bluuebunny/crossref_metadata_2025 huggingface dataset:
An elevated free cytosolic Ca2+ wave follows fertilization in eggs of the frog, Xenopus laevis.
Authors: Busa W B , Nuccitelli R
Published: May 2004

The eggs of most or all animals are thought to be activated after fertilization by a transient increase in free cytosolic Ca2+ concentration ([Ca2+]i). We have applied Ca2+-selective microelectrodes to detect such an increase in fertilized eggs of the frog, Xenopus laevis. As observed with an electrode in the animal hemisphere, [Ca2+]i increased from 0.4 to 1.2 microM over the course of 2 min after fertilization, and returned to its original value during the next 10 min. No further changes in [Ca2+]i were detected through the first cleavage division. In eggs impaled with two Ca2+ electrodes, the Ca2+ pulse was observed to travel as a wave from the animal to the vegetal hemisphere, propagating at a rate of approximately 10 microns/s across the animal hemisphere. The apparent delay between the start of the fertilization potential and initiation of the Ca2+ wave at the sperm entry site as approximately 1 min. Through these observations describe only the behavior of subcortical [Ca2+]i, we suggest that our data represent the subcortical extension of the cortical Ca2+ wave thought to trigger cortical granule exocytosis, and we present evidence that both the timing and magnitude of the Ca2+ pulse we observed are consistent with this identity. This first quantification of subcortical [Ca2+]i during fertilization indicates that the Ca2+ transient is available to regulate processes (e.g., protein synthesis) in the subcortical cytosol.
Topography of cell-glass apposition revealed by total internal reflection fluorescence of volume markers.
Authors: Gingell D , Todd I , Bailey J
Published: May 2004

We have developed a new method based on total internal reflection fluorescence to map the shape of the region between glass and the lower surface of a living cell spread upon it. Fluorescently labeled nonadsorbing volume marker molecules that cannot penetrate into the cell are locally stimulated so that they fluoresce only very near the glass/medium interface. The total fluorescence intensity at any point beneath the cell depends on the cell-to-glass separation. Focal contacts appear as dark areas owing to dye exclusion, whereas when the gap exceeds approximately 150 nm, fluorescence asymptotes to the bright background level. Our technique provides greater contrast than does interference reflection microscopy and is free from errors due to cytoplasmic thickness and refractive index inhomogeneities arising from cytoplasmic inclusions. We have shown that sufficiently large molecules suffer steric exclusion from regions accessible to small molecules, which gives new information about lateral penetrability in the apposition region.
Agents that activate protein kinase C reduce acetylcholine sensitivity in cultured myotubes.
Authors: Eusebi F , Molinaro M , Zani B M
Published: May 2004

We have examined acetylcholine (ACh)-elicited potentials or currents in current- or voltage-clamped cultured myotubes exposed to 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent tumor promoter that activates protein kinase C. Although this agent had little action on either membrane resting potential or electrical resistance, a reversible decrease in ACh sensitivity was induced on 3-4-d-old chick myotubes. Depression of transmitter action by TPA was extended to 7-8-d mouse myotubes only when they were treated with phosphatidylserine. Glyceryl dioleate had effects on myotubes similar to those of TPA but with a reduced efficacy. We conclude that the activation of protein kinase C might be involved with the capacity of ACh receptors to respond to transmitter stimulation.
Intermediate filament cytoskeleton of amnion epithelium and cultured amnion epithelial cells: expression of epidermal cytokeratins in cells of a simple epithelium.
Authors: Regauer S , Franke W W , Virtanen I
Published: May 2004

Using immunofluorescence microscopy and two-dimensional gel electrophoresis, we compared the cytoskeletal proteins expressed by human amnion epithelium in situ, obtained from pregnancies of from 10-wk to birth, with the corresponding proteins from cultured amnion epithelial cells and cultures of cells from the amniotic fluid of 16 week pregnancies. Epithelia of week 16 fetuses already display tissue-specific patterns of cytokeratin polypeptides which are similar, although not identical, to those of the corresponding adult tissues. In the case of the simple amnion epithelium, a complex and characteristic complement of cytokeratin polypeptides of Mr 58,000 (No. 5), 56,000 (No. 6), 54,000 (No. 7), 52,500 (No. 8), 50,000 (No. 14), 46,000 (No. 17), 45,000 (No. 18), and 40,000 (No. 19) is present by week 10 of pregnancy and is essentially maintained until birth, with the addition of cytokeratin No. 4 (Mr 59,000) and the disappearance of No. 7 (Mr 54,000) at week 16 of pregnancy. In full-term placentae, the amnion epithelium displays two morphologically distinct regions, i.e., a simple and a stratified epithelium, both of which express the typical amnion cytokeratin polypeptides. However, in addition the stratified epithelium also synthesizes large amounts of special epidermal cytokeratins such as No. 1 (Mr 68,000), 10 (Mr 56,500), and 11 (Mr 56,000). In culture amnion epithelial cells obtained from either 16-wk pregnancies or full-term placentae will continue to synthesize the amnion-typical cytokeratin pattern, except for a loss of detection of component No. 4. This pattern is considerably different from the cytokeratins synthesized by cultures of cells from amniotic fluids (cytokeratins No. 7, 8, 18, and 19, sometimes with trace amounts of No. 17) and from several so-called "amnion epithelial cell lines." In addition, amnion epithelial cells in situ as well as amnion epithelial cell cultures appear to be heterogeneous in that they possess some cells that co-express cytokeratins and vimentin. These observations lead to several important conclusions: In contrast to the general concept of recent literature, positively charged cytokeratins of the group No. 4-6 can be synthesized in a simple, i.e., one-layered epithelium. The change from simple to stratified amnion epithelium does not require a cessation of synthesis of cytokeratins of the simple epithelium type, but in this case keratins characteristic of the terminally differentiated epidermis (No. 1, 10, and 11) are also synthesized.(ABSTRACT TRUNCATED AT 400 WORDS)
Tubular structures in S49 mouse lymphoma are regulated through in vivo host-cell interaction and in vitro interferon treatment.
Authors: Hochman J , Mador N , Panet A
Published: May 2004

Malignant S49 mouse lymphoma cells that grow in suspension culture demonstrate in their cytoplasm characteristic tubular structures. These structures also appear in immunogenic, substrate-adherent variants of S49 cells that grow in culture. Upon transfer of both cell types into nude mice, the tubular structures of the adherent variants (and not the suspension-growing cells) undergo a profound alteration whereby their tubular components disappear and clusters of viruslike particles appear. These very closely resemble, on morphological grounds, precursors of B-type retroviruses. This specific in vivo interaction between the host and the S49 variant can be mimicked in culture by treatment of these cells for 24 h with 500 U/ml of mouse interferon. The suspension-growing S49 cells are unresponsive to interferon in this respect. Immunohistochemical analysis reveals that both tubular structures and the viruslike particles represent stages in the morphogenesis of mouse mammary tumor virus. A working hypothesis is advanced relating the regulation of the tubular system to the impaired tumorigenic potential of adherent S49 cells in syngeneic Balb/c hosts.
Calcium restriction prolongs metaphase in dividing Tradescantia stamen hair cells.
Authors: Hepler P K
Published: May 2004

Agents that lower extracellular calcium concentration (EGTA) or modulate calcium transport (lanthanum or D600) have been applied to dividing stamen hair cells of Tradescantia and analyzed for their ability to change the following: (a) the time required to progress from nuclear envelope breakdown to the onset of anaphase (metaphase transit time), (b) the time required to progress from anaphase to the initiation of the cell plate, and (c) the rate of chromosome motion in anaphase. Control cells complete metaphase in 32 min, initiate a cell plate in 19 min, and display a chromosome motion rate of 1.45 micron/min. If cells are treated with a calcium-EGTA buffer (pCa 8) for 4 h, the metaphase transit time is increased to 53 min without any change in the time of cell plate formation or the rate of chromosome motion. Lanthanum and D600, under conditions in which their access to the plasmalemma has been facilitated by pretreating the cells with cutinase, also markedly extend metaphase and in several instances permanently arrest cells. Lanthanum, however, produce little or no change in cell plate initiation or the rate of chromosome motion. Microscopic observations of the mitotic apparatus in calcium-stressed cells reveal normal chromatin condensation and metaphase progression. Chromosomes partly untwine but remain attached at their kinetochores. It is suggested that a flux of calcium, derived from the extracellular compartment, may cause the final splitting of sister chromosomes and trigger the onset of anaphase. However, once anaphase has begun, chromosome motion and cell plate initiation proceed normally even under conditions of extracellular calcium restriction.
Triton X-100 extraction of P815 tumor cells: evidence for a plasma membrane skeleton structure.
Authors: Apgar J R , Herrmann S H , Robinson J M , Mescher M F
Published: May 2004

It has been shown that a Triton X-100-insoluble protein matrix can be isolated from the plasma membranes of P815 tumor cells and murine lymphoid cells (Mescher, M. F., M. J. L. Jose and S. P. Balk, 1981, Nature (Lond.), 289:139-144). The properties of the matrix suggested that this set of proteins might form a membrane skeletal structure, stable in the absence of the lipid bilayer. Since purification of plasma membrane results in yields of only 20 to 40%, it was not clear whether the matrix was associated with the entire plasma membrane. To determine if a detergent-insoluble structure was present over the entire cell periphery and stable in the absence of the membrane bilayer or cytoskeletal components, we have examined extraction of whole cells with Triton X-100. Using the same conditions as those used for isolation of the matrix from membranes, we found that extraction of intact cells resulted in structures consisting of a continuous layer of protein at the periphery, a largely empty cytoplasmic space, and a nuclear remnant. Little or no lipid bilayer structure was evident in association with the peripheral layer, and no filamentous cytoskeletal structures could be seen in the cytoplasmic space by thin-section electron microscopy. Analysis of these Triton shells showed them to retain approximately 15% of the total cell protein, most of which was accounted for by low molecular weight nuclear proteins. 5'-Nucleotidase, a cell surface enzyme that remains associated with the plasma membrane matrix, was quantitatively recovered with the shells. Included among the polypeptides present in the shells was a set with mobilities identical to those of the set that makes up the plasma membrane matrix. The polypeptide composition of the shells further confirmed that cytoskeletal proteins were present to a very low extent, if at all, after the extraction. The results demonstrate that a detergent-insoluble protein matrix associated with the periphery of these cells forms a continuous, intact macrostructure whose stability is independent of the membrane bilayer or filamentous cytoskeletal elements, and thus has the properties of a membrane skeletal structure. Although not yet directly demonstrated, the results also strongly suggest that this peripheral layer is composed of the previously described set of plasma membrane matrix proteins. This article discusses possible roles for this proposed membrane skeletal structure in stabilizing the membrane bilayer and affecting the dynamics of other membrane proteins.
Contractile proteins in pericytes. I. Immunoperoxidase localization of tropomyosin.
Authors: Joyce N C , Haire M F , Palade G E
Published: May 2004

In these studies we have compared the relative amounts and isoforms of tropomyosin in capillary and postcapillary venule pericytes, endothelial cells, and vascular smooth muscle cells in four rat microvascular beds: heart, diaphragm, pancreas, and the intestinal mucosa. The results, obtained by in situ immunoperoxidase localization, indicate that (a) tropomyosin is present in capillary and postcapillary venule pericytes in relatively high concentration; (b) the tropomyosin content of pericytes appears to be somewhat lower than in vascular smooth muscle cells but higher than in endothelia and other vessel-associated cells; and (c) pericytes, unlike endothelia and other nonmuscle cells, contain detectable levels of tropomyosin immunologically related to the smooth muscle isoform. These results and our previous findings concerning the presence of a cyclic GMP-dependent protein kinase (Joyce, N., P. DeCamilli, and J. Boyles, 1984, Microvasc. Res. 28:206-219) in pericytes demonstrate that these cells contain significant amounts of at least two proteins important for contraction regulation. Taken together, the evidence suggests that pericytes are contractile elements related to vascular smooth muscle cells, possibly involved, as are the latter, in the regulation of blood flow through the microvasculature.
Cell-cell interactions promote mammary epithelial cell differentiation.
Authors: Levine J F , Stockdale F E
Published: May 2004

Mammary epithelium differentiates in a stromal milieu of adipocytes and fibroblasts. To investigate cell-cell interactions that may influence mammary epithelial cell differentiation, we developed a co-culture system of murine mammary epithelium and adipocytes and other fibroblasts. Insofar as caseins are specific molecular markers of mammary epithelial differentiation, rat anti-mouse casein monoclonal antibodies were raised against the three major mouse casein components to study this interaction. Mammary epithelium from mid-pregnant mice was plated on confluent irradiated monolayers of 3T3-L1 cells, a subclone of the Swiss 3T3 cell line that differentiates into adipocytes in monolayer culture and other cell monolayers (3T3-C2 cells, Swiss 3T3 cells, and human foreskin fibroblasts). Casein was synthesized by mammary epithelium only in the presence of co-cultured cells and the lactogenic hormone combination of insulin, hydrocortisone, and prolactin. Synthesis and accumulation of alpha-, beta-, and gamma-mouse casein within the epithelium was shown by immunohistochemical staining of cultured cells with anti-casein monoclonal antibodies, and the specificity of the immunohistochemical reaction was demonstrated using immunoblots. A competitive immunoassay was used to measure the amount of casein secreted into the culture medium. In a 24-h period, mammary epithelium co-cultured with 3T3-L1 cells secreted 12-20 micrograms beta-casein per culture dish. There was evidence of specificity in the cell-cell interaction that mediates hormone-dependent casein synthesis. Swiss 3T3 cells, newborn foreskin fibroblasts, substrate-attached material ("extracellular matrix"), and tissue culture plastic did not support casein synthesis, whereas monolayers of 3T3-L1 and 3T3-C2 cells, a subclone of Swiss 3T3 cells that does not undergo adipocyte differentiation, did. We conclude that interaction between mammary epithelium and other specific nonepithelial cells markedly influences the acquisition of hormone sensitivity of the epithelium and hormone-dependent differentiation.
The cercarial glycocalyx of Schistosoma mansoni.
Authors: Samuelson J C , Caulfield J P
Published: May 2004

Cercariae, the freshwater stage of Schistosoma mansoni infectious to man, are covered by a single unit membrane and an immunogenic glycocalyx. When cercariae penetrate the host skin, they transform to schistosomula by shedding tails, secreting mucous and enzymes, and forming microvilli over their surface. Here the loss of the glycocalyx from cercariae transforming in vitro was studied morphologically and biochemically. By scanning electron microscopy, the glycocalyx was a dense mesh composed of 15-30 nm fibrils that obscured spines on the cercarial surface. The glycocalyx was absent on organisms fixed without osmium and was partially lost when parasites aggregated in their own secretions before fixation. By transmission electron microscopy, a 1-2 microns thick mesh of 8-15-nm fibrils was seen on parasites incubated with anti-schistosomal antibodies or fixed in aldehydes containing tannic acid or ruthenium red. Cercariae transformed to schistosomula when tails were removed mechanically and parasites were incubated in saline. Within 5 min of transformation, organisms synchronously formed microvilli which elongated to 3-5 microns by 20 min and then were shed. However, considerable fibrillar material remained adherent to the double unit membrane surface of schistosomula. For biochemical labeling, parasites were treated with eserine sulfate, which blocked cercarial swimming, secretion, infectivity, and transformation to schistosomula. Material labeled by periodate oxidation and NaB3H4 was on the surface as shown by autoradiography and had an apparent molecular weight of greater than 10(6) by chromatography. Periodate-NaB3H4 glycocalyx had an isoelectric point of 5.0 +/- 0.4 and was precipitable with anti-schistosomal antibodies. More than 60% of the radiolabeled glycocalyx was released into the medium by transforming parasites in 3 h and was recovered as high molecular weight material. Parasites labeled with periodate and fluorescein-thiosemicarbazide and then transformed had a corona of fluorescence containing microvilli, much of which was shed onto the slide. Material on cercariae labeled by lodogen-catalyzed iodination was also of high molecular weight and was antigenic. In conclusion, the cercarial glycocalyx appears to be composed of acidic high molecular weight fibrils which are antigenic and incompletely cleared during transformation.
Calcium control of ciliary reversal in ionophore-treated and ATP-reactivated comb plates of ctenophores.
Authors: Nakamura S , Tamm S L
Published: May 2004

Previous work showed that ctenophore larvae swim backwards in high-KCl seawater, due to a 180 degrees reversal in the direction of effective stroke of their ciliary comb plates (Tamm, S. L., and S. Tamm, 1981, J. Cell Biol., 89: 495-509). Ion substitution and blocking experiments indicated that this response is Ca2+ dependent, but comb plate cells are innervated and presumably under nervous control. To determine whether Ca2+ is directly involved in activating the ciliary reversal mechanism and/or is required for synaptic triggering of the response, we (a) determined the effects of ionophore A23187 and Ca2+ on the beat direction of isolated nerve-free comb plates dissociated from larvae by hypotonic, divalent cation-free medium, and (b) used permeabilized ATP-reactivated models of comb plates to test motile responses to known concentrations of free Ca2+. We found that 5 microM A23187 and 10 mM Ca2+ induced dissociated comb plate cells to beat in the reverse direction and to swim counterclockwise in circular paths instead of in the normal clockwise direction. Detergent/glycerol-extracted comb plates beat actively in the presence of ATP, and reactivation was reversibly inhibited by micromolar concentrations of vanadate. Free Ca2+ concentrations greater than 10(-6)M caused reversal in direction of the effective stroke but no significant increase in beat frequency. These results show that ciliary reversal in ctenophores, like that in protozoa, is activated by an increase in intracellular free Ca2+ ions. This allows the unique experimental advantages of ctenophore comb plate cilia to be used for future studies on the site and mechanism of action of Ca2+ in the regulation of ciliary motion.
Microvillus 110K-calmodulin: effects of nucleotides on isolated cytoskeletons and the interaction of the purified complex with F-actin.
Authors: Verner K , Bretscher A
Published: May 2004

Microvilli isolated from intestinal epithelial cells contain a cytoskeletal Mr 110,000 polypeptide complexed with calmodulin (110K-CM) that is believed to link the microfilament core bundle laterally to the plasma membrane. Previous work has shown that physiological levels of ATP can partially solubilize the 110K-CM complex from isolated microvillus cytoskeletons or isolated microvilli. However, once extracted, the 110K-CM complex has been found to be difficult to maintain stably soluble in aqueous buffers. This is due to the presence of an endogenous ATPase (approximately 100 nmol Pi/min per mg at 37 degrees C) in microvillus cytoskeletal preparations that depletes the ATP with subsequent precipitation of 110K-CM. Addition of ATP to such precipitates resolubilizes 110K-CM. Inclusion of an ATP regenerating system in the solubilization of 110K-CM from cytoskeletons, or membrane-bound brush borders, increases the amount of 110K-CM solubilized. Solubilization of 110K-CM from microvillus cytoskeletons was found to require a divalent cation (Mg2+, Mn2+, or Co2+, but not Zn2+) and a nucleoside triphosphate (ATP, GTP, CTP, or ITP). ADP did not solubilize 110K-CM, but could partially inhibit ATP-dependent solubilization. Solubilized 110K was phosphorylated during extraction of microvillus cores with [gamma-32P]ATP, but this was unrelated to the solubilization of 110K-CM as the endogenous kinase was specific for ATP, whereas the solubilization was not. The 110K-CM was purified using ATP extraction of brush border cytoskeletons in the presence of an ATP regenerating system, gel filtration of the solubilized extract, an ATP depletion step to specifically precipitate 110K-CM with F-actin, and resolubilization followed by phosphocellulose chromatography. The purified complex was stably soluble in aqueous buffers both in the presence and absence of ATP. It bound almost quantitatively to F-actin in the absence of ATP, and showed nucleotide solubilization characteristics from F-actin similar to that found for solubilization of 110K-CM from microvillus cores. At low ATP levels, the binding to F-actin was increased in the presence of ADP. These results suggest that the purified complex has been isolated in a native form. The data confirm and extend the studies of Howe and Mooseker (1983, J. Cell Biol., 97:974-985) using a partially purified preparation of 110K-CM and further emphasize that 110K-CM is a stably water soluble complex and not an integral membrane protein.
Are cross-bridging structures involved in the bundle formation of intermediate filaments and the decrease in locomotion that accompany cell aging?
Authors: Wang E
Published: May 2004

Five different fibroblast strains derived from donors of a wide range of ages were used for investigation of senescence-associated changes in the organization of intermediate filaments (IFs) and the activity of cell locomotion. Results of immunofluorescence microscopy demonstrate that, in large and flat in vitro aged fibroblasts, vimentin-containing IFs are distributed as unusually organized large bundles. Electron microscopic examination shows that these large bundles are indeed composed of filaments of 8-10 nm. Such a profile of large bundles is rarely seen in young fibroblasts whose IFs are usually interdispersed among microtubules. Within the large filament bundles of senescent fibroblasts, cross-bridge-like extensions are frequently observed along the individual IFs. Immunogold labeling with antibody to one of the cross-bridging proteins, p50, further illustrates the abundance of interfilament links within the IF bundles. The senescence-related increase in interfilament association was also supported by the results of co-precipitation between vimentin and an associated protein of 50,000 D. Time-lapse cinematographic studies of cell locomotion reveal that accompanying aging, fibroblasts have a significantly reduced ability to translocate across a solid substratum. These results led me to suggest that the increased interfilament links via cross-bridges may in part contribute to the mechanism that orchestrates the formation of large filament bundles. The presence of enormous bundles in the cytoplasm may physically impede the efficiency of locomotion for these nondividing cells.
The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections.
Authors: Ris H
Published: May 2004

High voltage electron microscopy of intact cells prepared by the critical point drying (CPD) procedure has become an important tool in the study of three-dimensional relationships between cytoplasmic organelles. It has been claimed that critical point-dried specimens reveal a structure that is not visible in sections of plastic-embedded material; it has also been claimed that this structure, in association with known cytoplasmic filaments, forms a meshwork of tapering threads ("microtrabecular lattice"). Alternatively, this structure might be a surface tension artifact produced during CPD. To test possible sources of artifacts during CPD, model fiber systems of known structure were used. It was found that traces of water or ethanol in the CO2 caused distortions and fusion of fibers in pure muscle actin, fibrin, collagen, chromatin, and microtubules that produce a structure very similar to the proposed "microtrabecular lattice." These structures were, however, well preserved if water and ethanol were totally excluded from the CO2. The same results were obtained with whole mounts of cultured cells. A "microtrabecular lattice" was obtained if some water or ethanol was present in the pressure chamber. On the other hand, when water or ethanol were totally excluded from the CO2 during CPD, cytoplasmic filaments were uniform in thickness similar to their appearance in sections of plastic-embedded cells. It is concluded that the "microtrabecular lattice" is a distorted image of the cytoplasmic filament network produced during CPD by traces of water or ethanol in the CO2.
Isolation and characterization of a factor from calf serum that promotes the pigmentation of embryonic and transformed melanocytes.
Authors: Jerdan J A , Varner H H , Greenberg J H , Horn V J , Martin G R
Published: May 2004

A protein (Mr = 63,000) from calf serum that promotes the pigmentation of cultured chick neural crest and mouse melanoma cells has been partially isolated and characterized in this study. The stimulation of melanin synthesis in cultured cells was used to follow its activity during purification. The pigment-promoting factor was isolated by sequential column chromatography on dye-agarose matrices followed by hydroxyapatite and high pressure molecular sieve chromatography. The factor was found to stimulate melanin biosynthesis at 2-4 micrograms/ml and was specific for melanin-producing cells and their precursors. Antibodies raised in rabbits against the factor inhibited its pigment-promoting activity as well as that of whole calf serum. Enzyme-linked immunoadsorbent assays demonstrated that calf and bovine sera contain molecules that cross-react with the pigment-promoting factor. Horse, human, rat, and chicken sera, which lack the biological activity, also lacked immunological cross-reactivity. Extracts of certain tissues, particularly the submaxillary gland, were observed to be rich sources of pigment-promoting activity.
Source and sinks for the calcium released during fertilization of single sea urchin eggs.
Authors: Eisen A , Reynolds G T
Published: May 2004

The source and sinks for the intracellular calcium released during fertilization were examined in single eggs from the sea urchin, Arbacia punctulata. Single eggs were microinjected with the calcium photoprotein, aequorin. The calcium-aequorin luminescence was measured with a microscope-photomultiplier or observed with a microscope-image intensifier-video system. In the normal egg a propagated release has been observed. The source of the calcium was investigated in the organelle-stratified centrifuged egg and by the use of mitochondrial uncouplers. In the organelle-stratified centrifuged egg, the calcium-aequorin luminescence was found to originate from the clear zone. The principal constituent of the clear zone is the endoplasmic reticulum. Other potential sources of calcium are the mitochondria. Their contribution to the calcium transient was investigated by exposure of aequorin-injected eggs to mitochondrial uncouplers either before or after fertilization. There was no calcium released from the mitochondria before fertilization. A very large calcium store was released from the mitochondria after fertilization. Interestingly, eggs fertilized in the presence of uncouplers showed no increase in the calcium-aequorin luminescence over untreated eggs. Apparently, in the absence of mitochondrial uptake, other sinks for calcium with affinity and capacity similar to the mitochondria exist, but their nature is unknown. We suggest that the endoplasmic reticulum is the source of the intracellular calcium released upon fertilization and that the mitochondria are the principal sink. The results are discussed with regard to the metabolic activation of the egg.
Control by fibroblast growth factor of differentiation in the BC3H1 muscle cell line.
Authors: Lathrop B , Olson E , Glaser L
Published: May 2004

The regulation of creatine phosphokinase (CPK) expression by polypeptide growth factors has been examined in the clonal mouse muscle BC3H1 cell line. After arrest of cell growth by exposure to low concentrations of serum, BC3H1 cells accumulate high levels of muscle-specific proteins including CPK. The induction of this enzyme is reversible in the presence of high concentrations of fetal calf serum, which cause quiescent, differentiated cells to reenter the cell cycle. Under these conditions, the rate of CPK synthesis is drastically reduced. We show in the present communication that either pituitary-derived fibroblast growth factor (FGF) or brain-derived FGF are as effective as serum in repressing the synthesis of CPK when added to quiescent, differentiated cells. The decrease in the rate of synthesis of CPK occurs within 22 h after the addition of pituitary FGF to the cells. Pituitary FGF had very little effect, if any, on the rate CPK degradation. The overall rate of protein synthesis and the pattern of synthesis of the major polypeptides made by these cells was not altered by the addition of FGF. Although pituitary FGF was mitogenic for BC3H1 cells, the rate of cell growth was not absolutely correlated with the extent of repression of CPK. Brain-derived FGF fully repressed CPK induction under conditions where it showed no significant mitogenic activity. These results show that the expression of a muscle-specific protein, CPK, can be controlled by a single defined polypeptide growth factor in fully differentiated cultures, and that initiation of cell division is not required for their regulation to take place.
General organization of protein in HeLa 40S nuclear ribonucleoprotein particles.
Authors: Lothstein L , Arenstorf H P , Chung S Y , Walker B W , Wooley J C , LeStourgeon W M
Published: May 2004

The majority of the protein mass of HeLa 40S heterogeneous nuclear ribonucleoprotein monoparticles is composed of multiple copies of six proteins that resolve in SDS gels as three groups of doublet bands (A1, A2; B1, B2; and C1, C2) (Beyer, A. L., M. E. Christensen, B. W. Walker, and W. M. LeStourgeon. 1977. Cell. 11: 127-138). We report here that when 40S monoparticles are exposed briefly to ribonuclease, proteins A1, C1, and C2 are solubilized coincidentally with the loss of most premessenger RNA sequences. The remaining proteins exist as tetramers of (A2)3(B1) or pentamers of (A2)3(B1)(B2). The tetramers may reassociate in highly specific ways to form either of two different structures. In 0.1 M salt approximately 12 tetramers (derived from three or four monoparticles) reassemble to form highly regular structures, which may possess dodecahedral symmetry. These structures sediment at 43S, are 20-22 nm in width, and have a mass near 2.3 million. These structures possess 450-500 bases of slowly labeled RNA, which migrates in gels as fragments 200-220 bases in length. In 9 mM salt the tetramers reassociate to form 2.0 M salt-insoluble helical filaments of indeterminant length with a pitch near 60 nm and diameter near 18 nm. If 40S monoparticles are treated briefly with nuclease-free proteases, the same proteins solubilized by nuclease (A1, C1, and C2) are preferentially cleaved. This protein cleavage is associated with the dissociation of most of the heterogeneous nuclear RNA. Proteins A2 and B1 again reassemble to form uniform, globular particles, but these sediment slightly slower than intact monoparticles. These findings indicate that proteins A1, C1, and C2 and most of the premessenger sequences occupy a peripheral position in intact monoparticles and that their homotypic and heterotypic associations are dependent on protein-RNA interactions. Protein cross-linking studies demonstrate that trimers of A1, A2, and C1 exist as the most easily stabilized homotypic association in 40S particles. This supports the 3:1 ratio (via densitometry) of the A and C proteins to the B proteins and indicates that 40S monoparticles are composed of three or four repeating units, each containing 3(A1),3(A2),1(B1),1(B2),3(C1), and 1(C2).
Characterization of maturation-dependent extrinsic proteins of the rat sperm surface.
Authors: Rifkin J M , Olson G E
Published: May 2004

Mammalian spermatozoa must mature in the epididymis before they can fertilize an egg. It is known that modification of the protein composition of the sperm surface is an important part of the maturation process. In this paper, we present data on two related glycoproteins that can be extracted from mature but not immature spermatozoa. Cell surface radioiodination has shown that these proteins are on the sperm surface, and immunofluorescence microscopy, by use of monospecific antibodies to the proteins, has indicated that their localization is restricted to the periacrosomal region of the sperm head. We have also shown that in vitro, these proteins will bind to the identical region of immature sperm. Immunohistochemical localization of the proteins in the epididymis shows that they are produced and secreted by the cauda region. The significance of the addition of these proteins to the sperm surface in both maturation and fertilization is discussed.
Reversible chromosome condensation induced in Drosophila embryos by anoxia: visualization of interphase nuclear organization.
Authors: Foe V E , Alberts B M
Published: May 2004

We have studied the morphology of nuclei in Drosophila embryos during the syncytial blastoderm stages. Nuclei in living embryos were viewed with differential interference-contrast optics; in addition, both isolated nuclei and fixed preparations of whole embryos were examined after staining with a DNA-specific fluorescent dye. We find that: (a) The nuclear volumes increase dramatically during interphase and then decrease during prophase of each nuclear cycle, with the magnitude of the nuclear volume increase being greatest for those cycles with the shortest interphase. (b) Oxygen deprivation of embryos produces a rapid developmental arrest that is reversible upon reaeration. During this arrest, interphase chromosomes condense against the nuclear envelope and the nuclear volumes increase dramatically. In these nuclei, individual chromosomes are clearly visible, and each condensed chromosome can be seen to adhere along its entire length to the inner surface of the swollen nuclear envelope, leaving the lumen of the nucleus devoid of DNA. (c) In each interphase nucleus the chromosomes are oriented in the "telophase configuration," with all centromeres and all telomeres at opposite poles of the nucleus; all nuclei at the embryo periphery (with the exception of the pole cell nuclei) are oriented with their centromeric poles pointing to the embryo exterior.
The inhibition of neutrophil granule enzyme secretion and chemotaxis by pertussis toxin.
Authors: Becker E L , Kermode J C , Naccache P H , Yassin R , Marsh M L , Munoz J J , Sha'afi R I
Published: May 2004

Pertussis toxin treatment of rabbit peritoneal neutrophils causes a concentration-dependent inhibition of granule enzyme secretion induced by formylmethionyl-leucyl-phenylalanine, C5a, and leukotriene B4. It also inhibits chemotaxis induced by formylmethionyl-leucyl-phenylalanine. The same toxin treatment, however, has no effect on granule enzyme secretion induced by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate. Moreover, pertussis toxin treatment does not affect either the number or affinity of the formylpeptide receptors on the neutrophil nor does it have any effect on the unstimulated levels of cyclic AMP (cAMP) or the transient rise in cAMP induced by chemotactic factor stimulation in these cells. We hypothesize that pertussis toxin, as in other cells, interacts with a GTP binding regulatory protein identical with or analogous to either Ni or transducin which mediates the receptor-induced inhibition or activation of a target protein or proteins required in neutrophil activation. The nature of the target protein is unknown, but it is not the catalytic unit of adenylate cyclase. The target protein acts after binding of chemotactic factor to its receptor in the sequence that leads to the receptor-induced rise in intracellular Ca2+. It does not affect the responses elicited by the direct introduction of calcium into the cells or the activity of protein kinase C.
Role of myosin in terminal web contraction in isolated intestinal epithelial brush borders.
Authors: Keller T C , Conzelman K A , Chasan R , Mooseker M S
Published: May 2004

We have investigated the role of myosin in contraction of the terminal web in brush borders isolated from intestinal epithelium. At 37 degrees C under conditions that stimulate terminal web contraction (1 microM Ca++ and ATP), most (60-70%) of the myosin is released from the brush border. Approximately 80% of the myosin is also released by ATP at 0 degree C, in the absence of contraction. Preextraction of this 80% of the myosin from brush borders with ATP has no effect on either the time course or extent of subsequently stimulated contraction. However, contraction is inhibited by removal of all of the myosin with 0.6 M KCl and ATP. Contraction is also inhibited by an antibody to brush border myosin, which inhibits both the ATPase activity of brush border myosin and its ability to form stable bipolar polymers. These results indicate that although functional myosin is absolutely required for terminal web contraction only approximately 20% of the brush border myosin is actually necessary. This raises the possibility that there are at least two different subsets of myosin in the terminal web.
Association of the postsynaptic 43K protein with newly formed acetylcholine receptor clusters in cultured muscle cells.
Authors: Peng H B , Froehner S C
Published: May 2004

The postsynaptic membrane from Torpedo electric organ contains, in addition to the acetylcholine receptor (AChR), a major peripheral membrane protein of approximately 43,000 mol wt (43K protein). Previous studies have shown that this protein is closely associated with AChR and may be involved in anchoring receptors to the postsynaptic membrane. In this study, binding sites for monoclonal antibodies (mabs) to the 43K protein have been compared to the distribution of AChR in Xenopus laevis muscle cells in culture. In double label immunofluorescence experiments, clusters of AChR that occur spontaneously on these cells were stained with anti-43K mabs. Newly formed receptor clusters induced with positive polypeptide-coated latex beads were also stained with anti-43K mabs as early as 12 h after the application of the beads. Exact correspondence in the distribution of the anti-43K protein binding sites and the AChR was found in both types of clusters. These results suggest that the 43K protein becomes associated with AChR clusters during a period of active postsynaptic membrane differentiation. Thus, this protein may participate in the clustering process.
Localization of topoisomerase II in mitotic chromosomes.
Authors: Earnshaw W C , Heck M M
Published: May 2004

In the preceding article we described a polyclonal antibody that recognizes cSc-1, a major polypeptide component of the chicken mitotic chromosome scaffold. This polypeptide was shown to be chicken topoisomerase II. In the experiments described in the present article we use indirect immunofluorescence and immunoelectron microscopy to examine the distribution of topoisomerase II within intact chromosomes. We also describe a simple experimental protocol that differentiates antigens that are interspersed along the chromatin fiber from those that occupy restricted domains within the chromosome. These experiments indicate that the distribution of the enzyme appears to be independent of the bulk chromatin. Our data suggest that topoisomerase II is bound to the bases of the radial loop domains of mitotic chromosomes.
Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasmic transport.
Authors: Allen R D , Weiss D G , Hayden J H , Brown D T , Fujiwake H , Simpson M
Published: May 2004

Native microtubules prepared from extruded and dissociated axoplasm have been observed to transport organelles and vesicles unidirectionally in fresh preparations and more slowly and bidirectionally in older preparations. Both endogenous and exogenous (fluorescent polystyrene) particles in rapid Brownian motion alight on and adhere to microtubules and are transported along them. Particles can switch from one intersecting microtubule to another and move in either direction. Microtubular segments 1 to 30 microns long, produced by gentle homogenization, glide over glass surfaces for hundreds of micrometers in straight lines unless acted upon by obstacles. While gliding they transport particles either in the same (forward) direction and/or in the backward direction. Particle movement and gliding of microtubule segments require ATP and are insensitive to taxol (30 microM). It appears, therefore, that the mechanisms producing the motive force are very closely associated with the native microtubule itself or with its associated proteins. Although these movements appear irreconcilable with several current theories of fast axoplasmic transport, in this article we propose two models that might explain the observed phenomena and, by extension, the process of fast axoplasmic transport itself. The findings presented and the possible mechanisms proposed for fast axoplasmic transport have potential applications across the spectrum of microtubule-based motility processes.
Locations of Z-DNA in polytene chromosomes.
Authors: Lancillotti F , Lopez M C , Alonso C , Stollar B D
Published: May 2004

In polytene chromosomes of Drosophila hydei and D. melanogaster, Z-DNA was identified in varying distribution after different conditions for fixation were used. When salivary glands were fixed and squashed in 50% acetic acid alone, Z-DNA was found in the less dense DNA regions, such as interbands, some puffs, and a few of the less dense bands. Prefixation that combined ethanol and acetic acid exposure led to prominent immunofluorescent staining of the bands, generally but not strictly correlating with the total DNA content. Separate exposure to ethanol and acetic acid did not cause this band to stain, but if residual ethanol was present after ethanol fixation, subsequent exposure to acid did cause it. Under the more selective acid fixation conditions, Z-DNA reactivity was seen in portions of certain ecdysone-inducible puffs in the induced but not in the resting state; in other inducible regions, the Z-DNA immunoreactivity was not changed on induction. Z-DNA was also identified in polytene chromosomes within isolated nuclei that had been frozen and fixed in ethanol without exposure to acid; this Z-DNA was present in regions of low DNA density.
Electron microscopic characterization of chick embryonic skeletal muscle proteoglycans.
Authors: Pechak D G , Carrino D A , Caplan A I
Published: May 2004

In this article, proteoglycans from embryonic chick leg muscle are quantitatively and qualitatively compared with day 8 high density cell culture cartilage proteoglycans by electron microscopy of proteoglycan-cytochrome c monolayers. The visualized proteoglycan profiles were separated into four categories according to shape, size, and complexity. The two major categories were further characterized by lengths of core proteins, lengths of side projections, and distance between side projections. Two large proteoglycans are identifiable in spread leg muscle preparations. One group has a core protein (mean length of 205 nm) from which extend long thin side projections that we interpret to be groups of chondroitin sulfate glycosaminoglycans with a mean length of 79 nm. This large chondroitin sulfate proteoglycan is the only type found in muscle cultures as determined both biochemically in the past and now by electron microscopy and is referred to as muscle proteoglycan. The second large proteoglycan has a mean core protein length of 250 nm and side projections that are visibly shorter (mean length of 38 nm) and thicker than those of the muscle proteoglycan. This group is referred to as the mesenchymal proteoglycan since its biosynthetic origin is still uncertain. We compare these two profiles with the chick cartilage chondroitin sulfate proteoglycan that has a mean core protein length of 202 nm and side projections with a mean length of 50 nm. The data presented here substantiate the earlier biochemical characterization of these noncartilage proteoglycans and establish the unique structural features of the muscle proteoglycan as compared with the similar profiles of the cartilage and mesenchymal proteoglycans.
Ultrastructural and cytochemical demonstration of peroxisomes in cultured fibroblasts from patients with peroxisomal deficiency disorders.
Authors: Arias J A , Moser A B , Goldfischer S L
Published: May 2004

The oxidation of very long chain fatty acids and synthesis of ether glycerolipids (plasmalogens) occurs mainly in peroxisomes. Zellweger's cerebrohepatorenal syndrome (CHRS) is a rare, inherited metabolic disease characterized by an apparent absence of peroxisomes, an accumulation of very long chain fatty acids, and a decrease of plasmalogens in tissues and cultured fibroblasts from these patients. As peroxisomes are ubiquitous in mammalian cells, we examined normal and CHRS-cultured fibroblasts for their presence, using an electron microscopic histochemical procedure for the subcellular localization of catalase, a peroxisomal marker enzyme. Small (0.08-0.20 micron) round or slightly oval peroxisomes were seen in both normal and CHRS fibroblasts. The number of peroxisomes was analyzed morphometrically and found to be significantly reduced in all CHRS cell lines. These results are discussed in relation to the underlying defect in peroxisomal function and biogenesis in this disease.