bluuebunny/crossref_metadata_2025 huggingface dataset:
Mouse L cells expressing human prourokinase-type plasminogen activator: effects on extracellular matrix degradation and invasion.
Authors: Cajot J F , Schleuning W D , Medcalf R L , Bamat J , Testuz J , Liebermann L , Sordat B
Published: May 2004

A cosmid (cos pUK0322) harboring the complete human urokinase-type plasminogen activator (u-PA) gene and Geneticin resistance as a selectable marker was isolated from a human genomic library and characterized. After transfection of cos pUK0322 into mouse L cells and selection, several plasminogen activator (PA)-expressing clones were obtained and one (LuPA) was chosen for additional study. The PA expressed was identical to human pro-u-PA in enzymatic, electrophoretic, and antigenic properties. The expression of PA was stable over 50 population doublings. The regulation of the transfected gene was studied by treatment of the cells with various hormones and other effectors. Expression of PA activity was inhibited fivefold by dexamethasone and stimulated two- to threefold by agonists of the adenylate cyclase dependent pathway of signal transduction, such as dibutyryl cyclic AMP and cholera and pertussis toxins. The modulation of PA activity was associated with corresponding changes in mRNA steady-state levels. The phenotypic changes associated with pro-u-PA expression were analyzed in vitro by degradation of 3H-labeled extracellular matrix (ECM), invasion of a matrigel basement membrane analogue, and by light and electron microscopy. LuPA cells and reference HT-1080 fibrosarcoma cells, in contrast to control Lneo cells transfected with the neomycin resistance gene, degraded the ECM and invaded the matrigel basement membrane. Matrix degradation correlated with the modulation of pro-u-PA gene expression as it was inhibited by dexamethasone and promoted by dibutyryl cyclic AMP. Inhibition of PA or plasmin using anti-u-PA IgG or aprotinin prevented ECM degradation and invasion. These results demonstrate that u-PA expression alone is sufficient to confer to a cell an experimental invasive phenotype.
The GTP-binding protein Ypt1 is required for transport in vitro: the Golgi apparatus is defective in ypt1 mutants.
Authors: Bacon R A , Salminen A , Ruohola H , Novick P , Ferro-Novick S
Published: May 2004

The YPT1 gene encodes a raslike, GTP-binding protein that is essential for growth of yeast cells. We show here that mutations in the ypt1 gene disrupt transport of carboxypeptidase Y to the vacuole in vivo and transport of pro-alpha-factor to a site of extensive glycosylation in the Golgi apparatus in vitro. Two different ypt1 mutations result in loss of function of the Golgi complex without affecting the activity of the endoplasmic reticulum or soluble components required for in vitro transport. The function of the mutant Golgi apparatus can be restored by preincubation with wild-type cytosol. The transport defect observed in vitro cannot be overcome by addition of Ca++ to the reaction mixture. We have also established genetic interactions between ypt1 and a subset of the other genes required for transport to and through the Golgi apparatus.
Serum factors alter the extent of dephosphorylation of ligands endocytosed via the mannose 6-phosphate/insulin-like growth factor II receptor.
Authors: Einstein R , Gabel C A
Published: May 2004

Mouse L-cells that contain the cation-independent (CI) mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor endocytose acid hydrolases and deliver these enzymes to lysosomes. The postendocytic loss of the Man 6-P recognition marker from the cell-associated acid hydrolases was assessed by CI-Man 6-P receptor affinity chromatography. 125I-labeled acid hydrolases internalized by L-cells grown at high density were delivered to lysosomes but were not dephosphorylated. In contrast, the same 125I-labeled hydrolases internalized by L-cells maintained at low density were delivered to lysosomes and were extensively dephosphorylated. The dephosphorylation at low density required 5 h for completion suggesting that the phosphatase responsible for the dephosphorylation is located within the lysosomal compartment. Transition from the high to low density state was rapid and was not inhibited by cycloheximide. Medium substitution experiments indicated that serum factors were necessary to maintain the L-cells in the dephosphorylation-competent (low density) state, and that serum-free conditions led to a dephosphorylation-incompetent (high density) state. Addition of IGF II to cells in serum-free medium allowed acid hydrolases subsequently introduced by endocytosis to be dephosphorylated. The results indicate that the removal of the Man 6-P recognition marker from endocytosed acid hydrolases is regulated by serum factors in the growth medium, including IGF II.
ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells.
Authors: Anderson J M , Van Itallie C M , Peterson M D , Stevenson B R , Carew E A , Mooseker M S
Published: May 2004

We previously identified and characterized ZO-1 as a peripheral membrane protein specifically associated with the cytoplasmic surface of tight junctions. Here we describe the identification of partial cDNA sequences encoding rat and human ZO-1 and their use to study the assembly of tight junctions in the Caco-2 human intestinal epithelial cell line. A rat cDNA was isolated from a lambda-gtll expression library by screening with mAbs. Polyclonal antibodies were raised to cDNA-encoded fusion protein; several properties of these antibodies support this cDNA as encoding ZO-1. Expression of ZO-1 mRNA occurs in the rat and Caco-2 cells with a major transcript of approximately 7.5 kb. To disrupt tight junctions and study the subsequent process of assembly, Caco-2 cells were grown in suspension for 48 h in Ca++/Mg++-free spinner medium during which time they lose cell-cell contacts, become round, and by immunofluorescence microscopy show diffuse and speckled localization of ZO-1. Within hours of replating at confluent density in Ca++/Mg++-containing media, attached cells show discrete localization of ZO-1 at cell-cell contacts. Within 2 d, fully confluent monolayers form, and ZO-1 localizes in a continuous gasket-like fashion circumscribing all cells. ZO-1 mRNA levels are highest in cells in spinner culture, and upon replating rapidly fall and plateau at approximately 10% of initial levels after 2-3 wk in culture. ZO-1 protein levels are lowest in contact-free cells and rise five- to eightfold over the same period. In contrast, mRNA levels for sucrase-isomaltase, an apical membrane hydrolase, increase only after a confluent monolayer forms. Thus, in this model of contact-dependent assembly of the tight junction, there is both a rapid assembly beginning upon cell-cell contact, as well as a long-term modulation involving changes in expression of ZO-1 mRNA and protein levels.
The cortical microfilament system of lymphoblasts displays a periodic oscillatory activity in the absence of microtubules: implications for cell polarity.
Authors: Bornens M , Paintrand M , Celati C
Published: May 2004

For an understanding of the role of microtubules in the definition of cell polarity, we have studied the cell surface motility of human lymphoblasts (KE37 cell line) using video microscopy, time-lapse photography, and immunofluorescent localization of F-actin and myosin. Polarized cell surface motility occurs in association with a constriction ring which forms on the centrosome side of the cell: the cytoplasm flows from the ring zone towards membrane veils which keep protruding in the same general direction. This association is ensured by microtubules: in their absence the ring is conspicuous and moves periodically back and forth across the cell, while a protrusion of membrane occurs alternately at each end of the cell when the ring is at the other. This oscillatory activity is correlated with a striking redistribution of myosin towards a cortical localization and appears to be due to the alternate flow of cortical myosin associated with the ring and to the periodic assembly of actin coupled with membrane protrusion. The ring cycle involves the progressive recruitment of myosin from a polar accumulation, or cap, its transportation across the cell and its accumulation in a new cap at the other end of the cell, suggesting an assembly-disassembly process. Inhibition of actin assembly induces, on the other hand, a dramatic microtubule-dependent cell elongation with definite polarity, likely to involve the interaction of microtubules with the cell cortex. We conclude that the polarized cell surface motility in KE37 cells is based on the periodic oscillatory activity of the actin system: a myosin-powered equatorial contraction and an actin-based membrane protrusion are concerted at the cell level and occur at opposite ends of the cell in absence of microtubules. This defines a polarity which reverses periodically as the ring moves across the cell. Microtubules impose a stable cell polarity by suppressing the ring movement. A permanent association of the myosin-powered contraction and the membrane protrusion is established which results in the unidirectional activity of the actin system. Microtubules exert their effect by controlling the recruitment of cytoplasmic myosin into the cortex, probably through their direct interaction with the cortical microfilament system.
Three-dimensional image reconstruction of insect flight muscle. I. The rigor myac layer.
Authors: Taylor K A , Reedy M C , Córdova L , Reedy M K
Published: May 2004

We have obtained detailed three-dimensional images of in situ cross-bridge structure in insect flight muscle by electron microscopy of multiple tilt views of single filament layers in ultrathin sections, supplemented with data from thick sections. In this report, we describe the images obtained of the myac layer, a 25-nm longitudinal section containing a single layer of alternating myosin and actin filaments. The reconstruction reveals averaged rigor cross-bridges that clearly separate into two classes constituting lead and rear chevrons within each 38.7-nm axial repeat. These two classes differ in tilt angle, size and shape, density, and slew. This new reconstruction confirms our earlier interpretation of the lead bridge as a two-headed cross-bridge and the rear bridge as a single-headed cross-bridge. The importance of complementing tilt series with additional projections outside the goniometer tilt range is demonstrated by comparison with our earlier myac layer reconstruction. Incorporation of this additional data reveals new details of rigor cross-bridge structure in situ which include clear delineation of (a) a triangular shape for the lead bridge, (b) a smaller size for the rear bridge, and (c) density continuity across the thin filament in the lead bridge. Within actin's regular 38.7-nm helical repeat, local twist variations in the thin filament that correlate with the two cross-bridge classes persist in this new reconstruction. These observations show that in situ rigor cross-bridges are not uniform, and suggest three different myosin head conformations in rigor.
Three-dimensional image reconstruction of insect flight muscle. II. The rigor actin layer.
Authors: Taylor K A , Reedy M C , Córdova L , Reedy M K
Published: May 2004

The averaged structure of rigor cross-bridges in insect flight muscle is further revealed by three-dimensional reconstruction from 25-nm sections containing a single layer of thin filaments. These exhibit two thin filament orientations that differ by 60 degrees from each other and from myac layer filaments. Data from multiple tilt views (to +/- 60 degrees) was supplemented by data from thick sections (equivalent to 90 degrees tilts). In combination with the reconstruction from the myac layer (Taylor et al., 1989), the entire unit cell is reconstructed, giving the most complete view of in situ cross-bridges yet obtained. All our reconstructions show two classes of averaged rigor cross-bridges. Lead bridges have a triangular shape with leading edge angled at approximately 45 degrees and trailing edge angled at approximately 90 degrees to the filament axis. We propose that the lead bridge contains two myosin heads of differing conformation bound along one strand of F-actin. The lead bridge is associated with a region of the thin filament that is apparently untwisted. We suggest that the untwisting may reflect the distribution of strain between myosin and actin resulting from two-headed, single filament binding in the lead bridge. Rear bridges are oriented at approximately 90 degrees to the filament axis, and are smaller and more cylindrical, suggesting that they consist of single myosin heads. The rear bridge is associated with a region of apparently normal thin filament twist. We propose that differing myosin head angles and conformations consistently observed in rigor embody different stages of the power stroke which have been trapped by a temporal sequence of rigor cross-bridge formation under the constraints of the intact filament lattice.
Guanine nucleotide-induced polymerization of actin in electropermeabilized human neutrophils.
Authors: Therrien S , Naccache P H
Published: May 2004

The effects of exogenous guanine nucleotides on the polymerization of actin in human neutrophils were tested in an electropermeabilized cell preparation. Close to 40% permeabilization was achieved with a single electric discharge as measured by nucleic acid staining with ethidium bromide or propidium iodide with minimal (less than 2%) release of the cytoplasmic marker lactate dehydrogenase. In addition, electropermeabilized neutrophils retained their capacity to produce superoxide anions and to sustain a polymerization of actin in response to surface-receptor dependent stimuli such as chemotactic factors. Electropermeabilization produced a rapid and transient permeabilization that allowed the entry of guanine nucleotides into the cells. GTP and, to a larger extent, its nonhydrolyzable analog guanosine 5'-O-2-thiotriphosphate (GTP[S]), induced a time- and concentration-dependent polymerization of actin, as determined by increased staining with 7-nitrobenz-2-oxa-1,3-diazolylphallacidin. The effects of the aforementioned guanine nucleotides were antagonized by GDP[S], but were insensitive to pertussis toxin. Cholera toxin potentiated to a small degree the amount of actin polymerization induced by GTP[S]. These results provided direct evidence for the involvement of GTP-binding proteins in the regulation of the organization of the cytoskeleton of neutrophils, an event that is of crucial importance to the performance of the defense-oriented functions of these cells.
Influence of botulinum C2 toxin on F-actin and N-formyl peptide receptor dynamics in human neutrophils.
Authors: Norgauer J , Just I , Aktories K , Sklar L A
Published: May 2004

Stimulation of human neutrophils with the chemotactic N-formyl peptide causes production of oxygen radicals and conversion of monomeric actin (G-actin) to polymeric actin (F-actin). The effects of the binary botulinum C2 toxin on the amount of F-actin and on neutrophil cell responses were studied. Two different methods for analyzing the actin response were used in formyl peptide-stimulated cells: staining of F-actin with rhodamine-phalloidin and a transient right angle light scatter. Preincubation of neutrophils with 400 ng/ml component I and 1,600 ng/ml component II of botulinum C2 toxin for 30 min almost completely inhibited the formyl peptide-stimulated polymerization of G-actin and at the same time decreased the amount of F-actin in unstimulated neutrophils by an average of approximately 30%. Botulinum C2 toxin preincubation for 60 min destroyed approximately 75% of the F-actin in unstimulated neutrophils. Right angle light scatter analysis showed that control neutrophils exhibited the transient response characteristic of actin polymerization; however, after botulinum C2 toxin treatment, degranulation was detected. Single components of the binary botulinum C2 toxin were without effect on the actin polymerization response. Fluorescence flow cytometry and fluorospectrometric binding studies showed little alteration in N-formyl peptide binding or dissociation dynamics in the toxin-treated cells. However, endocytosis of the fluorescent N-formyl peptide ligand-receptor complex was slower but still possible in degranulating neutrophils treated with botulinum C2 toxin for 60 min. The half-time of endocytosis, estimated from initial rates, was 4 and 8 min in control and botulinum C2 toxin-treated neutrophils, respectively.
Probing the role of nonmuscle tropomyosin isoforms in intracellular granule movement by microinjection of monoclonal antibodies.
Authors: Hegmann T E , Lin J L , Lin J J
Published: May 2004

Chicken embryo fibroblast (CEF) cells were microinjected with several different monoclonal antibodies that recognize certain nonmuscle isoforms of tropomyosin. Immediately after injection, cells were recorded with a time-lapse video imaging system; later analysis of the tapes revealed that particles in cells injected with one of these antibodies (CG1, specific for CEF tropomyosin isoforms 1 and 3) showed a dramatic decrease in instantaneous speed while moving, distance moved per saltation, and proportion of time spent in motion. Injection of Fab fragments of CG1 resulted in similar changes in the pattern of granule movement. This inhibition of granule movement by CG1 antibody was reversible; at 2.5 h after injection, granules in injected cells had already reached three-fourths of normal speed. The speed of granule movement in cells injected either with antibody specific for tropomyosin isoforms not present in CEF cells, or with CG1 antibody preabsorbed with tropomyosin, was not significantly different from the speed of granules in uninjected cells. When cells were injected with CG1 or Fab fragments of CG1, fixed, and counter-stained with rabbit antibodies to reveal the microtubule, microfilament, and intermediate filament systems, no obvious differences from the patterns normally seen in uninjected cells were observed. Examination of the ultrastructure of injected cells by EM confirmed the presence of apparently intact and normal microtubule, actin, and intermediate filament networks. These experiments suggest that tropomyosin may play an important role in the movement of vesicles and organelles in the cell cytoplasm. Also, we have shown previously that the CG1 determinant can undergo a motility-dependent change in reactivity, that may be important for the regulatory function of nonmuscle tropomyosin (Hegmann, T. E., J. L.-C. Lin, and J. J.-C. Lin. 1988. J. Cell Biol. 106:385-393). Therefore, in addition to postulated microtubule-based motors, microfilaments may play a critical role in regulating granule movement in nonmuscle cells.
Characterization of intestinal microvillar membrane disks: detergent-resistant membrane sheets enriched in associated brush border myosin I (110K-calmodulin).
Authors: Mooseker M S , Conzelman K A , Coleman T R , Heuser J E , Sheetz M P
Published: May 2004

The actin bundle within each microvillus of the intestinal brush border (BB) is tethered laterally to the membrane by bridges composed of BB myosin I. Avian BB myosin I, formerly termed 110K-calmodulin, consists of a heavy chain with an apparent Mr of 110 kD and three to four molecules of calmodulin "light chains." Recent studies have shown that this complex shares many properties with myosin including mechanochemical activity. In this report, the isolation and characterization of a membrane fraction enriched in bound BB myosin I is described. This membrane fraction, termed microvillar membrane disks, was purified from ATP extracts of nonionic detergent-treated microvilli prepared from avian intestinal BBs. Ultrastructural analysis revealed that these membranes are flat, disk-shaped sheets with protrusions which are identical in morphology to purified BB myosin I. The disks exhibit actin-activated Mg-ATPase activity and bind and cross-link actin filaments in an ATP-dependent fashion. The mechanochemical activity of the membrane disks was assessed using the Nitella bead movement assay (Sheetz, M. P., and J. A. Spudich. 1983. Nature [Lond.]. 303:31-35). These preparations were shown to be free of significant contamination by conventional BB myosin. Latex beads coated with microvillar membrane disks move in a myosin-like fashion along Nitella actin cables at rates of 12-60 nm/s (average rate of 33 nm/s); unlike purified BB myosin I, the movement of membrane disk-coated beads was most reproducibly observed in buffers containing low Ca2+.
Formation and movement of myosin-containing structures in living fibroblasts.
Authors: McKenna N M , Wang Y L , Konkel M E
Published: May 2004

Gizzard myosin, fluorescently labeled with tetramethylrhodamine iodoacetamide, was microinjected into living 3T3 fibroblasts to label myosin-containing structures. The fluorophore was located predominantly on the heavy chain near the COOH terminus of the S1 head and on the 17-kD light chain. After microinjection of a tracer amount into living 3T3 cells, the fluorescent myosin showed a distribution identical to that revealed by immunofluorescence with antimyosin antibodies. Injected myosin became localized in small beads, which were found along large stress fibers, along fine fibers, and in a poorly organized form near the lamellipodia. De novo assembly of beads was observed continuously within or near the lamellipodia, suggesting that myosin molecules may undergo a constant cycling between polymerized and unpolymerized states. The nascent structures then moved away from lamellipodia and became organized into linear arrays. Similar movement was also observed for beads already associated with linear structures, and may represent a continuous flux of myosin structures. The dynamic reorganization of myosin may play an important role in cell movement and polarity.
Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro.
Authors: Yuspa S H , Kilkenny A E , Steinert P M , Roop D R
Published: May 2004

Epidermal differentiation is characterized by a series of coordinated morphological and biochemical changes which result in a highly specialized, highly organized, stratified squamous epithelium. Among the specific markers expressed in differentiating epidermis are (a) two early spinous cell proteins, keratins 1 and 10 (K1 and K10); and (b) two later granular cell proteins, filaggrin and a cornified envelope precursor (CE). In vitro, epidermal basal cells are selectively cultured in 0.05 mM Ca2+ medium, and terminal differentiation is induced when the Ca2+ concentration is increased to 1 mM. However, only a small fraction of the cells express the markers K1, K10, CE, or filaggrin in the higher Ca2+ medium. To explore the factors required for marker expression, cultured epidermal cells were exposed to intermediate Ca2+ concentrations and extracts were analyzed using specific antibody and nucleic acid probes for the four markers of interest. These studies revealed that marker expression was enhanced at a restricted concentration of Ca2+ in the medium of 0.10-0.16 mM. At this Ca2+ concentration, both protein and mRNA levels for each marker were substantially increased, whereas at higher or lower Ca2+ concentrations they were diminished or undetected. The percentage of cells expressing each marker was increased two- to threefold in the permissive Ca2+ medium as determined by immunofluorescence analysis. This optimal level of Ca2+ was required both to initiate and sustain marker expression. At the permissive Ca2+ concentration, expression of the markers was sequential and similar to the order of appearance in vivo. K1 was expressed within 8-12 h and K10 was expressed in the ensuing 12-24-h period. CE and filaggrin were expressed in the subsequent 24 h. Inhibition of K1 expression by cycloheximide suggested that an inducible protein was involved. Other investigators have determined that a shallow Ca2+ gradient exists in epidermis, where the basal cells and spinous cells are in a Ca2+ environment substantially below serum Ca2+ levels. These in vitro results suggest that the Ca2+ environment is a fundamental regulator of expression of epidermal differentiation markers and provide an explanation for the existence of the Ca2+ gradient in vivo.
Simultaneous measurements of cytosolic calcium and secretion in single bovine adrenal chromaffin cells by fluorescent imaging of fura-2 in cocultured cells.
Authors: Cheek T R , Jackson T R , O'Sullivan A J , Moreton R B , Berridge M J , Burgoyne R D
Published: May 2004

The cytosolic free calcium concentration ([Ca2+]i) and exocytosis of chromaffin granules were measured simultaneously from single, intact bovine adrenal chromaffin cells using a novel technique involving fluorescent imaging of cocultured cells. Chromaffin cell [Ca2+]i was monitored with fura-2. To simultaneously follow catecholamine secretion, the cells were cocultured with fura-2-loaded NIH-3T3t cells, a cell line chosen because of their irresponsiveness to chromaffin cell secretagogues but their large Ca2+ response to ATP, which is coreleased with catecholamine from the chromaffin cells. In response to the depolarizing stimulus nicotine (a potent secretagogue), chromaffin cell [Ca2+]i increased rapidly. At the peak of the response, [Ca2+]i was evenly distributed throughout the cell. This elevation in [Ca2+]i was followed by a secretory response which originated from the entire surface of the cell. In response to the inositol 1,4,5-trisphosphate (InsP3)-mobilizing agonist angiotensin II (a weak secretagogue), three different responses were observed. Approximately 30% of chromaffin cells showed no rise in [Ca2+]i and did not secrete. About 45% of the cells responded with a large (greater than 200 nM), transient elevation in [Ca2+]i and no detectable secretory response. The rise in [Ca2+]i was nonuniform, such that peak [Ca2+]i was often recorded only in one pole of the cell. And finally, approximately 25% of cells responded with a similar Ca2+-transient to that described above, but also gave a secretory response. In these cases secretion was polarized, being confined to the pole of the cell in which the rise in [Ca2+]i was greatest.(ABSTRACT TRUNCATED AT 250 WORDS)
The apolipoprotein A-I gene is actively expressed in the rapidly myelinating avian peripheral nerve.
Authors: LeBlanc A C , Földvári M , Spencer D F , Breckenridge W C , Fenwick R G , Williams D L , Mezei C
Published: May 2004

The expression of the apolipoprotein A-I (apo A-I) gene was investigated in the myelinating sciatic nerve. Hybridization analysis with an apo A-I cDNA probe obtained from a cDNA library of mRNA isolated from rapidly myelinating chick sciatic nerve indicated that apo A-I coding transcripts increase during development in the chick sciatic nerve in parallel with the increase of myelin lamellae. Substantial apo A-I-like immunoreactivity in chick sciatic nerve homogenates was detected by Western blotting. The amount of antigen increased from the 15-d embryonic stage to 1 d posthatch and then decreased. Two subcellular fractions corresponding to the cytoplasmic compartments were particularly enriched in apo A-I. apo A-I immunoreactivity was also found in highly purified myelin preparations. Immunohistochemical staining provided further evidence for the presence of apo A-I in the endoneurial compartment of the sciatic nerve. Electron microscopic examination of these fractions after negative staining showed the presence of spherical and disc-shaped particles resembling high density lipoproteins. The presence of apo A-I, cholesterol esters, phospholipids, and triacylglycerols in ultracentrifugal fractions corresponding to serum lipoproteins and the behavior of apo A-I on nondenaturing gradient gels implied that apo A-I was associated with lipid. Studies with short-term organ cultures of sciatic nerves from 1-d chicks strengthened the evidence for local synthesis and secretion of apo A-I and apo A-I-containing lipoproteins by this tissue. These results establish that the apo A-I gene is actively expressed in developing sciatic nerve during the period of rapid myelination. These findings support the hypothesis that apo A-I synthesized within the nerve participates in the local transport of lipids used in myelin biosynthesis.
Novel alpha-D-mannosidase of rat sperm plasma membranes: characterization and potential role in sperm-egg interactions.
Authors: Tulsiani D R , Skudlarek M D , Orgebin-Crist M C
Published: May 2004

During the course of a study of glycoprotein processing mannosidases in the rat epididymis, we have made an intriguing discovery regarding the presence of a novel alpha-D-mannosidase on the rat sperm plasma membranes. Unlike the sperm acrosomal "acid" mannosidase which has a pH optimum of 4.4, the newly discovered alpha-D-mannosidase has a pH optimum of 6.2, and 6.5 when assayed in sperm plasma membranes and intact spermatozoa, respectively. In addition, the two enzymes show different substrate specificity. The acrosomal alpha-D-mannosidase is active mainly towards synthetic substrate, p-nitrophenyl alpha-D-mannopyranoside, whereas the sperm plasma membrane alpha-D-mannosidase shows activity mainly towards mannose-containing oligosaccharides. Evidence is presented which suggest that the sperm plasma membrane alpha-D-mannosidase is different from several processing mannosidases previously characterized from the rat liver. The newly discovered alpha-D-mannosidase appears to be an intrinsic plasma membrane component, since washing of the purified membranes with buffered 0.4 M NaCl did not release the enzyme in soluble form. The enzyme requires nonionic detergent (Triton X-100) for complete solubilization. The enzyme is activated by Co2+ and Mn2+. However, Cu2+ and Zn2+ are potent inhibitors of the sperm plasma membrane alpha-D-mannosidase. At a concentration of 0.1 mM, these divalent cations caused nearly complete inactivation of the sperm enzyme. In addition methyl-alpha-D-mannoside, methyl-alpha-D-glucoside, mannose, 2-deoxy-D-glucose, and D-mannosamine are inhibitors of the sperm surface alpha-D-mannosidase. The physiological role of the newly discovered enzyme is not yet known. Several published reports in three species, including the rat, suggest that the sperm surface alpha-D-mannosidase may have a role in binding to mannose-containing saccharides presumably present on the zona pellucida.
Identification and characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin.
Authors: Wayner E A , Garcia-Pardo A , Humphries M J , McDonald J A , Carter W G
Published: May 2004

Using mAb technology (Wayner, E. A., W. G. Carter, R. Piotrowicz, and T. J. Kunicki. 1988. J. Cell Biol. 107:1881-1891), we have identified a new fibronectin receptor that is identical to the integrin receptor alpha 4 beta 1. mAbs P3E3, P4C2, and P4G9 recognized epitopes on the alpha 4 subunit and completely inhibited the adhesion of peripheral blood and cultured T lymphocytes to a 38-kD tryptic fragment of plasma fibronectin containing the carboxy-terminal Heparin II domain and part of the type III connecting segment (IIICS). The ligand in IIICS for alpha 4 beta 1 was the CS-1 region previously defined as an adhesion site for melanoma cells. The functionally defined mAbs to alpha 4 partially inhibited T lymphocyte adhesion to intact plasma fibronectin and had no effect on their attachment to an 80-kD tryptic fragment containing the RGD (arg-gly-asp) adhesion sequence. mAbs (P1D6 and P1F8) to the previously described fibronectin receptor, alpha 5 beta 1, completely inhibited T lymphocyte adhesion to the 80-kD fragment but had no effect on their attachment to the 38-kD fragment or to CS-1. Both alpha 4 beta 1 and alpha 5 beta 1 localized to focal adhesions when fibroblasts that express these receptors were grown on fibronectin-coated surfaces. These findings demonstrated a specific interaction of both receptors with fibronectin at focal contacts. In conclusion, these findings show clearly that cultured T lymphocytes use two independent receptors during attachment to fibronectin and that (a) alpha 5 beta 1 is the receptor for the RGD containing cell adhesion domain, and (b) alpha 4 beta 1 is the receptor for a carboxy-terminal cell adhesion region containing the Heparin II and IIICS domains. Furthermore, these data also show that T lymphocytes express a clear preference for a region of molecular heterogeneity in IIICS (CS-1) generated by alternative splicing of fibronectin pre-mRNA and that alpha 4 beta 1 is the receptor for this adhesion site.
Regulation of macrophage growth and antiviral activity by interferon-gamma.
Authors: Goldberg M , Belkowski L S , Bloom B R
Published: May 2004

Interferons, in addition to their antiviral activity, induce a multiplicity of effects on different cell types. Interferon (IFN)-gamma exerts a unique regulatory effect on cells of the mononuclear phagocyte lineage. To investigate whether the antiviral and antiproliferative effects of IFN-gamma in macrophages can be genetically dissociated, and whether IFN-alpha and IFN-gamma use the same cellular signals and/or effector mechanisms to achieve their biologic effects, we have derived a series of somatic cell genetic variants resistant to the antiproliferative and/or antiviral activities of IFN-gamma. Two different classes of variants were found: those resistant to the antiproliferative and antiviral effects of IFN-gamma against vesicular stomatitis virus (VSV) and those resistant to the antiproliferative effect, but protected against VSV and encephalomyocarditis virus (EMCV) lysis by IFN-gamma. In addition, a third class of mutants was obtained that was susceptible to the growth inhibitory activity, but resistant to the antiviral activity of IFN-gamma. Analysis of these mutants has provided several insights regarding the regulatory mechanisms of IFN-gamma and IFN-alpha on the murine macrophage cell lines. The antiproliferative activity of IFN-gamma on these cells, in contrast to that of IFN-alpha, is mediated by a cAMP-independent pathway. The antiproliferative and antiviral activities of IFN-gamma were genetically dissociated. Variants were obtained that are growth resistant but antivirally protected, or are growth inhibited but not antivirally protected against VSV or EMCV. The genetic analysis indicated that IFN-alpha and IFN-gamma regulate the induction of the dsRNA-dependent P1/eIF-2 alpha protein kinase and 2',5'-oligoadenylate synthetase enzymatic activities via different pathways. Finally, a unique macrophage mutant was obtained that was protected by IFN-gamma against infection by VSV, but not EMCV, suggesting that antiviral mechanisms involved in protection against these different types of RNA viruses must be distinct at some level.
Cytokine-induced respiratory burst of human neutrophils: dependence on extracellular matrix proteins and CD11/CD18 integrins.
Authors: Nathan C , Srimal S , Farber C , Sanchez E , Kabbash L , Asch A , Gailit J , Wright S D
Published: May 2004

Human polymorphonuclear leukocytes (PMN) released large quantities of hydrogen peroxide in response to tumor necrosis factor, but only when the cells were adherent to surfaces coated with extracellular matrix proteins. The PMN did not respond when exposed to cytokines and matrix proteins in suspension, or when exposed to cytokines while adherent to surfaces coated with stearic acid. PMN from children with genetic deficiency of the CD11/CD18 integrins underwent a normal respiratory burst upon adherence to uncoated polystyrene, but not in response to tumor necrosis factor when tested on polystyrene that was coated with serum, fibronectin, vitronectin, fibrinogen, thrombospondin, or laminin. Anti-CD18 antibodies, alone of sixteen antibodies tested, induced a similar defect in PMN from normal donors, when the PMN were tested on surfaces coated with serum, fibrinogen, thrombospondin, or laminin; no defect was induced by the anti-CD18 monoclonal antibody IB4 in normal PMN tested on surfaces coated with fibronectin or vitronectin. Thus, for cytokines to induce a respiratory burst in PMN, the cells must be able to use CD11/CD18 integrins and must interact with matrix proteins in the solid phase. CD11/CD18, which is already known to serve as a receptor for fibrinogen, may also be a receptor for thrombospondin and laminin. Finally, receptor(s) exist on PMN for fibronectin and vitronectin which are not blocked by the anti-CD18 antibody IB4 but which are nonetheless CD11/CD18 dependent.
Cellular heterogeneity in cultured human chondrocytes identified by antibodies specific for alpha 2(XI) collagen chains.
Authors: Swoboda B , Holmdahl R , Stöss H , von der Mark K
Published: May 2004

Collagen type XI is a component of hyaline cartilage consisting of alpha 1(XI), alpha 2(XI), and alpha 3(XI) chains; with 5-10% of the total collagen content, it is a minor but significant component next to type II collagen, but its function and precise localization in cartilaginous tissues is still unclear. Owing to the homology of the alpha 3(XI) and alpha 1(II) collagen chains, attempts to prepare specific antibodies to native type XI collagen have been unsuccessful in the past. In this study, we report on the preparation and use for immunohistochemistry of a polyclonal antibody specific for alpha 2(XI) denatured collagen chains. The antibody was prepared by immunization with the isolated alpha 2(XI) chain and reacts neither with native type XI collagen nor type I, II, V, or IX by ELISA or immunoblotting, nor with alpha 1(XI) or alpha 3(XI), but with alpha 2(XI) chains. Using this antibody, it was possible to specifically localize alpha 2(XI) in cartilage by pretreating tissue sections with 6 M urea. In double immunofluorescence staining experiments, the distribution of alpha 2(XI) as indicative for type XI collagen in fetal bovine and human cartilage was compared with that of type II collagen, using a monoclonal antibody to alpha 1(II). Type XI collagen was found throughout the matrix of hyaline cartilage. However, owing to cross-reactivity of the monoclonal anti-alpha 1(II) with alpha 3(XI), both antibodies produced the same staining pattern. Cellular heterogeneity was, however, detected in monolayer cultures of human chondrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Expression of mRNAs coding for the alpha 1 chain of type XIII collagen in human fetal tissues: comparison with expression of mRNAs for collagen types I, II, and III.
Authors: Sandberg M , Tamminen M , Hirvonen H , Vuorio E , Pihlajaniemi T
Published: May 2004

This paper describes the topographic distribution of the multiple mRNAs coding for a novel human short-chain collagen, the alpha 1 chain of type XIII collagen. To identify the tissues and cells expressing these mRNAs, human fetal tissues of 15-19 gestational wk were studied by Northern and in situ hybridizations. The distribution pattern of the type XIII collagen mRNAs was compared with that of fibrillar collagen types I, II, and III using specific human cDNA probes for each collagen type. Northern hybridization showed the bone, cartilage, intestine, skin, and striated muscle to contain mRNAs for type XIII collagen. An intense in situ hybridization signal was obtained with the type XIII collagen cDNAs in the epidermis, hair follicles, and nail root cells of the skin, whereas the fibrillar collagen mRNAs were detected in the dermis. Cells in the intestinal mucosal layer also appeared to contain high levels of alpha 1(XIII) collagen mRNAs, but contained none of the fibrillar collagen mRNAs. In the bone and striated muscle, alpha 1(XIII) collagen mRNAs were detected in the mesenchymal cells forming the reticulin fibers of the bone marrow and endomycium. The hybridization signal obtained with the alpha 1(XIII) collagen cDNA probe in cartilaginous areas of the growth plates was similar, but less intense, to that obtained with the type II collagen probe. A clear hybridization signal was also detected at the (pre)articular surfaces and at the margins of the epiphyses, whereas it was weaker in the resting chondrocytes in the middle of the epiphyses. The brain, heart, kidney, liver, lung, placenta, spleen, testis, tendon, and thymus did not appear to contain alpha 1(XIII) collagen mRNAs.
Yeast proteins that recognize nuclear localization sequences.
Authors: Silver P , Sadler I , Osborne M A
Published: May 2004

A variety of peptides can mediate the localization of proteins to the nucleus. We have identified yeast proteins of 70 and 59 kD that bind to nuclear localization peptides of SV-40 T antigen, Xenopus nucleoplasmin, and the yeast proteins Ga14 and histone H2B. These proteins are assayed by the binding of peptide-albumin conjugates to proteins immobilized on nitrocellulose filters. These binding proteins fractionate with nuclei and are extractable with salt but not detergent. Radiolabeled peptide-albumin conjugates also bind to isolated nuclei; the binding is saturable and can be extracted with salt. Different nuclear localization peptides compete with each other, implying that a single class of proteins is responsible for their recognition. The 70- and 59-kD proteins have the properties expected for a receptor that would act to direct proteins to the nucleus.
The RCC1 protein, a regulator for the onset of chromosome condensation locates in the nucleus and binds to DNA.
Authors: Ohtsubo M , Okazaki H , Nishimoto T
Published: May 2004

The RCC1 gene, a regulator for the onset of chromosome condensation was found to encode a protein with a molecular mass of 45 kD, determined using the antibody against the synthetic peptides prepared according to the amino acid sequence of the putative RCC1 protein. The p45 located in the nuclei was released from the isolated nuclei, either by DNase I digestion or by treatment with 0.3 M NaCl. Consistently, p45 bound to the DNA-cellulose column was eluted with 0.3 M NaCl. After sequential treatment with DNase I and 2 M NaCl, almost all of the RCC1 protein were released from the nuclei. Thus, RCC1 protein locates on the chromatin and is not a component of the nuclear matrix. In mitotic cells, p45 is dispersed into the cytoplasm. Presumably, RCC1 protein plays a role in regulating the onset of chromosome condensation, at the level of transcription or of mRNA maturation.
Proliferating cell nuclear antigen/cyclin in the ciliate Euplotes eurystomus: localization in the replication band and in micronuclei.
Authors: Olins D E , Olins A L , Cacheiro L H , Tan E M
Published: May 2004

Human autoimmune sera specific for proliferating cell nuclear antigen (PCNA)/cyclin (auxiliary protein for DNA polymerase delta) demonstrated the presence of epitopes within the macro- and micronuclei of the hypotrichous ciliated protozoa Euplotes eurystomus. Tightly bound PCNA/cyclin was localized at the site of DNA synthesis in macronuclei, the rear zone of the replication band. Starvation or heat shock, conditions that reduce macronuclear replication, resulted in a decrease of PCNA/cyclin in replication bands. Micronuclei also exhibited PCNA/cyclin localization which persisted for a large proportion of the vegetative cell cycle and exhibited significant resistance to adverse culture conditions. Immunoprecipitation of 35S-labeled soluble Euplotes proteins with PCNA/cyclin autoimmune sera revealed a spectrum of low molecular mass proteins. PCNA/cyclin-like proteins have now been observed in the widely divergent species: human, rat, amphibian, yeast, and ciliated protozoa.
N2,N2-dimethylguanosine-specific tRNA methyltransferase contains both nuclear and mitochondrial targeting signals in Saccharomyces cerevisiae.
Authors: Li J M , Hopper A K , Martin N C
Published: May 2004

The TRM1 gene of Saccharomyces cerevisiae encodes a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase, which modifies both mitochondrial and cytoplasmic tRNAs. The enzyme is targeted to mitochondria for the modification of mitochondrial tRNAs. Cellular fractionation and indirect immunofluorescence studies reported here demonstrate that this enzyme is also localized to the nucleus. Further, immunofluorescence experiments using strains that overproduce the enzyme show a staining at the periphery of the nucleus suggesting that the enzyme is found in a subnuclear destination near or at the nuclear membrane. There is no obvious cytoplasmic staining in these overproducing strains. Fusion protein technology was used to begin to localize sequences involved in the nuclear targeting of this enzyme. Indirect immunofluorescence studies indicate that sequences between the first 70 and 213 NH2-terminal amino acids of the methyltransferase are sufficient to target Escherichia coli beta-galactosidase to nuclei.
Protein transport from endoplasmic reticulum to the Golgi complex can occur during meiotic metaphase in Xenopus oocytes.
Authors: Ceriotti A , Colman A
Published: May 2004

We have previously shown that Xenopus oocytes arrested at second meiotic metaphase lost their characteristic multicisternal Golgi apparati and cannot secrete proteins into the surrounding medium. In this paper, we extend these studies to ask whether intracellular transport events affecting the movement of secretory proteins from the endoplasmic reticulum to the Golgi apparatus are also similarly inhibited in such oocytes. Using the acquisition of resistance to endoglycosidase H (endo H) as an assay for movement to the Golgi, we find that within 6 h, up to 66% of the influenza virus membrane protein, hemagglutinin (HA), synthesized from injected synthetic RNA, can move to the Golgi apparati in nonmatured oocytes; indeed after longer periods some correctly folded HA can be detected at the cell surface where it distributes in a nonpolarized fashion. In matured oocytes, up to 49% of the HA becomes endo H resistant in the same 6-h period. We conclude that movement from the endoplasmic reticulum to the Golgi can occur in matured oocytes despite the dramatic fragmentation of the Golgi apparati that we observe to occur on maturation. This observation of residual protein movement during meiotic metaphase contrasts with the situation at mitotic metabphase in cultured mammalian cells where all movement ceases, but resembles that in the budding yeast Saccharomyces cerevisiae where transport is unaffected.
Trimeric binding of the 70-kD uncoating ATPase to the vertices of clathrin triskelia: a candidate intermediate in the vesicle uncoating reaction.
Authors: Heuser J , Steer C J
Published: May 2004

Clathrin-coated vesicles were uncoated with the 70-kD "uncoating ATPase" from bovine brain, and the molecular products were visualized by freeze-etch electron microscopy. This yielded images of released clathrin triskelia with up to three 70-kD uncoating ATPase molecules bound to their vertices. Likewise, incubation of soluble clathrin triskelia with purified uncoating ATPase also led to trimeric binding of the ATPase to the vertices of clathrin triskelia. However, this occurred only when either EDTA or nonhydrolyzable analogues of ATP were present, in which case the ATPase also appeared to self-associate. When ATP was present instead, no 70-kD ATPases could be found on clathrin triskelia and all ATPases remained monomeric. These observations support the notion that ATP controls an allosteric conversion of the 70-kD uncoating ATPase between two different molecular conformations, an ATP-charged state in which the molecule has relatively low affinity for itself as well as low affinity for clathrin, and an ATP-discharged state in which both of these affinities are high. We presume that in vivo, the latter condition is brought about by ATP hydrolysis and product release, at which point the ATPase will bind tightly to clathrin and/or self-associate. We further propose that these reactions, when occurring in concert within a clathrin lattice, will tend to destabilize it by a mechanism we call "protein polymer competition". We stress the analogies between such a mechanism of uncoating and the ATP-driven events in muscle contraction. Finally, we show that under experimental conditions in which the uncoating ATPase fully removes the coats from brain coated vesicles, identical aliquots of the enzyme do not affect plasmalemmal coated pits in situ. This remarkable selectivity, the mechanism of which remains a complete mystery, is at least consistent with the idea that the 70-kD ATPase indeed plays a role in uncoating coated vesicles after they have formed in vivo.