissue known to carry high levels of p60c-src. The 38-kD protein is not calpactin I, II, or synaptophysin. Comparison of its peptide map showed a high degree of conservation among the different species and tissues examined. The interaction between p60c-src and the 38-kD protein involves disulphide bonds that are stable even when the cell fractionation is performed in the presence of a reducing agent. Since the presence of disulphide bonds among cytoplasmic proteins is very unlikely, the possibility of a noncovalent association between p60c-src and the 38-kD protein in vivo is discussed. The 38-kD protein may be involved in a function of p60c-src related to secretory organelles.
Selective degradation of T cell antigen receptor chains retained in a pre-Golgi compartment.
Authors: Chen C , Bonifacino J S , Yuan L C , Klausner R D
Published: May 2004

We have examined the fate of newly synthesized T cell antigen receptor (TCR) subunits in a T cell hybridoma deficient in expression of the clonotypic beta chain. Synthesis and assembly of the remaining chains proceed normally but surface expression of TCR chains is undetectable in these cells. A variety of biochemical and morphological techniques has been used to show that the TCR chains in these cells fail to be transported to any of the Golgi cisternae. Instead, they are retained in a pre-Golgi compartment which is either part of or closely related to the endoplasmic reticulum. The CD3-delta chain is degraded by a non-lysosomal process that is inhibited at temperatures at or below 27 degrees C. By contrast, the remaining chains (CD3-epsilon, CD3-gamma, and zeta) are very stable over 7 h. We propose possible mechanisms that may explain the differential fate of TCR chains retained in a pre-Golgi compartment.
Architecture of the sarcomere matrix of skeletal muscle: immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin filaments anchored at the Z line.
Authors: Wang K , Wright J
Published: May 2004

Nebulin, a giant myofibrillar protein (600-800 kD) that is abundant (3%) in the sarcomere of a wide range of skeletal muscles, has been proposed as a component of a cytoskeletal matrix that coexists with actin and myosin filaments within the sarcomere. Immunoblot analysis indicates that although polypeptides of similar size are present in cardiac and smooth muscles at low abundance, those proteins show no immunological cross-reactivity with skeletal muscle nebulin. Gel analysis reveals that nebulins in various skeletal muscles of rabbit belong to at least two classes of size variants. A monospecific antibody has been used to localize nebulin by immunoelectron microscopy in a mechanically split rabbit psoas muscle fiber preparation. Labeled split fibers exhibit six pairs of stripes of antibody-imparted transverse densities spaced at 0.1-1.0 micron from the Z line within each sarcomere. These epitopes maintain a fixed distance to the Z line irrespective of sarcomere length and do not exhibit the characteristic elastic stretch-response of titin epitopes within the I band domain. It is proposed that nebulin constitutes a set of inextensible filaments attached at one end to the Z line and that nebulin filaments are in parallel, and not in series, with titin filaments. Thus the skeletal muscle sarcomere may have two sets of nonactomyosin filaments: a set of I segment-linked nebulin filaments and a set of A segment-linked titin filaments. This four-filament sarcomere model raises the possibility that nebulin and titin might act as organizing templates and length-determining factors for actin and myosin respectively.