bluuebunny/crossref_metadata_2025 huggingface dataset:
Type VI collagen in extracellular, 100-nm periodic filaments and fibrils: identification by immunoelectron microscopy.
Authors: Bruns R R , Press W , Engvall E , Timpl R , Gross J
Published: May 2004

Filaments and fibrils that exhibit a 100-nm axial periodicity and occur in the medium and in the deposited extracellular matrix of chicken embryo and human fibroblast cultures have been tentatively identified with type VI collagen on the basis of their similar structural characteristics (Bruns, R. R., 1984, J. Ultrastruct. Res., 89:136-145). Using indirect immunoelectron microscopy and specific monoclonal and polyclonal antibodies, we now report their positive identification with collagen VI and their distribution in fibroblast cultures and in tendon. Primary human foreskin fibroblast cultures, labeled with anti-type VI antibody and studied by fluorescence microscopy, showed a progressive increase in labeling and changes in distribution with time up to 8 d in culture. With immunoelectron microscopy and monoclonal antibodies to human type VI collagen followed by goat anti-mouse IgG coupled to colloidal gold, they showed in thin sections specific 100-nm periodic labeling on extracellular filaments and fibrils: one monoclonal antibody (3C4) attached to the band region and another (4B10) to the interband region of the filaments and fibrils. Rabbit antiserum to type VI collagen also localized on the band region, but the staining was less well defined. Control experiments with antibodies to fibronectin and to procollagen types I and III labeled other filaments and fibrils, but not those with a 100-nm period. Heavy metal-stained fibrils with the same periodic and structural characteristics also have been found in both adult rat tail tendon and embryonic chicken tendon subjected to prolonged incubation in culture medium or treatment with adenosine 5'-triphosphate at pH 4.6. We conclude that the 100-nm periodic filaments and fibrils represent the native aggregate form of type VI collagen. It is likely that banded fibrils of the same periodicity and appearance, reported by many observers over the years in a wide range of normal and pathological tissues, are at least in part, type VI collagen.
Crystals of the Chlamydomonas reinhardtii cell wall: polymerization, depolymerization, and purification of glycoprotein monomers.
Authors: Goodenough U W , Gebhart B , Mecham R P , Heuser J E
Published: May 2004

Two of the three major outer layers of the Chlamydomonas reinhardtii cell wall (W6 and W4) can be solubilized from living cells with sodium perchlorate or other chaotropes and will repolymerize in vitro to form milligram amounts of wall crystals. Conditions for optimal crystalization are presented, and conditions that fail to induce polymerization are exploited to maintain monomers in aqueous solution for ion-exchange chromatography. The four major glycoproteins of the complex (GP1, 1.5, 2, and 3) have in this way been purified to apparent homogeneity and have been characterized morphologically by transmission electron microscopy using the quick-freeze, deep-etch technique and by amino acid composition. Three of the four are hydroxyproline-rich species that copolymerize to form the W6 layer. The fourth (GP1.5) is a glycine-rich species that binds to the interior of the in vitro crystal; it is apparently equivalent to the granules within the W4 layer in situ.
Cyclosporine A inhibits Ca2+-dependent stimulation of the Na+/H+ antiport in human T cells.
Authors: Rosoff P M , Terres G
Published: May 2004

The cyclic undecapeptide cyclosporine A (CsA) is a potent immunosuppressive agent that inhibits the initial activation of T lymphocytes. This agent appears to be most effective in blocking the action of mitogens such as concanavalin A and the calcium ionophore A23187, which cause an influx of Ca2+, but not those that may act by alternate mechanisms. These observations suggest that CsA may block a Ca2+-dependent step in T cell activation. We have shown that stimulation of the T3-T cell receptor complex-associated Ca2+ transporter activates the Na+/H+ antiport (Rosoff, P. M., and L. C. Cantley, 1985, J. Biol. Chem., 260: 14053-14059). The tumor-promoting phorbol esters, which are co-mitogenic for T cells, activate the exchanger by a separate pathway which is mediated by protein kinase C. Both the rise in intracellular Ca2+ and intracellular pH may be necessary for the successful triggering of cellular activation. In this report we show that CsA blocks the T3-T cell receptor-stimulated, Ca2+ influx-dependent activation of Na+/H+ exchange, but not the phorbol ester-mediated pathway in a transformed human T cell line. CsA inhibited mitogen-stimulation of interleukin-2 production in a separate cell line. CsA also inhibited vasopressin stimulation of the antiporter in normal rat kidney fibroblasts, but had no effect on serum or 12-O-tetradecanoyl phorbol 13-acetate stimulation. CsA did not affect serum or vasopressin or serum stimulation of normal rat kidney cell proliferation. CsA also had no effect on lipopolysaccharide or phorbol ester stimulation of Na+/H+ exchange activity or induction of differentiation in 70Z/3 pre-B lymphocytes in which these events are initiated by the protein kinase C pathway. These data suggest that mechanisms of activation of Na+/H+ exchange that involve an elevation in cytosolic Ca2+ are blocked by CsA but that C kinase-mediated regulation is unaffected. The importance of the Na+/H+ antiport in the regulation of growth and differentiation of T cells is discussed.
Blockage of cell-to-cell communication within pancreatic acini is associated with increased basal release of amylase.
Authors: Meda P , Bruzzone R , Knodel S , Orci L
Published: May 2004

To assess whether junctional coupling is involved in the secretory activity of pancreatic acinar cells, dispersed rat acini were incubated for 30 min in the presence of either heptanol (3.5 mM) or octanol (1.0 mM). Exposure to either alkanol caused a marked uncoupling of the acinar cells which, in control acini, were extensively coupled. Uncoupling was associated with an increased basal release of amylase that was at least twice that of controls. By contrast, carbamylcholine (10(-5) M)-induced maximal amylase secretion, cytosolic pH, and free Ca2+, as well as the structure of gap junctions joining the acinar cells, were unaffected. Both uncoupling and the alteration of basal secretion were already observed after only 5 min of exposure to heptanol, they both persisted throughout the 30-min exposure to the alkanols, and were reversible after removal of either heptanol or octanol. Since neither of the two uncouplers appeared to alter unspecifically the secretory machinery and the nonjunctional membrane of acinar cells, the data are consistent with the view that junctional coupling participates in the control of the basal secretion of acinar cells.
A nerve terminal anchorage protein from electric organ.
Authors: Carlson S S , Caroni P , Kelly R B
Published: May 2004

The nerve terminal and the postsynaptic receptor-containing membranes of the electric organ are both linked to the basal lamina that runs between them. We have identified an extracellular matrix protein whose physical properties suggest it anchors the nerve terminal to the basal lamina. The protein was identified because it shares an epitope with a proteoglycan component of electric organ synaptic vesicles. It too behaves like a proteoglycan. It is solubilized with difficulty from extracellular matrix fractions, elutes from DEAE Sephacel at pH 4.9 only at high ionic strength, and binds to a laminin affinity column from which it can be eluted with heparin. Under denaturing conditions it sediments rapidly and has a large excluded volume although it can be included in Sephacryl S-1000 columns. This large, highly charged extracellular matrix molecule can be readily reconstituted into liposomes consistent with the presence of a hydrophobic tail. By immunoelectron microscopy the antigen is found both in synaptic vesicles and on the plasma membrane of the nerve terminal. Since this is the first protein described that links the nerve terminal membrane to the extracellular matrix, we propose calling it terminal anchorage protein one (TAP-1).
Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. I. Ultrastructural autoradiographic localization and quantitation of distinct membrane acceptors for types A and B on motor nerves.
Authors: Black J D , Dolly J O
Published: May 2004

The labeling patterns produced by radioiodinated botulinum neurotoxin (125I-BoNT) types A and B at the vertebrate neuromuscular junction were investigated using electron microscopic autoradiography. The data obtained allow the following conclusions to be made. 125I-BoNT type A, applied in vivo or in vitro to mouse diaphragm or frog cutaneous pectoris muscle, interacts saturably with the motor nerve terminal only; silver grains occur on the plasma membrane, within the synaptic bouton, and in the axoplasm of the nerve trunk, suggesting internalization and retrograde intra-axonal transport of toxin or fragments thereof. 125I-BoNT type B, applied in vitro to the murine neuromuscular junction, interacts likewise with the motor nerve terminal except that a lower proportion of internalized radioactivity is seen. This result is reconcilable with the similar, but not identical, pharmacological action of these toxin types. The saturability of labeling in each case suggested the involvement of acceptors; on preventing the internalization step with metabolic inhibitors, their precise location became apparent. They were found on all unmyelinated areas of the nerve terminal membrane, including the preterminal axon and the synaptic bouton. Although 125I-BoNT type A interacts specifically with developing terminals of newborn rats, the unmyelinated plasma membrane of the nerve trunk is not labeled, indicating that the acceptors are unique components restricted to the nerve terminal area. BoNT types A and B have distinct acceptors on the terminal membrane. Having optimized the conditions for saturation of these binding sites and calibrated the autoradiographic procedure, we found the densities of the acceptors for types A and B to be approximately 150 and 630/micron 2 of membrane, respectively. It is proposed that these membrane acceptors target BoNT to the nerve terminal and mediate its delivery to an intracellular site, thus contributing to the toxin's selective inhibitory action on neurotransmitter release.
The acetylation of alpha-tubulin and its relationship to the assembly and disassembly of microtubules.
Authors: Maruta H , Greer K , Rosenbaum J L
Published: May 2004

A tight association between Chlamydomonas alpha-tubulin acetyltransferase (TAT) and flagellar axonemes, and the cytoplasmic localization of both tubulin deacetylase (TDA) and an inhibitor of tubulin acetylation have been demonstrated by the use of calf brain tubulin as substrate for these enzymes. A major axonemal TAT of 130 kD has been solubilized by high salt treatment, purified, and characterized. Using the Chlamydomonas TAT with brain tubulin as substrate, we have studied the effects of acetylation on the assembly and disassembly of microtubules in vitro. We also determined the relative rates of acetylation of tubulin dimers and polymers. The acetylation does not significantly affect the temperature-dependent polymerization or depolymerization of tubulin in vitro. Furthermore, polymerization of tubulin is not a prerequisite for the acetylation, although the polymer is a better substrate for TAT than the dimer. The acetylation is sensitive to calcium ions which completely inhibit the acetylation of both dimers and polymers of tubulin. Acetylation of the dimer is not inhibited by colchicine; the effect of colchicine on acetylation of the polymer can be explained by its depolymerizing effect on the polymer.
Reactivation of organelle movements along the cytoskeletal framework of a giant freshwater ameba.
Authors: Koonce M P , Schliwa M
Published: May 2004

The peripheral feeding network of the giant freshwater ameba Reticulomyxa can be easily and rapidly lysed to produce an extensive, stable, and completely exposed cytoskeletal framework of colinear microtubules and microfilaments. Most of the organelles that remain attached to this framework resume rapid saltatory movements at rates of up to 20 micron/s if ATP is added. This lysed model system is also capable of other forms of motility, namely an active splaying of microtubule bundles and bulk streaming. Reactivation does not occur with other nucleoside triphosphates, requires Mg ions, is insensitive to even high concentrations of erythro-9-(3-[2-hydroxynonyl]) adenine, is sensitive to vanadate only at concentrations of approximately 100 microM, and is inhibited by N-ethylmaleimide at concentrations greater than 100 microM. The physiology of this reactivation suggests an organelle transport motor distinct from cytoplasmic dynein and possibly the recently described kinesin. This system can serve as a model for elucidating the mechanisms of intracellular transport and, in addition, provides a unique opportunity to examine associations between microtubules and microfilaments.
Isolation and partial characterization of a 110-kD dimer actin-binding protein.
Authors: Ueno T , Korn E D
Published: May 2004

Two Triton-insoluble fractions were isolated from Acanthamoeba castellanii. The major non-membrane proteins in both fractions were actin (30-40%), myosin II (4-9%), myosin I (1-5%), and a 55-kD polypeptide (10%). The 55-kD polypeptide did not react with antibodies against tubulins from turkey brain, paramecium, or yeast. All of these proteins were much more concentrated in the Triton-insoluble fractions than in the whole homogenate or soluble supernatant. The 55-kD polypeptide was extracted with 0.3 M NaCl, fractionated by ammonium sulfate, and purified to near homogeneity by DEAE-cellulose and hydroxyapatite chromatography. The purified protein had a molecular mass of 110 kD and appeared to be a homodimer by isoelectric focusing. The 110-kD dimer bound to F-actin with a maximal binding stoichiometry of 0.5 mol/mol of actin (1 mol of 55-kD subunit/mol of actin). Although the 110-kD protein enhanced the sedimentation of F-actin, it did not affect the low shear viscosity of F-actin solutions nor was bundling of F-actin observed by electron microscopy. The 110-kD dimer protein inhibited the actin-activated Mg2+-ATPase activities of Acanthamoeba myosin I and myosin II in a concentration-dependent manner. By indirect immunofluorescence, the 110-kD protein was found to be localized in the peripheral cytoplasm near the plasma membrane which is also enriched in F-actin filaments and myosin I.
Axokinin phosphorylation by cAMP-dependent protein kinase is sufficient for activation of sperm flagellar motility.
Authors: Tash J S , Hidaka H , Means A R
Published: May 2004

Using a selective inhibitor of cAMP-dependent protein kinase, N-[2(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), the requirement for cAMP-dependent phosphoproteins in the initiation of dog sperm flagellar motility was examined. H-8 inhibited motility of live as well as reactivated sperm in a dose-dependent manner. The half-maximal inhibition of reactivated motility (32 microM) paralleled the inhibition of pure catalytic subunit of cAMP-dependent protein kinase (50 microM) measured under the same conditions. H-8 inhibited protein phosphorylation both in whole models and in isolated Nonidet P-40 (NP-40) extracts of sperm. Axokinin, the heat-stable NP-40-soluble protein whose phosphorylation is required for flagellar reactivation, represented 97% of the de novo phosphate incorporation in the NP-40 extract after stimulation by cAMP. 500 microM H-8 inhibited axokinin phosphorylation by 87%. When sperm were reactivated in the presence of up to 5 mM H-8 with NP-40 extract that had been prephosphorylated with cAMP-dependent protein kinase, then neither cAMP nor cAMP-dependent protein kinase activity was required for full flagellar reactivation. If sperm were rendered completely immotile by pretreatment with H-8, then the resulting model remained immotile in the continued presence of H-8 unless prephosphorylated axokinin was added. These results suggest that phosphorylated axokinin is not only required for flagellar reactivation but is sufficient as well.
The architecture of actin filaments and the ultrastructural location of actin-binding protein in the periphery of lung macrophages.
Authors: Hartwig J H , Shevlin P
Published: May 2004

A highly branched filament network is the principal structure in the periphery of detergent-extracted cytoskeletons of macrophages that have been spread on a surface and either freeze or critical point dried, and then rotary shadowed with platinum-carbon. This array of filaments completely fills lamellae extended from the cell and bifurcates to form 0.2-0.5 micron thick layers on the top and bottom of the cell body. Reaction of the macrophage cytoskeletons with anti-actin IgG and with anti-IgG bound to colloidal gold produces dense staining of these filaments, and incubation with myosin subfragment 1 uniformly decorates these filaments, identifying them as actin. 45% of the total cellular actin and approximately 70% of actin-binding protein remains in the detergent-insoluble cell residue. The soluble actin is not filamentous as determined by sedimentation analysis, the DNAase I inhibition assay, and electron microscopy, indicating that the cytoskeleton is not fragmented by detergent extraction. The spacing between the ramifications of the actin network is 94 +/- 47 nm and 118 +/- 72 nm in cytoskeletons prepared for electron microscopy by freeze drying and critical point drying, respectively. Free filament ends are rare, except for a few which project upward from the body of the network or which extend down to the substrate. Filaments of the network intersect predominantly at right angles to form either T-shaped and X-shaped overlaps having striking perpendicularity or else Y-shaped intersections composed of filaments intersecting at 120-130 degrees angles. The actin filament concentration in the lamellae is high, with an average value of 12.5 mg/ml. The concentration was much more uniform in freeze-dried preparations than in critical point-dried specimens, indicating that there is less collapse associated with the freezing technique. The orthogonal actin network of the macrophage cortical cytoplasm resembles actin gels made with actin-binding protein. Reaction of cell cytoskeletons and of an actin gel made with actin-binding protein with anti-actin-binding protein IgG and anti-IgG-coated gold beads resulted in the deposition of clusters of gold at points where filaments intersect and at the ends of filaments that may have been in contact with the membrane before its removal with detergent. In the actin gel made with actin-binding protein, 75% of actin-fiber intersections labeled, and the filament spacing between intersections is consistent with that predicted on theoretical grounds if each added actin-binding protein molecule cross-links two filaments to form an intersection in the gel.(ABSTRACT TRUNCATED AT 400 WORDS)
Endogenous lectins from cultured soybean cells: isolation of a protein immunologically cross-reactive with seed soybean agglutinin and analysis of its role in binding of Rhizobium japonicum.
Authors: Ho S C , Malek-Hedayat S , Wang J L , Schindler M
Published: May 2004

Incubation of Rhizobium japonicum with the cultured soybean cell line SB-1, originally derived from the roots of Glycine max, resulted in specific adhesion of the bacteria to the plant cells. This binding interaction appears to be mediated via carbohydrate recognition, since galactose can inhibit the heterotypic adhesion but glucose cannot. Affinity chromatography, on a Sepharose column derivatized with N-caproyl-galactosamine, of the supernatant fraction of a SB-1 cell suspension after enzymatic removal of cell wall yielded a single polypeptide (Mr approximately 30,000) on immunoblotting analysis with rabbit antibodies directed against seed soybean agglutinin. Fluorescently labeled rabbit anti-seed soybean agglutinin also yielded specific immunofluorescent staining on the cell wall and plasma membrane of the SB-1 cells. These results suggest that one likely candidate that may mediate the recognition between the Rhizobium and the soybean cells is the endogenously produced SB-1 lectin. This notion is supported by the observation that rabbit anti-seed soybean agglutinin blocked the Rhizobium-soybean cell adhesion, whereas control antibodies did not.
Distribution of a putative cell surface receptor for fibronectin and laminin in the avian embryo.
Authors: Krotoski D M , Domingo C , Bronner-Fraser M
Published: May 2004

The cell substratum attachment (CSAT) antibody recognizes a 140-kD cell surface receptor complex involved in adhesion to fibronectin (FN) and laminin (LM) (Horwitz, A., K. Duggan, R. Greggs, C. Decker, and C. Buck, 1985, J. Cell Biol., 101:2134-2144). Here, we describe the distribution of the CSAT antigen along with FN and LM in the early avian embryo. At the light microscopic level, the staining patterns for the CSAT receptor and the extracellular matrix molecules to which it binds were largely codistributed. The CSAT antigen was observed on numerous tissues during gastrulation, neurulation, and neural crest migration: for example, the surface of neural crest cells and the basal surface of epithelial tissues such as the ectoderm, neural tube, notochord, and dermomyotome. FN and LM immunoreactivity was observed in the basement membranes surrounding many of these epithelial tissues, as well as around the otic and optic vesicles. In addition, the pathways followed by cranial neural crest cells were lined with FN and LM. In the trunk region, FN and LM were observed surrounding a subpopulation of neural crest cells. However, neither molecule exhibited the selective distribution pattern necessary for a guiding role in trunk neural crest migration. The levels of CSAT, FN, and LM are dynamic in the embryo, perhaps reflecting that the balance of surface-substratum adhesions contributes to initiation, migration, and localization of some neural crest cell populations.
C3d fragment of complement interacts with laminin and binds to basement membranes of glomerulus and trophoblast.
Authors: Leivo I , Engvall E
Published: May 2004

Two mouse monoclonal antibodies generated against human placental homogenate were found to react specifically with human complement component C3. In immunofluorescence of human tissues, these antibodies gave a bright linear staining outlining the glomerular basement membrane of the adult kidney and the trophoblast basement membrane of placenta. An identical staining pattern was observed with a rabbit C3d antiserum which also prevented binding of the monoclonal antibodies to tissue sections. Only negligible basement membrane staining was observed in the same tissues with antisera to human C3c, C5, IgG, IgA, or IgM. When interactions of C3 with basement membrane proteins were tested in enzyme immunoassays and column chromatography, C3(H2O) was found to bind efficiently to solid-phase laminin. Native C3 from fresh plasma did not bind to laminin but C3 from plasma treated with methylamine bound efficiently. When C3 was cleaved with trypsin, C3b and C3d but not C3c bound to laminin-Sepharose. The interaction of C3 and laminin was inhibited by soluble laminin and by high ionic strength. The results indicate that C3d, a biologically active breakdown product of C3, can be found in glomerular and placental basement membranes in the absence of signs for ongoing local complement activation or immune complex deposition. It is possible that binding affinities between C3 and basement membrane molecules, especially laminin, are involved in the retention of C3d at these sites. Such interactions between C3 and components of the glomerular basement membrane could play important roles in complement-related pathological processes of the glomerulus.
Ultrastructural immunolocalization of lysyl oxidase in vascular connective tissue.
Authors: Kagan H M , Vaccaro C A , Bronson R E , Tang S S , Brody J S
Published: May 2004

The localization of lysyl oxidase was examined in calf and rat aortic connective tissue at the ultrastructural level using polyclonal chicken anti-lysyl oxidase and gold conjugated rabbit anti-chicken immunoglobulin G to identify immunoreactive sites. Electron microscopy of calf aortic specimens revealed discrete gold deposits at the interface between extracellular bundles of amorphous elastin and the microfibrils circumferentially surrounding these bundles. The antibody did not react with microfibrils which were distant from the interface with elastin. There was negligible deposition of gold within the bundles of amorphous elastin and those few deposits seen at these sites appeared to be associated with strands of microfibrils. Lysyl oxidase was similarly localized in newborn rat aorta at the interface between microfibrils and nascent elastin fibers. Gold deposits were not seen in association with extracellular collagen fibers even after collagen-associated proteoglycans had been degraded by chondroitinase ABC. However, the antibody did recognize collagen-bound lysyl oxidase in collagen fibers prepared from purified collagen to which the enzyme had been added in vitro. No reaction product was seen if the anti-lysyl oxidase was preadsorbed with purified lysyl oxidase illustrating the specificity of the antibody probe. The present results are consistent with a model of elastogenesis predicting the radial growth of the elastin fiber by the deposition and crosslinking of tropoelastin units at the fiber-microfibril interface.
The positive transcription factor of the 5S RNA gene proteolyses during direct exchange between 5S DNA sites.
Authors: Kmiec E B , Worcel A
Published: May 2004

We have examined the association, dissociation, and exchange of the 5S specific transcription factor (TFIIIA) with somatic- and oocyte-type 5S DNA. The factor associates faster with somatic than with oocyte 5S DNA, and the rate of complex formation is accelerated by vector DNA. Once formed, the TFIIIA-5S DNA complex is stable for greater than 4 h in the absence of free 5S DNA, and its dissociation is identical for somatic and for oocyte 5S DNA. In the presence of free 5S DNA, the factor transfers promptly from the complex to the free 5S DNA site. Unexpectedly, the direct exchange of factor between 5S DNA sites leads to proteolysis at the C-terminal arm of TFIIIA.
Microinjected Tetrahymena rDNA ends are not recognized as telomeres in Xenopus eggs.
Authors: Berg C A , Gall J G
Published: May 2004

Telomeres are essential structures that stabilize the ends of eukaryotic chromosomes and allow complete replication of linear DNA molecules. We examined the structure and replication of telomeres by observing the fate of the linear extrachromosomal rDNA of Tetrahymena after injection into unfertilized Xenopus eggs. The rDNA replicated efficiently as a linear extrachromosomal molecule, increasing in mass 30-50-fold by 15-20 h after injection. In addition, the molecules increased in length by addition of up to several kilobases of DNA to their termini. Sequence analysis demonstrated that the added DNA bore no resemblance to known telomeres. The junction between the rDNA and added DNA was apparently random, indicating that the addition reaction did not involve a site-specific recombination or integration event. Surprisingly, Southern blot analysis showed that the added DNA did not derive from Xenopus DNA, but rather from co-purifying and therefore co-injected Tetrahymena DNA. The nonspecific ligation of random DNA fragments to the rDNA termini suggests that microinjected Tetrahymena rDNA ends are not recognized as telomeres in Xenopus eggs.
Characterization of the junctional face membrane from terminal cisternae of sarcoplasmic reticulum.
Authors: Costello B , Chadwick C , Saito A , Chu A , Maurer A , Fleischer S
Published: May 2004

We have recently described a preparation of junctional terminal cisternae (JTC) from fast skeletal muscle of rabbit hind leg. The fraction differs from other heavy sarcoplasmic reticulum (SR) fractions in that it contains a substantial amount of junctional face membrane (JFM) (15-20% of the membrane) with morphologically well-defined junctional feet structures. In common with other heavy SR preparations, it contains predominantly the calcium pump membrane (80-85% of the membrane) and compartmental contents (CC), consisting mainly of calcium-binding protein (calsequestrin). In this study, a modified procedure for the preparation of JTC from frozen rabbit back muscle is described. The yield is substantially greater (threefold per weight of muscle), yet retaining characteristics similar to JTC from fresh hind leg muscles. Methodology has been developed for the disassembly of the JTC. This is achieved by selectively extracting the calcium pump membrane with 0.5% Triton X-100 in the presence of 1 mM CaCl2 to yield a complex of JFM with CC. The CC are then solubilized in the presence of EDTA to yield JFM. This fraction contains unidirectionally aligned junctional feet structures protruding from the cytoplasmic face of the membrane with repeat spacings comparable to that observed in JTC. The JFM contains 0.16 mumol phosphorus (lipid) per milligram protein. Characteristic proteins include 340 and 79-kD bands, a doublet at 28 kD, and a component that migrates somewhat slower than or equivalent to the calcium pump protein. Approximately 10% of the calcium-binding protein remains bound to the JFM after EDTA extraction, indicating the presence of a specific binding component in the JFM. The JFM, which is involved in junctional association with transverse tubule and likely in the Ca2+ release process in excitation-contraction coupling, is now available in the test tube.
Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia.
Authors: Stevenson B R , Siliciano J D , Mooseker M S , Goodenough D A
Published: May 2004

A tight junction-enriched membrane fraction has been used as immunogen to generate a monoclonal antiserum specific for this intercellular junction. Hybridomas were screened for their ability to both react on an immunoblot and localize to the junctional complex region on frozen sections of unfixed mouse liver. A stable hybridoma line has been isolated that secretes an antibody (R26.4C) that localizes in thin section images of isolated mouse liver plasma membranes to the points of membrane contact at the tight junction. This antibody recognizes a polypeptide of approximately 225,000 D, detectable in whole liver homogenates as well as in the tight junction-enriched membrane fraction. R26.4C localizes to the junctional complex region of a number of other epithelia, including colon, kidney, and testis, and to arterial endothelium, as assayed by immunofluorescent staining of cryostat sections of whole tissue. This antibody also stains the junctional complex region in confluent monolayers of the Madin-Darby canine kidney epithelial cell line. Immunoblot analysis of Madin-Darby canine kidney cells demonstrates the presence of a polypeptide similar in molecular weight to that detected in liver, suggesting that this protein is potentially a ubiquitous component of all mammalian tight junctions. The 225-kD tight junction-associated polypeptide is termed "ZO-1."
Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein.
Authors: Kumar N M , Gilula N B
Published: May 2004

An extended synthetic oligonucleotide (58-mer) has been used to identify and characterize a human liver gap junction cDNA. The cDNA is 1,574 bases long and contains the entire coding region for a gap junction protein. In vitro translation of the RNA products of this cDNA is consistent with it coding for a 32,022-D protein. Southern blot analysis indicates that the gap junction gene is present as a single copy, and that it can be detected in a variety of organisms using the human liver cDNA as a probe. The human cDNA has been used to screen a rat liver cDNA library, and a rat liver junction cDNA clone has been isolated. The rat liver clone is 1,127 bases in length, and it has strong sequence homology to the human cDNA in the protein-coding region, but less extensive homology in the 3'-untranslated region.
Cellular analysis by in situ hybridization and immunoperoxidase of alpha-fetoprotein and albumin gene expression in rat liver during the perinatal period.
Authors: Poliard A M , Bernuau D , Tournier I , Legrès L G , Schoevaert D , Feldmann G , Sala-Trepat J M
Published: May 2004

To analyze at the cellular level the decrease in alpha-fetoprotein (AFP) gene expression during the early postnatal growth, we searched for AFP gene transcripts by in situ hybridization using a specific cDNA probe, and for the corresponding protein by immunocytochemistry, on rat liver sections at various times of the perinatal period. The relative number of mRNA sequences was evaluated by Northern blot analysis. Albumin (ALB) gene expression was studied simultaneously with the same techniques. In 17-19-d-old fetuses all hepatocytes express simultaneously, for both genes, the mRNAs and the corresponding proteins. During the first postnatal weeks, at a time when the global number of AFP mRNA molecules decreases, all hepatocytes still contain cytoplasmic transcripts and protein. A zonal heterogeneity in the level of AFP gene expression develops around the first week, a higher number of gene products being detected in perivenous than in periportal hepatocytes. This heterogeneity persists until the fourth week when AFP mRNA sequences and protein are barely detectable. All hepatocytes express the ALB gene after birth, but at around the second week, a periportal intensification of the in situ hybridization signal and immunostaining becomes apparent. Our data indicate that co-expression of the AFP and ALB genes by all hepatocytes is a normal step in liver ontogeny; the diminution of AFP gene expression after birth is not the result of the disappearance of specialized cell clones; and zonal quantitative differences in the level of AFP and ALB gene expression are observed within the maturing liver lobule.
Monocyte-conditioned medium, interleukin-1, and tumor necrosis factor stimulate the acute phase response in human hepatoma cells in vitro.
Authors: Darlington G J , Wilson D R , Lachman L B
Published: May 2004

Human hepatoma cells mimic the acute phase response after treatment with monocyte-conditioned medium. Levels of secreted fibrinogen, alpha-1 acid glycoprotein, C-reactive protein, haptoglobin, and the third component of complement were elevated compared with control levels after 48 h of incubation with conditioned supernatant medium from an enriched fraction of normal peripheral monocytes. Albumin levels declined and alpha-1 antitrypsin remained unchanged. Levels of specific mRNA were measured by hybridization to slot blots and Northern blots and changed in correspondence with protein alterations. Interleukin-1 and tumor necrosis factor stimulated the third component of complement, but did not elevate any other member of the acute phase group and were therefore only partially active in this system. The identification of an in vitro model of the human acute phase response will permit analysis of the molecular basis for coordinate regulation of this group of facultative genes.
Schistosomula of Schistosoma mansoni use lysophosphatidylcholine to lyse adherent human red blood cells and immobilize red cell membrane components.
Authors: Golan D E , Brown C S , Cianci C M , Furlong S T , Caulfield J P
Published: May 2004

Human red blood cells (RBCs) adhere to and are lysed by schistosomula of Schistosoma mansoni. We have investigated the mechanism of RBC lysis by comparing the dynamic properties of transmembrane protein and lipid probes in adherent ghost membranes with those in control RBCs and in RBCs treated with various membrane perturbants. Fluorescence photobleaching recovery was used to measure the lateral mobility of two integral membrane proteins, glycophorin and band 3, and two lipid analogues, fluorescein phosphatidylethanolamine (Fl-PE) and carbocyanine dyes, in RBCs and ghosts adherent to schistosomula. Adherent ghosts manifested 95-100% immobilization of both membrane proteins and 45-55% immobilization of both lipid probes. In separate experiments, diamide-induced cross-linking of RBC cytoskeletal proteins slowed transmembrane protein diffusion by 30-40%, without affecting either transmembrane protein fractional mobility or lipid probe lateral mobility. Wheat germ agglutinin- and polylysine-induced cross-linking of glycophorin at the extracellular surface caused 80-95% immobilization of the transmembrane proteins, without affecting the fractional mobility of the lipid probe. Egg lysophosphatidylcholine (lysoPC) induced both lysis of RBCs and a concentration-dependent decrease in the lateral mobility of glycophorin, band 3, and Fl-PE in ghost membranes. At a concentration of 8.4 micrograms/ml, lysoPC caused a pattern of protein and lipid immobilization in RBC ghosts identical to that in ghosts adherent to schistosomula. Schistosomula incubated with labeled palmitate released lysoPC into the culture medium at a rate of 1.5 fmol/h per 10(3) organisms. These data suggest that lysoPC is transferred from schistosomula to adherent RBCs, causing their lysis.
Amino-terminal deletion mutants of the Rous sarcoma virus glycoprotein do not block signal peptide cleavage but can block intracellular transport.
Authors: Hardwick J M , Shaw K E , Wills J W , Hunter E
Published: May 2004

Protein sequence requirements for cleavage of the signal peptide from the Rous sarcoma virus glycoprotein have been investigated through the use of deletion mutagenesis. The phenotypes of these mutants have been characterized by expression of the cloned, mutated env genes in CV-1 cells using a late replacement SV40 vector. The deletion mutations were generated by Ba131 digestion at the XhoI site located near the 5' end of the coding sequence for the structural protein gp85, which is found at the amino terminus of the precursor glycoprotein, Pr95. The results of experiments with three mutants (X1, X2, and X3) are presented. Mutant X1 has a 14 amino acid deletion encompassing amino acids 4-17 of gp85, which results in the loss of one potential glycosylation site. In mutants X2 and X3 the amino terminal nine and six amino acids, respectively, of gp85 are deleted. During the biosynthesis of all three mutant polypeptides, the signal peptide is efficiently and accurately cleaved from the nascent protein, even though in mutants X2 and X3 the cleavage site itself has been altered. In these mutants the alanine/aspartic acid cleavage site has been mutated to alanine/asparagine and alanine/glutamine, respectively. These results are consistent with the concept that sequences C-terminal to the signal peptidase site are unimportant in defining the site of cleavage in eucaryotes. Mutants X2 and X3 behave like wild-type with respect to protein glycosylation, palmitic acid addition, cleavage to gp85 and gp37, and expression on the cell surface. Mutant X1, on the other hand, is defective in intracellular transport. Although it is translocated across the rough endoplasmic reticulum and core-glycosylated, its transport appears to be blocked at an early Golgi compartment. No terminal glycosylation of the protein, cleavage of the precursor protein to the mature products, or expression on the cell surface is observed. The deletion in X1 thus appears to destroy signals required for export to the cell surface.
Role of intracellular calcium and protein kinase C in the endocytosis of transferrin and insulin by HL60 cells.
Authors: Iacopetta B , Carpentier J L , Pozzan T , Lew D P , Gorden P , Orci L
Published: May 2004

The role of the cytosolic free calcium concentration ([Ca2+]i) and of protein kinase C on the internalization of transferrin and insulin in the human promyelocytic cell line HL60 was investigated. [Ca2+]i was selectively monitored and manipulated by the use of the fluorescent Ca2+ indicator and buffer quin2, while receptor-ligand internalization was studied directly by quantitative electron microscope autoradiography. Decreasing the [Ca2+]i up to 10-fold below resting level had no effect on the internalization of transferrin or insulin. Similarly, a 10-fold elevation of the [Ca2+]i using the calcium ionophore ionomycin caused little or no change in the endocytosis of the two ligands. In contrast, activation of protein kinase C by phorbol myristate acetate markedly stimulated the internalization of both occupied and unoccupied transferrin receptors, even in cells with very low [Ca2+]i. The insulin receptor was found to behave differently in response to phorbol myristate acetate, however, in that only the occupied receptors were stimulated to internalize. We conclude that the [Ca2+]i plays only a minor role in regulating receptor-mediated endocytosis, whereas protein kinase C can selectively modulate receptor internalization depending on receptor type and occupancy.
Toxoplasma modifies macrophage phagosomes by secretion of a vesicular network rich in surface proteins.
Authors: Sibley L D , Krahenbuhl J L , Adams G M , Weidner E
Published: May 2004

Modification of macrophage phagosomes begins shortly after formation as Toxoplasma cells secrete membranous vesicles that form a reticulate network within the vacuole. The Toxoplasma-modified compartments then resist normal endocytic processing and digestion. We have used the pronounced Ca++-dependent stability of the intraphagosomal membrane (IPM) network to purify and characterize the structural proteins of this assembly. In addition to the structural matrix, Toxoplasma secretes a discrete set of soluble proteins, including a newly described 22-kD calcium-binding protein. The IPM network adheres to intact Toxoplasma cells after host cell lysis in the presence of 1 mM Ca++; however, the network readily disperses in calcium-free buffer and was purified as vesicles that sedimented at 100,000 g. Purified IPM vesicles were specifically recognized by immune sera from mice with chronic Toxoplasma infection and consisted primarily of a 30-kD protein when analyzed by SDS PAGE. IPM network proteins share a major antigenic component located on the surface of extracellular Toxoplasma cells as shown by immunoperoxidase electron microscopy using a polyclonal antibody prepared against the IPM vesicles. Moreover, in Toxoplasma-infected macrophages, anti-IMP antibody confirmed that the extensive IPM array contains proteins also found on the Toxoplasma cell surface. Our results indicate the IMP network represents a unique structural modification of the phagosome comprised in part of Toxoplasma surface proteins.
Changes in the organization of the neuritic cytoskeleton during nerve growth factor-activated differentiation of PC12 cells: a serial electron microscopic study of the development and control of neurite shape.
Authors: Jacobs J R , Stevens J K
Published: May 2004

After exposure to nerve growth factor, PC12 cells differentiate within a period of only a few days into cholinergic sympathetic neurons. Using computer-assisted three-dimensional serial electron microscopic reconstruction, we describe the progressive cytoskeletal and structural changes of PC12 neurites at different stages in their differentiation. Developmental changes in these neurites can be characterized by two major transitions. First, microtubules (MTs), which define the longitudinal axis of the neurite, increase in number leading to a more cylindrical and uniform neurite shape. Second, there are major changes in the relative numbers of other organelle types, which reflect the functional specialization of the neurite. These changes do not in themselves seriously affect shape change of the neurite during development, however the presence of these organelles and their associated obligatory volumes (volumes surrounding organelle) account for well over 50% of the neurite's volume at all stages of development. The MT-MT distances and obligatory volumes associated with the organelles remain constant throughout development. Thus, we can conclude that many of the observed changes seen in developing PC12 neurites are due simply to the production of a greater number of MTs in the cell, and that many of the other important parameters that can be measured and contribute to neurite shape remain constant during development.
Experimental modification of PC12 neurite shape with the microtubule-depolymerizing drug Nocodazole: a serial electron microscopic study of neurite shape control.
Authors: Jacobs J R , Stevens J K
Published: May 2004

The microtubule-depolymerizing drug Nocodazole has been used to experimentally manipulate the form of PC12 neurites. Both time-lapse photography and serial electron microscopy demonstrate that microtubule depolymerization leads to varicosity formation due to a clustering of membranous organelles in young neurites (nerve growth factor activated within 7 d). Neurites that have been nerve growth factor activated 7 or more d before Nocodazole application are resistant to microtubule depolymerization. These data and data from previous papers has been combined in an attempt to predict quantitatively the volume and the shape of a neurite. The relationship is described mathematically by Vn = 4.52 Vo + 0.0054 MTl, where Vn is local neurite volume, Vo is organelle volume, and MTl is MT length (the constant, 0.0054 is micron2), and 4.52 is the obligatory volume constant derived from serial electron microscopic studies. The equation predicts the total volume of neurites despite alterations of morphology due to Nocodazole and despite changes in morphology during development.
Microtubule reassembly from nucleating fragments during the regrowth of amputated neurites.
Authors: Baas P W , Heidemann S R
Published: May 2004

We have proposed that stable microtubule (MT) fragments that resist depolymerization may serve as nucleating elements for the local control of MT dynamics in the axon (Heidemann, S. R., M. A. Hamborg, S. J. Thomas, B. Song, S. Lindley, and D. Chu, 1984, J. Cell Biol., 99:1289-1295). Here we report evidence that supports this proposal in studies on the role of MTs in the regrowth of neurites from the distal segments of amputated chick sensory neurites. Amputated neurites collapse to "beads" of axoplasm that rapidly regrow (Shaw, G., and D. Bray, 1977, Exp. Cell Res., 104:55-62). We examined both unarrested regrowth and regrowth after MT disassembly by either cold (-5 degrees C for 2 h) or nocodazole (0.1 microgram/ml for 15-20 min). In all these cases regrowth occurred at 3.5-4.5 micron/min with no delay times other than the times to reach 37 degrees C or rinse out the nocodazole. Electron micrographs of untreated beads show many MTs of varying lengths, while those of cold- and nocodazole-treated beads show markedly shorter MTs. The robust regrowth of neurites from beads containing only very short MTs argues against unfurling of intact MTs from the bead into the growing neurite. Electron micrographs of cold-treated beads lysed under conditions that cause substantial MT depolymerization in untreated intact neurites show persistent MT fragments similar to those in unlysed cold-treated beads. We interpret this as evidence that the MT fragments in cold-treated beads are somehow distinct from the majority of the MT mass that had depolymerized. Collapsed neurites treated with a higher dose of nocodazole (1.0 microgram/ml for 15-20 min) were completely devoid of MTs and regrew only after a 15-20 min delay in two cases but never regrew in 11 other cases. We found that MTs did not return in beads treated with 1.0 microgram/ml nocodazole even 30 min after removal of the drug. It was unlikely that the inability of these beads to reassemble MTs was due to incomplete removal of nocodazole in that a much higher dose (20 micrograms/ml nocodazole) could be quickly rinsed from intact neurites. Beads treated with 1.0 microgram/ml nocodazole could, however, be stimulated to reassemble MTs and regrow neurites by treatment with taxol. We conclude that the immediate, robust regrowth of neurites from collapsed beads of axoplasm requires MT nucleation sites to support MT reassembly.(ABSTRACT TRUNCATED AT 400 WORDS)