bluuebunny/crossref_metadata_2025 huggingface dataset:
The 46,000-dalton tyrosine protein kinase substrate is widespread, whereas the 36,000-dalton substrate is only expressed at high levels in certain rodent tissues.
Authors: Gould K L , Cooper J A , Hunter T
Published: May 2004

Proteins of molecular mass 46,000 (p46) and 34,000-39,000 (p36) daltons are phosphorylated at tyrosine in Rous sarcoma virus-transformed chicken and mouse fibroblasts. p46 has recently been identified as an isozyme of enolase but the function of p36 is unknown. The expression of these proteins in various mouse and rat tissues has been examined. In most tissues, except muscle, p46 is found at relatively constant levels. In muscle, a more basic, related protein is present. In contrast, the abundance of p36 varies more widely from tissue to tissue, suggesting that it has a function in some but not all differentiated cells. By SDS gel electrophoresis and immunoblotting, high levels of p36 (60-120% of its relative abundance in fibroblasts) were found in small intestine, lung, and thymus, and intermediate levels (20-50%) were found in spleen, lymph nodes, and testes. No p36 was detectable in brain and muscle. Where studied, p36 mRNA expression paralleled protein levels. The cell types within each tissue expressing p36 were identified by immunofluorescence and immunoperoxidase staining. These cell types include all endothelial cells and fibroblastic cells examined, as well as various epithelial cells, cardiac muscle cells, macrophages, and testicular interstitial cells. We were unable to detect p36 in skeletal or smooth muscle cells, erythrocytes, nerve cells, or lymphocytes in any of the examined tissues. p36 appears to be concentrated in the terminal web region of intestinal columnar epithelial cells.
Organization of acetylcholine receptor clusters in cultured rat myotubes is calcium dependent.
Authors: Bursztajn S , McManaman J L , Appel S H
Published: May 2004

The effect of extracellular Ca2+ concentration and myasthenic globulin on the distribution and appearance of acetylcholine receptor (AChR) clusters on rat myotubes was studied with tetramethyl-rhodamine-labeled alpha BTX. Low Ca2+ medium (2.5 X 10(-5) M) caused a time-dependent loss of AChR clusters, and a concomitant increase in small punctate areas of fluorescence. High Ca2+ concentrations (1.5 X 10(-2) M) increased the size of AChR clusters without altering AChR synthesis. These changes were not observed with other divalent ions. In the presence of myasthenic globulin, the rate of AChR turnover increases, and AChR clusters are rapidly dispersed. High Ca2+ concentration partially protects the AChR clusters from dispersal and decreases the rate of receptor turnover.
A new 185,000-dalton skeletal muscle protein detected by monoclonal antibodies.
Authors: Grove B K , Kurer V , Lehner C , Doetschman T C , Perriard J C , Eppenberger H M
Published: May 2004

The M line, which transverses the center of the thick filament region of skeletal muscle sarcomeres, appears to be a complex array of multiple structural elements. To date, two proteins have definitely been shown to be associated with the M line. They are MM-CK, localized in the M 4,4' substriations, and a 165,000-dalton (164 kd) protein, referred to as both M-protein and myomesin. Here we report the positive identification of a third M-line protein of 185 kd. In the course of making monoclonal antibodies (mAbs) against a 165-kd fraction, we also obtained mAbs that bound to the M line of isolated myofibrils as detected by indirect immunofluorescence, but recognized a protein band of 185 kd in immunoblotting experiments with either the original immunogen or low ionic strength myofibril extracts as antigenic targets. The evidence that the 185- and 165-kd proteins are distinct protein species is based on the separation of the two proteins into discrete peaks by ion exchange chromatography, the distinctive patterns of their degradation products, and non-cross-reactivity of any of seven mAbs. These mAbs recognize three unique antigenic determinants on the 185-kd molecule and at least two and probably four sites on the 165-kd molecule as determined from competitive binding and immunofluorescence experiments. To resolve the problem of multiple nomenclature for the 165-kd protein, the 185-kd protein will be referred to as myomesin and the 165-kd protein as M-protein.
Glucocorticoid inhibition of vascular smooth muscle cell proliferation: influence of homologous extracellular matrix and serum mitogens.
Authors: Longenecker J P , Kilty L A , Johnson L K
Published: May 2004

We examined the influence of glucocorticoid hormones on the proliferation of cultured adult bovine aortic smooth muscle cells (BASM) using both primary mass cultures and a cloned strain. Cloned BASM cells maintained on plastic culture dishes were inhibited by approximately 40% by dexamethasone treatment but showed no inhibition when grown of homologous extracellular matrix (ECM) coated dishes. Dexamethasone inhibited growth of primary cultures by 73% on plastic and by 45% on ECM. The inhibitory effect was specific for the glucocorticoids, dexamethasone, corticosterone, and cortisol and was not observed with progesterone, aldosterone, estradiol or 17-alpha OH progesterone. In cloned cells, the abolition of glucocorticoid inhibition by ECM was independent of seeding density and serum concentration. The inhibition on plastic was dependent on serum concentrations greater than 1% and resulted in both a slow rate of proliferation and a lower saturation density. A specific subset of peptides detected on two-dimensional gels was induced by glucocorticoids under growth inhibitory conditions but was not induced when the cells were grown on ECM. Primary cultures grown on ECM and exposed to Dulbecco's modified Eagle's Medium (DME) containing high density lipoprotein and transferrin grew at 40% of the rate observed for cultures exposed to DME with 10% serum. Both conditions showed growth inhibition of 70% in the presence of dexamethasone. The addition of epidermal and platelet-derived growth factors in DME containing high density lipoprotein and transferrin to cells grown on ECM resulted in growth rates comparable to that observed with cultures exposed to 10% serum and were inhibited 45% by dexamethasone. These results suggest that glucocorticoids inhibit smooth muscle proliferation by decreasing the sensitivity of the cells to mitogenic stimulation by high density lipoprotein when the cells are maintained on a homologous substrate.
Glucocorticoids stimulate collagen and noncollagen protein synthesis in cultured vascular smooth muscle cells.
Authors: Leitman D C , Benson S C , Johnson L K
Published: May 2004

The effect of glucocorticoids on collagen synthesis was examined in cultured bovine aortic smooth muscle (BASM) cells. BASM cells treated with 0.1 microM dexamethasone during their proliferative phase (11 d) were labeled with [3H]proline for 24 h, and the acid-precipitable material was incubated with bacterial collagenase. Dexamethasone produced an approximate twofold increase in the incorporation of proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP) in the cell layer and medium. The stimulation was present in both primary mass cultures and cloned BASM. An increase in CDP and NCP was detected at 0.1 nM, while maximal stimulation occurred at 0.1 microM. Only cells exposed to dexamethasone during their log phase of growth (1-6 d after plating) showed the increase in CDP and NCP when labeled 11 d after plating. The stimulatory effect was observed in BASM cells treated with the natural bovine glucocorticoid, cortisol, dexamethasone, and testosterone, but was absent in cells treated with aldosterone, corticosterone, cholesterol, 17 beta-estradiol, and progesterone. The increase in CDP and NCP was absent in cells treated with the inactive glucocorticoid, epicortisol, and totally abolished by the antagonist, 17 alpha-hydroxyprogesterone, suggesting that the response was mediated by specific cytoplasmic glucocorticoid receptors. Dexamethasone-treated BASM cells showed a 4.5-fold increase in the specific activity of intracellular proline, which was the result of a twofold increase in the uptake of proline and depletion of the total proline pool. After normalizing for specific activity, dexamethasone produced a 2.4- and 2.8-fold increase in the rate of collagen and NCP synthesis, respectively. Cells treated with dexamethasone secreted 1.7-fold more collagen protein in 24 h compared to control cultures. The BASM cells secreted 70% Type I and 30% Type III collagen into the media as assessed by two-dimensional gel electrophoresis. The ratio of these two types was not altered by dexamethasone. The results of the present study demonstrate that glucocorticoids can act directly on vascular smooth muscle cells to increase the synthesis and secretion of collagen and NCP.
Participation of calcium and calmodulin in the formation of acetylcholine receptor clusters.
Authors: Peng H B
Published: May 2004

The formation of acetylcholine receptor (AChR) clusters can be experimentally induced in cultured Xenopus myotomal muscle cells by positive polypeptide-coated latex beads (Peng, H.B., P.-C. Cheng, and P.W. Luther, 1981, Nature [Lond.], 292:831-834). This provides a simple procedure for studying the cellular process of AChR clustering. In this study, the involvement of calcium and calmodulin in this process was examined. A deprivation in extracellular calcium by calcium-free medium or by the addition of calcium antagonists such as divalent cations Co2+ and Ni2+ (1-5 mM) or organic compounds verapamil and D-600 (0.1-0.5 mM) suppressed the formation of AChR clusters induced by the latex beads in a largely reversible manner. Antagonists against calmodulin, including trifluoperazine (1-5 microM) and the naphthalene sulfonamide W-7 (20 microM), also suppressed AChR clustering. However, the effect of W-7 was much weaker than that of trifluoperazine (TFP). Although the formation of AChR clusters is inhibited by these drugs, the stability of the existent clusters is relatively insensitive to them. These data suggest that the clustering of AChR involves a Ca2+ and calmodulin-activated process. Immunofluorescence studies using an antibody against calmodulin indicate that calmodulin is diffusely distributed in the cytoplasm in addition to its localization at the I-bands. Thus I propose that a local rise in intracellular calcium caused by a locally applied stimulus, exemplified here by the polypeptide-coated latex beads, may trigger the formation of AChR clusters. Furthermore, the cellular machinery for this process may involve calmodulin and is diffusely distributed in the muscle cell.
A time-lapse video image intensification analysis of cytoplasmic organelle movements during endosome translocation.
Authors: Herman B , Albertini D F
Published: May 2004

Vital fluorescence staining has been used in conjunction with time-lapse video image intensification microscopy to analyze the distribution and movement of endosomes, lysosomes, and mitochondria in cultured rat ovarian granulosa cells. Exposure of 5-d granulosa cell cultures to pyrene-concanavalin A (P-Con A) or 3,3'-dioctadecylindocarbocyanine-labeled low-density lipoprotein (dil-LDL) at 4 degrees C results in the formation of randomly distributed endosomes 10 min after warming to 37 degrees C that exhibit saltatory motion for 20 min. If granulosa cells are labeled at 4 degrees C with both P-Con A and dil-LDL and warmed to 37 degrees C, both ligands are found within the same endosomes which migrate centripetally to the cell center where label accumulates within phase-dense structures by 60 min. The initial endosome saltations occur over short distances (mean distance = 4.6 micron) with a mean velocity of 0.03 micron/s. Endosome saltations then cease and are followed by a gradual centriptal migration of endosomes to the cell center where they accumulate and fuse with phase-dense structures. The second phase of movement involves a continuous, unidirectional migration of endosomes over distances ranging from 5 to 40 micron at a mean velocity of 0.05 micron/s. Lysosomes were simultaneously visualized as acridine orange-staining, phase-dense structures in control cells and cells exposed to fluorescent ligands. In untreated cells, lysosomes are dispersed throughout the cytoplasm and undergo bidirectional saltations covering a mean distance of 8.7 micron with a mean velocity of 0.3 micron/s. Lysosomes redistribute centripetally to the perinuclear region of the cell by saltatory movement within 20 min of exposure to ligand. Mitochondria were visualized with the fluorescent dye rhodamine 123 in granulosa cells labeled with P-Con A and were found to redistribute to the cell center coincident with endosomes. The microtubule-disrupting agent nocodazole was found to inhibit lysosome saltations and all phases of endosome movement. Taxol, a microtubule-stabilizing agent, partially impaired lysosome movement and led to a redistribution of lysosomes into linear aggregates surrounding the nucleus. Taxol was also found to inhibit endosome movement. The data indicate that (a) endosome movement proceeds initially by saltation and later by a nonsaltatory centripetal migration in association with mitochondria, that (b) lysosomes and endosomes undergo a temporally distinct but spatially similar change in cytoplasmic distribution, and that (c) microtubules are required for the directed translocation of endosomes and lysosomes towards the cell center.
Association of the cyclic AMP chemotaxis receptor with the detergent-insoluble cytoskeleton of Dictyostelium discoideum.
Authors: Galvin N J , Stockhausen D , Meyers-Hutchins B L , Frazier W A
Published: May 2004

Treatment of 6-h differentiated Dictyostelium discoideum cells with the nonionic detergent Triton X-100 dissolves away membranes and soluble components, as judged by marker enzyme distributions, leaving intact a cytoskeletal residue that contains approximately 10% of the cell protein and 50% of the actin. Nitrobenzooxadiazo-phallacidin staining for F-actin and electron microscopy of detergent-extracted whole-mounts indicate that the cytoskeletons retain the size and shape of intact cells and contain F-actin in cortical meshworks. The cytoskeletons contain little if any remaining membrane material by morphological criteria, and the plasma membrane enzymes cyclic nucleotide phosphodiesterase and alkaline phosphatase are absent from the insoluble residue, which retains only 15% of the membrane concanavalin A-binding glycoproteins. This detergent-insoluble residue retains a specific [3H]cAMP-binding site with the nucleotide specificity, rapid kinetics and approximate affinity of the cAMP receptor on intact cells. Upon detergent extraction of cells, the number of cAMP-binding sites increases 20-70%. The binding site is attached to the insoluble residue whether or not the cAMP receptor is occupied at the time of detergent addition. The pH dependence for recovery of the insoluble cAMP-binding site is much sharper than that on intact cells or membranes with an optimum at pH 6.1. Conditions of pH and ionic composition that lead to disruption of the cytoskeleton upon detergent treatment also result in the loss of cAMP binding. During differentiation, the detergent-insoluble cAMP binding increases in parallel with cell surface cAMP receptors and chemotaxis to cAMP.
A lipophilic iron chelator can replace transferrin as a stimulator of cell proliferation and differentiation.
Authors: Landschulz W , Thesleff I , Ekblom P
Published: May 2004

Of the different growth supplements used in chemically defined media, only transferrin is required for differentiation of tubules in the embryonic mouse metanephros. Since transferrin is an iron-carrying protein, we asked whether iron is crucial for tubulogenesis. Differentiation of metanephric tubules both in whole embryonic kidneys and in a transfilter system was studied. The tissues were grown in chemically defined media containing transferrin, apotransferrin, the metal-chelator complex ferric pyridoxal isonicotinoyl hydrazone (FePIH), and excesses of ferric ion. Although we found that apotransferrin was not as effective as iron-loaded transferrin in promoting proliferation in the differentiating kidneys, excess ferric ion at up to 100 microM, five times the normal serum concentration, could not promote differentiation or proliferation. However, iron coupled to the nonphysiological, lipophilic iron chelator, pyridoxal isonicotinoyl hydrazone, to form FePIH, could sustain levels of cell proliferation and tubulogenesis similar to those attained by transferrin. Thus, the role of transferrin in cell proliferation during tubulogenesis is solely to provide iron. Since FePIH apparently bypasses the receptor-mediated route of iron intake, the use of FePIH as a tool for investigating cell proliferation and its regulation is suggested.
Generation of flagella by cultured mouse spermatids.
Authors: Gerton G L , Millette C F
Published: May 2004

During the short-term culturing of mouse spermatogenic cells, flagella were generated by round spermatids previously lacking tails. Unseparated germ cells were obtained by enzymatic treatments and round spermatids (greater than 90% pure) were purified by unit gravity sedimentation. As determined by Nomarski or phase-contrast microscopy, no cells had flagella immediately after isolation; flagella were first clearly detected after 6 1/2 h of culture in Eagle's minimal essential medium containing 10% fetal bovine serum and 6 mM lactate. After 24 h, approximately 20% of round spermatids had formed flagella. Multinucleated round spermatids often formed multiple flagella, the number never exceeding the number of nuclei per symplast. Round spermatids were the only spermatogenic cells capable of tail formation. Flagella elongation was blocked by 1 microM demecolcine, an inhibitor of tubulin polymerization. Indirect immunofluorescence localized tubulin in the flagella. As seen by scanning electron microscopy, flagella developed as early as 2 h after culture and continued to elongate over the next 20 h, reaching lengths of at least 19 micron. Transmission electron microscopy demonstrated that flagella formed in culture resembled flagella from Golgi-phase round spermatids in situ; the flagella consisted of "9+2" axonemes lacking other accessory structures such as outer dense fibers and the fibrous sheath. As determined by acridine orange staining of the developing acrosomes, all spermatids that formed flagella in culture were Golgi-phase spermatids. By these criteria, the structures are indeed true flagella, corresponding in appearance to what others have described for early mammalian spermatid flagella in situ. We believe this is the first substantiated report of limited in vitro differentiation by isolated mammalian spermatids.
Temperature dependence of protein transport across lymphatic endothelium in vitro.
Authors: O'Morchoe C C , Jones W R , Jarosz H M , O'Morchoe P J , Fox L M
Published: May 2004

The purpose of the work was to develop an in vitro model for the study of lymphatic endothelium and to determine, using this model, whether or not a cytoplasmic process may be involved in transendothelial transport. Segments of canine renal hilar lymphatics were dissected clean, cannulated at both ends, and transferred to a perfusion chamber for measurement of transendothelial protein transport and for ultrastructural tracer studies. The segments were subsequently processed for light and electron microscopy. By both structural and functional criteria the lymphatics were judged to have retained their integrity. At 37 degrees C, 36 lymphatics showed a mean rate of protein transport of 3.51 +/- 0.45 (SEM) micrograms/min per cm2 of lymphatic endothelium. The rate was influenced by the temperature of the system, being significantly reduced by 49% +/- 4.8, 31% +/- 5.3, and 29% +/- 3.9 when the temperature was lowered to 4 degrees, 24 degrees, and 30 degrees C, respectively. When the temperature was raised to 40 degrees C, the rate was significantly increased by 48% +/- 12.2. The vesicular system and the intercellular regions in vessels with increased or reduced rates of transport were analyzed quantitatively to ascertain whether the rate changes could be correlated with ultrastructurally demonstrable changes in either of these postulated pathways. No significant changes in junctional or vesicular parameters were found between the control lymphatics and those perfused at 24 degrees, 30 degrees, and 40 degrees C. At 4 degrees C, the temperature at which the rate of protein transport was maximally reduced, vesicular size decreased, and the number of free cytoplasmic vesicles increased, whereas the number associated with the abluminal and luminal surfaces decreased. We concluded that isolated perfused lymphatic segments transport protein at a relatively constant rate under control conditions, and that this transendothelial transport comprises both temperature-dependent and temperature-independent mechanisms. The findings were considered in terms of the different theories of lymph formation and were interpreted as providing support for the vesicular theory.
The hagfish slime gland thread cell. I. A unique cellular system for the study of intermediate filaments and intermediate filament-microtubule interactions.
Authors: Downing S W , Spitzer R H , Koch E A , Salo W L
Published: May 2004

Thread cell differentiation in the slime gland of the Pacific hagfish Eptatretus stouti has been studied using light microscopy and scanning and transmission electron microscopy. Thread cell differentiation is remarkable in that the life history of the cell is largely dedicated to the production of a single, tapered, cylindrical, highly coiled, and precisely packaged cytoplasmic thread that may attain lengths of 60 cm and diameters approaching 1.5 micron. Each tapered thread, in turn, is comprised almost entirely of large numbers of intermediate filaments (IFs) bundled in parallel. During differentiation of the thread, the IFs become progressively more tightly packed. Various numbers of microtubules (MTs) are found among the bundled IFs during differentiation of the thread but disappear during the latter stages of thread differentiation. Observations of regularly spaced dots in longitudinal bisections of developing threads, diagonal striations in tangential sections of developing threads, and circumferentially oriented, filament-like structures observed at the periphery of developing threads cut in cross section have led us to postulate a helically oriented component(s) wrapped around the periphery of the developing thread. The enormous size of the fully differentiated thread cell, its apparent singular dedication to the production of IFs, the ease of isolating and purifying the threads and IF subunits (see accompanying paper), and the unique position of the hagfish in the phylogenetic scheme of vertebrate evolution all contribute to the attractiveness of the hagfish slime gland thread cell as a potential model system for studying IF subunit synthesis, IF formation from IF subunits, aggregation of IFs into IF bundles and the interaction(s) of IFs and MTs.
Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction.
Authors: Miller T M , Heuser J E
Published: May 2004

Frog nerve-muscle preparations were quick-frozen at various times after a single electrical stimulus in the presence of 4-aminopyridine (4-AP), after which motor nerve terminals were visualized by freeze-fracture. Previous studies have shown that such stimulation causes prompt discharge of 3,000-6,000 synaptic vesicles from each nerve terminal and, as a result, adds a large amount of synaptic vesicle membrane to its plasmalemma. In the current experiments, we sought to visualize the endocytic retrieval of this vesicle membrane back into the terminal, during the interval between 1 s and 2 min after stimulation. Two distinct types of endocytosis were observed. The first appeared to be rapid and nonselective. Within the first few seconds after stimulation, relatively large vacuoles (approximately 0.1 micron) pinched off from the plasma membrane, both near to and far away from the active zones. Previous thin-section studies have shown that such vacuoles are not coated with clathrin at any stage during their formation. The second endocytic process was slower and appeared to be selective, because it internalized large intramembrane particles. This process was manifest first by the formation of relatively small (approximately 0.05 micron) indentations in the plasma membrane, which occurred everywhere except at the active zones. These indentations first appeared at 1 s, reached a peak abundance of 5.5/micron2 by 30 s after the stimulus, and disappeared almost completely by 90 s. Previous thin-section studies indicate that these indentations correspond to clathrin-coated pits. Their total abundance is comparable with the number of vesicles that were discharged initially. These endocytic structures could be classified into four intermediate forms, whose relative abundance over time suggests that, at this type of nerve terminal, endocytosis of coated vesicles has the following characteristics: (a) the single endocytotic event is short lived relative to the time scale of two minutes; (b) earlier forms last longer than later forms; and (c) a single event spends a smaller portion of its lifetime in the flat configuration soon after the stimulus than it does later on.
Microspectrofluorometry by digital image processing: measurement of cytoplasmic pH.
Authors: Tanasugarn L , McNeil P , Reynolds G T , Taylor D L
Published: May 2004

An interface of our microspectrofluorometer with an image processing system performs microspectrofluorometric measurements in living cells by digital image processing. Fluorescence spectroscopic parameters can be measured by digital image processing directly from microscopic images of cells, and are automatically normalized for pathlength and accessible volume. Thus, an accurate cytoplasmic "map" of various spectroscopic parameters can be produced. The resting cytoplasmic pH of fibroblasts (3T3 cells) has been determined by measuring the ratio of fluorescein fluorescence exited by two successive wavelengths (489 and 452 nm). Fluorescein-labeled dextran microinjected into the cells is used as a pH indicator, since it is trapped in the cytoplasm but is excluded from the nucleus and other organelles. The average cytoplasmic pH is 6.83 (+/- 0.38). However, cytoplasmic pH exhibits a nonunimodal distribution, the lower mean pH being 6.74 (+/- 0.23). When 3T3 cells pinocytose medium containing fluorescein dextran, pinosomes peripheral to the nucleus exhibit a lower pH than those closer to the ruffling edge of the cell. The present image processing system is analyzed for linearity of detection, light scattering artifacts, signal to noise ratio, standard curves, and spatial resolution. The results obtained from digital image analysis are shown to be comparable to the results from standard microspectrofluorometry. We also discuss several other applications of this ratio imaging technique in cell biology.
Recycling of the asialoglycoprotein receptor and the effect of lysosomotropic amines in hepatoma cells.
Authors: Schwartz A L , Bolognesi A , Fridovich S E
Published: May 2004

Receptor-mediated uptake and degradation of 125I-asialoorosomucoid (ASOR) in human hepatoma HepG2 cells is inhibited by the lysosomotropic amines chloroquine and primaquine. In the absence of added ligand at 37 degrees C, these amines induce a rapid (t1/2 5.5-6 min) and reversible loss of cell surface 125I-ASOR binding sites as well as a rapid decrease in 125I-ASOR uptake and degradation. There is no effect of these amines on the binding of 125I-ASOR to the cell surface at 4 degrees C or on the rate of internalization of prebound 125I-ASOR. The loss of 125I-ASOR surface binding at 37 degrees C is not attributable to altered affinity of ligand-receptor binding. In the presence of added ligand at 37 degrees C, there is a more rapid (t1/2 2.5-3 min) loss of hepatoma cell surface receptors. In addition, the amines inhibit the rapid return of the internalized receptor to the cell surface. We examined the nature of this loss of 125I-ASOR surface binding sites by following the fate of receptor molecules after biosynthetic labeling and after cell surface iodination. At 37 degrees C, chloroquine and primaquine induce a loss of asialoglycoprotein receptor molecules from the hepatoma cell surface to an internal pool.
A rapid decrease in epidermal growth factor-binding capacity accompanies the terminal differentiation of mouse myoblasts in vitro.
Authors: Lim R W , Hauschka S D
Published: May 2004

Specific mitogens stimulate the proliferation and repress the differentiation of mouse myoblasts (MM14). When mitogens are depleted, MM14 cells cease proliferation, commit to terminal differentiation, and become refractory to growth stimulation. The behavior of mitogen receptors during the transition from a proliferative to a permanently postmitotic state was examined using the epidermal growth factor receptor (EGFR) as a model system. Whereas proliferating myoblasts bound substantial amounts of EGF, their binding capacity declined rapidly upon exposure to low-mitogen medium. The decline became irreversible when a cell differentiated. Within 24 h, less than 5% of the original EGF binding capacity remained. Since the ability to internalize and degrade bound EGF was unaffected, the change presumably reflected a decrease in EGFR availability. Several observations indicated that loss of EGFR following mitogen removal is related to differentiation rather than the result of starvation or cell-cycle arrest. First, the decline is correlated with the absence of a single mitogen (fibroblast growth factor) and is independent of serum concentrations. Second, myoblasts that are either cycling through G1 or arrested at G0, but prevented from differentiating, all bind large amounts of EGF. These findings suggest that specific reduction in mitogen receptors could be part of a mechanism whereby terminally differentiating cells become refractory to mitogenic stimulation.
Peripheral neurons and Schwann cells secrete plasminogen activator.
Authors: Krystosek A , Seeds N W
Published: May 2004

The secretion of the protease plasminogen activator (PA) by cells of developing peripheral nerve was demonstrated. Fetal and early postnatal dorsal root ganglia were established in culture as explants or as individual neurons and Schwann cells. A fibrin overlay assay was used to visualize the locations of PA secretion. Fibrinolytic zones formed around the somata of explants and were skewed in the direction of maximal fiber outgrowth. Individual growth cones at the tips of long fasiculated fiber bundles also released PA. Approximately 50% of individual neurons showed PA secretion; especially pronounced release occurred at some growth cones. Culture of nerve growth factor-independent adult neurons showed that PA expression was independent of effects of this growth hormone. A subpopulation of Schwann cells was also active in PA secretion, which could be detected at the soma, at the bipolar processes, or along the entire cell length. Possible functions of neural PA in development and regeneration are discussed.
Identification of a calcium-regulated insulinoma cell phosphoprotein as an islet cell keratin.
Authors: Schubart U K , Fields K L
Published: May 2004

This report describes the cytoskeleton nature of a 60,000-mol-wt protein, P60, previously shown to undergo Ca2+ influx-induced phosphorylation concomitant with insulin release in hamster insulinoma cells. Four lines of evidence suggest that P60 is an intermediate filament protein of the keratin class. (a) As previously described (Schubart, U.K., 1982, J. Biol. Chem. 257:12231-12238), Triton X-100-insoluble cytoskeletons are enriched for P60; (b) these cytoskeletons contain 7-11-nm filaments as determined by negative staining; (c) immunoblot analysis revealed that all proteins detected in the insulinoma cell cytoskeletons are recognized by a monoclonal antibody that interacts with a common determinant in all intermediate filament proteins; and (d) P60 was shown, by its identical migration on two-dimensional electrophoresis and by its immunologic relatedness, to be analogous to a known keratin present in HeLa cells. An antibody specific for P60, as judged by immunoblotting, was developed in a rabbit. In indirect immunofluorescence studies on insulinoma cells, this anti-P60 antibody produced a filamentous staining pattern. The antibody also permitted the identification of P60 in normal pancreatic islets as determined both by immunoblotting of hamster islet proteins resolved by two-dimensional electrophoresis and by indirect immunofluorescence microscopy on cryostat sections of hamster pancreas. In addition, the antibody recognized an antigen in the epithelial layer of pancreatic exocrine ducts, as determined by indirect immunofluorescence. The data have implications for the embryonic origin of pancreatic islets. Together with the phosphorylation data, these findings suggest that this islet cell cytokeratin may be involved in the regulation of insulin release.
Specific binding of fibronectin--antifibronectin immune complexes to procollagen: a new pitfall in immunostaining.
Authors: McDonald J A , Kelley D G
Published: May 2004

We observed intense intracellular immunofluorescence of rat lung fibroblasts stained with hybridoma culture supernatant containing monoclonal antibodies to human plasma fibronectin, but no pericellular matrix staining. Immunoprecipitation and absorption experiments revealed that this intracellular staining by hybridoma-conditioned medium was due to binding of fibronectin-antifibronectin immune complexes via the fibronectin to intracellular procollagen. The anomalous staining patterns we encountered were not revealed by the usual controls for immunohistochemical specificity, and also occurred in rat tissue sections. This general phenomena--binding of serum antigens present in hybridoma medium to cellular components--could in principle result in artifactual staining with monoclonal antibodies to other serum components, so investigators using monoclonal antibodies should be aware of this new artifact. Our results also demonstrate that fibronectin binds specifically to native procollagen. Monoclonal antibodies may be useful for studying fibronectin-procollagen and other macromolecular interactions.
Down regulation of epidermal growth factor receptors: direct demonstration of receptor degradation in human fibroblasts.
Authors: Stoscheck C M , Carpenter G
Published: May 2004

The metabolism of the receptor for epidermal growth factor (EGF) has been measured by labeling the receptor in vivo with radioactive amino acid precursors and then determining, by immunoprecipitation with specific anti-EGF receptor antisera, the rate of degradation of the receptor when the cells are placed in a nonradioactive medium. In human fibroblasts the rate of EGF receptor degradation (t1/2 = 10.1 h) was faster than the rate of degradation of total cell protein. When EGF was added to the nonradioactive medium, the half-life of prelabeled receptor was decreased to 1.2 h in human fibroblasts. These data demonstrate by direct analysis of receptor protein that during "down regulation" the EGF receptor is rapidly degraded. Enhanced receptor degradation was observed 5-10 min after the addition of EGF. The EGF-induced degradation of the receptor was blocked by methylamine, chloroquine, iodoacetate, or incubation at 25 degrees C. We have also shown that EGF-induced down regulation in human fibroblasts results in a decrease in the total amount of EGF receptor protein present. The amount of EGF receptor protein has been quantitated by radiolabeling cellular protein and immunoprecipitation of the receptor. The EGF receptor constitutes approximately 0.0035% of the cellular protein in human fibroblasts.
Separation of the osmotically driven fusion event from vesicle-planar membrane attachment in a model system for exocytosis.
Authors: Akabas M H , Cohen F S , Finkelstein A
Published: May 2004

We demonstrate that there are two experimentally distinguishable steps in the fusion of phospholipid vesicles with planar bilayer membranes. In the first step, the vesicles form a stable, tightly bound pre-fusion state with the planar membrane; divalent cations (Ca++) are required for the formation of this state if the vesicular and/or planar membrane contain negatively charged lipids. In the second step, the actual fusion of vesicular and planar membranes occurs. The driving force for this step is the osmotic swelling of vesicles attached (in the pre-fusion state) to the planar membrane. We suggest that osmotic swelling of vesicles may also be crucial for biological fusion and exocytosis.
Attachment of vimentin filaments to desmosomal plaques in human meningiomal cells and arachnoidal tissue.
Authors: Kartenbeck J , Schwechheimer K , Moll R , Franke W W
Published: May 2004

Desmosomal proteins are co-expressed with intermediate-sized filaments (IF) of the cytokeratin type in epithelial cells, and these IF are firmly attached to the desmosomal plaque. In meningiomal and certain arachnoidal cells, however, vimentin IF are attached to desmosomal plaques. Meningiomas obtained after surgery, arachnoid "membranes", and arachnoid granulations at autopsy, as well as meningiomal cells grown in short-term culture have been examined by single and double immunofluorescence and immunoelectron microscopy using antibodies to desmoplakins, vimentin, cytokeratins, glial filament protein, neurofilament protein, and procollagen. In addition, two-dimensional gel electrophoresis of the cytoskeletal proteins has been performed. Using all of these techniques, vimentin was the only IF protein that was detected in significant amounts. The junctions morphologically resembling desmosomes of epithelial cells have been identified as true desmosomes by antibodies specific for desmoplakins and they provided the membrane attachment sites for the vimentin IF. These findings show that anchorage of IF to the cell surface at desmosomal plaques is not restricted to cytokeratin IF as in epithelial cells and desmin IF as in cardiac myocytes, suggesting that binding to desmosomes and hemidesmosomes is a more common feature of IF organization. The co-expression of desmosomal proteins and IF of the vimentin type only defines a new class of cell ("desmofibrocyte") and may also provide an important histodiagnostic criterion.
Extracellular Na+ and initiation of DNA synthesis: role of intracellular pH and K+.
Authors: Burns C P , Rozengurt E
Published: May 2004

Initiation of DNA synthesis in confluent quiescent 3T3 cell cultures stimulated by epidermal growth factor (EGF), vasopressin, and insulin was abolished by removing extracellular Na+. The inhibition was reversible, time- and Na+-concentration-dependent, and not due to an effect on binding or internalization of 125I-EGF. Stimulation by combinations of other growth factors with different mechanisms of action was also affected by decreasing extracellular Na+, but with different half-maximal Na+ concentrations. When choline was used as an osmotic substitute for Na+, the decrease in DNA synthesis was correlated with the decrease in intracellular K+. In contrast, when sucrose was used there was stimulation of the Na+-K+ pump and maintenance of intracellular K+ that resulted in a somewhat higher rate of DNA synthesis at lowered extracellular Na+ compared to choline. Mitogenesis induced by epidermal growth factor, vasopressin, and insulin led to cytoplasmic alkalinization as determined by an increase in uptake of the weak acid 5,5-dimethyloxazolidine-2,4-dione. Experimental decrease in extracellular Na+ blocked this cellular alkalinization. Therefore, under some conditions the supply of extracellular Na+ may limit cellular proliferation because of a reduction in the provision of Na+ to the Na+/H+ antiport and resultant failure of alkalinization. We conclude that Na+ flux and its effect on intracellular K and pH has a major role in the complex system that regulates proliferation.
Studies on the expression of the microtubule-associated protein, tau, during mouse brain development, with newly isolated complementary DNA probes.
Authors: Drubin D G , Caput D , Kirschner M W
Published: May 2004

Tau protein is a collection of closely related polypeptides that associate with microtubules in vivo and stimulate their assembly in vitro. Using an affinity-purified antiserum against bovine brain tau protein, we found that the number and amount of tau polypeptides changes dramatically during mouse brain development. The different forms appear to result from changes in tau mRNA since in vitro translation products reflect the qualitative and quantitative changes found in vivo. To study the mRNA and genomic complexity of tau protein, we used tau mRNA, purified from polysomes with tau antiserum, to isolate embryonic mouse tau complementary DNA clones. With these probes we have determined that embryonic tau protein is translated from a 6-kb mRNA that persists throughout brain development.
Failure to release iron from transferrin in a Chinese hamster ovary cell mutant pleiotropically defective in endocytosis.
Authors: Klausner R D , van Renswoude J , Kempf C , Rao K , Bateman J L , Robbins A R
Published: May 2004

A Chinese hamster ovary cell mutant defective in the receptor-mediated endocytosis of several unrelated ligands (Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064-1071) failed to accumulate iron provided in the form of diferric transferrin. Analysis of the steps of the transferrin cycle indicated that binding and internalization of transferrin proceeded normally in mutant cells. However, the mutant appeared unable to dissociate iron from transferrin, as evidenced by release of diferric transferrin from the mutant versus apotransferrin from the parent. Uptake of ferric ions from the growth medium was enhanced in the mutant.
Immunocytochemical localization of the intrinsic factor-cobalamin receptor in dog-ileum: distribution of intracellular receptor during cell maturation.
Authors: Levine J S , Allen R H , Alpers D H , Seetharam B
Published: May 2004

Absorption of cobalamin is facilitated by the binding of the intrinsic factor-cobalamin complex (IF-cbl) to specific receptors in the ileum. The physical and biochemical characteristics of this ligand-receptor binding reaction have been extensively studied, but little is known about the cellular mechanisms or receptor synthesis, intracellular transport, and expression on the microvillus surface membrane. We attempted to delineate these mechanisms by using ultrastructural immunocytochemistry to localize the IF-cbl receptor in the crypt, mid-villus, and villus tip regions of mucosal biopsies obtained from the ileum of anesthetized dogs. Prior to initiating the ileal localization studies, the antisera to purified canine IF-cbl receptor that was employed in our studies was shown to have specificity for site (e.g., ileal enterocytes vs. other cells within the gastrointestinal tract) and immunohistochemical specificity. Receptor synthesis in endoplasmic reticulum begins in crypt enterocytes, but continues in cells throughout the villus. In the mid-villus region synthesized receptor translocates vectorially to the microvillus surface associated with membranous vesicles and then inserts into the microvillus pit. Receptor remains fixed to the microvillus pit and does not distribute uniformly over the brush border membrane. All villus tip enterocytes contained IF-cbl receptor in microvillus pits, vesicles, and endoplasmic reticulum, but in addition extensive perinuclear membrane staining was evident as well as re-internalized receptor associated with multivesicular bodies. Basolateral membranes contained no receptor at any level of the villus. These observations suggest that the IF-cbl receptor (a) translocates to the apical cell surface at the mid-villus region by transport in vesicles, (b) directly inserts into and then remains fixed in microvillus pits, (c) is elaborated on the luminal surface most extensively in villus tip cells, and (d) although reinternalized, does not move IF and/or cbl to the basolateral cell surface.
Transcription of alpha- and beta-tubulin genes in vitro in isolated Chlamydomonas reinhardi nuclei.
Authors: Keller L R , Schloss J A , Silflow C D , Rosenbaum J L
Published: May 2004

Removal of the flagella of Chlamydomonas results in increases in both flagellar protein synthesis and tubulin messenger RNA accumulation. These observations led us to examine whether flagellar protein gene sequences are transcribed differentially in nuclei isolated before and after deflagellation. A nuclear isolation protocol was developed using the cell wall-less strain of Chlamydomonas, CW 15, after cell lysis with 0.5% Nonidet P-40. Transcriptional activity of isolated nuclei was determined by incorporating [32P]UTP into TCA-precipitable and phenol-extractable RNA, and by hybridizing newly transcribed RNA to complementary DNA clones containing alpha- and beta-tubulin sequences. Nuclei from deflagellated cells are more active in transcribing sequences that hybridize with alpha- and beta-tubulin complementary DNA probes than are nuclei from nondeflagellated cells. In addition, while total [32P]UTP incorporation is inhibited 45% by alpha-amanitin concentrations of 1.0 micrograms/ml, tubulin RNA synthesis in this system is completely inhibited by this concentration of alpha-amanitin. This demonstration of differential transcription in nuclei before and after cell deflagellation provides the means to study in vitro the mechanisms that signal and regulate flagellar protein gene activity.
Monoclonal antibodies as probes of the distribution of ZP-2, the major sulfated glycoprotein of the murine zona pellucida.
Authors: East I J , Dean J
Published: May 2004

Three sulfated glycoproteins (ZP-1, ZP-2, and ZP-3) make up the zona pellucida, an extracellular glycocalyx that surrounds mouse oocytes. We have produced five monoclonal antibodies specific to the zona. All five immunoprecipitated ZP-2, and in addition, two of the antibodies immunoprecipitated ZP-3. This suggests the presence of either a common antigenic site or one made up in part by each of the two glycoproteins. The monoclonal antibodies bound to approximately 1.3 X 10(8) binding sites per ovulated mouse egg which represents 2% of the total number of ZP-2 molecules present in the zona. ZP-2 appeared to be present throughout the zona and indirect immunofluorescence revealed a fibrous pattern with no evidence of localization. Furthermore, this pattern of distribution, which was identical for all five monoclones, remained constant after fertilization at the two-cell embryo stage. Laser photobleaching demonstrated that ZP-2 is stably integrated in the extracellular matrix of the zona pellucida. No mouse tissue other than the ovary contained ZP-2 and ZP-2 is antigenically distinct from other previously described extracellular matrix proteins.
Insulin-induced formation of ruffling membranes of KB cells and its correlation with enhancement of amino acid transport.
Authors: Goshima K , Masuda A , Owaribe K
Published: May 2004

Insulin induced the formation of ruffling membranes in cultured KB cells (a cell strain derived from human epidermoid carcinoma) within 1-2 min after its addition. The ruffled regions were stained strongly with antibody to actin but not that to tubulin. Pretreatment of KB cells with agents disrupting microfilaments (cytochalasins), but not with those disrupting microtubules (colcemid, nocodazole, and colchicine) completely inhibited the formation of ruffling membranes. Pretreatment of KB cells with dibutyryl cyclic AMP, but not with dibutyryl cyclic GMP, also inhibited the formation of ruffling membranes. Addition of insulin enhanced Na+-dependent uptake of a system A amino acid (alpha-amino isobutyric acid; AIB) by the cells within 5 min after the addition, and decreased the cyclic AMP content of the cells. Treatments that inhibited insulin-induced formation of ruffling membranes of KB cells also inhibited insulin-induced enhancement of their AIB uptake. From these observations, the mechanism of insulin-induced formation of ruffling membranes and its close correlation with AIB transport are discussed.