bluuebunny/crossref_metadata_2025_split huggingface dataset:
Light chains of sea urchin kinesin identified by immunoadsorption
Authors: Johnson Catharine S. , Buster Dan , Scholey Jonathan M. 
Published: February 2005

AbstractPrevious studies with monoclonal antibodies indicate that sea urchin kinesin contains two heavy chains arranged in parallel such that their N‐terminal ends fold into globular mechanochemical heads attached to a thin stalk ending in a bipartitetail [Scholey et al. 1989]. In the present, complementary study, we have used the monoclonal antikinesin. SUK4, to probe the quaternary structure of sea urchin (Strongylocentrotus purpuratus) kinesin. Kinesin prepared from sea urchin cyto‐sol sedimented at 9.6 S on sucrose density gradients and consisted of 130‐kd heavy chains plus an 84‐kd/78 kd doublet (1 mol heavy chain: 1 mol doublet determined by gel densitomctry). Low levels of 110‐kd and 90‐kd polypeptides were sometimes present as well. The 84‐kd/78 kd polypeptides are thought to be light chains because they were precipitated from the kinesin preparation at a stoichiometry of one mol doublet per 1 mol heavy chain using SUK4‐Sepharose immunoaffinity resins. The 110‐kd and 90‐kd peptides, by contrast, were removed using this immunoadsorption method. SUK4‐Sepharose immunoaffinity chromatography was also used to purify the 130‐kd heavy chain and 84‐kd/78‐kd doublet (1 mol heavy chain: 1 mol doublet) directly from sea urchin egg cytosolic extracts, and from a MAP (microtubule‐associated protein) fraction eluted by ATP from microtubules prepared in the presence of AMPPNP but not from microtubules prepared in ATP. The finding that sea urchin kinesin contains equi‐molar quantities of heavy und light chains, together with the aforementioned data on kinesin morphology, suggests that native sea urchin kinesin is a tetramer assembled from two light chains and two heavy chains.
Identification and characterization of a novel mammalian intermediate filament‐associated protein
Authors: Brown Kevin D. , Binder Lester I. 
Published: February 2005

AbstractA novel monoclonal antibody, designated M1.4, recognizes the high molecular weight microtubule‐associated protein MAPIA (ca. Mr 380 kD) in both bovine and rat brain. In HeLa cells, however, M1.4 binds to a 240 kD polypeptide on immunoblots and co‐localizes with both vimentin and cytokeratin filaments using double‐label immunofluorescence microscopy. Immunoelectron microscopy indicates that the 240 kD polypeptide localizes along bundled intermediate filaments in a periodic manner. Two‐dimensional electrophoretic analysis indicates that the 240 kD polypeptide has a basic pI of 7.7. When HeLa cell intermediate filaments are isolated using standard non‐ionic detergent/high‐salt conditions the 240 kD polypeptide does not sediment with the intermediate filaments, unlike the established intermediate filament‐associated protein plectin. Immunoblot analysis with M1.4 shows the 240 kD polypeptide is expressed in a number of mammalian cell lines. Additionally, double‐label immunofluorescence shows the 240 kD polypeptide to associate with vimentin filaments in African Green Monkey kidney (CV‐1) and JC neuroblastoma cells. Due to its unique biochemical and biological characteristics, the 240 kD polypeptide is clearly a novel intermediate filament‐associated protein for which we have proposed the designation gyronemin (Gr.gyros: around: nemin: filament).
Neurite elongation is blocked if microtubule polymerization is inhibited in PC12 cells
Authors: Keith Charles H. 
Published: February 2005

AbstractWe have injected process‐bearing PC12 cells with colchicine‐tubulin mixed with either fluorescein‐dextran or a rhodamine‐labelled tubulin analogue to determine the role of microtubule polymerization in neurite elongation. Colchicine‐tubulin is a specific, substoichiometric poison of microtubule assembly. We have shown that colchicine‐tubulin does not cause existing PC12 microtubules to disassemble, and yet can inhibit the assembly of rhodamine‐tubulin injected along with it. In population s'udies of neurite outgrowth in injected and uninjected cells, we find that colchicine‐tubulin substantially inhibits neurite extension from injected cells over a wide variety of concentrations. In acute time‐course studies of injected cells, we find that colchicine‐tubulin does not block neurite outgrowth until the injectate reaches the neurite tip. Thereafter, however, it blocks process elongation completely. Thus we can conclude that microtubule polymerization in the region of the growth cone is an important element in neurite elongation. While polymerization at the cell body may be important in supplying subunits to the distal neurite, it does not play a direct role in process extension.
Stoichiometry of estramustine phosphate binding to MAP2 measured by the disassembly of chick brain MAP2: Tubulin microtubules
Authors: Burns Roy G. 
Published: February 2005

AbstractThe concentration of estramustine phosphate required to inhibit the assembly or to induce the disassembly of chick brain MAP2:tubulin microtubules is markedly dependent upon the microtubule protein concentration. Analysis of this relationship shows that estramustine phosphate and tubulin compete for common MAP2 sites, that MAP2 can bind 5–6 moles‐mole−1 estramustine phosphate, and that the Kd of these sites is ≏ 20 μM estramustine phosphate. It is proposed that two molecules of estramustine phosphate interact with each of the three tubulin‐binding sites of MAP2 and inhibit the MAP2:tubulin interaction by neutralising two highly conserved basic residues.
Computerized analysis of flagellar motility by digitization and fitting of film images with straight segments of equal length
Authors: Brokaw Charles J. 
Published: February 2005

AbstractMethods are described for computerized analysis of digitized images obtained by scanning photomicrographs of swimming sperm flagella. After storing a series of image frames in computer memory, the entire series is analyzed automatically. For each sperm image, the sperm head is located to obtain a starting point for analysis of the flagellum. This location is obtained by minimizing image intensity along a model of the sperm head outline. The flagellum is modelled by a series of straight segments of equal length: 0.5 or 1 μm. The angles between these segments are adjusted to give minimum image intensity along the line of the model as well as minimizing smoothing functions. Extensions to analyze a series of images in each frame, and to measure the positions of beads attached to the flagellar microtubules, are also described.
Identification of surface components of mammalian respiratory tract cilia
Authors: Hastie Annette T. , Krantz Mori J. , Colizzo Frank P. 
Published: February 2005

AbstractCilia isolation methods were modified to retain respiratory tract ciliary membranes and to identify accessible surface components. Prior to isolation of cilia, halves of cow tracheae were treated with the extended spacer arm analog of N‐hydroxy succinimido‐biotin (NHS‐LC‐biotin) to label accessible membrane constituents. Mechanical disruption of the epithelium and substitution of CHAPS for Triton X‐100 provided a good yield of cilia with membranes and with minimal contamination. Subsequent extraction of these cilia with Triton X‐100 solubilized the membranes and released soluble matrix proteins. Proteins of membrane + matrix and axoneme fractions were analyzed after electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The major biotin‐labeled components in the membrane + matrix fraction were 105, 98, and 92 kd, were glycosylated, and remained with reconstituted, pelleted membrane vesicles along with the major non‐biotinylated protein at 51 kd. Other membrane+matrix proteins at 126 and 76 kd bound streptavidin even from noniabeled trachea, but remained soluble. Several biotin‐labeled proteins distinct from those in the membrane fraction remained with Triton X‐100‐extracted axonemes. Streptavidin‐colloidal‐gold (SAG) particles appeared to bind randomly along the length of cilia. The peripheral join between A and B microtubules was a predominant nonspecific location of SAG on axonemes. Axonemes with biotin label also bound significant numbers of SAG to outer dynein arms, confirming the streptavidin reaction with separated proteins on transfers. These results suggest close association of the membrane with the axoneme in respiratory tract cilia and a membrane composition somewhat different from protozoan cilia.
Alpha‐actinin and actin in the outer retina: A double immunoelectron microscopic study
Authors: Arikawa Kentaro , Williams David S. 
Published: February 2005

AbstractActin has many diverse functions in the outer retina. To help elucidate its organization in this area, we have investigated the extent of its association with the actin cross‐linking protein alpha‐actinin. Ultrathin sections of chicken retina were double‐immunolabelled with monospecific antibodies against actin and alpha‐actinin. The highest relative amount of alpha‐actinin to actin label was measured in the adherens junctions between the individual retinal pigmented epithelial (RPE) cells and between the photoreceptor and Mueller cells; in the photoreceptor myoid; and in the RPE basal microvilli. The lowest amount was in the Mueller cell microvilli, the RPE apical processes, and in the photoreceptor ellipsoid. It is likely that the areas containing the highest ratio of alpha‐actinin to actin labelling are where the actin filaments are most highly cross‐linked into bundles and linked to the plasma membrane by alpha‐actinin. Actin filaments terminate in these areas, and, except for the myoid region, they are involved in cell‐cell or cell‐substrate adherens junctions.
Cell division in Dictyostelium with special emphasis on actomyosin organization in cytokinesis
Authors: Fukui Yoshio , Inoué Shinya 
Published: February 2005

AbstractThis study focuses on the dynamic reorganization of actin and myosin (“conventional” myosin, or myosin‐II) during cytokinesis in D. discoideum. This is the first study identifying the birefringence of the spindle microtubules as well as three sets of microfilamentous structure in Dictyostelium. The change of organization in these fibrillar structures was followed in real‐time with video microscopy, using a Universal Polarizing Microscope equipped with polarized‐light (POL) and differential interference contrast (DIC) optics combined with digital image processing. High‐frequency mitotic cells were obtained by semi‐synchronous culture, and high‐resolution observations were made by utilizing the agar‐overlay method (Yumura et al.: Journal of Cell Biology 99:894–899, 1984). The molecular identity of the birefringent structures was determined by fluorescence microscopy. Through‐focus observations were performed with an axial resolution of 0.3 μm depth of field.The actomyosin fibrils show a dramatic reorganization throughout mitosis. The fibrils at the leading lamellipodia disappear, and there is a striking assembly of the cortical actomyosin in pro‐metaphase, which is accompanied by a decrease in cell volume. The cortical actomyosin gradually increases through anaphase. After late anaphase, very active polar lamellipodia, with an average life of less than 1 minute, are formed. We confirmed that the polar lamellipodia include actin, but not myosin‐II. At the cleavage furrow, the microfilaments form two distinctive structures: circular contractile ring at the equator, and a cortical filament array parallel to the polar axis. Myosin is localized in the contractile ring, but not associated with the axial array of F‐actin. Actomyosin in the contractile ring gradually transforms into cortical network at the posterior region of daughter cells. The constriction of the furrow is accompanied by a drastic efflux of water as evidenced by highly active contractile vacuole formation and turbulent motion of minute vesicles connected to the furrow. This study demonstrates the presence of a new microfilament structure, as well as the dynamic property of the contractile ring, and sheds new light on the contractile mechanisms underlying cytoki‐nesis.
Production and specificity of monoclonal antibodies against calmodulin from dictyostelium discoideum
Authors: Hulen Durvis , Baron Andre , Salisbury Jeffrey , Clarke Margaret 
Published: February 2005

AbstractMonoclonal antibodies were raised against calmodulin purified from Dictyostelium discoideum. To increase its antigenicity, the calmodulin was conjugated to keyhole limpet hemocyanin; mice were immunized with the conjugate. Hybridomas producing antibodies against calmodulin were identified by screening culture supernatants with calmodulin coupled to bovine serum albumin. The specificity of antibodies from hybridoma culture supernatants was tested by Western blot of Dictyostelium cell lysates. For this purpose, methods were developed that permitted sensitive detection of calmodulin bound to membranes. The key elements of the blotting protocol were use of PVDF membrane, transfer conducted in phosphate buffer, and glutaraldehyde fixation after transfer. These methods permitted detection of as little as 0.1 ng of calmodulin spotted directly onto the membrane, or 10 ng transferred from an SDS polyacrylamide gel. Ten calmodulin‐specific antibodies were identified; most of these reacted preferentially with the calcium‐containing form of Dictyostelium calmodulin. Several of the monoclonal antibodies cross‐reacted with calmodulin from bovine brain.
Calcium sensors in sea urchin sperm flagella
Authors: Brokaw Charles J. 
Published: February 2005

AbstractThe asymmetry of ATP‐reactivated flagellar bending waves of Triton‐demem‐brated sea urchin spermatozoa has been measured over a range of free Ca2+ ion concentrations from 10−9 to 10−4 M. Detailed examination of the gradual response of asymmetry to Ca2+ ion concentration over this wide range indicates the presence of two Ca2+ sensors. A high‐affinity sensor operates at Ca2+ concentrations near 10−7.5 M. A lower‐affinity sensor operates at Ca2+ concentrations above 10−6 M, in the typical range for calmodulin‐mediated responses. Incubation of demembranated sperm flagella at high Ca2+ concentrations to release calmodulin is required to enable these Ca2+ responses to be observed. This treatment also causes a decrease in the apparent affinity of the flagella for cal‐modulin, as determined by measuring the increase in asymmetry in response to addition of exogenous calmodulin at low Ca2+ concentration.
Chromosome fiber dynamics and congression oscillations in metaphase PtK2 Cells at 23°C
Authors: Wise Dwayne , Cassimeris Lynne , Rieder Conly L. , Wadsworth Patricia , Salmon Edward D. 
Published: February 2005

AbstractA bioriented chromosome is tethered to opposite spindle poles during congression by bundles of kinetochore microtubules (kMts). At room temperature, kinetochore fibers are a dominant component of mitotic spindles of PtK2 cells. PtK2 cells at room temperature were injected with purified tubulin covalently bound to DTAF and congression movements of individual chromosomes were recorded in time lapse. Congression movements of bioriented chromosomes between the poles occur over distances of 4.5 μm or greater. DTAF‐tubulin injection had no effect on either the velocity or extent of these movements. Other cells were lysed, fixed, and the location of DTAF‐tubulin incorporation was detected from digitally processed images of indirect immunofluorescence of an antibody to DTAF. Microtubules were labeled with an anti‐beta tubulin antibody. At 2‐5 minutes after injection, concentrated DTAF‐tubulin staining was seen in the kinetochore fibers proximal to the kinetochores; a low concentration of DTAF‐tubulin staining occurred at various sites through the remaining length of the fibers toward the pole. Kinetochore fibers in the same cell displayed different lengths (0.2 to 4 μm) of concentrated DTAF‐tubulin incorporation proximal to the kinetochore, as did sister kinetochore fibers. Ten minutes after injection, the lengths of DTAF‐containing chromosomal fibers were greater than expected if incorporation resulted solely from the lengthening of kinetochore microtubules due to congression movements of the chromosomes. Besides incorporation as a result of chromosome movement, two other mechanisms might explain the length of the DTAF‐containing segments: (1) a poleward flux of tubulin subunits (Mitchison, 1989) or (2) capture of DTAF‐containing nonkinetochore microtubules.
A monoclonal antibody recognizing a common antigen on Drosophila embryos and human fibroblasts
Authors: Callaini Giuliano , Riparbelli Maria Giovanna 
Published: February 2005

AbstractWe used a monoclonal antibody specific for vimentin from human fibroblasts to stain whole mounts of Drosophila embryos. In immunofluorescence observations this antibody cross‐reacts with an antigenic determinant localized throughout mitosis at the nuclear boundary. Double fluorescence observations with the Rb188 antibody that specifically recognizes a centrosomal protein of the Drosophila embryo [Whitfield et al., 1988] showed that the anti‐vimentin antibody cross‐reacts with an antigen localized in the centrosomal region.
Discrete subcellular localization of a cytoplasmic and a mitochondrial isozyme of creatine kinase in intestinal epithelial cells
Authors: Keller Thomas C. S. , Gordon Phillip V. 
Published: February 2005

AbstractTwo isozymes of creatine kinase have been purified differentially from mitochondrial and cytoplasmic subfractions of intestinal epithelial cells. These intestinal epithelial cell creatine kinases were indistinguishable from the cytoplasmic (B‐CK) and mitochondrial (Mi‐CK) creatine kinase isozymes of brain when compared by SDS‐PAGE, cellulose polyacetate electrophoresis, and peptide mapping. In intestinal epithelial cells, immunolocalization of the Mi‐CK isozyme indicates that it is associated with long, thin mitochondria, which are excluded from the brush border at the apical end of each cell. In contrast, immunolocalization of the B‐CK isozyme indicates that it is concentrated distinctly in the brush border terminal web domain. Although absent from the microvilli, B‐CK also is distributed diffusely throughout the cytoplasm. Terminal web localization of B‐CK was maintained in glycerol‐permeabilized cells and in isolated brush borders, indicating that B‐CK binds to the brush border structure. The abundance and localization of the mitochondrial and cytoplasmic creatine kinase isozymes suggest that they are part of a system that temporally and/or spatially buffers dynamic energy requirements of intestinal epithelial cells.
Unidirectional microtubule assembly in cell‐free extracts of Spisula solidissima oocytes is regulated by subtle changes in pH
Authors: Suprenant Kathy A. 
Published: February 2005

AbstractMost, if not all, microtubules in vivo grow unidirectionally from a nucleation site such as the centrosome. This organized growth of microtubules can generate and maintain the radially symmetrical array of interphase microtubules as well as the bipolar mitotic apparatus. To investigate the regulation of polarized microtubule growth, we have prepared a cell‐free extract from surf clam oocytes that exhibits unidirectional microtubule assembly. Immunofluorescence microscopy was used to visualize the net assembly of microtubules onto the fast (plus)‐ and slow (minus)‐ growing ends of isolated ciliary axonemes. All detectable microtubule growth in these cytoplasmic extracts occurred at the plus (+) ends and the extent of (+) end growth was regulated by subtle changes in pH. Microtubule assembly in these crude extracts was highly favored at pH 7.3, the pH of the post‐fertilization cytoplasm. In contrast, when tubulin was purified from these oocyte extracts, integral components were lost, and microtubule growth became predominantly bidirectional and was favored at acidic pH. These results indicate that cytoplasmic factors may inhibit bidirectional growth in vivo and that temporal or local changes in cytoplasmic pH may influence microtubule assembly during the cell cycle.
Synchronous triggering of trout sperm is followed by an invariable set sequence of movement parameters whatever the incubation medium
Authors: Cosson Marie‐Paule , Cosson Jacky , Billard Roland 
Published: February 2005

AbstractThe movement of live trout spermatozoa is very brief (25 sec at 20°C) and conditions have been developed to get synchronous initiation of sperm motility which allowed quantification of the major parameters of sperm movement during the motility phase.Recorded flagellar beat frequencies decreased steadily from values of 55 Hz at the beginning to 20 Hz at the end of the motility phase. Sperm forward velocities followed a similar pattern from 250 to 20 μm.sec−1 in the same conditions and the diameters of sperm trajectories were reduced from 370 to 40 μm. Thus none of the characteristics of sperm movement was constant during the motile phase which ended abruptly by a straightening of the flagella.The decrease in flagellar beat frequencies and sperm velocities are much greater than what could be extrapolated from the decrease of intracellular ATP (Christen R. et al: Eur. J. Biochem, 166:667–671, 1987) or from measurements of ATP‐dependence of reactivated sperm velocities (Okuno M. and Morisawa N.: In Biological Functions of Microtubules and Related Structures. New York: Academic Press, pp. 151–162, 1982). Therefore, the cessation of flagellar beating at 25 sec is not directly the result of the low concentration of intracellular ATP.The decrease in the diameters of sperm trajectories which occurred during the first part of the motility phase was correlated with [Ca]i measurements (Cosson M.P. et al, Cell Motil. Cytoskeleton, 14:424–434, 1989). The effect of Ca2+ at the axonemal level does not indicates that Ca2+ influx is previous to flagellar beating but rather suggests a classical Ca2+ regulation of the flagellar assymetry.The short duration of the motility phase and the characteristics of sperm movement were very similar in various conditions (high external K+, low pH media) where increased external Ca2+ or divalent ions were shown to overcome K+ and H+ inhibition of sperm motility, both conditions which have been shown to depolarize the plasma membrane potential (Gatti J.L. et al: J. Cell Physiol., 143:546–554, 1990).The present study of the parameters of sperm movement suggests that once motility is initiated, a defined set of axonemal events will take place whatever the external conditions.