bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of a novel microtubule-binding domain in microtubule-associated protein 1A (MAP1A)
Authors: Cravchik Anibal , Reddy David , Matus Andrew 
Published: April 2021

ABSTRACT
Several microtubule-associated proteins (MAPs) have been shown to bind to microtubules via short sequences with repeated amino acids motifs. A microtubule-binding domain has hitherto not been defined for the adult brain microtubule-associated protein 1A (MAP1A). We have searched for a microtubule-binding domain by expressing different protein regions of MAP1A in cultured cell lines using cDNA constructs. One construct included an area with homology to the microtubule-binding domain of MAP1B (Noble et al. (1989)J. Cell Biol. 109, 437-448), but this did not bind to microtubules in transfected cells. Further investigation of other areas of MAP1A revealed a protein domain, capable of autonomously binding to micro-tubules, which bears no homology to any previously described microtubule-binding sequence. This MAP1A domain is rich in charged amino acids, as are other mammalian microtubule-binding domains, but unlike them has no identifiable sequence repeats. Whereas all previously described mammalian microtubule-binding domains are basic, this novel microtubule-binding domain of MAP1A is acidic. The expression of this polypeptide in cultured cell lines led to a rearrangement of the microtubules in a pattern distinct from that produced by MAP2 or tau, and increased their resistance to treatment with the microtubule depolymerising agent nocodazole. When the MAP1A microtubule-binding domain was co-expressed in cultured cell lines together with MAP2c, the MAP1A microtubule-binding domain was able to bind to the MAP2c-induced microtubule bundles. These results suggest that different microtubule-binding sequences have a common ability to stabilise microtubules but differ in their influence on microtubule arrangement in the cell. This may be significant in neurons, where microtubule-associated proteins with different microtubule-binding sequences are expressed in different cell compartments and at different times during development.
A molecular analysis of the origin of the Crepis capillaris B chromosome
Authors: Jamilena M. , Rejón C. Ruiz , Rejón M. Ruiz 
Published: April 2021

ABSTRACT
The origin of the B chromosome of Crepis capillaris has been studied by using in situ hybridization with different DNA probes. Genomic in situ hybridization (GISH) with DNA from plants with and without Bs as probes indicates that the B chromosome has many DNA sequences in common with A chromosomes, showing no region rich in B-specific sequences. Six additional DNA probes were used to test the possible origin of this B from the standard NOR chromosome (chromosome 3). In the short arm of the NOR chromosome, we detected not only 18 S + 25 S rDNA, but also 5 S rDNA and a specific repetitive sequence from the NOR chromosome (pCcH32); in the heterochromatic bands of the long arm, we found two different repetitive sequences (pCcE9 and pCcD29). In the B chromosome, however, only the 18 S + 25 S rDNA and the telomeric sequences from Arabidopsis thaliana were observed. Our in situ hybridization data with telomeric repeats indicate that the two telomeres of the B are larger than those of the A chromosomes, confirming the isochromosomal nature of this B. Hybridizations of 18 S + 25 S rDNA and telomeric repeats to blots of DNA from plants with and without Bs reveal a high homology between A and B 18 S + 25 S rDNA genes, but some sequence dissimilarities between A and B telomeres. Taken as a whole, these data indicate that the entire B of C. capillaris, although possibly having originated from the standard genome, did not derive directly from the NOR chromosome.
Neutralising antibody to TGF-β1,2 reduces cutaneous scarring in adult rodents
Authors: Shah Mamta , Foreman David M. , Ferguson Mark W. J. 
Published: April 2021

ABSTRACT
Scarring is a major cause of many clinical problems. Scar tissue interferes with growth, impairs function and is aesthetically unpleasant. However, scarring does not appear to be a problem of embryonic life. Embryonic wounds heal with a lower inflammatory and angiogenic response and have a different growth factor profile compared to adult wounds. We have used neutralising antibody to transforming growth factor-β1,2(TGF-β1,2) to alter the growth factor profile of cutaneous wounds in adult rodents and studied the effect on scar tissue formation. This paper extends our preliminary report that neutralising antibody to TGF-β reduces cutaneous scarring in adult rodents. To be effective, the neutralising antibody to TGF-β needs to be administered at the time of wounding or soon thereafter. The antiscarring effects of this neutralising antibody to TGF-β were dose dependent. Exogenous addition of neutralising antibody to TGF-β to incisional wounds reduced the inflammatory and angiogenic responses and reduced the extracellular matrix deposition in the early stages of wound healing without reducing the tensile strength of the wounds. Importantly, the architecture of the neodermis of wounds treated with neutralising antibody to TGF-β resembled more closely that of normal dermis compared to the unmanipulated control wounds, which healed with an abnormal neodermal architecture resulting in obvious scarring. This study suggests a novel therapeutic approach to reducing scarring in post-natal life.
Rearrangements of intranuclear structures involved in RNA processing in response to adenovirus infection
Authors: Puvion-Dutilleul Francine , Bachellerie Jean-Pierre , Visa Neus , Puvion Edmond 
Published: April 2021

ABSTRACT
We have studied in HeLa cells at the electron microscope level the response to adenovirus infection of the RNA pro- cessing machinery. Components of the spliceosomes were localized by in situ hybridization with biotinylated U1 and U2 DNA probes and by immunolabeling with Y12 anti-Sm monoclonal antibody, whereas poly(A)+ RNAs were localized by specific binding of biotinylated poly(dT) probe. At early stages of nuclear transformation, the distribution of small nuclear RNPs was similar to that previously described in non-infected nuclei (Visa, N., Puvion-Dutilleul, F., Bachellerie, J. P. and Puvion, E., Eur. J. Cell Biol. 60, 308-321, 1993; Visa, N., Puvion-Dutilleul, F., Harper, F., Bachellerie, J. P. and Puvion, E., Exp. Cell Res. 208, 19-34, 1993). As the infection progresses, the large virus-induced inclusion body consists of a central storage site of func- tionally inactive viral genomes surrounded by a peripheral shell formed by clusters of interchromatin granules, compact rings and a fibrillogranular network in which are embedded the viral single-stranded DNA accumulation sites. Spliceosome components and poly(A)+ RNAs were then exclusively detected over the clusters of interchro- matin granules and the fibrillogranular network whereas the viral single-stranded DNA accumulation sites and compact rings remained unlabeled, thus appearing to not be directly involved in splicing. Our data, therefore, suggest that the fibrillogranular network, in addition to being the site of viral transcription, is also a major site of viral RNA splicing. Like the clusters of interchromatin granules, which had been already involved in spliceosome assembly, they could also have a role in the sorting of viral spliced polyadenylated mRNAs before export to the cytoplasm. The compact rings, which contain non- polyadenylated viral RNA, might accumulate the non-used portions of the viral transcripts resulting from differential poly(A)+ site selection.
Independent localization of dystrophin N- and C-terminal regions to the sarcolemma of mdx mouse myofibres in vivo
Authors: Dunckley Matthew G. , Wells Kim E. , Piper Tony A. , Wells Dominic J. , Dickson George 
Published: April 2021

ABSTRACT
Dystrophin has been proposed to associate with the skeletal muscle membrane by way of a glycoprotein complex that interacts with its C-terminal domains. Transfection of mdx mouse myotubes in culture or myofibres in vivo with recombinant genes encoding human dystrophin deletion mutants shows, however, that not only the C terminus of dystrophin but also its N-terminal actin-binding domain can locate independently to the muscle sarcolemma. This observation suggests that lack of sarcolemma-associated dystrophin in Duchenne muscular dystrophy (DMD) muscle may result from enhanced degradation of truncated mutation products rather than their inability per se to associate with the sarcolemma.
Talin, vinculin and DRP (utrophin) concentrations are increased at mdx myotendinous junctions following onset of necrosis
Authors: Law Douglas J. , Allen David L. , Tidball James G. 
Published: April 2021

ABSTRACT
Duchenne muscular dystrophy (DMD) and the myopathy seen in the mdx mouse both result from absence of the protein dystrophin. Structural similarities between dys- trophin and other cytoskeletal proteins, its enrichment at myotendinous junctions, and its indirect association with laminin mediated by a transmembrane glycoprotein complex suggest that one of dystrophin’s functions in normal muscle is to form one of the links between the actin cytoskeleton and the extracellular matrix. Unlike Duchenne muscular dystrophy patients, mdx mice suffer only transient muscle necrosis, and are able to regenerate damaged muscle tissue. The present study tests the hypoth- esis that mdx mice partially compensate for dystrophin’s absence by upregulating one or more dystrophin-indepen- dent mechanisms of cytoskeleton-membrane association. Quantitative analysis of immunoblots of adult mdx muscle samples showed an increase of approximately 200% for vinculin and talin, cytoskeletal proteins that mediate thin filament-membrane interactions at myotendinous junctions. Blots also showed an increase (143%) in the dys- trophin-related protein called utrophin, another myotendi- nous junction constituent, which may be able to substitute for dystrophin directly. Muscle samples from 2-week-old animals, a period immediately preceding the onset of muscle necrosis, showed no significant differences in protein concentration between mdx and controls. Quanti- tative analyses of confocal images of myotendinous junctions from mdx and control muscles show significantly higher concentrations of talin and vinculin at the myotendi- nous junctions of mdx muscle. These findings indicate that mdx mice may compensate in part for the absence of dys- trophin by increased expression of other molecules that subsume dystrophin’s mechanical function.
Concentration of pp125 focal adhesion kinase (FAK) at the myotendinous junction
Authors: Baker Lauren P. , Daggett David F. , Peng H. Benjamin 
Published: April 2021

ABSTRACT
Focal adhesion kinase is a recently characterized tyrosine kinase that is concentrated at focal contacts in cultured cells. It is thought to play an important role in the the reg- ulation of the integrin-based signal transduction mechanism involved in the assembly of this membrane spe- cialization. In this study, we examined the immunocyto- chemical distribution of focal adhesion kinase in Xenopus skeletal muscle and its role in the formation of two sar- colemmal specializations, the myotendinous junction and the neuromusuclar junction, using a monoclonal antibody (2A7) against this protein. Immunoprecipitation of Xenopus embryonic tissues with this antibody demon- strated a single band at a relative molecular mass of 116 kDa. A distinct concentration of immunolabeling for focal adhesion kinase was observed at the myotendinous junction of muscle fibers in vivo. At this site, the labeling for this protein is correlated with an accumulation of phos- photyrosine immunolabeling. Focal adhesion kinase was not concentrated at the neuromuscular junction in muscle cells either in vivo or in vitro. However, it was localized at spontaneously formed acetylcholine receptor clusters in cultured Xenopus myotomal muscle cells, although its dis- tribution was not exactly congruent with that of the receptors. In these cells, the accumulation focal adhesion kinase was induced by polystyrene microbeads. In addition, beads also induce the formation of acetylcholine receptor clusters and myotendinous junction-like special- izations. By following the appearance of the focal adhesion kinase relative to the formation of these sarcolemmal spe- cializations at bead-muscle contacts in cultured muscle cells, we conclude that the accumulation of this protein was in pace with the development of the myotendinous junction, but occurred well after the clustering of acetylcholine receptors. These results suggest that focal adhesion kinase may be involved in the development and/or maintenance of the myotendinous junction through an integrin-based signaling system. Although it can accumulate at acetyl- choline receptor clusters formed in culture, it does not appear to be involved in the development of the neuro- muscular junction.
A single p34cdc2 protein kinase (encoded by nimXcdc2) is required at G1 and G2 in Aspergillus nidulans
Authors: Osmani Aysha H. , Peij Noel van , Mischke Michelle , O’Connell Matthew J. , Osmani Stephen A. 
Published: April 2021

ABSTRACT
We have cloned and sequenced a homolog of cdc2 from Aspergillus nidulans that can complement the Schizosac-charomyces pombe cdc2-33 mutation. The gene was deleted and is required for continued nuclear DNA replication but not for mitochondrial DNA replication. Three different temperature-sensitive alleles were generated by reverse genetics. All of the mutations generate the nim phenotype of A. nidulans. The new gene was designated nimXcdc2 as it is not allelic to any of the other nim genes (nimA to nimW) of A. nidulans. Reciprocal shift experiments place an essential function for nimXcdc2 in G1 and G2. Antipeptide antibodies were generated that detect NIMXcdc2, and antisera were also generated to detect NIMEcyclinB. The two p34cdc2 protein species previously detected in A. nidulans, p34 and p37, both precipitate using NIMXcdc2 C-terminus-specific antibodies but only p34 co-precipitates with NIMEcyclinB. Dephosphorylation of denatured p34 converts it to the p37 form, showing p37 to be the non-phosphory-lated form of NIMXcdc2. The phosphorylation of p34 is therefore associated with its interaction with NIMEcyclinB.
Accumulation of delta 2-tubulin, a major tubulin variant that cannot be tyrosinated, in neuronal tissues and in stable microtubule assemblies
Authors: Paturle-Lafanechère Laurence , Manier Monique , Trigault Nathalie , Pirollet Fabienne , Mazarguil Honoré , Job Didier 
Published: April 2021

ABSTRACT
Tubulin is the major protein component of brain tissue. It normally undergoes a cycle of tyrosination-detyrosination on the carboxy terminus of its α-subunit and this results in subpopulations of tyrosinated tubulin and detyrosinated tubulin. Brain tubulin preparations also contain a third major tubulin subpopulation, composed of a non-tyrosinatable variant of tubulin that lacks a carboxy-terminal glutamyl-tyrosine group on its α-subunit (delta 2-tubulin). Here, the abundance of delta 2-tubulin in brain tissues, its distribution in developing rat cerebellum and in a variety of cell types have been examined and compared with that of total α-tubulin and of tyrosinated and detyrosinated tubulin. Delta 2-tubulin accounts for approximatively 35% of brain tubulin. In rat cerebellum, delta 2-tubulin appears early during neuronal differentiation and is detected only in neuronal cells. This apparent neuronal specificity of delta 2-tubulin is confirmed by examination of its distribution in cerebellar cells in primary cultures. In such cultures, neuronal cells are brightly stained with anti-delta 2-tubulin antibody while glial cells are not. Delta 2-tubulin is apparently present in neuronal growth cones. As delta 2-tubulin, detyrosinated tubulin is enriched in neuronal cells, but in contrast with delta 2-tubulin, detyrosinated tubulin is not detectable in Purkinje cells and is apparently excluded from neuronal growth cones.
In a variety of cell types such as cultured fibroblasts or primary culture of bovine adrenal cortical cells, delta 2-tubulin is confined to very stable structures such as centrosomes and primary cilia. Treatment of such cells with high doses of taxol leads to the appearence of delta 2-tubulin in microtubule bundles. Delta 2-tubulin also occurs in the paracrystalline bundles of protofilamentous tubulin formed after vinblastine treatment. Delta 2-tubulin is present in sea urchin sperm flagella and it appears in sea urchin embryo cilia during development.
Thus, delta 2-tubulin is apparently a marker of very long-lived microtubules. It might represent the final stage of α-tubulin maturation in long-lived polymers.
Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: identification of a vinculin and pp125Fak- binding region
Authors: Turner* Christopher E. , Miller John T. 
Published: April 2021

ABSTRACT
Paxillin is a cytoskeletal protein involved in actin- membrane attachment at sites of cell adhesion to the extra- cellular matrix. Extensive tyrosine phosphorylation of this protein occurs during integrin-mediated cell adhesion, embryonic development, fibroblast transformation and following stimulation of cells by mitogens that operate through the family of seven membrane-spanning G- protein-coupled receptors. Paxillin binds in vitro to the focal adhesion protein vinculin as well as to the SH3 domain of c-src and, when tyrosine phosphorylated, to the SH2 domain of v-crk. Here, we report the complementary DNA, and derived amino acid sequence, that codes for approximately 90% of the paxillin protein. We have iden- tified a region in the amino-terminal half of the protein that supports the binding of both vinculin and the focal adhesion tyrosine kinase, pp125Fak. Although there is no significant overall homology with other identified proteins, the carboxyl third of paxillin contains one LIM domain and three LIM-like sequences. The LIM motif is common to a number of transcription factors and to two other focal adhesion proteins, zyxin and cysteine-rich protein. In addition to several potential tyrosine phosphorylation sites there are five tyrosine-containing sequences that conform to SH2-binding motifs. The protein also contains a short proline-rich region indicative of a SH3-binding domain. Taken together, these data suggest that paxillin is a unique cytoskeletal protein capable of interaction with a variety of intracellular signaling, and structural, molecules important in growth control and the regulation of cytoskeletal organization. The homology with zyxin and cysteine-rich protein defines a novel family of LIM-con- taining molecules associated with sites of actin-membrane attachment.
Immunolocalization of tropomodulin, tropomyosin and actin in spread human erythrocyte skeletons
Authors: Ursitti Jeanine A. , Fowler Velia M. 
Published: April 2021

ABSTRACT
The human erythrocyte membrane skeleton consists of a network of short actin filaments cross-linked into a hexagonal network by long, flexible spectrin molecules. The lengths of the short actin filaments (33±5 nm) at the central junctions are proposed to be stabilized and limited by asso- ciation with tropomyosin and the tropomyosin-binding protein, tropomodulin. Here, we use immunogold labelling followed by negative staining to specifically localize tropo- modulin, tropomyosin and actin to the sites of the central junctions in spread membrane skeletons. In addition to negative staining, immunogold labelling for tropomodulin at the sites of the central junctions was also visualized by a quick-freeze, deep-etch, rotary-replication technique. These experiments confirm previous indirect evidence that the short filaments at the central junctions are indeed actin filaments and provide the first direct evidence that tropo- modulin and tropomyosin are associated with the erythro- cyte actin filaments in situ.
Probing the action of Clostridium difficile toxin B in Xenopus laevis oocytes
Authors: Just Ingo , Richter Hans-Peter , Prepens Ulrike , Eichel-Streiber Christoph von , Aktories Klaus 
Published: April 2021

ABSTRACT
Clostridium difficile toxin B and Clostridium botulinum C3 exoenzyme caused comparable morphological alteration of CHO cells, which was accompanied by disaggregation of the microfilamental cytoskeleton. The cytotoxic effect of toxin B was correlated with a decrease in C3-catalyzed ADP-ribosylation of the low-molecular-mass GTP-binding protein Rho, which is involved in the regulation of the actin cytoskeleton. We used Xenopus laevis oocytes as a model to study the toxin effect on Rho in more detail. Toxin B treatment of oocytes caused a decrease in subsequent ADP-ribosylation of cytoplasmic Rho by C3. This decrease was observed when toxin B was applied externally or after microinjection. Besides endogenous Rho, microinjected recombinant Rho-glutathione S-transferase fusion protein was affected. Impaired ADP-ribosylation of Rho was neither due to altered guanine nucleotide binding nor to complexation with the guanine nucleotide dissociation inhibitor, which is known to inactivate Rho and to prevent Rho modification by C3. Proteolytical degradation of Rho was excluded by immunoblot analysis. In intact oocytes toxin B caused neither ADP-ribosylation nor phosphorylation of Rho. The data indicate that C. difficile toxin B acts on Rho proteins in Xenopus oocytes to inhibit ADP-ribosylation by C3. It is suggested that toxin B mediates its cytotoxic effect via functional inactivation of Rho.
Evidence that the 16 kDa proteolipid (subunit c) of the vacuolar H+-ATPase and ductin from gap junctions are the same polypeptide in Drosophila and Manduca: molecular cloning of the Vha16k gene from Drosophila
Authors: Finbow M. E. , Goodwin S. F. , Meagher L. , Lane N. J. , Keen J. , Findlay J. B. C. , Kaiser K. 
Published: April 2021

ABSTRACT
The 16 kDa proteolipid (subunit c) of the eukaryotic vacuolar H+-ATPase (V-ATPase) is closely related to the ductin polypeptide that forms the connexon channel of gap junctions in the crustacean Nephrops norvegicus. Here we show that the major protein component of Manduca sexta gap junction preparations is a 16 kDa polypeptide whose N-terminal sequence is homologous to ductin and is identical to the deduced sequence of a previously cloned cDNA from Manduca (Dow et al., Gene, 122, 355-360, 1992). We also show that a Drosophila melanogaster cDNA, highly homologous to the Manduca cDNA, can rescue Saccharomyces cerevisiae, defective in V-ATPase function, in which the corresponding yeast gene, VMA3, has been inactivated. Evidence is presented for a single genetic locus (Vha16) in Drosophila, which in adults at least contains a single transcriptional unit. Taken together, the data suggest that in Drosophila and Manduca, the same polypeptide is both the proteolipid subunit c component of the V-ATPase and the ductin component of gap junctions. The intron/exon structure of the Drosophila Vha16 is identical to that of a human Vha16 gene, and is consistent with an ancient duplication of an 8 kDa domain. A pilot study for gene inactivation shows that transposable P-elements can be easily inserted into the Drosophila ductin Vha16 gene. Although without phenotypic consequences, these can serve as a starting point for generation of null alleles.