bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetics of granulocyte phagocytosis: Rate limited by cytoplasmic viscosity and constrained by cell size
Authors: Evans Evan 
Published: February 2005

AbstractMicromanipulation of yeast particles and blood granulocytes has been used to study the kinetics of single phagocytosis events. The ingestion process was quantitated by observation of sequential adhesion and encapsulation times. Both adherence and encapsulation times were found to increase greatly as the temperature was reduced below 37°C calcium in solution facilitated adhesion of the particle to the phagocyte but not encapsulation; both adhesion and encapsulation processes required a minimum level of plasma components (presumably complement). The general nature of these observations were confirmatiory of previous studies, but this study is unique in that the specific time course of single particle ingestion was quantitated. It was immediately apparent that the phagocytosis process was 100% efficient above the threshold concentrations required for plasma and temperature, but variations in times from cell to cell indicated heterogeneity in the population. The total time for ingestion varied from as low as 2 sec/particle at 37°C to above several min/particle below 15°C. Encapsulation times for particles were normalized by estimates of particle surface areas to establish a specific time/unit area of particle surface: from 0.5 sec/10−8 cm2 at 37°C to greater than 8 sec/10−8 cm2 at 15°C. The temperature dependence of the encapsulation time correlated well with the temperature dependence of the “apparent” viscosity for granulocytes measured by micropipet aspiration. As such, the kinetic properties observed in these phagocytosis tests are consistent with a model that both assembly of the contractile system and the displacement of the surface by active contraction in phagocytosis are limited by viscous dissipation in the cell. Based on temperature dependence of the adhesion time, the activation energy associated with “turning on” the contractile system is quite large, with a value of about 31 kcal/mole. Finally, it was found that serial “feeding” of yeast to a single granulocyte satiated the phagocyte only after six to eleven particles had been encapsulated. The number limit to ingestion was directly proportional to inital size of the granulocyte.
The cytoskeleton of the naked green flagellate Spermatozopsis similis: Isolation, whole mount elecron microscopy, and preliminary biochemical and immunological characterization
Authors: Lechtreck K.‐F. , McFadden G. I. , Melkonian M. 
Published: February 2005

AbstractThe cytoskeleton of the naked, biflagellate green alga Spermatozopsis similis Preisig & Melkonian was isolated by treatment of cells with Nonidet P‐40 (0.1%) in lysis buffer (30 mM HEPES, 5 mM EGTA, 15 mM KCl, pH 7) and studied in detail by whole‐mount electron microscopy. Isolated cytoskeletons retain the twisted shape of live cells and consist of the two axonemes, the basal apparatus with 4 microtubular and two fibrous roots, and 8–10 secondary cytoskeletal microtubules (SCMT's). The four microtubular flagellar roots differ in number of microtubules (two types with 2 or 5 microtubules, respectively), in their association with fibrous roots of the system I‐type (two‐stranded roots), in total length (two roots with an average of 4.5 μm and two roots with and average of 7.5 μm), and in length of individual root microtubules. Certain of the root microtubules and most of the SCMT's extend to the posterior end of the cell where they converge, terminate and are interconnected by a fibrous cap‐like structure, the rhizosyndesmos. This novel structure consists of a network of 2 nm filaments that presumably lacks centrin as indicated by double immunofluorescence (anti‐α‐tubulin and anti‐centrin) of isolated cytoskeletons. Two‐dimensional gel electrophoresis of isolated, purified basal apparatuses of S. similis identifies among other proteins two isoforms of centrin and α‐ and β‐tubulin as intrinsic components.
Formation of miniasters in the cytoplasm of hexyleneglycol‐treated sea urchin eggs
Authors: Endo Sachiko , Toriyama Masaru , Ohta Kunihiro , Sakai Hikoichi 
Published: February 2005

AbstractMiniasters formed in mitotic sea urchin egg after treatment with 5% hexylene‐glycol were investigated with the combined techniques of indirect immunofluo‐rescence using anti‐tubulin and anti‐51 kD protein antibodies and electron microscopy.The formation of miniasters was dependent on the mitotic cycle. In the cytoplasm of eggs treated with hexyleneglycol at early prometaphase, a small number of microtubule fragments was observed, whereas in those treated at pro‐metaphase, many miniasters and microtubule fragments were seen. When treated at metaphase, we found a great number of miniasters: 250–350 in one egg. In contrast, no miniasters were seen in eggs treated at anaphase, although many long microtubules that spread throughout the cytoplasm were observed. In the eggs treated at telophase, we scarcely noticed microtubule structures in the cytoplasm. In the center of miniasters, granules were found, showing the same size and electron density as those of the microtubule‐organizing granules (MTOGs). Furthermore, the 51 kD protein, a component of the centrosome and mitotic spindle, was observed to be localized in the region of miniasters.
Identification and immunolocalization of cytoplasmic dynein in dictyostelium
Authors: Koonce Michael P. , Richard McIntosh J. 
Published: February 2005

AbstractA high molecular weight microtubule binding protein has been isolated from homogenates of Dictyostelium. Because of its sedimentation velocity (20s), ATP‐sensitive binding to microtubules. UV‐vanadate‐ATP mediated fragmentation, prominent CTPase activity, and its ability to produce limited microtubule movement in vitro, we consider this protein to be a form of cytoplasmic dynein. A polyclonal antibody monospecific to this protein was produced, and dynein's intracellular distribution in ameboid cells was examined by immunofluorescence. The antibody labels a punctate cytoplasmic pattern, localizes to a spherical region adjacent to the nucleus, and also appears to label the nuclei. The punctate staining pattern is consistent with cytoplasmic dynein's proposed function in organelle transport. The spherical juxtanuclear object stained is coincident with this cell's microtubule organizing center, an obvious termination point for minus‐end directed microtubule motors. By immunofluorescence, there does not appear to be a substantial amount of dynein in the intranuclear mitotic spindles of Dictyostelium, These data provide evidence for localization of cytoplasmic dynein in cells, and suggest that Dictyostelium will be a useful system in which to study the molecular biology of microtubule‐associated motor enzymes.
In vitro induction of crawling in the amoeboid sperm of the nematode parasite, Ascaris suum
Authors: Sepsenwol Sol , Taft Stephen J. 
Published: February 2005

AbstractIn a highly synchronous process, the immotile spermatids of Ascaris suum extend pseudopods and become rapidly crawling sperm when treated with an extract from the glandular vas deferens of the male under strict anaerobic conditions. Within 9–12. min, a pseudopod develops, elongates rapidly, and exhibits a continuous flow of membrane specializations, the villipodia, from tip toward base. When attached to acid‐washed glass, the pseudopod pulls the cell body along at speeds exceeding 70 μm/min. The pseudopod length remains constant while retrograde flow of villipodia proceeds at the same rate as the sperm's forward movement. Cohorts of about 15 villipodia form at the leading edge, move reaward together, and disappear at the junction of pseudopod and cell body. These are the termination of branched, refringent fibers, which extend the length of the pseudopod. The latter are the fiber complexes that form its cytoskeleton (Sepsenwol et al.: Journal of Cell Biology 108:55–66, 1989). Locomoting cells sometimes change direction when another crawls by and follow each other. When cells are exposed to air, forward movement ceases in a predictable pattern: the forward extension of the leading edge ceases, the pseudopod shortens from the base, and the cell body continues to be pulled forward. These data contribute to a model for Ascaris sperm amoeboid motility in which independent processes of continuous extension at the leading edge and continuous shortening at the base of the pseudopod act to propel the cell forward.
Keratin filaments restrict organelle migration into the forming spindle of newt pneumocytes
Authors: Mandeville Elizabeth C. , Rieder Conly L. 
Published: February 2005

AbstractWhen viewed by light microscopy the mitotic spindle of newt pneumocytes appears to assemble in an optically clear area of cytoplasm, virtually devoid of mitochondria and other organelles, which is often much larger than the spindle. This clear area is also frequently larger than the region previously occupied by the nucleus. It forms even in prometaphase cells depleted of microtubules prior to nuclear envelope breakdown by colchicine treatment. Time‐lapse video microscopy reveals that as prometaphase proceeds this clear area slowly and progressively collapses around the forming spindle so that it is greatly diminished or nonexistent by the onset of anaphase. The sharply defined nature of the boundary between the clear area and the remaining cytoplasm and the fact that organelles accumulate at its periphery suggest that a structural barrier is present at the boundary that limits organelle migration into the forming spindle. Immunofluo‐ rescence and electron microscopy, of cells previously followed in the living state, reveal that the periphery of the clear area contains prominent bundles of keratin filaments but lacks microtubules and actin. From our observations we conclude that keratin filaments form a loosely organized cage that surrounds the forming newt pneumocyte spindle. We propose that this cage functions, in part, to restrict the dispersion of chromosomes during nuclear envelope breakdown and to impede the bulk migration of organelles into the forming spindle.
The outer arm dynein α‐heavy chains of sea urchin sperm flagella and embryonic cilia are different
Authors: Ogawa Kazuo , Yokota Etsuo , Hamada Yoshio , Wada Shigeo , Okuno Makoto , Nakajima Yoko 
Published: February 2005

AbstractSea urchin sperm outer arm dynein is a multi‐subunit protein composed of heavy chains, intermediate chains, and light chains. We prepared monoclonal and affinity‐purified polyclonal antibodies to heavy and intermediate chain subunits and examined whether the embryonic ciliary axonemes ofthe same species contain the polypeptides sharing epitopes with them. Ciliary axonemes contained a high molecular weight polypeptide with the exact same mobility as flagellar β‐heavy chain. This polypeptide also shared epitopes with it. In contrast, no polypeptide had the exact same molecular weight as flagellar α‐heavy chain and shared epitopes with it. Western blots showed that ciliary axonemes also contain three polypeptides sharing epitopes with the respective flagellar intermediate chain. The present results revealed that the α‐heavy chains of flagellar and ciliary outer arm dyneins are different.
Nuclear and mitotically enhanced epitope
Authors: Chriss Hoffman John , Michael Mullins J. 
Published: February 2005

AbstractSalt‐extracted proteins of taxol‐stabilized microtubules from Chinese hamster ovary cells arrested at mitosis were used to immunize mice for hybridoma production. From a group of related monoclonal antibodies (MAbs), one, C9, recognized an epitope on antigens localized by immunofluorescence microscopy to interphase centrosomes and nuclei. The availability of the nuclear antigen was cell cycle‐dependent; however, permeabilization of cells before fixation revealed that the antigen was present throughout the cell cycle. The nuclear antigen was exposed during prophase and was released from the nucleus upon nuclear envelope breakdown filling the cytoplasm of the mitotic cell. Antigenic material re‐accumulated at daughter nuclei and was concealed during Gl phase. Detergent extraction of the cytoplasmic antigen from mitotic cells enabled localization of antigens to centrosomes, kinetochores, and the furrowing region/midbody. Immunoblot analysis of cells of a variety of species of origin identified an approximate 250 kD polypeptide as corresponding to the nuclear antigen, whereas polypeptides of 107/117 kD as well as approximately 250 kD accounted for the mitotic cytoplasmic antigens. No polypeptides could be associated with antigens at centrosomes, kinetochores, or midbodies. This MAb joins the antibody preparations previously reported that describe nuclear antigens, or epitopes on antigens, enhanced at mitosis.
T‐1, a mitotic arrester, alters centrosome configurations in fertilized sea urchin eggs
Authors: Itoh Tomohiko J. , Schatten Heide , Schatten Gerald , Mazia Daniel , Kobayashi Akio , Sato Hidemi 
Published: February 2005

AbstractT‐1 induces modifications in the shape of the centrosome at division in fertilized eggs of the North American sea urchin, Lytechinus pictus. Phase contrast microscopy observations of mitotic apparatus isolated from T‐1treated (1.7–8.5μM) eggs at first division shows that the centrosomes already begin to spread or to separate by prophase and that the mitotic spindle is barrel‐shaped. When eggs are fertilized with sperm that have been pretreated with T‐1, the centrosomes become flattened; the spindles are of normal length. Immunofluorescence microscopy using an anti‐centrosomal monoclonal antibody reveals that T‐l modifies the structure of the centrosome so that barrel‐shaped spindles with broad centrosomes are observed at metaphase, rather than the expected focused poles and fusiform spindle. Higher concentrations of T‐l induce fragmentation of centrosomes, causing abnormal accumulation of microtubules in polar regions. These results indicate that T‐l directly alters centrosomal configuration from a compact structure to a flattened or a spread structure. T‐l can be classified as a new category of mitotic drugs that may prove valuable in dissecting the molecular nature of centrosomes.
Propranolol, a β‐adrenergic receptor blocker, affects microfilament organization, but not microtubules, during the first division in sea urchin eggs
Authors: Nicotra Antonietta , Schatten Gerald 
Published: February 2005

AbstractPropranolol, a β‐adrenergic receptor blocker, blocks the formation of the cleavage furrow, while karyokinesis is unaffected during first division in the sea urchins Paracentrotus lividus or Lytcchinus pictus. This effect is reversed by adrenalin, indicating that it is mediated by an adrenergic mechanism. The staining of F‐actin microfilaments by rhodamine phalloidin in eggs in which the cleavage is blocked by the drug has revealed that propranolol affects both the distribution and the organization of actin microfilaments. A low‐voltage scanning electron microscopy (LVSEM) study of microvilli in these eggs shows an extensive rearrangement of the egg surface. Anti‐tubulin immunofluorescence microscopy of eggs treated with propranolol shows that they form normal mitotic asters. This indicates that while cleavage is affected, mitotic spindle formation is not. These results suggest that neurotransmitter monoamines known to be present in the sea urchin egg might be involved in the reorganization of the actin cytoskeleton underlying the formation of the cleavage furrow.
1,2‐dioctanoylglycerol accelerates or retards mitotlc progression in Tradescantia stamen hair cells as a function of the time of its addition
Authors: Larsen Paul M. , Wolniak Stephen M. 
Published: February 2005

AbstractWe have treated living, intact stamen hair cells from the spiderwort plant, Tra‐descantia virginiana, with 0.5 μg/ml or 60 μg/ml 1,2‐dioctanoylglycerol, a potent and permeant activator of protein kinase C, and have observed the rates of progression of mitosis from prophase through anaphase. We have found that in addition to the concentration used, the time of initial treatment with 1,2‐di‐octanoylglycerol defines the response by the cells. The cells rapidly undergo nuclear envelope breakdown when this diglyceride is added in very late prophase, 0 to ∼8 min prior to the time of normal nuclear envelope breakdown. Anaphase onset occurs 28 min after nuclear envelope breakdown, rather than after the 33 min interval observed in untreated cells. Rapid progression through metaphase is also observed if cells are treated with 0.5 μg/ml 1,2‐dioctanoylglycerol during prometaphase, up to 15 min after nuclear envelope breakdown. The addition of 0.5 μg/ml 1,2‐dioctan oylglycerol in late metaphase, ∼26 min after nuclear envelope breakdown, results in sister chromatid separation slightly ahead of its normal time, 33 min after nuclear envelope breakdown, and in precocious cell plate vesicle aggregation, 3–5 min earlier than that observed in untreated cells. Treatment of cells with 60 μg/ml of 1,2‐dioctanoylglycerol at any point during the interval from 0 to ∼5 min prior to nuclear envelope breakdown results in precocious entry into anaphase. If cells are treated with either 0.5 μg/ml or 60 μg/ml 1,2‐dioctanoylglycerol earlier than 20 min before nuclear envelope breakdown, they do not enter mitosis, but instead revert to interphase without dividing. When 1,2‐dioctanoylglycerol is added atother times during mitosis, the rate of subsequent mitotic progression is dramatically slowed; the cells require >55 min to progress from nuclear envelope breakdown to anaphase onset, though once in anaphase, the cells progress onward to cytokinesis at normal rates. Treatments of cells with 1,3‐dioctanoylglycerol at any point during prophase, prometaphase, or metaphase are without effect on the rate of subsequent mitotic progression. The shifts in response by cells treated at specific times with 1,2‐dioctanoylglycerol during mid‐ and late metaphase may be indicative of the existence of one or more regulatory switch points (i.e., checkpoints) just prior to anaphase onset.
Increasing tubC β‐tubulin synthesis by placing it under the control of a benA β‐Tubulin upstream sequence causes a reduction in benA β‐tubulin level but has no effect on microtubule function
Authors: May Gregory S. , Waring Richard B. , Morris N. Ronald 
Published: February 2005

AbstractWe have constructed a chimeric β‐tubulin gene that places the structural gene for the tubC β‐tubulin of Axpergillus nidulans under the control of the benA β‐tubulin promoter. Introduction of cither this chimeric gene or a second wild‐type ben.A gene into a benomyl‐resistant benA22 strain causes it to become benomyl sensitive, indicating that the introduced genes are functional. Analysis of the tubulin proteins synthesized in benA22 strains into which a second wild‐type benA β‐tubulin gene was transformed showed that the total amount of β‐tubulin protein was the same as in the parental strain with a single benA gene. Thus the level of β‐tubulin must be regulated. This was also true of transformants carrying an extra copy of the chimeric β‐tubulin gene. The total amount of β‐tubulin was the same as in the parental strain. Two‐dimensional gel analysis showed that the endoge‐nous benA22 and the introduced chimeric tubC gene contributed equally to the total β‐tubulin pool. Th; fact that one‐half of the benA β‐tubulin could be replaced by tubC β‐tubulin with no effect on the growth of the cells suggests that the benA and tubC β‐tubulins are functionally interchangeable.
Heterogeneity of microtubules in dividing sea urchin eggs revealed by immunofluorescence microscopy: Spindle microtubules are composed of tubulin isotypes different from those of astral microtubules
Authors: Oka Mikako T. , Arai Takao , Hamaguchi Yukihisa 
Published: February 2005

AbstractThe heterogeneity of mitotic microtubules in dividing sea urchin eggs was investigated by indirect immunofluorescence using five anti‐α‐tubulin (YL1/2, DM1A, E3B8, D2D6, and 6‐11B‐1) and two anti‐β‐tubulin (E6B6 and DM1B) antibodies. These antibodies were divided into four classes in regard to the different immunofluorescent staining patterns: class I, which strongly stained both the spindle and aster (YL1/2, DM1A, E3B8 and E6B6); class II, which strongly stained the spindle but weakly stained the aster (D2D6); class III, which stained only the aster (DM1B); and class IV, which did not stain the mitotic apparatus (6‐11B‐1). These results suggest that tubulin isotypes are distributed differently in the sea urchin mitotic microtubules and that α‐tubulin isotype(s) recognized by D2D6 is (are) localized mainly in spindle microtubules, whereas β‐tubulin isotype(s) recognized by DM1B is (are) found only in astral microtubules.
Depolarization‐controlled parameters of the ciliary cycle and axonemal function
Authors: Sugino Kazuyuki , Machemer Hans 
Published: February 2005

AbstractDepolarization‐induced cycles of a frontal cirrus of Stylonychia were investigated by applying methods of axial‐view analysis of the cilia, high‐speed microcinématography, and step voltage‐clamp. Rising depolarization (from 3 mV to 7ge; 30mV) increased the rate of beating from zero to maximally 58 Hz. During cyclic activity, the axis of the beat cone of a proximal segment of the cirrus was inclined by 60° (0° = perpendicular to cell surface), and was always oriented 90° counterclockwise to the power stroke. With the stimulus amplitude rising, the orientations of the power stroke and inclination were increasingly shifted in more counterclockwise directions by up to 80° After correction for inclination ( = normalization), and following planification of the track of the segment, we determined the following properties of the cycle during depolarization: The course of the cycle tended to be rounded, i.e., the ratio of major over minor amplitudes (= spatial polarity) approximated a value of 1.6 which is only two thirds of maximal spatial polarity observed during hyperpolarization. The angular velocity generally increased with rising steps of depolarization; up to +5 mV (and comparable to hyperpolarization‐induced responses), the velocity maximum occurred during the return stroke. With depolarizations ≥7 mV the angular velocity maximum shifted to the power stroke so that the temporal polarity (rates of power stroke over rates of return stroke) increased from 0.4 to 1.6. Calculations of the angular velocity as referred to the proximal ciliary segment level suggest active sliding rates (between 5 and 30 nm/ms) of identified pairs of doublet microtubules. Ciliary frequency is a function of the rate of reorientation of the cyclic track; this parameter, which corresponds to the rate of translocation of active sliding between pairs of doublets, grew with the amplitude of depolarization. Translocation rates were high during transitions between the beat phases (power stroke, return stroke), and were reduced during these phases. Orientational polarograms of the mean rates of both active sliding and sliding translocation show properties of discreteness as well as continuity. The depolarization‐induced changes in inclination, and the inferred patterns of sliding rate and sliding translocation rate, are compared with previous results from hyperpolarization‐dependent activation of the same motor organelle.