bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reversion analysis of dynein intermediate chain function
Authors: Mitchell David R. , Kang Yong 
Published: April 2021

ABSTRACT
The ODA6 locus of Chlamydomonas reinhardtii encodes a 70 kDa intermediate chain protein of the flagellar outer row dynein ATPase, and mutations at this locus prevent assembly of the entire outer row dynein arm complex. To initiate a structure-function analysis of the 70 kDa protein, we used transformation with chimeric mutant/wild-type genes to localize the defect in one assembly mutation, oda6-95. Sequence analysis revealed a frame-shift mutation in codon 53, which is followed by a stop codon after 13 amino acids in the new reading frame. By selecting intragenic pseudorevertants of this mutation we obtained 11 new oda6 alleles. Many of these pseudorevertants encode intermediate chain proteins that permit assembly of outer row arms but do not restore full wild-type motility. Revertant strains fall into two phenotypic classes, one with average beat frequencies of 54 Hz (similar to wild type) and one with average frequencies of 27 Hz (compared with 24 Hz for oda6-95) during normal forward swimming. Low beat frequency strains also display abnormalities during photophobic reversal (symmetric waveform). Amplification and sequence analysis of revertant alleles indicated that each reversion caused a second frame-shift, within a 115 nt interval, which restored the original reading frame, and that phenotypic severity was related to both direction (5’ or 3’ and distance between the original mutation and the reversion event. On the basis of immunoblot analysis of outer arm proteins, we conclude that revertant motility defects do not correlate with deficits in assembly of a specific dynein heavy chain or intermediate chain polypeptide, and electron microscopy confirms that revertants have normal outer arm structures. These results suggest that the 70 kDa intermediate chain plays a direct role in outer arm function distinct from its role in the assembly process.
Molecular cloning and cellular localization of a bip homologue in Trypanosoma brucei: Divergent er retention signals in a lower eukaryote
Authors: Bangs James D. , Uyetake Lyle , Brickman Marla Jo , Balber Andrew E. , Boothroyd John C. 
Published: April 2021

ABSTRACT
Using the polymerase chain reaction with degenerate primers, three new members of the hsp70 gene family of Trypanosoma brucei have been identified. A genomic clone of one of these, gA, has been fully sequenced and the corresponding gene product has been characterized using antibody to recombinant gA fusion protein. gA is the trypanosomal homologue of BiP, an endoplasmic reticulum resident hsp70 gene family member, based on four lines of evidence: (1) gA protein has 64% deduced amino acid identity with rat BiP; (2) the deduced amino acid sequence has a putative secretory signal peptide; (3) the gA gene product is a soluble luminal resident of a trypanosomal microsome fraction; (4) the gA polypeptide does not cofractionate with mitochondrial markers. Trypanosomes are the most primitive eukaryote yet in which BiP has been identified. The gA polypeptide has been used as a specific marker for the direct visualization of endoplasmic reticulum in trypanosomes by both indirect immunofluorescence and cryoimmuno electron microscopy. The endoplasmic reticulum is seen as a tubular network that extends throughout the cell excluding the flagellum. The C-terminal tetrapeptide of gA is MDDL, which, together with the C-terminal tetrapeptide (KQDL) of a trypanosome protein disulfide isomerase homologue (Hsu et al. (1989)Biochemistry 28, 6440-6446), indicates that endoplasmic reticulum retrieval signals in trypanosomes may be as divergent and heterogeneous as any seen in the other eukaryotes yet studied.
Evidence for two intracellular calcium pools in Dictyostelium: the campinduced calcium influx is directed into a nbd-cland 2,5-di-(tert-butyl)-1,4 hydroquinone-sensitive pool
Authors: Flaadt H. , Jaworski E. , Malchow D. 
Published: April 2021

ABSTRACT
Signal transduction in Dictyostelium for oriented movement and differentiation involves a fine tuning of the cytosolic Ca2+ concentration. We have previously shown that cAMP binding to the cell surface receptor elicits two cellular events: (i) to enhance Ca2+ entry across the plasma membrane; (ii) to increase Ca2+ uptake into Ca2+-sequestering organelles. Here we used permeabilised cells to show that cAMP-induced Ca2+ uptake in these cells was sensitive to the Ca2+ transport ATPase blocker 2,5-di-(tert-butyl)-1,4-hydroquinone (BHQ) and the vacuolar H+-ATPase inhibitor NBD-Cl. By contrast, bafilomycin A1 and vanadate, inhibitors of Ca2+ uptake into acidosomes in Dictyostelium, did not reduce the cAMP-induced Ca2+ uptake of permeabilised cells. GTPγS served as a tool to measure Ins(l,4,5) P3-(InsP3)-sensitive Ca2+ release. Following NBD-Cl or BHQ treatment Ca2+ release was reversibly inhibited. We conclude that the cAMP-controlled Ca2+ influx is directed into a NBD-Cl and BHQ-sensitive compartment, which comprises the InsP3-releasable pool. The acidosomal Ca2+ store seems to provide for additional Ca2+ if required.
Association of the brain anion exchanger, AE3, with the repeat domain of ankyrin
Authors: Morgans Catherine W. , Kopito Ron R. 
Published: April 2021

ABSTRACT
The 89 kDa NH2-terminal domain of erythrocyte ankyrin is composed almost entirely of 22 tandem repeats of a 33 amino acid sequence and constitutes the binding site for the cytoplasmic NH2-terminal domain of the erythrocyte anion exchanger, AE1. We have developed an assay to evaluate the in vivo interaction between a fragment of ankyrin corresponding to this domain (ANK90) and two non-erythroid anion exchangers, AE2 and AE3, that share considerable structural homology with AE1. Association was assessed by coimmunoprecipitation of ANK90-anion exchanger complexes from detergent extracts of cells cotransfected with plasmids encoding the ankyrin fragment and the anion exchanger or mutants thereof. ANK90 was coimmunoprecipitated with AE1 but not with an AE1 deletion mutant lacking the cytoplasmic NH2-terminal domain. Using this assay, we show that the brain anion exchanger AE3, but not the closely related homologue, AE2, is capable of binding to ankyrin.
Cell-to-cell transport via motile tubules in growing hyphae of a fungus
Authors: Shepherd V. A. , Orlovich D. A. , Ashford A. E. 
Published: April 2021

ABSTRACT
The system of pleiomorphic, motile tubules and vacuoles in growing hyphal tips of Pisolithus tinctorius has been shown to play a role in intracellular transport. Here we show that the same system also exchanges material between adjacent cells. This exchange is most obvious between terminal and penultimate cells following nuclear division in the tip cell and just before dissolution of the cell wall between the clamp connection and penultimate cell. At this stage the two new dolipore septa are complete. The process was studied in living hyphae using confocal and conventional fluorescence microscopy. Tubules could move in either direction across the septum and often extended and retracted several times and penetrated for some distance (e.g. 40 µm) into the receiving cell. Movements appeared co-ordinated and during the exchange tubules transiently interconnected vacuoles in adjacent cells and by peristaltic movements appeared to transfer material between them.
The fluorescent tubules occupied a specific plane in the vicinity of the septum and remained in this plane for the duration of their movement, suggesting that their orientation and direction of movement is controlled. In freeze-substituted hyphae, tubular cisternae of similar dimensions to fluorescent tubules passed through the parenthesome pores perpendicular to the septum and in some cases entered the mouth of the septal pore. This indicates that the septal pore is of an appropriate dimension to accommodate the tubules and that they can cross the septal pore to exchange material between vacuole systems of adjacent cells. This is the first direct demonstration of such intercellular transport via a sub-cellular compartment.
The highly divergent α- and β-tubulins from Dictyostelium discoideum are encoded by single genes
Authors: Triviños-Lagos Leda , Ohmachi Tetsuo , Albrightson Caryn , Burns Roy G. , Ennis Herbert L. , Chisholm Rex L. 
Published: April 2021

ABSTRACT
As a step in the characterization of the microtubule system of Dictyostelium discoideum, we have isolated and sequenced full-length cDNA clones that encode the Dic tyostelium a- and β-tubulins, as well as the Dictyostelium a -tubulin gene. Southern blot analysis suggests that Dic tyostelium is unusual in that its genome contains single α- and β-tubulin genes, rather than the multi-gene family common in most eukaryotic organisms. The complete α-tubulin cDNA contains 1558 nucleotides, with an open reading frame, that encode a protein of 457 amino acids. The complete β-tubulin cDNA contains 1572 nucleotides and encodes a protein of 456 amino acids. Analysis of the deduced protein sequences indicates that while there is a significant degree of sequence similarity between the Dictyostelium tubulins and other known tubulins, the Dictyostelium α-tubulin displays the greatest sequence divergence yet described. Single α- and β-tubulin transcripts are detected by northern blot analysis during all stages of Dictyostelium development. The highest levels of message accumulate late in germinating spores and vegetative amoebae. Despite changes in α - and β-tubulin mRNA levels, protein levels remain constant throughout development. We have expressed the carboxy-terminal two-thirds of the α- and β-tubulins as trpE fusions in Escherichia coli and used this protein to produce polyclonal antisera specific for the Dic tyostelium ơ- and β-tubulins. These antisera recognize one ex- and two β-tubulin spots on western blots of 2-D gels and, by indirect immunofluorescence, both recognize the interphase and mitotic microtubule arrays in vegetative amoebae.
The small gtp-binding protein, Rab6p, is associated with both golgi and post-golgi synaptophysin-containing membranes during synaptogenesis of hypothalamic neurons in culture
Authors: Tixier-Vidal Andrée , Barret Alain , Picart Renée , Mayau Véronique , Vogt Dorothée , Wiedenmann Bertram , Goud Bruno 
Published: April 2021

ABSTRACT
We have recently localized a small GTP-binding protein (Rab6p) thought to be involved in vesicular membrane transport, to the medial and trans-cisternae of the Golgi apparatus in NRK (normal rat kidney) cells. Here, we have localized and quantified Rab6p during the development in culture of embryonic neurons, up to synapse formation, and compared its subcellular distribution and level of expression to that of synaptophysin, a major integral membrane protein of small synaptic vesicles. Using immunocytochemistry (laser scanning confocal microscopy, immunoelectron microscopy), fractionation and immunoisolation methods, we show that during the early phase of synaptogenesis, Rab6p is associated with synaptophysin-containing membranes of a trans-Golgi subcompartment, post-Golgi vesicles and small synaptic vesicles or their precursors. Concomitantly, Rab6p undergoes translocation from cytosol to membranes and its level of expression increases. However, at late stages, the association of Rab6p to small synaptic vesicles sharply decreases and its level of expression plateaus. These findings suggest a role for Rab6p in the post-Golgi transport of synaptophysin, at an early step of the biogenesis of small synaptic vesicles.
Implanted myoblasts not only fuse with myofibers but also survive as muscle precursor cells
Authors: Yao Shou-Nan , Kurachi Kotoku 
Published: April 2021

ABSTRACT
Intramuscular implanted myoblasts can fuse with existing myofibers. Here we report that implanted primary myoblasts marked with retroviral transgenes can also persist as muscle precursor cells. These cells can be recovered as viable myoblasts from muscles of recipent mice even months after myoblast implantation, and they can fully resume expression of the transgenes in culture. Upon re-implantation into muscles, they again not only fuse with existing myofibers, but also survive as muscle precursor cells in the tissue. These reserve myogenic cells should be able to contribute to host myofibers in muscle regeneration when the recombinant myofibers are damaged, providing an additional mechanism to maintain a persistent expression of transgenes delivered by myoblast-mediated gene transfer.
The spatial distribution and relative abundance of gap-junctional connexin40 and connexin43 correlate to functional properties of components of the cardiac atrioventricular conduction system
Authors: Gourdie Robert G. , Severs Nicholas J. , Green Colin R. , Rothery Stephen , Germroth Patricia , Thompson Robert P. 
Published: April 2021

ABSTRACT
Electrical coupling between heart muscle cells is mediated by specialised regions of sarcolemmal interaction termed gap junctions. In previous work, we have demonstrated that connexin42, a recently identified gapjunctional protein, is present in the specialised conduction tissues of the avian heart. In the present study, the spatial distribution of the mammalian homologue of this protein, connexin40, was examined using immunofluorescence, confocal scanning laser microscopy and quantitative digital image analysis in order to determine whether a parallel distribution occurs in rat. Con-nexin40 was detected by immunofluorescence in all main components of the atrioventricular conduction system including the atrioventricular node, atrioventricular bundle, and Purkinje fibres. Quantitation revealed that levels of connexin40 immunofluorescence increased along the axis of atrioventricular conduction, rising over 10-fold between atrioventricular node and atrioventricular bundle and a further 10-fold between atrioventricular bundle and Purkinje fibres. Connexin40 and con-nexin43, the principal gap-junctional protein of the mammalian heart, were co-localised within atrioventricular nodal tissues and Purkinje fibres. By applying a novel photobleach/double-labelling protocol, it was demonstrated that connexin40 and connexin43 are colocalised in precisely the same Purkinje fibre myocytes. A model, integrating data on the spatial distribution and relative abundance of connexin40 and connexin43 in the heart, proposes how myocyte-type-specific patterns of connexin isform expression account for the electrical continuity of cardiac atrioventricular conduction.
Organization of the double-stranded rna-activated protein kinase dai and virus-associated va rnai in adenovirus-2-infected hela cells
Authors: Jiménez-García Luis F. , Green Simon R. , Mathews Michael B. , Spector David L. 
Published: April 2021

ABSTRACT
We have examined the cellular distribution of the double-stranded RNA-activated protein kinase DAI in adenovirus 2 (Ad2) -infected and uninfected HeLa cells. In uninfected cells DAI was found to be concentrated in the cytoplasm. In addition, DAI was localized in the nucleoli and diffusely distributed throughout the nucleoplasm. Cells treated with α-interferon displayed a similar pattern of distribution for DAI. When RNA polymerase I activity was inhibited by the drug actinomycin D, nucleoli segregated and DAI was found to colocalize with the dense fibrillar region of the nucleoli. During mitosis, the distribution of DAI paralleled that of rRNA. In adenovirus-infected cells the localization of DAI was similar to that in uninfected interphase cells. VA RNAI was detected in Ad2-infected cells by 10-14 hours postinfection as fine dots in the nucleoplasm. By 18-24 hours post-infection, VA RNAI appeared in bigger and more abundant dots in the nucleoplasm and the cytoplasm was intensively labeled. Transient expression of the VA RNAI gene in uninfected cells resulted in a similar localization of the RNA. Our results are consistent with a role for DAI and VA RNAI in protein synthesis and suggest that DAI may play an early role in ribosome biogenesis in the nucleolus in addition to its cytoplasmic role in translation.
Regional variation in distribution of egf receptor in developing and adult corneal epithelium
Authors: Zieske James D. , Wasson Michael 
Published: April 2021

ABSTRACT
Epidermal growth factor receptor has been localized to the proliferative cell layers in a variety of stratified squamous epithelia. In the current study, the rat cornea was used as an experimental model to determine if epidermal growth factor receptor is concentrated in epithelial stem cells. Epidermal growth factor receptor was localized using immunofluorescence microscopy in adult and neonatal (1-day to 4-week) rat corneas. Antibody binding to epidermal growth factor receptor was present in basal cells across the adult cornea but was more intense in the limbal zone. In rats 1 day to 1 week of age, the corneal epithelium consisted of one or two layer of cells that were intensely labeled by anti-epidermal growth factor receptor. Following epithelial stratification, which occurred just prior to eyelid opening (∼12 days), expression of epidermal growth factor receptor was greatly reduced in central corneal epithelium and gained an adult pattern by 3 weeks of age. Expression of epidermal growth factor receptor was also examined by incubating 1 mm slices of adult corneas with 125I-epidermal growth factor (4 nM) for 90 minutes, followed by washing and autoradiography. Basal cells in the limbal zone contained 4.5-fold more silver grains per cell than did basal cells in the central cornea. These data suggest that cells with high potential for proliferation, i.e. limbal basal cells and all basal cells in developing rats, express high epidermal growth factor receptor levels. High levels of receptor may allow these cells to be rapidly stimulated by growth factors to undergo cell division during development and following wounding in adult corneas. High epidermal growth factor receptor levels may also provide a mechanism whereby limbal basal cells are maintained in an undifferentiated stem cell state rather than entering the pathway of terminal differentiation.
Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions
Authors: Watt Fiona M. , Kubler M.-Dominique , Hotchin Neil A. , Nicholson Linda J. , Adams Josephine C. 
Published: April 2021

ABSTRACT
Suspension-induced terminal differentiation of human epidermal keratinocytes can be inhibited by fibronectin through binding to the α5β1 integrin. We have investigated the effect of fibronectin on expression of integrins and proteins of the actin cytoskeleton and have explored the nature of the differentiation stimulus by testing different combinations of anti-integrin monoclonal antibodies or extracellular matrix proteins in the suspension assay. Fibronectin prolonged cell surface expression of β1 integrins but did not overcome the inhibition of intracellular transport of integrins that occurs when keratinocytes are placed in suspension. Fibronectin did not prevent the suspension-induced decline in the level of mRNAs encoding the β1 integrin subunit, actin, filamin and α-actinin; furthermore, the inhibition of terminal differentiation did not depend on the state of assembly of microfilaments or microtubules. Terminal differentiation could be partially inhibited by an adhesion-blocking monoclonal antibody to the β1 integrin subunit or by a combination of adhesion blocking antibodies recognising the α subunits that associate with α1 (α2, α3 and α5). Although laminin and type IV collagen do not inhibit terminal differentiation individually, they were inhibitory when added to cells in combination with a low concentration of fibronectin. We conclude that the proportion of keratinocyte β1 integrins occupied by ligand can regulate the initiation of terminal differentiation independently of the state of assembly of the actin cytoskeleton.
Evidence that vault ribonucleoprotein particles localize to the nuclear pore complex
Authors: Chugani Diane C. , Rome Leonard H. , Kedersha Nancy L. 
Published: April 2021

ABSTRACT
Vaults are cytoplasmic ribonucleoprotein organelles that are highly conserved among diverse eukaryotic species. Their mass (12.9 MDa), diameter (26-35 nm) and shape (two halves, each with eightfold radial symmetry) have recently been determined and are similar to those ascribed to the central plug (or transporter) of the nuclear pore complex (NPC). The size and eightfold symmetry of the vault particle make it conducive to interacting physically in a complementary manner with NPCs. The present study demonstrates that vaults specifically associate with nuclei by both immunoblotting and immunofluorescence. Immunogold EM confirmed that vaults associate with the nuclear envelope in tissue sections and with NPCs of isolated nuclei.
In vivo expression and molecular characterization of the porcine slow myosin heavy chain
Authors: Chang Kin-Chow , Fernandes Kenneth , Goldspink Geoffrey 
Published: April 2021

ABSTRACT
We report on the molecular characterization of the porcine slow-myosin heavy chain (HC) β gene and the isolation of its 5’ end cDNA. In vivo expression study, by in situ hybridization and histochemistry, revealed a highly regular rosette pattern of fiber arrangement, with a slow fiber occupying the central core, in all the skeletal muscles examined. This feature can be advantageous in the distinction of primary and secondary fibers in myogenic lineage studies. In the neonatal heart, ß isoform expression is diffuse, with higher expression occurring in the ventricle than in the atrium. Transient transfection assays showed the porcine promoter functions in a muscle- and differentiation stage-specific manner. In the 5’ regulatory region are several putative positive and negative regulatory elements, including a positive and a negative element in close proximity to each other in intron 1.
Cell binding specificity of mouse r-cadherin and chromosomal mapping of the gene
Authors: Matsunami Hiroaki , Miyatani Seiji , Inoue Takayoshi , Copeland Neal G. , Gilbert Debra J. , Jenkins Nancy A. , Takeichi Masatoshi 
Published: April 2021

ABSTRACT
R-cadherin was originally identified as a chicken cad- herin expressed by the retina. Here, we describe the identification of a mouse homologue of R-cadherin. We isolated mouse cDNAs encoding a cadherin with 94% identity in amino acid sequence to the chicken R-cad- herin, and defined this molecule as mouse R-cadherin. L cells transfected with the mouse R-cadherin cDNA acquired a cadherin-mediated cell-cell adhesiveness as found for other cadherins. To examine the binding specificity of mouse R-cadherin, L cells expressing this cadherin (mRL) were mixed with L cells expressing chicken R-cadherin (cRL), mouse N-cadherin (mNL), mouse E-cadherin (mEL) and mouse P-cadherin (mPL). While mRL cells randomly intermixed with cRL cells, those cells aggregated separately from mEL or mPL cells. Mixing of mRL with mNL cells gave an intermediate result; that is, they formed both separate and chimeric aggregates, suggesting that R- and N-cadherin can interact with each other although each has a preference to bind to its own type. Similar properties were previously found for chicken R-cadherin. Thus, the cell binding specificity of R-cadherin is entirely conserved between the two species, suggesting a conserved role for this protein in morphogenesis. We also located the mouse R-cadherin gene to chromosome 2.