bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional histone antibody fragments traverse the nuclear envelope.
Authors: Einck L , Bustin M
Published: May 2004

Factors important in the translocation process of proteins across the nuclear membrane were studied by microinjecting either fluoresceinated nonimmune IgG and F(ab)2 or the corresponding molecules, prepared from antisera to histones, into the nucleus and cytoplasm of human fibroblasts. Intact IgG from both preparations remained at the site of injection regardless of whether it was injected into the nucleus or the cytoplasm. In contrast, nonimmune F(ab)2 distributed uniformly throughout the cell. The F(ab)2 derived from affinity-pure antihistone moves into the nucleus after cytoplasmic injection and remains in the nucleus after nuclear microinjection. The migration of the antihistone F(ab)2 into the nucleus results in inhibition of uridine incorporation in the nuclei of the microinjected cells. We conclude that non-nuclear proteins, devoid of specific signal sequences, traverse the nuclear membrane and accumulate in the nucleus provided their radius of gyration is less than 55A and the nucleus contains binding sites for these molecules. These findings support the model of "quasibifunctional binding sites" as a driving force for nuclear accumulation of proteins. The results also indicate that active F(ab)2 fragments, microinjected into somatic cells, can bind to their antigenic sites suggesting that microinjection of active antibody fragments can be used to study the location and function of nuclear components in living cells.
Physical, immunochemical, and functional properties of Acanthamoeba profilin.
Authors: Tseng P C , Runge M S , Cooper J A , Williams R C , Pollard T D
Published: May 2004

Acanthamoebe profilin has a native molecular weight of 11,700 as measured by sedimentation equilibrium ultracentrifugation and an extinction coefficient at 280 nm of 1.4 X 10(4) M-1cm-1. Rabbit antibodies against Acanthamoeba profilin react only with the 11,700 Mr polypeptide among all other ameba polypeptides separated by electrophoresis. These antibodies react with a 11,700 Mr polypeptide in Physarum but not with any proteins of Dictyostelium or Naeglaria. Antibody-binding assays indicate that approximately 2% of the ameba protein is profilin and that the concentration of profilin is approximately 100 mumol/liter cells. During ion exchange chromatography of soluble extracts of Acanthamoeba on DEAE-cellulose, the immunoreactive profilin splits into two fractions: an unbound fraction previously identified by Reichstein and Korn (1979, J. Biol. Chem., 254:6174-6179) and a tightly bound fraction. Purified profilin from the two fractions is identical by all criteria tested. The tightly bound fraction is likely to be attached indirectly to the DEAE, perhaps by association with actin. By fluorescent antibody staining, profilin is distributed uniformly throughout the cytoplasmic matrix of Acanthamoeba. In 50 mM KCl, high concentrations of Acanthamoeba profilin inhibit the elongation rate of muscle actin filaments measured directly by electron microscopy, but the effect is minimal in KCl with 2 MgCl2. By using the fluorescence change of pyrene-labeled Acanthamoeba actin to assay for polymerization, we confirmed our earlier observation (Tseng, P. C.-H., and T. D. Pollard, 1982, J. Cell Biol. 94:213-218) that Acanthamoeba profilin inhibits nucleation much more strongly than elongation under physiological conditions.
Binding and degradation of platelet thrombospondin by cultured fibroblasts.
Authors: McKeown-Longo P J , Hanning R , Mosher D F
Published: May 2004

Thrombospondin was purified from human platelets and labeled with 125I, and its metabolism was quantified in cell cultures of human embryonic lung fibroblasts. 125I-Thrombospondin bound to the cell layer. The binding reached an apparent steady state within 45 min. Trichloroacetic acid-soluble radioactivity was detected in the medium after 30 min of incubation; the rate of degradation of 125I-thrombospondin was linear for several hours thereafter. Degradation of 125I-thrombospondin was saturable. The apparent Km and Vmax for degradation at 37 degrees C were 6 X 10(-8) M and 1.4 X 10(5) molecules per cell per minute, respectively. Degradation was inhibited by chloroquine or by lowering the temperature to 4 degrees C. Experiments in which cultures were incubated with thrombospondin for 45 min and then incubated in medium containing no thrombospondin revealed two fractions of bound thrombospondin. One fraction was localized by indirect immunofluorescence to punctate structures; these structures were lost coincident with the rapid degradation of 50-80% of bound 125I-thrombospondin. The second fraction was localized to a trypsin-sensitive, fibrillar, extracellular matrix. 125I-Thrombospondin in the matrix was slowly degraded over a period of hours. Binding of 125I-thrombospondin to the extracellular matrix was not saturable and indeed was enhanced at thrombospondin concentrations greater than 3 X 10(-8) M. The ability of 125I-thrombospondin to bind to extracellular matrix was diminished tenfold by limited proteolytic cleavage with trypsin. Degradation of trypsinized 125I-thrombospondin was also diminished, although to a lesser extent than matrix binding. Heparin inhibited both degradation and matrix binding. These results suggest that thrombospondin may play a transitory role in matrix formation and/or organization and that specific receptors on the cell surface are responsible for the selective removal of thrombospondin from the extracellular fluid and matrix.
Genetic dissection of the central pair microtubules of the flagella of Chlamydomonas reinhardtii.
Authors: Dutcher S K , Huang B , Luck D J
Published: May 2004

Mutations at two loci, which cause an altered mobility of the flagella, affected the central pair microtubule complex of Chlamydomonas reinhardtii flagella. The mutations at both loci primarily affected the C1 microtubule of the complex. Three alleles at the PF16 locus affected the stability of the C1 microtubule in isolated axonemes. This phenotype has allowed us to determine that at least ten polypeptides of the central pair complex are unique to the C1 microtubule. The motility defect was correlated with the failure to assemble three of these ten polypeptides in vivo. The structural gene product of the PF16 locus was a polypeptide with molecular weight 57,000 as shown by analysis of five intragenic revertants and by analysis of axonemes from dikaryon rescue experiments. Three alleles at the PF6 locus affected the assembly of one of the two projections of the C1 microtubule and this projection was formed by at least three polypeptide components, which are a subset of polypeptides missing in isolated pf16 axonemes. No structural gene product has been identified for the PF6 locus. The gene product is probably not one of the identified projection constituents as shown by analysis of dikaryon rescue experiments. Chemical extraction of isolated wild-type axonemes suggests that at least seven polypeptide components are unique to the C2 microtubule.
Surface antigens of brain synapses: identification of minor proteins using polyclonal antisera.
Authors: Matus A , Ng M , Pehling G , Ackermann M , Hauser K
Published: May 2004

Antigenic proteins of brain synaptic plasma membranes (SPM) and postsynaptic densities (PSD) were characterized using antisera raised against SPM. Immunostaining of brain sections showed that the antigens were restricted to synapses, and electron microscopy revealed staining at both presynaptic terminals and PSDs. In primary brain cell cultures the antisera were also neuron-specific but the antigens were distributed throughout the entire neuronal plasma membrane, suggesting that some restrictive influence present in whole tissue is absent when neurons are grown dispersed. The antigenic proteins with which these antisera react were identified using SDS gel immunoblots. SPM and PSD differed from one another in their characteristic antigenic proteins. Comparison with amido-black stained gel blots showed that in both cases most of these did not correspond to known abundant proteins of SPM or PSDs revealed by conventional biochemical techniques. None of the antigens revealed by the polyclonal antisera were detected by any of a large series of monoclonal antibodies against SPM.
Intracellular transport of influenza virus hemagglutinin to the apical surface of Madin-Darby canine kidney cells.
Authors: Rodriguez-Boulan E , Paskiet K T , Salas P J , Bard E
Published: May 2004

The intracellular pathway followed by the influenza virus hemagglutinin (HA) to the apical surface of Madin-Darby canine kidney cells was studied by radioimmunoassay, immunofluorescence, and immunoelectron microscopy. To synchronize the migration, we used a temperature-sensitive mutant of influenza WSN, ts61, which, at the nonpermissive temperature, 39.5 degrees C, exhibits a defect in the HA that prevents its exit from the endoplasmic reticulum. Upon transfer to permissive temperature, 32 degrees C, the HA appeared in the Golgi apparatus after 10 min, and on the apical surface after 30-40 min. In the presence of cycloheximide, the expression was not inhibited, indicating that the ts defect is reversible; a wave of HA migrated to the cell surface, where it accumulated with a half time of 60 min. After passage through the Golgi apparatus the HA was detected in a population of smooth vesicles, about twice the size of coated vesicles, located in the apical half of the cytoplasm. These HA-containing vesicles did not react with anti-clathrin antibodies. Monensin (10 microM) delayed the surface appearance of HA by 2 h, but not the transport to the Golgi apparatus. Incubation at 20 degrees C retarded the migration to the Golgi apparatus by approximately 30 min and blocked the surface appearance by acting at a late stage in the intracellular pathway, presumably at the level of the post-Golgi vesicles. The initial appearance of HA on the apical surface was in the center; no preference was observed for the tight-junctional regions.
The kinetochore is part of the metaphase chromosome scaffold.
Authors: Earnshaw W C , Halligan N , Cooke C , Rothfield N
Published: May 2004

We used antisera from patients with the CREST syndrome of scleroderma (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) to show that an antigenic component of the kinetochore present in metaphase chromosomes is also present in nonhistone chromosome scaffolds isolated following extensive digestion of the DNA and extraction of the bulk of chromosomal protein. All sera from 12 scleroderma CREST patients previously shown by immunofluorescence microscopy to have circulating antikinetochore antibodies recognise a protein of Mr 77,000 (CREST-77) in an immunoblotting assay. 9 of the 12 sera also recognise an antigen of Mr 110,000 (CREST-110). These proteins are present in isolated chromosomes and nonhistone scaffolds derived from them by two different procedures. Sera of five scleroderma CREST patients who are antikinetochore negative (by immunofluorescence) bind to neither protein in immunoblots. These data suggest that CREST-77 (and possibly CREST-110) is a component of the human kinetochore, and that the kinetochore is an integral part of the mitotic chromosome scaffolding.
Genes required for completion of import of proteins into the endoplasmic reticulum in yeast.
Authors: Ferro-Novick S , Hansen W , Schauer I , Schekman R
Published: May 2004

Yeast secretory mutants sec53 and sec59 define a posttranslational stage in the penetration of glycoprotein precursors into the endoplasmic reticulum (ER). In the previous report we showed that at the restrictive temperature (37 degrees C) these mutants accumulate enzymatically inactive and incompletely glycosylated forms of the secretory enzyme invertase and the vacuolar enzyme carboxypeptidase Y. Cell fractionation experiments reveal that these precursor forms remain firmly bound to the ER membrane. However, upon return to the permissive temperature (24 degrees C), the invertase precursors are glycosylated, become partially active, and are secreted. Thermoreversible conversion does not require protein synthesis, but does require energy. In contrast to the effect of these mutations, inhibition of oligosaccharide synthesis with tunicamycin at 37 degrees C causes irreversible accumulation of unglycosylated invertase. The effect of the drug is exaggerated by high temperature since unglycosylated invertase synthesized in the presence of tunicamycin at 25 degrees C is secreted. A portion of the invertase polypeptide accumulated at 37 degrees C is preserved when membranes from sec53 and sec59 are treated with trypsin. In the presence of Triton X-100 or saponin, the invertase is degraded completely. The protected fragment appears to represent a portion of the invertase polypeptide that is embedded in or firmly associated with the ER membrane. This association may develop early during the synthesis of invertase, so that in the absence of translocation, some of the completed polypeptide chain remains exposed on the cytoplasmic surface of the ER.
Intracellular segregation of asialoglycoproteins and their receptor: a prelysosomal event subsequent to dissociation of the ligand-receptor complex.
Authors: Wolkoff A W , Klausner R D , Ashwell G , Harford J
Published: May 2004

Rat hepatocytes in monolayer culture rapidly internalized asialoglycoproteins and the receptors to which they are bound. Subsequent to endocytosis, the receptor-ligand complex is dissociated within an acidic endosome (Harford, J., K. Bridges, G. Ashwell, and R. D. Klausner, 1983, J. Biol. Chem. 258:3191-3197; Harford, J., A. W. Wolkoff, G. Ashwell, and R. D. Klausner, 1983, J. Cell Biol. 96:1824-1828). Here we show that addition of the proton ionophore monensin to the cells after dissociation has occurred results in intracellular rebinding of ligand molecules. With increasing time inside the cell, the ability of ligand to reassociate with receptor progressively decreases consistent with a segregation of receptor and ligand. The combination of colchicine and cytochalasin B appears to retard the process of segregation. In contrast, removal of sodium from the medium, while inhibiting degradation of ligand, does not affect the decrease in monensin-mediated rebinding. Nonetheless, both sodium deprivation and treatment with colchicine plus cytochalasin B result in the ligand remaining in a low density, nonlysosomal subcellular fraction. Thus, segregation, like dissociation, appears to occur in a pre-lysosomal endocytic compartment. Perturbation of the endocytic pathway by reduced temperature (18 degrees C) was also explored. Our data are consistent with two temperature-sensitive steps: receptor-ligand dissociation is inhibited and there is an independent temperature-sensitive step involved in delivery of ligand to lysosomes. This second effect was localized as being beyond the point in the pathway sensitive to sodium deprivation.
1,25-Dihydroxyvitamin D3-induced differentiation in a human promyelocytic leukemia cell line (HL-60): receptor-mediated maturation to macrophage-like cells.
Authors: Mangelsdorf D J , Koeffler H P , Donaldson C A , Pike J W , Haussler M R
Published: May 2004

The human-derived promyelocytic leukemia cell line, HL-60, is known to differentiate into mature myeloid cells in the presence of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). We investigated differentiation by monitoring 1,25(OH)2D3-exposed HL-60 cells for phagocytic activity, ability to reduce nitroblue tetrazolium, binding of the chemotaxin N-formyl-methionyl-leucyl-[3H]phenylalanine, development of nonspecific acid esterase activity, and morphological maturation of Wright-Giemsa-stained cells. 1,25(OH)2D3 concentrations as low as 10(-10) M caused significant development of phagocytosis, nitroblue tetrazolium reduction, and the emergence of differentiated myeloid cells that had morphological characteristics of both metamyelocytes and monocytes. These cells were conclusively identified as monocytes/macrophages based upon their adherence to the plastic flasks and their content of the macrophage-characteristic nonspecific acid esterase enzyme. The estimated ED50 for 1,25(OH)2D3-induced differentiation based upon nitroblue tetrazolium reduction and N-formyl-methionyl-leucyl-[3H]phenylalanine binding was 5.7 X 10(-9) M. HL-60 cells exhibited a complex growth response with various levels of 1,25(OH)2D3: less than or equal to 10(-10) M had no detectable effect, 10(-9) M stimulated growth, and greater than or equal to 10(-8) M sharply inhibited proliferation. We also detected and quantitated the specific receptor for 1,25(OH)2D3 in HL-60 and HL-60 Blast, a sub-clone resistant to the growth and differentiation effects of 1,25(OH)2D3. The receptor in both lines was characterized as a DNA-binding protein that migrated at 3.3S on high-salt sucrose gradients. Unequivocal identification was provided by selective dissociation of the 1,25(OH)2D3-receptor complex with the mercurial reagent, p-chloromercuribenzenesulfonic acid, and by a shift in its sedimentation position upon complexing with anti-receptor monoclonal antibody. On the basis of labeling of whole cells with 1,25(OH)2[3H]D3 in culture, we found that HL-60 contains approximately 4,000 1,25(OH)2D3 receptor molecules per cell, while the nonresponsive HL-60 Blast is endowed with approximately 8% of that number. The concentration of 1,25(OH)2D3 (5 X 10(-9) M) in complete culture medium, which facilitates the saturation of receptors in HL-60 cells, is virtually identical to the ED50 for the sterol's induction of differentiation. This correspondence, plus the resistance of the relatively receptor-poor HL-60 Blast, indicates that 1,25(OH)2D3-induced differentiation of HL-60 cells to monocytes/macrophages is occurring via receptor-mediated events.
Differential inhibition of nerve growth factor and epidermal growth factor effects on the PC12 pheochromocytoma line.
Authors: Seeley P J , Rukenstein A , Connolly J L , Greene L A
Published: May 2004

Tests have been made of the action of the methyltransferase inhibitors 5'-S-methyl adenosine, 5'-S-(2-methyl-propyl)-adenosine, and 3-deaza-adenosine +/- L-homocysteine thiolactone, on nerve growth factor (NGF)-dependent events in the rat pheochromocytoma line PC12. Each of these agents inhibited NGF-dependent neurite outgrowth at concentrations of the order of millimolar. Slow initiation of neurite outgrowth over several days and more rapid regeneration of neurites (congruent to 1 d) were blocked, as was the priming mechanism necessary for genesis of neurites. The inhibitions were reversible in that PC12 cells maintained for several days in the presence of inhibitors grew neurites normally after washout of these agents. Other NGF-dependent responses of the PC12 line (i.e., induction of ornithine decarboxylase activity [over 4 h], enhancement of tyrosine hydroxylase phosphorylation [over 1 h], and rapid changes in cell surface morphology [30 s onward]) were inhibited by each of the agents. In contrast, corresponding epidermal growth factor-dependent responses in ornithine decarboxylase activity, phosphorylation, and cell surface morphology were not blocked, but instead either unaffected or enhanced, by the methylation inhibitors. These inhibitors did not act by blockade of binding of NGF to high- or low-affinity cell surface receptors, though they partially inhibited internalization of [125I]NGF. The inhibition of rapidly-induced NGF-dependent events and the differential inhibition of responses to NGF and epidermal growth factor imply that the methyltransferase inhibitors specifically block one of the first steps in the mechanistic pathway for NGF.
Synthesis and assembly of the cytoskeleton of Naegleria gruberi flagellates.
Authors: Walsh C
Published: May 2004

When Naegleria gruberi flagellates were extracted with nonionic detergent and stained by the indirect immunofluorescence method with AA-4.3 (a monoclonal antibody against Naegleria beta-tubulin), flagella and a network of cytoskeletal microtubules (CSMT) were seen. When Naegleria amebae were examined in the same way, no cytoplasmic tubulin-containing structures were seen. Formation of the flagellate cytoskeleton was followed during the differentiation of amebae into flagellates by staining cells with AA-4.3. The first tubulin containing structures were a few cytoplasmic microtubules that formed at the time amebae rounded up into spherical cells. The formation of these microtubules was followed by the appearance of basal bodies and flagella and then by the formation of the CSMT. The CSMT formed before the cells assumed the flagellate shape. In flagellate shaped cells the CSMT radiate from the base of the flagella and follow a curving path the full length of the cell. Protein synthetic requirements for the formation of CSMT were examined by transferring cells to cycloheximide at various times after initiation. One-half the population completed the protein synthesis essential for formation of CSMT 61 min after initiation of the differentiation. This is 10 min after the time when protein synthesis for formation of flagella is completed and 10-15 min before the time when the protein synthesis necessary for formation of the flagellate shape is completed.
Changes in the distribution of the 34-kdalton tyrosine kinase substrate during differentiation and maturation of chicken tissues.
Authors: Greenberg M E , Brackenbury R , Edelman G M
Published: May 2004

We examined the distribution of the 34-kilodalton (34-kD) tyrosine kinase substrate in tissues of adult and embryonic chicken using both a mouse monoclonal antibody and a rabbit polyclonal antibody raised against the affinity purified 34 kD protein. We analyzed the localization by immunoblotting of tissue extracts, by immunofluorescence staining of frozen tissue sections, and by staining sections of paraffin-embedded organs by the peroxidase antiperoxidase method. The 34-kD protein was present in a variety of cells, including epithelial cells of the skin, gastrointestinal, and respiratory tracts, as well as in fibroblasts and chondrocytes of connective tissue and mature cartilage, and endothelial cells of blood vessels. The 34-kD protein was also found in subpopulations of cells in thymus, spleen, bone marrow, and bursa. The protein was not detected in cardiac, skeletal, or smooth muscle cells, nor in epithelial cells of liver, kidney, pancreas, and several other glands. Although most neuronal cells did not contain the 34-kD protein, some localized brain regions did contain detectable amounts of this protein. The 34-kD protein was not detected in actively dividing cells of a number of tissues. Changes in the distribution of the 34-kD protein were observed during the differentiation or maturation of cells in several tissues including epithelial cells of the skin and gastrointestinal tract, fibroblasts of connective tissue, and chondroblasts.
Evidence that coated vesicles transport acetylcholine receptors to the surface membrane of chick myotubes.
Authors: Bursztajn S , Fischbach G D
Published: May 2004

Coated vesicles are present in the myoplasm of embryonic chick myotubes grown in vitro. They are most numerous beneath regions of the surface membrane that contain a high density of acetylcholine receptors (AChR). Prolonged exposure of myotubes to saline extract of chick brain increases the number of intracellular AChR and the number of coated vesicles. This suggests that coated vesicles contain AChR, and this hypothesis was tested with horseradish peroxidase-alpha-bungarotoxin (HRP-alpha BTX) conjugates. The conjugates enter saponin-permeabilized cells and, as judged by the inhibition of [125I] alpha BTX binding, they label the entire intracellular AChR pool. Approximately 50% of the coated vesicles contained HRP-alpha BTX reaction product. In addition, reaction product was detected in Golgi cisternae and along membranes that bound a subsurface tubulovesicular network. The majority of labeled vesicles are probably involved in exocytosis rather than endocytosis of AChR because very few coated vesicles were labeled when HRP-alpha BTX was added to the medium bathing intact cells. Moreover, inhibition of protein synthesis with puromycin resulted in a large decrease in the number of labeled vesicles. These results suggest that a subpopulation of coated vesicles ferry newly synthesized AChR to the cell surface.
Structure and transmembrane nature of the acetylcholine receptor in amphibian skeletal muscle as revealed by cross-reacting monoclonal antibodies.
Authors: Sargent P B , Hedges B E , Tsavaler L , Clemmons L , Tzartos S , Lindstrom J M
Published: May 2004

A collection of 126 monoclonal antibodies (mAbs) made against acetylcholine receptors (AChRs) from the electric organs of Torpedo californica or Electrophorus electricus was tested for cross-reactivity with AChRs in cryostat sections of skeletal muscle from Rana pipiens and Xenopus laevis by indirect immunofluorescence. 49 mAbs (39%) cross-reacted with AChRs from Rana, and 25 mAbs (20%) cross-reacted with AChRs from Xenopus. mAbs specific for each of the four subunits of electric organ AChR (alpha, beta, gamma, delta) cross-reacted with AChRs from each amphibian species. mAbs cross-reacting with Xenopus AChRs were, with one exception, a subset of the mAbs cross-reacting with Rana AChRs. The major difference detected between the two species was in binding by mAbs specific for the main immunogenic region (MIR) of the alpha-subunit. Whereas 22 of 33 anti-MIR mAbs tested cross-reacted with Rana AChRs, only one of these mAbs cross-reacted with Xenopus AChRs. Some (32) of the cross-reacting mAbs were tested for binding to AChRs in intact muscle. 21 of these mAbs bound to AChRs only when membranes were made permeable with saponin. Electron microscopy using immunoperoxidase or colloidal gold techniques revealed that these mAbs recognize cytoplasmic determinants and that mAbs that do not require saponin in order to bind AChRs in intact muscle recognize extracellular determinants. These results suggest that AChRs in skeletal muscle of Rana and Xenopus are composed of subunits corresponding to the alpha-, beta-, gamma-, and delta-subunits of AChRs from fish electric organs. The subunit specificity of mAbs whose binding was examined by electron microscopy suggests that parts of each subunit (alpha, beta, gamma, delta) are exposed on the cytoplasmic surface and that, as in AChRs from fish electric organs and mammalian muscle, the MIR on alpha-subunits of Rana AChRs is exposed on the extracellular surface.
Hagfish slime gland thread cells. II. Isolation and characterization of intermediate filament components associated with the thread.
Authors: Spitzer R H , Downing S W , Koch E A , Salo W L , Saidel L J
Published: May 2004

The slime glands of hagfish have two major cell types, gland thread cells (GTCs) and gland mucous cells (GMCs), both of which upon contact with water contribute to the formation of an abundant quantity of viscous mucus. In previous studies we reported a method for the isolation of GTCs and showed that each ellipsoidal thread cell normally contains a single tapered thread which is uniquely coiled into a space-saving conformation and occupies most of the cell volume. Subsequently, the developing thread was found to consist mainly of intermediate filaments (IFs) aligned in parallel not only to one another but also to a far fewer number of interspersed microtubules (see accompanying paper). In the present report, urea extracts of GTCs were purified and characterized to establish the properties of thread components. One major (alpha) and two minor (beta, gamma) components prepared by anion exchange chromatography were shown to have similar apparent molecular weights of 63,500 +/- 500 daltons but different isoelectric pH values (alpha, 7.56; beta, 5.67; gamma, 5.31). Although the amino acid content of alpha differed significantly from beta and gamma, each of the three was highest in Gly, relatively high in Glx, Ser, Thr, Asx, Ala, Val, and Leu, and relatively low in Cys/2 and Trp. The amino acid compositions of beta and gamma were very similar, and only beta showed evidence of carbohydrate. The threonine content of the alpha component was higher than has been reported for IFs of different origin, and the high content of hydroxyamino acids (18, 19 residues per 100) in alpha, beta, and gamma has been approached only by several IF polypeptides from human or bovine epidermal keratins. Mixtures of the purified components formed 9-11-nm filaments in vitro. The results indicate that the hagfish thread cell is a rich source of IFs, which have a structure that facilitates formation of macrofibrils within the cell.
Transient kinetics of a cGMP-dependent cGMP-specific phosphodiesterase from Dictyostelium discoideum.
Authors: Van Haastert P J , Van Lookeren Campagne M M
Published: May 2004

Chemotactic stimulation of Dictyostelium discoideum cells induces a fast transient increase of cGMP levels which reach a peak at 10 s. Prestimulation levels are recovered in approximately 30 s, which is achieved mainly by the action of a guanosine 3',5'-monophosphate cGMP-specific phosphodiesterase. This enzyme is activated about fourfold by low cGMP concentrations. The phosphodiesterase has two distinct cGMP-binding sites: a catalytic site and an activator site. cAMP does not bind to either site; inosine 3',5'-monophosphate (cIMP) binds only to the catalytic site, whereas 8-bromoguanosine 3',5'-monophosphate (c-b8-GMP) preferentially binds to the activator site. For detailed kinetical measurements we have used [3H]cIMP as the substrate and c-b8-GMP as the activator. c-b8-GMP activated the hydrolysis of [3H]cIMP by reducing the Km, whereas the Vmax was not altered. The hydrolysis of [3H]cIMP was measured at 5-s intervals by using a new method for the separation of 5'-nucleotides from cyclic nucleotides. The hydrolysis of [3H]cIMP by nonactivated enzyme or by preactivated enzyme was linear with time, which indicates that a steady state is reached at the catalytic site within 5 s after addition of the substrate. In contrast, the hydrolysis of [3H]cIMP immediately after activation by 0.1 microM c-b8-GMP was not linear with time, but increased in a quasi-exponential manner with a time constant of 21 s. This suggests that a steady state at the activator site is only reached in 30-45 s after addition of the activator. The on-rate of activation (k1) was 3 X 10(5) M-1s-1 for c-b8-GMP and 1.4 X 10(5) M-1s-1 for cGMP. The off-rate of activation (k-1) was 0.03 s-1 for both c-b8-GMP and cGMP. The significance of these kinetic constants for the chemoattractant-mediated cGMP response in vivo is discussed.
Unphosphorylated gelsolin is localized in regions of cell-substratum contact or attachment in Rous sarcoma virus-transformed rat cells.
Authors: Wang E , Yin H L , Krueger J G , Caliguiri L A , Tamm I
Published: May 2004

Regions associated with cell-substratum contact or attachment in Rous sarcoma virus (RSV)-transformed rat fibroblasts (RR1022 cells) were identified by reflection-interference microscopy. Electron microscopy of such regions revealed the presence of discrete membrane-associated structures composed of a paracrystalline lattice of hexagons and pentagons to which actin filaments appear to be attached. Staining of actin by biotin-labeled heavy meromyosin showed that transformed cells, unlike normal fibroblasts, lack prominent actin fibers, and that, instead, much of the fluorescence is concentrated in loci corresponding to locations of transient association between the cell and the substratum. In stationary cells, such loci were found in rosette formation, predominantly in the region beneath the nucleus. In cells engaged in active movement, such as during migration into a wound, the actin-containing spots were concentrated in the region of the leading edge. A similar pattern of staining was observed with antibody to gelsolin, a 91,000-dalton Ca2+-dependent actin filament-shortening protein. Since the action of gelsolin on actin is reversible and dependent on physiologically relevant changes in calcium concentration, the localization of gelsolin, together with actin-bundling proteins such as alpha-actinin, in the regions containing many small microfilament bundles on the ventral side of cytoplasm suggests that gelsolin may be a component of the mechanism for the disassembly and assembly of actin during the dissolution and reformation of structures for cell-substratum contact during cell locomotion. Regulation of gelsolin activity was not dependent on protein phosphorylation, as shown by lack of 32P-incorporation into gelsolin in either transformed or normal fibroblasts.
Immunocytochemical localization of microtubule-associated protein 1 in rat cerebellum using monoclonal antibodies.
Authors: Huber G , Matus A
Published: May 2004

Immunohistochemical staining with monoclonal antibodies showed that microtubule-associated protein 1 (MAP1) has a restricted cellular distribution in the rat cerebellum. Anti-MAP1 staining was found only in neurons, where it was much stronger in dendrites than in axons. There were striking variations in the apparent concentration of MAP1 in different classes of neurons. Purkinje cells were the most strongly labeled, while granule cell neurons gave a faint, threshold-level reaction with the antibody. The reaction of Golgi neurons was intermediate between these two extremes. Equivalent results were obtained using two different methods of tissue preparation. Thus MAP1 appears to be a neuron-specific protein that is highly concentrated in dendrites and occurs at markedly different levels in different types of neurons. These observations provide further indications of heterogeneity among brain microtubules.
4F2 monoclonal antibody recognizes a surface antigen on spread human fibroblasts of embryonic but not of adult origin.
Authors: Azzarone B , Suarez H , Mingari M C , Moretta L , Fauci A S
Published: May 2004

The 4F2 monoclonal antibody (mAb) has been shown to recognize a 120-kilodalton glycoprotein expressed on the cell surface of human peripheral blood monocytes, activated (but not resting) T or B cells, and T and B lymphoblastoid cell lines. In this report we show that 4F2 mAb specifically binds to the surface of adherent human embryonic fibroblasts but fails to bind to normal adult fibroblasts. Moreover, 4F2 antigen was expressed on sarcoma-derived or SV40-transformed adult fibroblastic cells. Finally, addition of 4F2 mAb inhibited the growth of cultured HT-1080 fibrosarcoma cell line, but had no inhibitory effect on various embryonic and adult normal or transformed fibroblasts.
PC12 cells as a source of neurite-derived cell surface mitogen, which stimulates Schwann cell division.
Authors: Ratner N , Glaser L , Bunge R P
Published: May 2004

Primary cultures of rat dorsal root ganglion Schwann cells were used to assay the efficacy of PC12 cells in stimulating Schwann cell proliferation. Co-cultures of PC12 cells and Schwann cells assayed by [3H]thymidine labeling followed by autoradiography showed proliferation of Schwann cells only where contact occurred between PC12 neurites and Schwann cells. Membranes derived from PC12 cells were shown to have many characteristics similar to membranes derived from sensory neurons; both could mimic whole cells in stimulating Schwann cell division; both were inactivated by mild heat treatment and by trypsinization, and both elevated intracellular cyclic AMP concentrations in Schwann cells 16 h after addition of membranes. We conclude that PC12 cells will be a valuable source for the isolation of the neuronal cell surface component which controls proliferation of Schwann cells during development of the peripheral nervous system.
Substrate adhesion of rat hepatocytes: on the mechanism of attachment to fibronectin.
Authors: Johansson S , Höök M
Published: May 2004

We examined the mechanisms of cell attachment to fibronectin-coated substrates. Inhibition of cell attachment was obtained by species-specific antifibronectin antibodies, which presumably recognize a distinct antigenic structure in the protein located at, or in the immediate vicinity of, the cell-binding site. The inhibiting antibodies could be adsorbed on a column of Sepharose substituted with plasma fibronectin. The initial phase of cell attachment was also inhibited by addition of soluble fibronectin to the incubation medium in a reaction that exhibited specificity and concentration dependence. These data suggest that cell-binding sites are available in an active form on the surface of soluble fibronectin. However, the inhibitory effect of fibronectin was greatly enhanced by adding the protein together with heparin, heparan sulfate, collagen, or a fibronectin-binding collagen peptide (CB-7), which is consistent with an "activation" of fibronectin on binding to these matrix components. A similar activation of fibronectin was obtained by cleaving the protein with trypsin. We discuss these findings in relation to conformational rearrangements in the fibronectin molecule. Data is presented supporting a mechanism of cell attachment to fibronectin involving multiple weak interactions between cellular receptors and substrate molecules, although some steps in the attachment process appear to disobey the requirements for such a mechanism.
Membrane-associated actin in the rhabdomeral microvilli of crayfish photoreceptors.
Authors: de Couet H G , Stowe S , Blest A D
Published: May 2004

Infiltration of compound eyes of crayfish, Cherax destructor, with the thiol protease inhibitor Ep-475 or with trifluoperazine prior to fixation for electron microscopy was found to stabilize an axial filament of 6-12 nm diam within each rhabdomeral microvillus of the photoreceptors. Rhabdoms isolated from retinal homogenates by sucrose gradient centrifugation under conditions that stabilize cytoskeletal material contained large amounts of a 42-kd polypeptide that co-migrated with insect flight muscle actin in one- and two-dimensional PAGE, inhibited pancreatic DNase l, and bound to vertebrate myosin. Vertebrate skeletal muscle actin added to retinal homogenates did not co-purify with rhabdoms, implying that actin was not a contaminant from nonmembranous structures. DNase l inhibition assays of detergent-lysed rhabdoms indicated the presence of large amounts of filamentous actin provided ATP was present. Monomeric actin in such preparations was completely polymerizable only after 90 min incubation with equimolar phalloidin. More than half of the actin present could be liberated from the membrane by sonication, indicating a loose association with the membrane. However, a large proportion of the actin was tightly bound to the rhabdomeral membrane, and washing sonicated membrane fractions with solutions of a range of ionic strengths and nonionic detergents failed to remove it. Antibodies to scallop actin only bound to frozen sections of rhabdoms after gentle permeabilization and very long incubation periods, probably because of steric hindrance and the hydrophobicity of the structure. The F-actin probe nitrobenzoxadiazol phallacidin bound to rhabdoms and labeled F-actin aggregates in other retinal components, but rhabdom fluorescence was not abolished by preincubation with phalloidin. The biochemical data indicate the existence of two distinct actin-based cytoskeletal systems, one being closely membrane associated. The other may possibly constitute the axial filament, although the evidence for this is equivocal.
Distribution of urokinase-type plasminogen activator immunoreactivity in the mouse.
Authors: Larsson L I , Skriver L , Nielsen L S , Grøndahl-Hansen J , Kristensen P , Danø K
Published: May 2004

Immunocytochemistry, using rabbit antibodies to a urokinase-type 48-Kdalton Mr mouse plasminogen activator, showed that enzyme immunoreactivity is widely distributed in the normal mouse. Strong staining was obtained in widely disseminated connective tissue cells with a fibroblast-like morphology. Such cells occurred in high numbers in the lamina propria mucosae of the gastrointestinal tract, and in moderate numbers in the connective tissue septa of the pancreas. A few such cells were detected around the larynx, trachea, and bronchi. Immunoreactivity also occurred in epithelial cells of the proximal and distal kidney tubules, the ductus deferens, and in pulmonary pneumocytes. In addition, presumably extracellular staining was seen irregularly along the basement membrane and fibrillar structures in the lamina propria of the small and large intestines. Moreover, decidual cells of the mouse placenta stained strongly, and a moderate staining was observed in epithelial cells of involuting mammary glands, but not in those of noninvoluting glands. No immunoreactivity was observed in endothelial cells. Control experiments included absorption of the antibodies against highly-purified mouse plasminogen activator and the corresponding proenzyme, and the finding of a good correspondence between the number of immunoreactive cells and measurable enzymatic activity determined in adjacent tissue sections. Separation by SDS PAGE followed by immunoblotting revealed only one immunochemically stainable protein band with Mr approximately 48 Kdaltons in extracts from tissues showing immunoreactivity.
Visualization of early intramembranous ossification by electron microscopic and spectroscopic imaging.
Authors: Arsenault A L , Ottensmeyer F P
Published: May 2004

We present electron microscopic and electron spectroscopic images of putative nucleation sites and early mineral deposits during intramembranous ossification of the murine perichondrial ring. Electron spectroscopic imaging (ESI) permits the quantitative determination and direct visualization of spatial distribution of atomic elements within specimens at high spatial resolution. In this study ESI was used to determine the elemental distributions of phosphorus, sulfur, and calcium. Nucleation and subsequent mineralization in the perichondrial ring occurred sequentially along the longitudinal axis. Proximal regions of the ring contained a matrix with only a few nucleation sites that are characterized in conventional electron micrographs as small loci of low-density material in which dense particles are located. Elemental maps of these sites that we obtained by ESI reveal a sulfur-containing matrix in which localized concentrations of phosphorus occur. With further maturation the loci became centers for the genesis of numerous dense rods or crystals. These mineral deposits contained increased concentrations of P, S, and Ca, compared with the surrounding matrix. The appearance of S at nucleation sites and its persistence in developing mineral deposits suggests that a sulfur-containing moiety may serve as a locus within the osteoid matrix to attain high local concentrations of Ca and P, which leads to the controlled local formation of calcium phosphates. Calcification of the perichondrial ring has been found to occur in the absence of matrix vesicles, which illustrates that these membrane-bounded organelles are not obligatory sites for nucleation in this matrix.
Structure and properties of an under-sulfated heparan sulfate proteoglycan synthesized by a rat hepatoma cell line.
Authors: Robinson J , Viti M , Höök M
Published: May 2004

A rat hepatoma cell line was shown to synthesize heparan sulfate and chondroitin sulfate proteoglycans. Unlike cultured hepatocytes, the hepatoma cells did not deposit these proteoglycans into an extracellular matrix, and most of the newly synthesized heparan sulfate proteoglycans were secreted into the culture medium. Heparan sulfate proteoglycans were also found associated with the cell surface. These proteoglycans could be solubilized by mild trypsin or detergent treatment of the cells but could not be displaced from the cells by incubation with heparin. The detergent-solubilized heparan sulfate proteoglycan had a hydrophobic segment that enabled it to bind to octyl-Sepharose. This segment could conceivably anchor the molecule in the lipid interior of the plasma membrane. The size of the hepatoma heparan sulfate proteoglycans was similar to that of proteoglycans isolated from rat liver microsomes or from primary cultures of rat hepatocytes. Ion-exchange chromatography on DEAE-Sephacel indicated that the hepatoma heparan sulfate proteoglycans had a lower average charge density than the rat liver heparan sulfate proteoglycans. The lower charge density of the hepatoma heparan sulfate can be largely attributed to a reduced number of N-sulfated glucosamine units in the polysaccharide chain compared with that of rat liver heparan sulfate. Hepatoma heparan sulfate proteoglycans purified from the culture medium had a considerably lower affinity for fibronectin-Sepharose compared with that of rat liver heparan sulfate proteoglycans. Furthermore, the hepatoma proteoglycan did not bind to the neoplastic cells, whereas heparan sulfate from normal rat liver bound to the hepatoma cells in a time-dependent reaction. The possible consequences of the reduced sulfation of the heparan sulfate proteoglycan produced by the hepatoma cells are discussed in terms of the postulated roles of heparan sulfate in the regulation of cell growth and extracellular matrix formation.
Monoclonal antibodies to laminin reveal the heterogeneity of basement membranes in the developing and adult mouse tissues.
Authors: Wan Y J , Wu T C , Chung A E , Damjanov I
Published: May 2004

Two monoclonal antibodies raised against laminin isolated from a mouse parietal yolk sac cell line were used for immunohistochemical studies of basement membranes of the mouse embryo and various fetal and adult tissues. No immunoreactivity with either of the two monoclonal antibodies could be detected in the preimplantation-stage embryos, although it has been shown that these embryos contain extracellular laminin reactive with the conventional polyclonal antilaminin antibodies. Reichert's membrane in early postimplantation stages of development reacted with the monoclonal antibody LAM-I but not with the antibody LAM-II. However, from day 8 of pregnancy onward the Reichert's membrane reacted with both antibodies. Basement membranes of the embryo proper were unreactive with both monoclonal antibodies until day 12 of pregnancy. By day 14 some basement membranes of the fetal tissues became reactive with one or both monoclonal antibodies, whereas others remained still unreactive. In the 17-d fetus and the newborn mouse most of the basement membranes reacted with both monoclonal antibodies, whereas others still reacted with only one. Similar heterogeneity in the immunoreactivity of basement membranes of various tissues was noted in the adult mouse as well. These results indicate that the immunoreactivity of laminin in the extracellular matrix changes during development and that the basement membranes in various anatomic locations display heterogeneity even in the adult mouse.
Identification of distinct messenger RNAs for nuclear lamin C and a putative precursor of nuclear lamin A.
Authors: Laliberté J F , Dagenais A , Filion M , Bibor-Hardy V , Simard R , Royal A
Published: May 2004

The lamins are the major components of the nuclear matrix and are known as lamins A, B, and C with Mr 72,000, 68,000, and 62,000 when analysed by SDS PAGE. These three polypeptides are very similar, as determined by polypeptide mapping and immunological reactivity. Lamins A and C are so homologous that a precursor-product relationship has been proposed. Using an antiserum against nuclear matrix proteins that specifically immunoprecipitates the three lamins, we examined their synthesis in the rabbit reticulocytes lysate. Four bands of Mr 62,000, 68,000, 70,000, and 74,000 were specifically immunoprecipitated when polysomes or polyadenylated RNA were translated in vitro. By two-dimensional gel electrophoresis, the 68,000- and the 62,000-mol-wt proteins were identified as lamins B and C, respectively, and the 74,000-mol-wt polypeptide had properties of a precursor of lamin A. The mRNAs of lamin C and of the putative precursor of lamin A were completely separated by gel electrophoresis under denaturing conditions, and their respective sizes were determined. These results suggest that lamin A is not a precursor of lamin C.