bluuebunny/crossref_metadata_2025_split huggingface dataset:
The identification and localization of actin and actin‐like filaments in lactating guinea pig mammary gland alveolar cells
Authors: Amato Philip A. , Loizzi Robert F. 
Published: February 2005

AbstractCytochalasin B, a microfilament‐altering drug, inhibits lactose synthesis in lactating guinea pig mammary gland [Biochim. Biophys. Acta 392:20, 1975] but not primarily by inhibiting glucose transport [Eur. J. Cell Biol. 20:150, 1979]. In order to study the possible role of microfilaments in lactose synthesis and secretion, we isolated both the alveolar (milk‐secreting) and myoepithelial (contractile) cells from lactating mammary gland. Light microscopy shows that the alveolar cell fraction (viability approximately 71%) is homogenous and that the cells retain strong polarity of secretory structures in the apical region. Two proteins were extracted from the alveolar cell fraction. One (mol wt 42,000) comigrates with skeletal muscle actin on SDS‐PAGE gels. The other, a high‐molecular‐weight (180,000) protein (HMWP) may be analogous to actin‐binding protein or clathrin. An extract from the myoepithelial cell fraction also contains a protein that comigrates with actin but no HMWP. Whole tissue extract contains the 42K protein, and a 185K HMWP. Examination of the alveolar cell extract by electron microscopic (EM) negative staining revealed meshworks of multistranded, interconnecting filaments, with attached globular structures (100–200 A) (possibly the HMWP) and single filaments (40–60 A diameter) branching off. To localize these filamentous structures in situ, whole tissue was glycerinated and incubated with rabbit skeletal muscle heavy meromyosin (HMM). Masses of filaments in myoepithelial cells served as convenient standards for HMM decoration. Decorated filaments have cross‐arms or projections, unlike the narrow, smooth filaments of control tissue. Decorated filaments in alveolar cells are located beneath the plasma membrane, in close association with secretory vacuoles, and near the Golgi apparatus; filaments near the latter two are often oriented perpendicular to the plasma membrane. Microvesicles are embedded in meshworks under the plasmalemma and near the Golgi apparatus. Intermediate‐sized (85–115 A diameter), non‐decorated filaments diverge from the meshworks of decorated filaments. Microvesicles are associated with intermediate‐sized filaments as well. The association of actin‐like filaments with secretory vacuoles and microvesicles and their location in areas of the cell concerned with biosynthetic activities suggest a possible function in the intracellular transport of secretory products.
Quantitative studies on polarization optical properties of living cells III: Cortical birefringence of the dividing sea urchin egg
Authors: Shôji Yôko , Hamaguchi Yukihisa , Hiramoto Y. 
Published: February 2005

AbstractBirefringence (BR) at the cell surface of fertilized eggs of the sand‐dollar, Clypeaster japonicus, during mitosis and cleavage was determined with a photoelectric BR detection apparatus [Hiramoto et al, 1981a]. The cortex of about 2 μm thickness is birefringent positive with respect to the normal to the cell surface. The hyaline layer is negatively birefringent. The halo‐layer consisting of a row of microvilli surrounding the egg is positively birefringent in normal Ca‐free sea water, while it is negatively birefringent in Ca‐free sea water with high refractive index. The BR of the cortex gradually increases over the entire surface during mitosis until the onset of cleavage. The BR of the cortex at the polar region reaches a maximum shortly after the onset of cleavage and then decreases, while the BR of the cortex at the equatorial region begins to decrease shortly before the onset of cleavage, reaches a minimum shortly after the cleavage starts, and then increases again as the cleavage furrow advances. The coefficient of birefringence of the cortex is about 2.5 × 10−5 at the maximum. The BR change of the cortex during mitosis and cleavage is interpreted as a passive deformation caused by the constriction of the contractile ring as well as an active structural change of the cortex occurring in the dividing cell.
Simultaneous oscillations of Ca2+ efflux and tension generation in the permealized plasmodial strand of physarum
Authors: Yoshimoto Y. , Matsumura F. , Kamiya N. 
Published: February 2005

AbstractCalcium is now generally thought to play a key role in regulating a variety of cellular movements. When the plasmodium of Physarum polycephalum was treated with the calcium‐ionophore A23187 or the quasi‐ionophore amphotericin B, Ca2+ leaked out. Ca2+ efflux into the ambient solution from the plasmodial strand segment was measured by the luminescence of a photoprotein aequorin, and the tensile force production was recorded simultaneously. Ca2+ efflux oscillated with the same period as the cycle of tension generation in the strand, but the phase of cyclic changes in Ca2+ efflux was opposite to that of tension generation. That is, Ca2+ efflux fell in the increasing tension phase and rose in the decreasing tension phase. Cyclic changes in efflux of Ca2+ are provisionally interpreted as reflecting corresponding changes in concentrations of free Ca2+ in the cytoplasm.
Nucleated assembly of mitotic microtubules in living PTK2 cells after release from nocodazole treatment
Authors: De Brabander Marc , Geuens Gustaaf , Mey Jan De , Joniau Marcel 
Published: February 2005

AbstractThe reassembly of microtubules is described in mitotic cells after release from nocodazole‐induced block. The formation of microtubules was followed by light microscopic immunocytochemical staining using the PAP method, combined with to‐luidine blue staining of the chromatin. The light microscopic observations on whole cells were compared with ultrastructural observations on thin sections. This step is essential to ascertain complete destruction of microtubules during the nocodazole treatment and to correlate immunocytochemical staining with the presence of microtubules.Removal of nocodazole (10 or 1 μg/ml) after a sufficiently long incubation to induce a complete disappearance of microtubules resulted in the appearance of tubulin staining specifically associated with the centromeres and with one or two isolated points in the cytoplasm. Electron microscopy confirmed that the staining was due to the massive accumulation of small microtubules at the kinetochores and centrosomes. Kinetochore nucleation was seen only in association with condensed metaphase‐stage chromosomes and not with the less‐condensed prophase chromosomes.In a second type of experiment cells were allowed to enter mitosis in the presence of an incompletely active concentration of nocodazole (0.1 μg/ml). The construction of the mitotic spindle was arrested; however, short microtubules were assembled at the kinetochores and centrosomes.These experiments demonstrate that in living mitotic PTK2 cells the kinetochores, as well as the centrosomes, exert a nucleating action on tubulin assembly.The further elongation of microtubules after removal of nocodazole was seen to occur preferentially along axes between the centrosomes and the kinetochores. This resulted in the construction of normal metaphases that evolved through anaphase and telophase. We have attempted to formulate a hypothesis that may explain the oriented assembly that seems to be essential in the construction of the spindle.
Coupling of photomovement and photosynthesis in desmids
Authors: Häder Donat‐P. 
Published: February 2005

AbstractThe effects of the uncouplers CCCP and DNP on photokinesis, phototaxis, and the photophobic response in the desmid Cosmarium have been studied both in population systems and by videomicrographic, single‐cell analysis. Light‐dependent motility is specifically inhibited by both uncouplers, indicating that photokinesis is driven by photophosphorylation. In population experiments, phototaxis and accumulations in light traps due to photophobic responses are inhibited by drug concentrations comparable to those that inhibit photokinesis. Analysis of single‐cell behavior demonstrated, however, that neither photophobic responses elicited by an increase in light intensity (step‐up response) nor by a decrease (stepdown response) are inhibited, as long as the reduced motility allows the organisms to cross a light‐‐dark border. Phototactic orientation is not impaired by DNP in the single cell analysis, but CCCP significantly reduced the degree of orientation. The results indicate that, although chlorophyll is the photoreceptor for all three photoresponses, at least the photophobic response is independent of both the photosynthetic electron transport chain and photophosphorylation.
Effects of phalloidin microinjection and localization of fluorescein‐labeled phalloidin in living sand dollar eggs
Authors: Hamaguchi Yukihisa , Mabuchi Issei 
Published: February 2005

AbstractEffects of microinjection of phalloidin on fertilization and cleavage of sand dollar (Clypeaster japonicus and Scaphechinus mirabilis) eggs were studied. The drug, previously injected into unfertilized eggs, showed no effect on the elevation of the fertilization membrane upon insemination up to an intracellular concentration of 50 μM. However, the movement of the egg pronucleus to the sperm pronucleus was inhibited and the fusion of pronuclei did not occur. The subsequent development no longer took place. When phalloidin was injected into fertilized eggs, the thickness of the cortical layer increased and the microvilli became conspicuous. Both nuclear division and cleavage were inhibited at the intracellular concentration of more than 20 μM, though the latter seemed to be more sensitive to phalloidin than the former.Fluorescein‐labeled phalloidin (FL‐phalloidin) was injected into eggs in order to investigate F‐actin localization by fluorescence microscopy. In both unfertilized and fertilized eggs, FL‐phalloidin was localized in the cortical layer within 1 min after injection. It was also localized in the cortical layer as radially oriented rodlike structures when injected into fertilized eggs before the disappearance of the nuclear membrane. No distinct fluorescence was detected in the mitotic apparatus or in the cleavage furrow. FL‐phalloidin redistributed gradually into egg cytoplasm. In unfertilized eggs, fluorescent rods were found especially in the egg pronucleus 30 min after injection.
Organization of the cytoskeleton in square fibroblasts
Authors: Marek L. F. , Kelley R. O. , Perdue B. D. 
Published: February 2005

AbstractThe relationship between the cytoskeleton, stress fiber formation, and cell shape has been difficult to determine in fibroblasts grown in tissue culture. Vagaries in cell shape are complicated, as well, by stochastic cell movements. We dictated the attachment sites and shape of fibroblasts by growing them on square adhesive substrates surrounded by nonadhesive substrates. Cytoskeletal models were made by treating the cells with buffered Triton X‐100 and glycerol. The residues were then examined by scanning electron microscopy followed by light microscopy of the same cells. The cytoskeletons of randomly moving cells were examined with whole mount transmission microscopy to confirm images seen with scanning microscopy. The cells thus examined demonstrated definite relationships between ruffling activity and stress fiber terminations, which were limited to the more adhesive, palladium substrate. No stress fibers were seen to end on the lesser adhesive substrate, agarose, and ruffling did not occur across the agarose. Cells too small to fill an entire square tended to extend across one diagonal of the square, and the stress fibers ran parallel to the longest axis of these cells. Larger cells were able to completely fill their squares. The cytoskeletons of these cells were organized in a spatial relation to the square shape of the cells. The cortical meshwork was aligned circularly and diagonally within the cells. Stress fibers appeared to form from the microfilaments of the meshwork and were aligned diagonally across the cells. We conclude that the diagonal arrangement of the stress fibers and cortical meshwork is caused by the same mechanism by which smaller cells spread over the longest axis of a square. Regions of cells where the meshwork was absent or where stress fibers were tightly bundled were occupied by more randomly arranged cytoskeletal components. Regions of tighyly bundled stress fibers did not seem to coincide with regions of cortical meshwork as seen by either whole mount transmission or scanning electron microscopy. Stress fibers were revealed in the light microscope to course beneath more randomly oriented cytoskeletal elements. These “lacework‐like” elements were found frequently in square cells. Conspicuous structures in this random lacework were focal points of radially arranged filaments. Our observations suggest a continuity between stress fibers and the cortical microfilaments. The orientation of fibers and filaments was, in turn, dependent on cell shape for organization within the cell.
Cell locomotion in vitro by Xenopus laevis gastrula mesodermal cells
Authors: Nakatsuji Norio , Johnson Kurt E. 
Published: February 2005

AbstractProspective mesodermal cells of Xenopus laevis gastrulae showed substantial locomotion in vitro, averaging 4.3 μM/min, when dissociated and cultured on a glass surface coated with collagen and fetal bovine serum. The cell translocate by making lamellipodia and filopodia whereas the main cell body remains rounded. When two mesodermal cells made contact with each other, they showed contact paralysis of lamellipodial activity. In contrast, when mesodermal cells contact ectodermal cells, contact paralysis does not occur. Rather, migrating mesodermal cells continue to translocate. The locomotion in vitro appears to mimic that in vivo during gastrulation, because of the similarities of the rate of movement and the cell shape in culture and in embryos. Neither prospective ectodermal cells from gastrulae nor prospective mesodermal cells from blastulae showed locomotion under the same culture conditions.
Capping of cross‐linked concanavalin a receptors on the surface of substrate‐spread platelets
Authors: Alexandrova A. Y. , Vasiliev J. M. 
Published: February 2005

AbstractLigand‐induced redistribution of concanavalin A (Con A) receptors on the surfaces of substrate‐spread rabbit platelets was studied. These receptors were diffusely distributed on the surface of prefixed cells. Incubation of living cells with Con A and anti‐Con A antiserum led to redistribution of corresponding receptors: These receptors were patched, removed from the upper surface of substrate‐attached pseudopodia and lamellipodia, and accumulated on the surface of central cell parts. Capping of receptors was inhibited by cytochalasins B and D and by cold. Thus, capping of surface receptors can take place not only in large nucleated cells but also in platelets, that is, in small anucleate cell fragments. It is suggested that the cells of various types in the course of their attachment to the substratum form pseudopodia and lamellipodia that have a number of common characteristics, including the ability to move the cross‐linked surface receptors centripetally.
Directional protrusive pseudopodial activity and motility in macrophages induced by extracellular electric fields
Authors: Orida Norman , Feldman Joseph D. 
Published: February 2005

AbstractExtracellularly applied electric fields (< 12 V/cm) strongly influence murine resident peritoneal macrophages (Mø) to undergo directional protrusive pseudopodial activity to wards the positive pole of the electric fields in the absence of exogenously applied chemotactic ligands. Internal and external morphological features were not grossly disrupted by the fields. Directional motility induced by the electric fields was inhibited in the presence of 1.0 mM La3+ or 2.5 mM Mg2+ and 5.0 mM EGTA. Effects of the fields were latent in the inhibited cells and directional motility was expressed after termination of the field and removal of the inhibitors. Receptors for the lectins concanavalin A (Con A) and phytohemagglutinin (PHA‐L) were uniformly distributed on the surfaces of Mø with no exposure to electric fields. After exposure to the fields, Con A receptors were preferentially distributed on regions of the Mø surface facing the negative pole and PHA‐L receptors were preferentially distributed on those regions facing the positive pole. The possibility that directional Mø motility is regulated by the molecular topography of the cell surface is discussed.
Persistence of the expression of β‐tropomyosin in dystrophic avian pectoral muscle
Authors: Feit Howard , Domke Robert 
Published: February 2005

AbstractThe isotype pattern of tropomyosin was investigated in normal and dystrophic avian pectroal muscle using two‐dimensional gel electrophoresis. Previous reports have shown that adult pectoral muscle of chickens contains only the α‐subunit of tropomyosin and a breast‐type troponin‐T (TN‐T), whereas pectoral fetal muscle contains both α‐ and β‐tropomyosin and leg‐type TN‐T. The change from the fetal to the adult forms begins shortly after hatching. It has been previously reported that avian dystrophic pectoral muscle contains both the leg‐ and breast‐type TN‐T; we show that in avian dystrophic muscle there is also persistent expression of the β‐subunit of tropomyosin.
Effect of various extraction solutions and thrombin activation on the composition of the platelet cytoskeleton
Authors: Rosenberg Sharon , Lawrence John , Stracher Alfred 
Published: February 2005

AbstractWhen human blood platelets were immersed in an ice‐cold solution containing 1% Triton ×‐1200, 40 mM KCl, 10 mM EGTA, 10 mM imidazole‐HCl, and 2 mM NaN3 pH 7.0, a flocculent precipitate appeared immediately in the tube. This precipitate was collected at 3,000g and SDS‐polyacrylamide gel analysis showed it to consist mainly of actin, α‐actinin, actin‐binding protein (ABP), and varying amounts of myosin.Any modifications of this solution used to isolate the platelets' Triton‐insoluble cytoskeleton caused profound changes in the nature of the cytoskeleton isolated. Increasing the KCl concentration resulted in a lower yield of cytoskeletal actin and ABP. Inclusion of EDTA in the solution resulted in an increased amount of myosin associated with the cytoskeleton, whereas including MgATP decreased the myosin yield.Experiments with the purified proteins showed that ABP and myosin can each protect the actin from depolymerizing when dialyzed into the Triton solubilization solution. In addition, it was found that when platelets were stimulated with thrombin for 2 min prior to the addition of the Triton solution, 3–4 times more myosin was associated with the cytoskeletal precipitate.The results suggest, therefore, that any variations in solution conditions used for isolating the cytoskeleton from resting platelets, which results in alterations in the amount of ABP, may have profound effects on the state of actin polymerization. Likewise, in thrombin‐activated platelets, it is suggested that the increased association of myosin with the cytoskeleton results in a greater stabilization of the F‐actin associated with the cytoskeleton. These factors must be considered when interpreting the results regarding the nature of actin transformations in the resting and activated platelet.
Effect of fragmin on actin polymerization: Evidence for enhancement of nucleation and capping of the barbed end
Authors: Sugino Hiroaki , Hatano Sadashi 
Published: February 2005

AbstractAs reported previously, fragmin isolated form Physarum plasmodia restricts the polymerization of actin to produce short F‐actin filaments in the presence of Ca2+ ions. Here it is shown that when actin is polymerized at low concentrations of salts, fragmin increases the critical concentration of actin for polymerization. This effect of fragmin on the critical concentration is independent of the molar ratio of fragmin to actin. The addition of actin monomers onto heavy meromyosin‐decorated F‐actin fragments treated with fragmin occurs unidirectionally at the pointed end of each fragment. These results suggest that fragmin binds to the barbed ends of F‐actin filaments and inhibits association and dissociation of actin monomers at this end. Fragmin accelerates the initial stage of polymerization of actin. When a constant amount of G‐actin is polymerized in the presence of small amounts of fragmin, the inverse of the half‐polymerization time increases in proportion to the square root of the amount of fragmin added. This means that fragmin acts as a potent promoter of the nucleation step in actin polymerization. Both functions of fragmin–promotion of nucleation and capping at the barbed end of F‐actin–require micromolar concentrations of Ca2+.
Evidence for a role of 13S axonemal ATPase in modulation of ciliary microtubule sliding
Authors: Zanetti Nina C. , Warner F. D. 
Published: February 2005

AbstractWe recently demonstrated that elevated concentrations (≥ 20 μM) of the dynein substrate MgATP2− inhibit the spontaneous ATP‐induced sliding disintegration of isolated, Triton‐demembranted Tetrahymena cilia. We have used a turbidimetric assay (ΔA350 nm) and electron microscopy to examine the effect of ATP on sliding disintegration when activated by other divalent cations. Mg2+, Ca2+, and Mn2+ are each capable of activating sliding, but only with Mg2+ and Mn2+ is disintegration inhibited by elevated ATP concentrations (≥ 1 mM). The two major ATPase activities obtained by KCI extraction of Tetrahymena axonemes differ in their cation specificities such that Mg2+ and Ca2+ activate the 21S dynein ATPase with equal efficiency, whereas the 13S axonemal ATPase activity is reduced by ∼ 50% when CaATP2− replaces MgATP2− as substrate. With 1 mM MgATP2− as substrate, 10−7 to 10−2M added CaC12 alleviates the ATP‐dependent inhibition of disintegration and likewise represses 13S MgATPase activity. In contrast, free Ca2+ has no effect on either the disintegration response or MgATPase activity. In contrast to Triton‐treated cilia, glycerinated cilia, which beat in 1 mM MgATP2−, are inhibited from beating by high CaATP2− concentrations. These substrate specificities suggest that concentration‐dependent, substrate inhibition of sliding disintegration may be a manifestation of a physiological mechanism that is mediated by the 13S axonemal ATPase and that may function to modulate sliding during bend formation. However, the effects of added CaCl2 probably do not reflect a physiological mechanism for regulating beat parameters, but rather may result from CaATP2− competing for MgATP2− binding sites on the 13S ATPase, thereby blocking expression of the 13S ATPase.
Taxol binds differentially to flagellar outer doublets and their reassembled microtubules
Authors: Parness Jerome , Asnes Clara F. , Horwitz Susan Band 
Published: February 2005

AbstractTaxol induces the in vitro assembly of calcium stable microtubules from flagellar tubulin solubilized from sea urchin (Strongylocentrotus purpuratus) sperm tail outer doublets by sonication. Assembly occurs in the presence or absence of exogenous GTP. The drug (10 μM) reduces the critical concentration of protein required for assembly to ⩽0.04 mg/ml. 3H‐Taxol binds specifically to both isolated flagellar outer doublets and to reassembled microtubules with calculated maximal binding ratios of 0.25 and 1.32 moles taxol/mole polymerized flagellar tubulin dimer, respectively. We suggest that the discrepancy in maximal binding ratios may result from the presence of an endogenous molecule(s) along the surface of outer doublet microtubules that restricts taxol binding to that structure.