bluuebunny/crossref_metadata_2025_split huggingface dataset:
Actin in the developing stomatal complex of winter rye: A comparison of actin antibodies and Rh‐phalloidin labeling of control and CB‐treated tissues
Authors: Cho Soon‐Ok , Wick Susan M. 
Published: February 2005

AbstractActin localization during stomatal complex formation in rye leaf epidermis was compared by three different labeling procedures. When leaf segments are fixed with formaldehyde prior to staining microfilament (MF) patterns visualized with actin antibodies and those with rhodamine‐phalloidin (Rh‐ph) are basically identical in controls. Likewise, on tissues treated with cytochalasin B (CB), actin antibodies and Rh‐ph produce very similar labeling patterns. Compared to MF alignments in fixed samples, additional sets of MFs are observed at the very cortical regions of epidermal cells that are stained with Rh‐ph without aldehyde fixation. Cortical MFs are also present in a variety of mitotic cells; MFs of meristematic cells and guard mother cells are more concentrated near the walls facing spindle poles, whereas a fine meshwork of MFs is observed along the entire periclinal surface of subsidiary mother cells. Although exactly how MFs are involved in control of the division site in higher plant cells is still to be determined, the presence of MFs during mitosis and the abnormal division observed in some stomatal cells after treatment with CB suggest that MFs are necessary for normal orientation of division in these cells, and thus normal morphogenesis.
Characterization of a nucleotide‐sensitive high molecular weight microtubule‐associated protein in the ovary of a hemipteran insect
Authors: Anastas Angela , Hunt Cherryl , Stebbings Howard 
Published: February 2005

AbstractWe have identified a 260 kD polypeptide as being the major microtubule‐associated protein (MAP) in the ovaries of the hemipteran insect, Oncopeltus fasciatus. The 260 kD insect ovarian MAP resembles certain mammalian brain MAPs by remaining soluble after boiling and promoting the assembly of tubulin into microtubules. It differs from most MAPs by exhibiting nucleotide‐sensitivity, being removed from microtubules by both ATP and GTP. Antibodies specific for the 260 kD MAP allowed its immmunofluorescent localization to the massive micro‐tubule aggregates forming the translocation systems which in hemipterans link the developing oocytes with anteriorly positioned nutritive cells. Such antibodies, in conjunction with electrophoretic methods, also demonstrated the 260 kD MAP to be species‐ and, to an extent at least, tissue‐specific. The Oncopeltus 260 kD MAP was not present in the ovaries of either Notonecta or Corixa, hemipterans which have similar microtubule systems to Oncopeltus but MAPs of slightly different molecular weight. The 260 kD MAP from the ovaries of Oncopeltus was not present in neuronal ganglia of the same species. The significance of the species‐ and tissue‐specificity of the 260 kD MAP, as well as its nucleotide‐sensitivity, are speculated upon and discussed.
Site‐specific antibodies block kinase a phosphorylation of desmin in vitro and inhibit incorporation of myoblasts into myotubes
Authors: Tao Jing‐Xiang , Ip Wallace 
Published: February 2005

AbstractDesmin and vimentin are two type III intermediate filament (IF) proteins, which can be phosphorylated in vitro by cAMP‐dependent kinase (kinase A) and protein kinase C, and the in vitro phosphorylation of these proteins appears to favor the disassembled state. The sites of phosphorylation for desmin and vimentin have been mapped to their amino‐terminal headpiece domains; in chicken smooth muscle desmin the most kinase A‐reactive residues are ser‐29 and ser‐35. In this study we have examined the phosphorylation of desmin by the catalytic subunit of kinase A by using anti‐peptide antibodies directed against residues 26–36. The antibodies, which we call anti‐D26, recognize both native and denatured desmin and can discriminate between intact desmin and those derivatives that do not possess residues 26–36. Pre‐incubation of desmin with affinity purified anti‐D26 blocks total kinase A catalyzed incorporation of 32P into desmin by 75–80%. When antibody‐treated IFs are subjected to phosphorylation, no filament breakdown is observed after 3 hours. Thus anti‐D26 antibodies block phosphorylation of IF in vitro. We have also explored the role of desmin phosphorylation in skeletal muscle cell differentiation using these antibodies. Quail embryo cells, induced to differentiate along the myogenic pathway by infection with avian SKV retroviruses expressing the ski oncogene, were microinjected with affinity purified anti‐D26 at the mononucleated, myoblast stage. By 24 h post‐injection, the vast majority of uninjected cells had fused into multinucleated myotubes, but all microinjected cells were arrested in the process of incorporating into myotubes and remained mononucleated. This observation suggests that kinase A phosphorylation‐induced dynamic behavior of the desmin/vimentin IF cytoskeleton may be one of the many cytoskeletal restructuring events that must take place during myoblast fusion.
Subcellular compartmentalization of myosin isoforms in embryonic chick heart ventricle myocytes during cytokinesis
Authors: Conrad Abigail H. , Clark William A. , Conrad Gary W. 
Published: February 2005

AbstractEmbryonic chick heart ventricle myocytes retain the ability to alternate between proliferation and functional differentiation. A cytoplasmic isoform of myosin is present in cleavage furrows of various nonmuscle cells during cytokinesis, whereas one or more of the cardiac myosin isoforms are localized in sarcomeres of beating cardiomyocytes. Antibodies were employed to reveal the subcellular localizations of cytoplasmic and cardiac myosin isoforms in embryonic chick ventricle cardiomyocytes during cytokinesis. Monoclonal anticytoplasmic myosin antibodies were prepared against myosin purified from brains of 1‐day posthatched chickens and shown to react with chick brain myosin heavy chain by Western blots and/or ELISA tests. One monoclonal antibrain myosin antibody also cross‐reacted with chick cardiac myosin but not with skeletal or smooth muscle myosins. Two antichick cardiac myosin monoclonal antibodies and one antichick skeletal myosin polyclonal antibody that cross‐reacts with cardiac myosin were employed to identify cardiac sarcomeric myosin.Cells were isolated from day 8 embryonic chick heart ventricles, enriched for myocytes, grown in vitro for 3 days, and then examined by immunofluorescence techniques. Monoclonal antibodies against cytoplasmic myosin preferentially localized in the cleavage furrows of both cardiofibroblasts and cardiomyocytes in all stages of cytokinesis. In contrast, antibodies that recognize cardiac myosin were distributed throughout cardiomyocytes during early stages of cytokinesis, but became progressively excluded from the furrow area during middle and late stages of cytokinesis. These data suggest that in cells that contain both cytoplasmic and sarcomeric myosin isoforms, only cytoplasmic myosin isoforms are mobilized to form the contractile ring for cytokinesis.
Comparative kinetics of short and long sperm in sperm dimorphic Drosophila species
Authors: Bressac C. , Joly D. , Devaux J. , Serres C. , Feneux D. , Lachaise D. 
Published: February 2005

AbstractAll species of the Drosophila obscura group exhibit within‐ejaculate sperm length dimorphism. The present work is a contribution to the understanding of sperm competition through a comparative study of sperm kinetic parameters in four of these species. Videomicrographic observations at 200 frames per second of sperm from males and females, out of the storage organ, prior or after storage were made. Drosophila sperm display both major and minor waves. The former is analysed by measuring coiling diameter (μm) and the latter by recording both beat frequency (s−1) and wave propagation velocity (μm·s−1). Results show that the ‘behaviour’ of short and long spermatozoa noticeably differ: short sperm kinetics remains unaltered after storage while both major and minor waves of long spermatozoa are markedly modified. Thus, evidence is provided here of a sort of “differential activation” which is assumed to result in different survival abilities of short and long sperm within the storage organ of females.
Diversity of microtubular doublets in insect sperm tails: A computer‐aided image analysis
Authors: Afzelius B. A. , Bellon P. L. , Dallai R. , Lanzavecchia S. 
Published: February 2005

AbstractAxonemal doublets of some insect spermatozoa were fixed in a mixture of glutaraldehyde and tannic acid, post‐fixed in uranyl acetate, and examined by electron microscopy, in order better to characterize the protofilament pattern. Most species had outer and inner dynein arms; others had only the inner one or none. Electron micrographs show the individual protofilaments to be well resolved and to be separated by an electron dense material. A certain “noise” inherent in the electron‐microscopical technique was found and is believed to be due to irregularities in fixation, embedding, and section staining, and to beam damage. The noise level was reduced by using a computer program in which similar picture elements are averaged. The resulting averaged images of the axonemal doublets show a few widened “gaps” in the wall of protofilaments. These widened gaps coincide with the location of dynein arms, spokes, or intertubular material. There were, on the other hand, no widened gaps at the level of attachement of the accessory tubules. We tentatively conclude that at least some of the proteins that associate with microtubules are inserted deep inside the microtubular wall rather than having a superficial attachement. The internal structure of the A‐subtubule is rather constant in species where both sets of dynein arms are present, whereas that of the B‐tubule is more variable.
Control of ciliary orientation in ciliated sheets from Paramecium–differential distribution of sensitivity to cyclic nucleotides
Authors: Noguchi Munenori , Nakamura Yoichi , Okamoto Ken‐Ichi 
Published: February 2005

AbstractCiliated sheets of cell cortex were prepared from Triton‐glycerol‐extracted Paramecium to observe directly the change of ciliary orientation. The observation of the ciliary responses revealed the modes of ciliary control by Ca2+ and cyclic nucleotides. The cilia changed their pointing direction clockwise from 11–12 to 5 o'clock (with the anterior of the cell defined as 12 o'clock) in the horizontal plane of cell surface when Ca2+ concentration was decreased from 10−6 M to 10−7 M. Cyclic AMP competed with Ca2+ ion in determining the orientation of the cilia. On the other hand, cGMP tended to change the ciliary orientation toward 3 o'clock. Ciliary sensitivity to cyclic nucleotides depended on their location on the cell surface. The cilia on the left‐hand field of the cell were more sensitive to cyclic nucleotide than those on the right‐hand field. The differential distribution of ciliary sensitivity within a single cell seems to be functional in the sophisticated turning mechanism in the behavioral response of Paramecium.
Cloning, sequencing, and mapping of an α‐actinin gene from the nematode Caenorhabditis elegans
Authors: Barstead Robert J. , Kleiman Lawrence , Waterston Robert H. 
Published: February 2005

AbstractThe dense‐bodies in the body wall muscle of the nematode Caenorhabditis elegans function to anchor the actin thin filaments to the adjacent sarcolemma. One of the major components of the dense‐bodies is the actin‐binding protein α‐actinin. To facilitate a genetic analysis of α‐actinin, we have cloned a cDNA encoding the nematode protein, identified its position on the nematode physical map, and developed a unique PCR based approach to test the position of the cloned gene relative to known genetic deletions. The peptide sequence deduced from the cDNA shows that, apart from a few exceptional regions, the nematode protein shows strong similarity to other known α‐actinins. Its position on the genetic map shows that none of the known muscle affecting mutations identified in C. elegans are in this α‐actinin gene. This gene has been given the name atn‐1 (α‐actinin‐1).
Dictyostelium discoideum essential myosin light chain: Gene structure and characterization
Authors: Pollenz Richard S. , Chisholm Rex L. 
Published: February 2005

AbstractWe have used a Dictyostelium essential myosin light chain (EMLC) cDNA clone to isolate additional cDNA clones which supply a different 3′ sequence from that previously described. The revised cDNA sequence encodes a polypeptide of 150 amino acids. Amino acid residues 147–167 of the previously reported sequence are replaced by new residues 147 to 150. The new cDNA encodes a polypeptide with 66% amino acid sequence identity with the Physarum polycephalum EMLC, and approximately 30% identity with mammalian EMLC sequences. These new cDNA clones were used to isolate two genomic DNA fragments which contain the entire EMLC gene. The Dictyostelium EMLC gene contains a single intron located immediately 3′ of the translation initiation codon and encodes a product most similar to MLC3 isoform of vertebrates. Primer extension analysis places the transcription initiation site approximately 90 nucleotides upstream of the translation initiation site. A DNA fragment containing 350 bases of sequence upstream of the putative transcription initiation site is sufficient to drive expression of a reporter gene upon reintroduction into growing Dictyostelium cells. In addition, the CAT reporter mRNA produced by this construct showed a pattern of developmental regulation similar to that previously reported for the endogenous EMLC mRNA. Based on comparison with published EMLC sequences from a variety of sources, the Dictyostelium EMLC shows slightly higher similarity to vertebrate EMLCs from striated musele sources than nonmuscle sources. While Dictyostelium and human nonmuscle sequences display only 28% identity over their entire sequence, the region from residue 88 to 108 shows much higher identity (67%). The high evolutionary conservation of this region of the EMLC suggests it may play an important role in EMLC function, and as such, represents a good target for future mutagenesis studies.
The identification of mammalian centrosomal antigens using human autoimmune anticentrosome antisera
Authors: Balczon Ron , West Kelly 
Published: February 2005

AbstractHuman autoimmune sera were screened for the presence of anticentrosome autoantibodies. Two high titer sera were identified that reacted with HeLa, CHO, and PtK2 centrosomes by immunofluorescence, although the fluorescent patterns that were obtained using the two antisera were separate and distinct. Serum obtained from patient IJ contained antibodies that reacted with epitopes present only in mitotic centrosomes; staining of interphase centrosomes was never detected uing IJ antiserum. Immunoblot analysis demonstrated that antibodies present in IJ antiserum reacted with a 190 kD spindle pole antigen. Immunofluorescent staining of cultured mammalian cells demonstrated that antibodies present in serum obtained from patient SPJ reacted with both interphase and mitotic centrosomes. Characterization of SPJ antiserum by immunoblotting demonstrated that antibodies present in the SPJ serum recognized proteins of Mrs of 39, 185, and 220 kD, although the possibility that the 185 kD polypeptide was a proteolytic breakdown product of the 220 kD protein has not been eliminated. Neither antiserum was able to inhibit microtubule nucleation from centrosomes in a lysed cell system in which pure 6S tubulin was added to permeabilized cells following pretreatment of the cells with either SPJ or IJ antiserum. These antisera should be useful probes for studying the biochemistry of the mammalian centrosome.
Analysis of inducible contractile rings suggests a role for protein kinase C in embryonic cytokinesis and wound healing
Authors: Bement William M. , Capco David G. 
Published: February 2005

AbstractA semi‐in vitro system derived from Xenopus oocytes which allows induction of contractile ring (CR) formation and closure is described and exploited to elucidate regulatory and structural features of cytokinesis. The inducible CRs (ICRs) are composed of actin filaments and closure is actin filament‐dependent as is cytokinesis in vivo. ICR closure in this system is calcium‐dependent and pH‐sensitive, as is cytokinesis in permeabilized cells (Cande: Journal of Cell Biology 87:326, 1980). Closure of ICRs proceeds at a rate and with a kinetic pattern similar to embryonic cytokinesis. Collectively, these data demonstrate that this system is a faithful mimic of cytokinesis in vivo. ICR formation and closure is protein kinase C (PKC)‐dependent and neomycin‐sensitive, indicating that the PKC branch of the polyphosphoinositide pathway regulates formation of the actomyosin ring which is the effector of cytokinesis. Kinetic measurements show that the rate of ICR closure reaches a peak of 4–8 μm/sec. Since the maximum measured velocity of actin filament translocation by vertebrate, non‐muscle myosins is 0.04 μm/sec, the later observations support a model in which the CR is segmented, containing multiple sites where filaments overlap in a “sliding filament” fashion. Because the rate decreases after reaching a peak, the results also suggest that the number of overlap sites decrease with time.
α‐, β‐, and γ‐tubulins: Sequence comparisons and structural constraints
Authors: Burns Roy G. 
Published: February 2005

AbstractComparison of ≏ 160 α‐, β‐, and γ‐tubulins, and excluding the highly divergent C‐terminal peptide, indicates that the three subclasses have similar tertiary structures. Conserved sequences within or between the subclasses have been identified, together with the locations of known epitopes, chemical modifications, and mutations. Evidence is also reviewed concerning the identity of the GTP‐binding sites, about which residues are exposed in the assembled microtubule and at subunit:subunit interfaces. These characteristics constrain the possible tertiary structure of the tubulin subunit.
Centripetal flow and directed reassembly of the major sperm protein (MSP) cytoskeleton in the amoeboid sperm of the nematode, Ascaris suum
Authors: Roberts Thomas M , King Karen L. 
Published: February 2005

AbstractThe cytoskeleton of the amoeboid spermatozoa of Ascaris suum consists of major sperm protein (MSP) filaments arranged into long, branched fiber complexes that span the length of the pseudopod and treadmill rearward continuously due to assembly and disassembly at opposite ends of the complexes (Sepsenwol et al., Journal of Cell Biology 108:55–66, (1989)). Examination by video‐enhanced microscopy showed that this cytoskeletal flow is tightly coupled to sperm locomotion. The fiber complexes treadmilled reaward at the same rate (10–50 μm/ min) as the cell crawled forward. Only fiber complexes with their plasmalemmal ends within a limited sector along the leading edge of the pseudopod underwent continuous assembly. Thus, the location of this sector, which occupies about 50% of the pseudopod perimeter, determined the direction of sperm locomotion. Treatment of sperm with agents that lower intracellular pH, such as, weak acids and protonophores, caused the fiber complexes to disassemble completely in 4–5 sec. Removal of these compounds resulted in reassembly of the cytoskeleton in a pattern that mimicked treadmilling in intact sperm. The fiber complexes were reconstructed by assembly at their plasmalemmal ends so that within 30–60 sec the entire filament system reformed and the cell resumed locomotion. Both cytoskeletal reassembly and treadmilling required exogenous HCO3−. These results suggest that variation in intracellular pH may help regulate cytoskeletal treadmilling and thereby play a significant role in sperm locomotion.
Properties of the spectrin‐like structural element of smooth‐muscle α‐actinin
Authors: Kahana Edith , Gratzer W. B. 
Published: February 2005

AbstractThe fragment of smooth muscle α‐actinin, comprising the four spectrin‐like structural repeating units, has a high α‐helix content, similar to that of spectrin, and a hydrodynamic frictional coefficient, indicative of an elongated, probably bent or kinked rod‐like structure, as found for spectrin dimer and tetramer. The fragment exists in solution as an extremely stable dimer, which is dissociated only under denaturing conditions and is much more resistant to dissociation by urea than is the spectrin heterodimer. High‐resolution proton magnetic resonance spectra reveal that a part of the polypeptide chain gives rise to sharp resonances; this is also true of spectrin and it implies that the individual structural repeating units contain segmentally mobile elements, which may be required to generate the elastic properties of the spectrin family of proteins. Again like spectrin, the α‐actinin fragment contains multiple binding sites for long‐chain fatty acids, as revealed by quenching of tryptophan fluorescence by 2‐bromostearate (though not by 9(10)‐bromostearate). The results point to extensive structural and functional similarities between the repeating units of all the proteins of the spectrin family.
Conserved β‐tubulin binding domain for the microtubule‐associated motors underlying sperm motility and fast axonal transport
Authors: Goldsmith Matthew , Connolly Joe A. , Kumar Norm , Wu Jie , Yarbrough Lynwood R. , van der Kooy Derek 
Published: February 2005

AbstractAn antiserum against tubulin, NS20, has been previously shown to inhibit anterograde and retrograde axonal transport by 50% in vivo and in vitro. We report here that Protein A purified NS20 antibodies also attenuate sperm motility by 50% in demembranated sea urchin sperm. This inhibition is absorbed out by preincubating the NS20 antibodies with a biochemically purified porcine microtubule preparation, with recombinant Trypanosoma β‐ (but not α‐) tubulin and most specifically, with a 37 amino acid (a.a.) synthetic peptide corresponding to a domain near (but not including) the porcine β‐tubulin C terminus. Furthermore, addition of this β‐tubulin peptide alone is sufficient to attenuate motility by 50% in demembranated sperm, indicating that this critical 37a.a. NS20 antigen is a motor binding domain. Together, the results suggest that at least two phenotypically distinct forms of microtubule‐based motility, axonal transport and flagellar beating, are homologous at the fundamental level of the microtubule domains (the β‐tubulin peptide and we suggest a distinct but similarly located α‐tubulin domain) mediating the attachment of tubulin‐associated motors.