bluuebunny/crossref_metadata_2025 huggingface dataset:
Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse.
Authors: Clarke H J , Masui Y
Published: May 2004

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.
On the surface lattice of microtubules: helix starts, protofilament number, seam, and handedness.
Authors: Mandelkow E M , Schultheiss R , Rapp R , Müller M , Mandelkow E
Published: May 2004

The tubulin monomers of brain microtubules reassembled in vitro are arranged on a 3-start helix, irrespective of whether the number of protofilaments is 13 or 14. The dimer packing is that of the B-lattice described for flagellar microtubules. This implies that the tubulin core of microtubules contains at least one helical discontinuity. Neither 5-start nor 8-start helices have a physical significance and thus cannot be implicated in models of microtubule elongation, but the structure is compatible with elongation of protofilaments by dimers or protofilamentous oligomers. The inner and outer surfaces of the microtubule wall can be visualized by propane jet freezing, freeze fracturing, and metal replication, at a resolution of at least 4 nm. The 3-start helix is left-handed, in contrast to a previous study based on negative staining and shadowing. The reasons for this discrepancy are discussed.
Bridgelike interconnections between thick filaments in stretched skeletal muscle fibers observed by the freeze-fracture method.
Authors: Suzuki S , Pollack G H
Published: May 2004

The ultrastructure of frog semitendinosus muscle was explored using the freeze-fracture, deep-etch, rotary-shadowing technique. Mechanically skinned fibers were stretched to decrease or eliminate the overlap of thick and thin filaments before rapid freezing with liquid propane. In relaxed, contracting, and rigor fibers, a significant number of bridgelike interconnections, distinct from those observed in the M-region, were observed between adjacent thick filaments in the non-overlap region. Their half-length and diameter corresponded approximately to the known dimensions of the cross-bridge (or myosin S-1). The interconnection may thus be formed by the binding of two apposed cross-bridges projecting from adjacent thick filaments. Fixation with 0.5% glutaraldehyde for 5-10 min before freezing effectively preserved these structures. The results indicate that the interconnections are genuine structures that appear commonly in stretched muscle fibers. They may play a role in stabilizing the thick filament lattice, and possibly in the contractile process.
Further analysis of pemphigus autoantibodies and their use in studies on the heterogeneity, structure, and function of desmosomes.
Authors: Jones J C , Yokoo K M , Goldman R D
Published: May 2004

Pemphigus is an autoimmune disease that causes blistering of human epidermis. We have recently shown that autoantibodies in the serum of three pemphigus patients bind to desmosomes (Jones, J. C. R., J. Arnn, L. A. Staehelin, and R. D. Goldman, 1984, Proc. Natl. Acad. Sci. USA., 81:2781-2785), and we suggested that pemphigus blisters form, at least in part, from a specific antibody-induced disruption of desmosomes in the epidermis. In this paper, experiments are described that extend our initial observations. 13 pemphigus serum samples, which include four known pemphigus vulgaris (Pv) and four known pemphigus foliaceus (Pf) serum samples, have been analyzed by both immunofluorescence and by immunoblotting using cell-free desmosome preparations. Tissue sections of mouse skin processed for double indirect immunofluorescence using each of the pemphigus serum samples and a rabbit antiserum directed against a component of the desmosomal plaque (desmoplakin) show similar punctate cell surface staining patterns. This suggests that all 13 pemphigus serum samples contain autoantibodies that recognize desmosomes. These autoantibodies appear specific for stratified squamous epithelial cell desmosomes and do not recognize desmosomes of other tissues (e.g., mouse heart and mouse intestine). Cultured mouse keratinocytes, which possess well-defined desmosomes, were processed for indirect immunofluorescence using the pemphigus serum samples. Eight of the 13 sera (including the four known Pv samples but not the known Pf sera) stain desmosomes in these preparations. By double indirect immunofluorescence the desmoplakin antiserum stains a double fluorescent line along the contacting edges of cultured keratinocytes, whereas the positive pemphigus serum samples stain a single fluorescent line along this same border. We believe that these pemphigus autoantibodies recognize extracellular antigens located somewhere within the region between the two apposing membranes that comprise the desmosome. The pemphigus sera exhibit positive immunoblotting reactions with desmosome-enriched fractions obtained from bovine tongue epithelium. Three serum samples (including two of the four known Pf serum samples) react with 160- and 165-kD desmosome-associated polypeptides (Koulu, L., A. Kusimi, M. S. Steinberg, V. Klaus-Kovtun, and J. R. Stanley, 1984, J. Exp. Med., 160:1509-1518). Another eight serum samples (including the four known Pv sera) recognize a 140-kD desmosome-associated polypeptide. We propose that the antigens recognized by these human autoantibodies may play important roles in the adhesion of cells within the epidermis.(ABSTRACT TRUNCATED AT 400 WORDS)
Distribution of tyrosinated and nontyrosinated alpha-tubulin during mitosis.
Authors: Gundersen G G , Bulinski J C
Published: May 2004

The C-terminus of alpha-tubulin undergoes a reversible posttranslational tyrosination/detyrosination. The distributions of the tyrosinated (Tyr) and nontyrosinated (Glu) species during mitosis of cultured cells have been investigated by immunofluorescence using antibodies directed against the C-terminus of either Tyr or Glu tubulin. The distribution of Tyr tubulin differed from that of Glu tubulin at each stage of mitosis; in general, the distribution of Tyr tubulin was similar to that of total tubulin, whereas Glu tubulin had a more restricted distribution. The Glu species was found in half-spindle fibers but was not detected in astral fibers at any stage and was seen in the interzone only during telophase. These results were confirmed by a direct comparison of the distributions of Tyr and Glu tubulin in cells double-labeled with the two antibodies. Evidence for the occurrence of Tyr and Glu tubulin in each class of half-spindle fibers (kinetochore and polar) was obtained from the staining patterns of the two antibodies in cold-treated cells. Immunoblots of extracts prepared from synchronous mitotic cells showed that Glu tubulin was a minor species of the total tubulin in the spindle; no changes in the amount of either Tyr or Glu tubulin were detected at any stage of mitosis. These results show that Tyr tubulin is the major species in the mitotic spindle and is found in all classes of spindle fibers, whereas Glu tubulin is present in small amounts and shows a more restricted distribution. The presence of two biochemically distinct forms of alpha-tubulin in the spindle may be important for spindle function.
Localization of the human NCAM gene to band q23 of chromosome 11: the third gene coding for a cell interaction molecule mapped to the distal portion of the long arm of chromosome 11.
Authors: Nguyen C , Mattei M G , Mattei J F , Santoni M J , Goridis C , Jordan B R
Published: May 2004

cDNA clones containing sequences coding for the murine neural cell adhesion molecule (N-CAM) were used in Southern hybridizations on human genomic DNA and demonstrated approximately 90% homology between human and murine NCAM genes. In situ hybridization with one of these clones was performed on human metaphase chromosomes and allowed the localization of the human NCAM gene to band q23 of chromosome 11. The genes for two other cell surface molecules believed to be involved in cell-cell interactions, Thy-1 and the delta chain of the T3-T cell receptor complex, have recently been localized to the same region of chromosome 11 in man. Moreover, this region of the human chromosome 11 appears to be syntenic to a region of murine chromosome 9 that also contains the staggerer locus: staggerer mice show abnormal neurological features which may be related to abnormalities in the conversion of the embryonic to the adult forms of the N-CAM molecule.
Molecular forms of N-CAM and its RNA in developing and denervated skeletal muscle.
Authors: Covault J , Merlie J P , Goridis C , Sanes J R
Published: May 2004

The neural cell adhesion molecule (N-CAM) is present in both embryonic and perinatal muscle, but its distribution changes as myoblasts form myotubes and axons establish synapses (Covault, J., and J. R. Sanes, 1986, J. Cell Biol., 102:716-730). Levels of N-CAM decline postnatally but increase when adult muscle is denervated or paralyzed (Covault, J., and J. R. Sanes, 1985, Proc. Natl. Acad. Sci. USA., 82:4544-4548). To determine the molecular forms of N-CAM and N-CAM-related RNA during these different periods we used immunoblotting and nucleic acid hybridization techniques to analyze N-CAM and its RNA in developing, cultured, adult, and denervated adult muscle. As muscles develop, the extent of sialylation of muscle N-CAM decreases, and a 140-kD desialo form of N-CAM (generated by neuraminidase treatment) is replaced by a 125-kD form. This change in the apparent molecular weight of desialo N-CAM is paralleled by a change in N-CAM RNA: early embryonic muscles express a 6.7-kb RNA species which hybridizes with N-CAM cDNA, whereas in neonatal muscle this form is largely replaced by 5.2- and 2.9-kb species. Similar transitions in the desialo form of N-CAM, but not in extent of sialylation, accompany differentiation in primary cultures of embryonic muscle and in cultures of the clonal muscle cell lines C2 and BC3H-1. Both in vivo and in vitro, a 140-kD desialo form of N-CAM and a 6.7-kb N-CAM RNA are apparently associated with myoblasts, whereas a 125-kD desialo form and 5.2- and 2.9-kb RNAs are associated with myotubes and myofibers. After denervation of adult muscle, a approximately 12-15-fold increase in the levels of N-CAM is accompanied by a approximately 30-50-fold increase in N-CAM RNA, suggesting that N-CAM expression is regulated at a pretranslational level. Forms of N-CAM and its RNA in denervated muscle are similar to those seen in perinatal myofibers.
Aggregating factor from Torpedo electric organ induces patches containing acetylcholine receptors, acetylcholinesterase, and butyrylcholinesterase on cultured myotubes.
Authors: Wallace B G
Published: May 2004

A factor in extracts of the electric organ of Torpedo californica causes the formation of clusters of acetylcholine receptors (AChRs) and aggregates of acetylcholinesterase (AChE) on myotubes in culture. In vivo, AChRs and AChE accumulate at the same locations on myofibers, as components of the postsynaptic apparatus at neuromuscular junctions. The aim of this study was to compare the distribution of AChRs, AChE, and butyrylcholinesterase (BuChE), a third component of the postsynaptic apparatus, on control and extract-treated myotubes. Electric organ extracts induced the formation of patches that contained high concentrations of all three molecules. The extract-induced aggregation of AChRs, AChE, and BuChE occurred in defined medium, and these components accumulated in patches simultaneously. Three lines of evidence indicate that a single factor in the extracts induced the aggregation of all three components: the dose dependence for the formation of patches of AChRs was the same as that for patches of AChE and BuChE; the AChE- and BuChE-aggregating activities co-purified with the AChR-aggregating activity; and all three aggregating activities were immunoprecipitated at the same titer by a monoclonal antibody against the AChR-aggregating factor. We have shown previously that this monoclonal antibody binds to molecules concentrated in the synaptic cleft at neuromuscular junctions. Taken together, these results suggest that during development and regeneration of myofibers in vivo, the accumulation at synaptic sites of at least three components of the postsynaptic apparatus, AChRs, AChE, and BuChE, are all triggered by the same molecule, a molecule similar if not identical to the electric organ aggregating factor.
Brain peptides and glial growth. I. Glia-promoting factors as regulators of gliogenesis in the developing and injured central nervous system.
Authors: Giulian D , Allen R L , Baker T J , Tomozawa Y
Published: May 2004

Glia-promoting factors (GPFs) are peptides of the central nervous system which accelerate the growth of specific glial populations in vitro. Although these factors were first discovered in the goldfish visual system (Giulian, D., Y. Tomozawa, H. Hindman, and R. Allen, 1985, Proc. Natl. Acad. Sci. USA., 83:4287-4290), we now report similar peptides are found in mammalian brain. The cerebral cortex of rat contains oligodendroglia-stimulating peptides, GPF1 (15 kD) and GPF3 (6 kD), as well as astroglia-stimulating peptides, GPF2 (9 kD) and GPF4 (3 kD). The concentrations of specific GPFs increase in brain during periods of gliogenesis. For example, GPF1 and GPF3 are found in postnatal rat brain during a peak of oligondendroglial growth while GPF2 and GPF4 are first detected at a time of astroglial proliferation in the embryo. Stab wound injury to the cerebral cortices of rats stimulates astroglial proliferation and induces marked elevations in levels of GPF2 and GPF4. Our findings suggest that two distinct classes of GPFs, those acting upon oligodendroglia and those acting upon astroglia, help to regulate cell growth in the developing and injured central nervous system.
Brain peptides and glial growth. II. Identification of cells that secrete glia-promoting factors.
Authors: Giulian D , Young D G
Published: May 2004

Glia-promoting factors (GPFs) are brain peptides which stimulate growth of specific macroglial populations in vitro. To identify the cellular sources of GPFs, we examined enriched brain cell cultures and cell lines derived from the nervous system for the production of growth factors. Ameboid microglia secreted astroglia-stimulating peptides, while growing neurons were the best source of the oligodendroglia-stimulating factors. These secretion products co-purified by gel filtration, anion exchange chromatography, and reverse-phase high performance liquid chromatography with GPFs isolated from goldfish and rat brain. Our findings suggest that glial growth in the central nervous system is regulated in part by a signaled release of peptides from specific secretory cells.
The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells.
Authors: Richter-Landsberg C , Jastorff B
Published: May 2004

Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differential effects on the activation of cAMP-dependent protein kinases, i.e., (Sp)-cAMPS behaves as a cAMP agonist and (Rp)-cAMPS behaves as a cAMP antagonist. Our data show that the establishment of a neuritic network, as observed from PC12 cells treated with NGF alone, could not be induced by either forskolin, cAMP, or cAMP analogues alone. The presence of NGF in combination with forskolin or cAMP or its agonistic analogues potentiated the initiation of neurite outgrowth from PC12 cells. The (Sp)-cAMPS-induced stimulation of NGF-mediated process formation was successfully blocked by the (Rp)-cAMPS diastereomer. On the other hand, NGF-stimulated neurite outgrowth was not inhibited by the presence of the cAMP antagonist (Rp)-cAMPS. We conclude that the morphological differentiation of PC12 cells stimulated by NGF does not require cAMP as a second messenger. The constant increase of intracellular cAMP, caused by either forskolin or cAMP and the analogues, in combination with NGF, not only rapidly stimulated early neurite outgrowth but also exerted a maintaining effect on the neuronal network established by NGF.
PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor.
Authors: Green S H , Rydel R E , Connolly J L , Greene L A
Published: May 2004

Four mutant PC12 pheochromocytoma cell lines that are nerve growth factor (NGF)-nonresponsive (PC12nnr) have been selected from chemically mutagenized cultures by a double selection procedure: failure both to grow neurites in the presence of NGF and to survive in NGF-supplemented serum-free medium. The PC12nnr cells were deficient in all additional NGF responses surveyed: abatement of cell proliferation, changes in glycoprotein composition, induction of ornithine decarboxylase, rapid changes in protein phosphorylation, and cell surface ruffling. However, PC12nnr cells closely resembled non-NGF-treated PC12 cells in most properties tested: cell size and shape; division rate; protein, phosphoprotein, and glycoprotein composition; and cell surface morphology. All four PC12nnr lines differed from PC12 cells in three ways in addition to failure of NGF response: PC12nnr cells failed to internalize bound NGF by the normal, saturable, high-affinity mechanism present in PC12 cells. The PC12nnr cells bound NGF but entirely, or nearly entirely, at low-affinity sites only, whereas PC12 cells possess both high- and low-affinity NGF binding sites. The responses to dibutyryl cyclic AMP that were tested appeared to be enhanced or altered in the PC12nnr cells compared to PC12 cells. Internalization of, and responses to, epidermal growth factor were normal in the PC12nnr cells ruling out a generalized defect in hormonal binding, uptake, or response mechanisms. These findings are consistent with a causal association between the presence of high-affinity NGF receptors and of NGF responsiveness and internalization. A possible relationship is also suggested between regulation of cAMP responses and regulation of NGF responses or NGF receptor affinity.
Functional expression of the polymeric immunoglobulin receptor from cloned cDNA in fibroblasts.
Authors: Deitcher D L , Neutra M R , Mostov K E
Published: May 2004

The polymeric immunoglobulin receptor, a transmembrane protein, is made by a variety of polarized epithelial cells. After synthesis, the receptor is sent to the basolateral surface where it binds polymeric IgA and IgM. The receptor-ligand complex is endocytosed, transported across the cell in vesicles, and re-exocytosed at the apical surface. At some point the receptor is proteolytically cleaved so that its extracellular ligand binding portion (known as secretory component) is severed from the membrane and released together with the polymeric immunoglobulin at the apical surface. We have used a cDNA clone coding for the rabbit receptor and a retroviral expression system to express the receptor in a nonpolarized mouse fibroblast cell line, psi 2, that normally does not synthesize the receptor. The receptor is glycosylated and sent to the cell surface. The cell cleaves the receptor to a group of polypeptides that are released into the medium and co-migrate with authentic rabbit secretory component. Cleavage and release of secretory component do not depend on the presence of ligand. The cells express on their surface 9,600 binding sites for the ligand, dimeric IgA. The ligand can be rapidly endocytosed and then re-exocytosed, all within approximately 10 min. Very little ligand is degraded. At least some of the ligand that is released from the cells is bound to secretory component. The results presented indicate that we have established a powerful new system for analyzing the complex steps in the transport of poly-Ig and the general problem of membrane protein sorting.
The effects of basic substances and acidic ionophores on the digestion of exogenous and endogenous proteins in mouse peritoneal macrophages.
Authors: Ohkuma S , Chudzik J , Poole B
Published: May 2004

Basic substances and acidic ionophores that increase the lysosomal pH in cultured macrophages (Ohkuma, S., and B. Poole, 1978, Proc. Natl. Acad. Sci. USA., 75:3327-3331; Poole, B., and S. Ohkuma, 1981, J. Cell Biol., 90:665-669) inhibited the digestion of heat-denatured acetylated bovine serum albumin (BSA) taken up by the cells. For several substances, the shift in pH sufficed to explain the inhibition of proteolysis. Additional effects, presumably on enzyme activities, have to be postulated for tributylamine, amantadine, and chloroquine. Sodium fluoride (10 mM) had no significant effect on the breakdown of BSA by macrophages. The breakdown of endogenous macrophage proteins, whether short lived or long lived, was inhibited approximately 40% by 10 mM NaF and 30%, or sometimes less in the case of long-lived proteins, by 100 microM chloroquine. When the cells were supplied with BSA, a mixture of cell proteins, or even inert endocytosible materials, the breakdown of endogenous long-lived proteins and the inhibitory effect of chloroquine on this process were selectively reduced. Inhibition of endocytosis by cytochalasins B or D did not affect the chloroquine-sensitive breakdown of endogenous proteins, indicating that the proteins degraded by this process were truly endogenous and not taken in from the outside by cellular cannibalism. On the other hand, when macrophage proteins were supplied extracellularly, their breakdown occurred at the same rate for short-lived and long-lived proteins, and it was strongly inhibited by chloroquine and not by NaF. It is concluded from these results that the breakdown of endogenous proteins, both short-lived and long-lived, probably takes place partly (approximately 30%) in lysosomes and partly through one or more nonlysosomal mechanism(s) unaffected by chloroquine and presumably susceptible to inhibition by fluoride. A difference must exist between short-lived and long-lived proteins in the manner in which they reach lysosomes or are handled by these organelles; this difference would account for the selective effect of the supply of endocytosible materials on the lysosomal processing of long-lived proteins.
Ultrastructure of the intact skeleton of the human erythrocyte membrane.
Authors: Shen B W , Josephs R , Steck T L
Published: May 2004

Filamentous skeletons were liberated from isolated human erythrocyte membranes in Triton X-100, spread on fenestrated carbon films, negatively stained, and viewed intact and unfixed in the transmission electron microscope. Two forms of the skeleton were examined: (a) basic skeletons, stripped of accessory proteins with 1.5 M NaCl so that they contain predominantly polypeptide bands 1, 2, 4.1, and 5; and (b) unstripped skeletons, which also bore accessory proteins such as ankyrin and band 3 and small plaques of residual lipid. Freshly prepared skeletons were highly condensed. Incubation at low ionic strength and in the presence of dithiothreitol for an hour or more caused an expansion of the skeletons, which greatly increased the visibility of their elements. The expansion may reflect the opening of spectrin from a compact to an elongated disposition. Expanded skeletons appeared to be organized as networks of short actin filaments joined by multiple (5-8) spectrin tetramers. In unstripped preparations, globular masses were observed near the centers of the spectrin filaments, probably corresponding to complexes of ankyrin with band 3 oligomers. Some of these globules linked pairs of spectrin filaments. Skeletons prepared with a minimum of perturbation had thickened actin protofilaments, presumably reflecting the presence of accessory proteins. The length of these actin filaments was highly uniform, averaging 33 +/- 5 nm. This is the length of nonmuscle tropomyosin. Since there is almost enough tropomyosin present to saturate the F-actin, our data support the hypothesis that tropomyosin may determine the length of actin protofilaments in the red cell membrane.
Ultrastructural localization of the progesterone receptor by an immunogold method: effect of hormone administration.
Authors: Perrot-Applanat M , Groyer-Picard M T , Logeat F , Milgrom E
Published: May 2004

The progesterone receptor has been localized in the rabbit uterus by immunocytochemistry at the electron microscopic level, using monoclonal antibodies and the protein A-gold technique. The progesterone receptor in uterine stromal cells was mainly localized in the nucleus; however, a small fraction of antigen was present in the cytoplasm, where it was associated with the rough endoplasmic reticulum and with free ribosomes. The plasma membrane was not labeled. In the nucleus, the receptor was always associated with condensed chromatin or areas surrounding condensed chromatin, whereas the nuceolus was not labeled. In the chromatin, receptor distribution varied according to the hormonal state: in the absence of progesterone, the receptor was randomly scattered over the clumps of condensed chromatin; after administration of the progestin R5020, it was mainly detected in the border regions between condensed chromatin and nucleoplasm and, to a lesser extent, over dispersed chromatin in the nucleoplasm. These areas have been shown to be the most active sites of gene transcription.
Cell-mediated co-action of transforming growth factors: incubation of type beta with normal rat kidney cells produces a soluble activity that prolongs the ruffling response to type alpha.
Authors: Myrdal S E , Twardzik D R , Auersperg N
Published: May 2004

Intense, continuous ruffling is a characteristic of many transformed cells, but untransformed cells ruffle intensely only briefly after exposure to growth factors. We reported previously that cells of a normal rat kidney (NRK) cell line transformed by Kirsten murine sarcoma virus secrete their own ruffle-inducing agent(s) that cause sustained ruffling in either themselves or untransformed NRK cells. In the present study, we examined the roles of the transforming growth factors TGF-alpha and TGF-beta in the induction and maintenance of ruffling in untransformed NRK cells and observed the following: TGF-alpha caused a transient epidermal growth factor (EGF)-like response, which could be blocked by prior exposure of cells to EGF or by antiserum directed against the COOH-terminus of TGF-alpha. TGF-beta caused no ruffling and did not itself prolong TGF-alpha ruffling. A new, buffer-soluble (transferable) mediator activity produced by incubation of TGF-beta with NRK cells for 6-h extended the duration of maximal TGF-alpha-induced ruffling by several-fold. This study demonstrates that TGF-alpha alone causes an EGF-like, transient ruffling response, but neither TGF-alpha or TGF-beta alone, nor the two together, cause transformation-associated sustained ruffling. Rather, TGF-alpha acts in concert with a new, TGF-beta-dependent activity. This new activity appears to inhibit normal cellular off-regulation of TGF-alpha-induced ruffling. Inhibition of the cellular off-regulation of a growth factor response could play a key role in the unregulated growth associated with malignancy.
Sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells.
Authors: Gottlieb T A , Gonzalez A , Rizzolo L , Rindler M J , Adesnik M , Sabatini D D
Published: May 2004

Previous studies (Rindler, M. J., I. E., Ivanov, H. Plesken, and D. D. Sabatini, 1985, J. Cell Biol., 100: 136-151; Rindler, M. J., I. E. Ivanov, H. Plesken, E. J. Rodriguez-Boulan, and D. D. Sabatini, 1984, J. Cell Biol., 98: 1304-1319) have demonstrated that in polarized Madin-Darby canine kidney cells infected with vesicular stomatitis virus (VSV) or influenza virus the viral envelope glycoproteins G and HA are segregated to the basolateral and apical plasma membrane domains, respectively, where budding of the corresponding viruses takes place. Furthermore, it has been shown that this segregation of the glycoproteins reflects the polarized delivery of the newly synthesized polypeptides to each surface domain. In transfection experiments using eukaryotic expression plasmids that contain cDNAs encoding the viral glycoproteins, it is now shown that even in the absence of other viral components, both proteins are effectively segregated to the appropriate cell surface domain. In transfected cells, the HA glycoprotein was almost exclusively localized in the apical cell surface, whereas the G protein, although preferentially localized in the basolateral domains, was also present in lower amounts, in the apical surfaces of many cells. Using transfected and infected cells, it was demonstrated that, after reaching the cell surface, the G protein, but not the HA protein, undergoes interiorization by endocytosis. Thus, in the presence of chloroquine, a drug that blocks return of interiorized plasma membrane proteins to the cell surface, the G protein was quantitatively trapped in endosome- or lysosome-like vesicles. The sequestration of G was a rapid process that was completed in many cells by 1-2 h after chloroquine treatment. The fact that in transfected cells the surface content of G protein was not noticeably reduced during a 5-h incubation with cycloheximide, a protein synthesis inhibitor that did not prevent the effect of chloroquine, implies that normally, G protein molecules are not only interiorized but are also recycled to the cell surface.
The lateral mobility of the (Na+,K+)-dependent ATPase in Madin-Darby canine kidney cells.
Authors: Jesaitis A J , Yguerabide J
Published: May 2004

Fluorescence microphotolysis (recovery after photobleaching) was used to determine the lateral mobility of the (Na+,K+)ATPase and a fluorescent lipid analogue in the plasma membrane of Madin-Darby canine kidney (MDCK) cells at different stages of development. Fluorescein-conjugated Fab' fragments prepared from rabbit anti-dog (Na+,K+)ATPase antibodies (IgG) and 5-(N-hexadecanoyl)aminofluorescein (HEDAF) were used to label the plasma membrane of confluent and subconfluent cultures of MDCK cells. Fractional fluorescence recovery was 50% and 80-90% for the protein and lipid probes, respectively, and was independent of developmental stage. The estimated diffusion constants of the mobile fraction were approximately 5 X 10(-10) cm2/s for the (Na+,K+)ATPase and approximately 2 X 10(-9) cm2/s for HEDAF. Only HEDAF diffusion showed dependency on developmental stage in that D for confluent cells was approximately twice that for subconfluent cells. These results indicate that (Na+,K+)ATPase is 50% immobilized in all developmental stages, whereas lipids in confluent MDCK cells are more mobile than in subconfluent cells. They suggest, furthermore, that the degree of immobilization of the (Na+,K+)ATPase is insufficient to explain its polar distribution, and they support restricted mobility of the ATPase through the tight junctions as the likely mechanism for preventing the diffusion of this protein into the apical domain of the plasma membrane in confluent cell cultures.
Specific binding sites for albumin restricted to plasmalemmal vesicles of continuous capillary endothelium: receptor-mediated transcytosis.
Authors: Ghitescu L , Fixman A , Simionescu M , Simionescu N
Published: May 2004

The interaction of homologous and heterologous albumin-gold complex (Alb-Au) with capillary endothelium was investigated in the mouse lung, heart, and diaphragm. Perfusion of the tracer in situ for from 3 to 35 min was followed by washing with phosphate-buffered saline, fixation by perfusion, and processing for electron microscopy. From the earliest time examined, one and sometimes two rows of densely packed particles bound to some restricted plasma membrane microdomains that appeared as uncoated pits, and to plasmalemmal vesicles open on the luminal front. Morphometric analysis, using various albumin-gold concentrations, showed that the binding is saturable at a very low concentration of the ligand and short exposure. After 5 min, tracer-carrying vesicles appeared on the abluminal front, discharging their content into the subendothelial space. As a function of tracer concentration 1-10% of plasmalemmal vesicles contained Alb-Au particles in fluid phase; from 5 min on, multivesicular bodies were labeled by the tracer. Plasma membrane, coated pits, and coated vesicles were not significantly marked at any time interval. Heparin or high ionic strength did not displace the bound Alb-Au from vesicle membrane. No binding was obtained when Alb-Au was competed in situ with albumin or was injected in vivo. Gold complexes with fibrinogen, fibronectin, glucose oxidase, or polyethyleneglycol did not give a labeling comparable to that of albumin. These results suggest that on the capillary endothelia examined, the Alb-Au is adsorbed on specific binding sites restricted to uncoated pits and plasmalemmal vesicles. The tracer is transported in transcytotic vesicles across endothelium by receptor-mediated transcytosis, and to a lesser extent is taken up by pinocytotic vesicles. The existence of albumin receptors on these continuous capillary endothelia may provide a specific mechanism for the transport of albumin and other molecules carried by this protein.
A lens intercellular junction protein, MP26, is a phosphoprotein.
Authors: Johnson K R , Lampe P D , Hur K C , Louis C F , Johnson R G
Published: May 2004

The major protein present in the plasma membrane of the bovine lens fiber cell (MP26), thought to be a component of intercellular junctions, was phosphorylated in an in vivo labeling procedure. After fragments of decapsulated fetal bovine lenses were incubated with [32P]orthophosphate, membranes were isolated and analyzed by SDS PAGE and autoradiography. A number of lens membrane proteins were routinely phosphorylated under these conditions. These proteins included species at Mr 17,000 and 26,000 as well as a series at both 34,000 and 55,000. The label at Mr 26,000 appeared to be associated with MP26, since (a) boiling the membrane sample in SDS led to both an aggregation of MP26 and a loss of label at Mr 26,000, (b) the label at 26,000 was resistant to both urea and nonionic detergents, and (c) two-dimensional gels showed that a phosphorylated Mr 24,000 fragment was derived from MP26 with V8 protease. Studies with proteases also provided for a localization of most label within approximately 20 to 40 residues from the COOH-terminus of MP26. Published work indicates that the phosphorylated portion of MP26 resides on the cytoplasmic side of the membrane, and that this region of MP26 contains a number of serine residues. The same region of MP26 was labeled when isolated lens membranes were reacted with a cAMP-dependent protein kinase prepared from the bovine lens. After the in vivo labeling of lens fragments, phosphoamino acid analysis of MP26 demonstrated primarily labeled serines, with 5-10% threonines and no tyrosines. Treatments that lowered the intracellular calcium levels in the in vivo system led to a selective reduction of MP26 phosphorylation. In addition, forskolin and cAMP stimulated the phosphorylation of MP26 and other proteins in concentrated lens homogenates. These findings are of interest because MP26 appears to serve as a protein of cell-to-cell channels in the lens, perhaps as a lens gap junction protein.
A long-lived fusogenic state is induced in erythrocyte ghosts by electric pulses.
Authors: Sowers A E
Published: May 2004

Treatment of erythrocyte ghosts in random positions in a suspension with membrane fusion-inducing direct current electric field pulses causes the membranes to become fusogenic. Significant fusion yields are observed if the membranes are dielectrophoretically aligned into membrane-membrane contact with a weak alternating electric field as much as 5 min after the application of the pulses. This demonstrates that a long-lived membrane structural alteration is involved in this fusion mechanism. Other experiments indicate that the areas on the membrane which become fusogenic after treatment with the pulses may be very highly localized. The locations of these fusogenic areas coincide with where the trans-membrane electric field strength was greatest during the pulse. The fusogenic membrane alteration, or components thereof, in these areas laterally diffuses very slowly or not at all, or, to be fusogenic, must be present at concentrations in the membrane above a certain threshold. The loss of soluble 0.9-3-nm-diameter fluorescent probes from resealed cytoplasmic compartments of randomly positioned erythrocyte ghosts occurs through electric field pulse-induced pores only during a pulse but not between pulses or after a train of pulses if the probe diameter is 1.2 nm or greater. For a given pulse treatment of membranes in random positions in suspensions, an increase in ionic strength of the medium results in (a) a decrease in loss during the pulse, (b) no difference in loss between pulses, and (c) an increase in fusion yield when membrane-membrane contact is established. The latter two results (b and c) are incompatible with a fusion mechanism that proposes a simple relationship between electric field-induced pores and fusion.
Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm.
Authors: Bleil J D , Wassarman P M
Published: May 2004

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O-linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only bacKground levels to heads of both acrosome-intact and -reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.
Changes in sperm plasma membrane lipid diffusibility after hyperactivation during in vitro capacitation in the mouse.
Authors: Wolf D E , Hagopian S S , Ishijima S
Published: May 2004

We have used the technique of fluorescence recovery after photobleaching to measure the diffusibility of the fluorescent lipid analogue, 1,1'-dihexadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate on the morphologically distinct regions of the plasma membranes of mouse spermatozoa, and the changes in lipid diffusibility that result from in vitro hyperactivation and capacitation with bovine serum albumin. We found that, as previously observed on ram spermatozoa, lipid analogue diffusibility is regionalized on mouse spermatozoa, being fastest on the flagellum. The bovine serum albumin induced changes in diffusibility that occur with hyperactivation are also regionalized. Specifically, if we compare serum incubated in control medium, which maintains normal motility, with those hyperactivated in capacitating medium, we observe with hyperactivation an increase in lipid analogue diffusion rate in the anterior region of the head, the midpiece, and tail, and a decrease in diffusing fraction in the anterior region of the head.
Effects of mechanical tension on protrusive activity and microfilament and intermediate filament organization in an epidermal epithelium moving in culture.
Authors: Kolega J
Published: May 2004

Mechanical tension influences tissue morphogenesis and the synthetic, mitotic, and motile behavior of cells. To determine the effects of tension on epithelial motility and cytoskeletal organization, small, motile clusters of epidermal cells were artificially extended with a micromanipulated needle. Protrusive activity perpendicular to the axis of tension was dramatically suppressed. To determine the ultrastructural basis for this phenomenon, cells whose exact locomotive behavior was recorded cinemicrographically were examined by transmission electron microscopy. In untensed, forward-moving lamellar protrusions, microfilaments appear disorganized and anisotropically oriented. But in cytoplasm held under tension by micromanipulation or by the locomotive activity of other cells within the epithelium, microfilaments are aligned parallel to the tension. In non-spreading regions of the epithelial margin, microfilaments lie in tight bundles parallel to apparent lines of tension. Thus, it appears that tension causes alignment of microfilaments. In contrast, intermediate filaments are excluded from motile protrusions, being confined to the thicker, more central part of the cell. They roughly follow the contours of the cell, but are not aligned relative to tension even when microfilaments in the same cell are. This suggests that the organization of intermediate filaments is relatively resistant to physical distortion and the intermediate filaments may act as passive structural support within the cell. The alignment of microfilaments under tension suggests a mechanism by which tension suppresses protrusive activity: microfilaments aligned by forces exerted through filament-surface or filament-filament interconnections cannot reorient against such force and so cannot easily extend protrusions in directions not parallel to tension.
Localization of low-sulfur keratin proteins in the wool follicle using monoclonal antibodies.
Authors: French P W , Hewish D R
Published: May 2004

Monoclonal antibodies that recognize components of the low-sulfur keratin proteins extracted from Merino wool have been used to locate these components within the wool follicle. Immunoblotting procedures showed that all of the monoclonal antibodies bound more than one of the eight low-sulfur protein components, indicating that these proteins have antigenic determinants in common. Immunofluorescence studies showed that those antibodies specific for the component 7 family of the low-sulfur proteins bound to the developing wool fiber, whereas those antibodies recognizing the component 8 family bound to areas throughout the wool follicle, particularly the inner and outer root sheaths, but also to the fiber, the cuticle, and the epidermis. One of the monoclonal antibodies also bound to intermediate filament networks of cultured human epithelial cells.
Actin at receptor-rich domains of isolated acetylcholine receptor clusters.
Authors: Bloch R J
Published: May 2004

Acetylcholine receptor (AChR) clusters of cultured rat myotubes, isolated by extraction with saponin (Bloch, R. J., 1984, J. Cell Biol. 99:984-993), contain a polypeptide that co-electrophoreses with purified muscle actins. A monoclonal antibody against actin reacts in immunoblots with this polypeptide and with purified actins. In indirect immunofluorescence, the antibody stains isolated AChR clusters only at AChR domains, strips of membrane within clusters that are rich in receptor. It also stains the postsynaptic region of the neuromuscular junction of adult rat skeletal muscle. Semiquantitative immunofluorescence analyses show that labeling by antiactin of isolated analyses show that labeling by antiactin of isolated AChR clusters is specific and saturable and that it varies linearly with the amount of AChR in the cluster. Filaments of purified gizzard myosin also bind preferentially at AChR-rich regions, and this binding is inhibited by MgATP. These experiments suggest that actin is associated with AChR-rich regions of receptor clusters. Depletion of actin by extraction of isolated clusters at low ionic strength selectively releases the actin-like polypeptide from the preparation. Simultaneously, AChRs redistribute within the plane of the membrane of the isolated clusters. Similarly, brief digestion with chymotrypsin reduces immunofluorescence staining and causes AChR redistribution. Treatments that deplete AChR from clusters in intact cells also reduce immunofluorescent staining for actin in isolated muscle membrane fragments. Upon reversal of these treatments, cluster reformation occurs in regions of the membrane that also stain for actin. I conclude that actin is associated with AChR domains and that changes in this association are accompanied by changes in the organization of isolated AChR clusters.
Is a rise in intracellular concentration of free calcium necessary or sufficient for stimulated cytoskeletal-associated actin?
Authors: Sha'afi R I , Shefcyk J , Yassin R , Molski T F , Volpi M , Naccache P H , White J R , Feinstein M B , Becker E L
Published: May 2004

The addition of the calcium ionophore A23187 to rabbit neutrophils increases the amount of actin associated with the cytoskeleton regardless of the presence or absence of calcium in the incubation medium. In the presence of extracellular calcium, the effect of A23187 is biphasic with respect to concentration. The action of the ionophore is rapid, transient, and is inhibited by pertussis toxin, hyperosmolarity, and quinacrine. On the other hand, the addition of pertussis toxin or hyperosmolarity has small if any, effect on the rise in intracellular calcium produced by A23187. While quinacrine does not affect the fMet-Leu-Phe-induced increase in cytoskeletal actin and the polyphosphoinositide turnover, its addition inhibits completely the stimulated increase in Ca-influx produced by the same stimulus. The results presented here suggest that a rise in the intracellular concentration of free calcium is neither necessary nor sufficient for the stimulated increase in cytoskeletal-associated actin. A possible relationship between the lipid remodeling stimulated by chemoattractants and the increased cytoskeletal actin is discussed.