bluuebunny/crossref_metadata_2025_split huggingface dataset:
The A/B domain of truncated retinoic acid receptors can block differentiation and promote features of malignancy
Authors: Aneskievich Brian J. , Fuchs Elaine 
Published: April 2021

ABSTRACT
Recently, we discovered that stable introduction of a carboxyl-terminally truncated retinoic acid receptor gamma (tRARγ) into an epidermal keratinocyte line blocked the ability of these cells to differentiate, as judged by their failure to express late markers of squamous differ-entiation. We now demonstrate a correlation between the level of residual endogenous RAR activity of tRARγ-expressing keratinocyte lines and degree of terminal differ-entiation. Mutagenesis studies localize the effects to the A/B subdomain of the truncated receptor. Despite tRARγ’s capacity to interfere with RAR-mediated transactivation of retinoic acid response elements (RAREs) in keratinocytes, the effects of the truncated receptor are independent of its ability to bind DNA and directly interact with endogenous RARs. tRARα also inhibits RARE-mediated gene expression in keratinocytes, even though its full-length counterpart enhances RARE activity in these cells. Intrigu-ingly, both tRARγ and RARγ suppress keratin promoter activity in epidermal cells, although for tRARγ, the effect is mediated through the A/B domain whereas for RARγ, the effects require DNA binding. Taken together, these findings suggest that the truncation allows for new and aberrant interactions with transcriptional proteins/co-factors that participate in governing RARE activity. This discovery may have relevance in tumorigenesis, where genetic lesions can result in mutant RARs or in loss of receptor expression.
Dermal fibroblasts convert to a myogenic lineage in mdx mouse muscle
Authors: Gibson Alexander J. , Karasinski Janusz , Relvas João , Moss Jill , Sherratt Tim G. , Strong Peter N. , Watt Diana J. 
Published: April 2021

ABSTRACT
Duchenne muscular dystrophy is a primary muscle disease that manifests itself in young boys as a result of a defect in a gene located on the X-chromosome. This gene codes for dystrophin, a normal muscle protein that is located beneath the sarcolemma of muscle fibres. Therapies to alleviate this disease have centred on implanting normal muscle precursor cells into dystrophic fibres to compensate for the lack of this gene and its product. To date, donor cells for implantation in such therapy have been of myogenic origin, derived from paternal biopsies. Success in human muscle, however, has been limited and may reflect immune rejection problems. To overcome this problem the patient’s own myogenic cells, with the dystrophin gene inserted, could be used, but this could lead to other problems, since these cells are those that are functionally compromised by the disease. Here, we report the presence of high numbers of dystrophin-positive fibres after implanting dermal fibroblasts from normal mice into the muscle of the mdx mouse -the genetic homologue of Duchenne muscular dystrophy. Dystrophin-positive fibres were also abundant in mdx muscle following the implantation of cloned dermal fibroblasts from the normal mouse. Our results suggest the in vivo conversion of these non-myogenic cells to the myogenic pathway resulting in the formation of dystrophin-positive muscle fibres in the deficient host. The use of dermal fibroblasts may provide an alternative approach to the previously attempted myoblast transfer therapy, which in human trials has yielded disappointing results.
The role of protein phosphorylation in the assembly of a replication competent nucleus: investigations in Xenopus egg extracts using the cyanobacterial toxin microcystin-LR
Authors: Murphy Jacqueline , Crompton Catherine M. , Hainey Sandra , Codd Geoffrey A. , Hutchison Christopher J. 
Published: April 2021

ABSTRACT
Cell-free extracts of Xenopus eggs support nuclear assembly and DNA replication in vitro. Extracts supple-mented with the protein phosphatase inhibitor micro-cystin-LR displayed various inhibitory effects at different concentrations of the toxin. In the presence of cycloheximide, additions of microcystin did not induce histone H1-kinase activity. Nevertheless, increasing concentrations of microcystin did sequentially prevent DNA replication, nuclear lamina assembly and nuclear envelope assembly. DNA replication was prevented when microcystin was added at 250 nM. Furthermore, this effect could be reversed after the addition of the catalytic sub-unit of protein phosphatase 2A to inhibited extracts. At a concentration of 250 nM microcystin, nuclear membrane assembly, nuclear lamina assembly and nuclear transport all occurred in egg extracts. In addition single-stranded M13 DNA replication was also permitted. However, it appeared that replicase assembly was not completed, since nuclei assembled in microcystin-treated extracts displayed an unusual distribution of proliferating cell nuclear antigen (PCNA). Although PCNA was located at sites that resembled pre-replication foci, this nuclear protein was readily solubilised when nuclei were isolated and extracted sequentially with Triton, nucleases and salts. Despite this, nuclei containing pre-assembled replication forks could synthesise DNA when transferred into microcystin-treated extracts.
Divergent modes of autophagy in the methylotrophic yeast Pichia pastoris
Authors: Tuttle Daniel L. , Dunn William A. 
Published: April 2021

ABSTRACT
The budding yeast Pichia pastoris responds to methanolic media by synthesizing high levels of cytosolic enzymes (e.g. formate dehydrogenase) and peroxisomal enzymes (e.g. alcohol oxidase), which are necessary to assimilate this carbon source. Major alterations in cellular metabolism are initiated upon a shift in carbon source to ethanol or glucose. These alterations require the synthesis of new proteins and the rapid degradation of those enzymes no longer needed for methanol utilization. In this study, we have measured cytosolic and peroxisomal enzyme activities and examined the fate of morphologically distinct peroxisomes to assess the degradative response of this yeast during nutrient adaptation. Utilizing biochemical, morphological and genetic approaches, we have shown that there exist in P. pastoris at least two pathways for the sequestration of peroxisomes into the vacuole for degradation. The ethanol-induced pathway is independent of protein synthesis and includes an intermediate stage in which individual peroxisomes are sequestered into autophagosomes by wrapping membranes, which then fuse with the vacuole. This process is analogous to macroau-tophagy. The glucose-induced pathway invokes the engulfment of clusters of peroxisomes by finger-like protrusions of the vacuole by a process analogous to microautophagy. Unlike ethanol adaptation, glucose stimulated the degradation of formate dehydrogenase as well. Peroxisomes remained outside the vacuoles of glucose-adapted cycloheximide-treated normal cells, suggesting that protein synthesis is required for peroxisome entry into the yeast vacuole. Two complementary mutants (gsa1 and gsa2) that are unable to degrade peroxisomes or formate dehydrogenase during glucose adaptation were isolated. The mutated gene products appear to function in one or more events upstream of degradation within the vacuole, since ethanol-induced peroxisome degradation proceeded normally in these mutants and peroxisomes were found outside the vacuoles of glucoseadapted gsa2 cells. Mutants lacking vacuolar proteinases A and B were unable to degrade alcohol oxidase or formate dehydrogenase during ethanol or glucose adaptation. Peroxisomes were found to accumulate within the vacuoles of these proteinase mutants during adaptation. Combined, the results suggest that there exist in Pichia pastoris two independent pathways for the sequestration of peroxisomes into the vacuole, the site of degradation.
A yeast protein that bidirectionally affects nucleocytoplasmic transport
Authors: Singleton David R. , Chen Shaoping , Hitomi Midori , Kumagai Chino , Tartakoff Alan M. 
Published: April 2021

ABSTRACT
We have identified a temperature-sensitive mutant of Sac-charomyces cerevisiae (npl3) that accumulates polyadeny-lated RNA in the nucleus at 37°C, as judged by in situ hybridization. The strong nuclear signal is not simply due to increased cytoplasmic turnover of mRNA, as reincuba-tion at 37°C with an RNA polymerase inhibitor shows no diminution in the in situ signal. Over several hours at 37°C, the average poly(A) tail length increases and a character-istic ultrastructural alteration of the nucleoplasm occurs. Cloning and sequencing indicate that the corresponding gene is NPL3/NOP3, which codes for a nucleolar/nuclear protein implicated in protein import into the nucleus (Bossie et al. (1992). Mol. Biol. Cell 3, 875-893) and in rRNA maturation (Russell and Tollervey (1992). J. Cell Biol. 119, 737-747). NPL3 includes bipartite RNA recognition motifs (RRM) and a Gly-Arg repeat domain, as in several nucleolar proteins. A point mutation adjacent to one of the RRM has been identified in the ts copy of the gene. Although this protein is not concentrated in nuclear pores, NPL3 is implicated in both import and export from the nucleus. Judging from the site of the npl3 mutation and since the block in RNA export can be detected prior to an obvious nuclear import defect in npl3, the defect in RNA export may be primary. Since other mutants that interrupt RNA export do not block protein import, the NPL3 protein itself appears to be implicated in protein import.
Lamin A precursor is localized to intranuclear foci
Authors: Sasseville A. Marie-Josée , Raymond Yves 
Published: April 2021

ABSTRACT
Lamin A is synthesized in the cytoplasm as a precursor bearing a carboxyl-terminal CaaX box or isoprenylation signal. This precursor is post-translationally processed through multiple steps: isoprenylation with a farnesyl residue on the cysteine of the CaaX box, proteolytic removal of the last three amino acids, carboxymethylation of the cysteine residue and, finally, proteolytic removal of 15 amino acids from the carboxyl terminus. This last step gives rise to mature lamin A from which the isoprenylated terminus has been removed. Isoprenylation is a prerequisite for all other steps of processing. The subcellular location of these processing steps for lamin A is still a matter of debate. We have produced an antibody specific to the 18 amino acid carboxyl terminus of the lamin A precursor that does not recognize mature lamin A. This antibody detects intranuclear foci by immunofluorescence. Larger amounts of lamin A precursor were accumulated by treating cells with mevinolin (MVN), an inhibitor of iso-prenoid synthesis. In MVN-treated cells, the lamin A precursor accumulated most strikingly in the peripheral nuclear lamina where it was assembled, while intranuclear foci were maintained. The addition of an excess of meval-onate (MVA), which restores isoprenylation activity, to MVN-treated cells led to a progressive disappearance of the lamin A precursor from the peripheral lamina. This process was completed after 4 hours of MVA treatment, after which the lamin A precursor was restricted to intranuclear foci. We conclude from these results that the non-isoprenylated lamin A precursor appears competent for assembly into the peripheral nuclear lamina, and that all the processing steps leading to mature lamin A can occur within the nuclear space.
Interleukin-8 induces motile behavior and loss of focal adhesions in primary fibroblasts
Authors: Dunlevy Jane R. , Couchman John R. 
Published: April 2021

ABSTRACT
Interleukin-8 (IL-8) is a proinflammatory cytokine that promotes neutrophil migration. Although fibroblasts are known to secrete IL-8, the actions of this cytokine on fibroblasts have not been previously reported. We have found that in subconfluent populations of cultured primary fibroblasts, IL-8 causes an increase in the percentage of cells lacking focal adhesions. Most of the IL-8-stimulated cells not only exhibit a lack of focal adhesions but also have a migratory phenotype that includes a protrusive leading edge and trailing tail. In addition, IL-8 was found to promote primary fibroblast chemotaxis in modified Boyden chambers as well as chemokinesis on serum-coated cover-slips. Human primary fibroblasts were also found to specif-ically bind to IL-8 with high affinity. We have previously shown that a lack of focal structures in primary fibroblasts can be used as an index of chemokinetic locomotion and have fully characterized this system using newborn rat heart conditioned medium. The main stimulus in heart con-ditioned medium that is responsible for the lack of focal adhesions in the majority of cells can be immunoprecipi-tated using a polyclonal antibody against recombinant human IL-8. Additionally, video microscopy assays using heart conditioned medium depleted with the IL-8 antibody show an increase in the percentage of stationary cells, a consequent decrease in the percentage of migrating cells, and a twofold increase in the mitotic rate.
Interleukin-1α and tumor necrosis factor-α, which are early inflammatory cytokines, have been previously shown to stimulate IL-8 production in macrophages, fibroblasts, endothelial and epithelial cells. Our findings indicate that these two cytokines also cause an increase in the percent-age of fibroblasts without focal adhesions. Additionally, this increase in cells lacking focal structures can be largely attributed to the production and subsequent autocrine action of a factor immunoprecipitated with an IL-8 antibody. Conversely, GRO-α, which has a high homology with IL-8, does not cause a similar increase in the percent-age of cells lacking focal adhesions, but was not antagonis-tic to the effects of IL-8.
Stabilization and bundling of subtilisin-treated microtubules induced by microtubule associated proteins
Authors: Saoudi Yasmina , Paintrand Isabelle , Multigner Luc , Job Didier 
Published: April 2021

ABSTRACT
The acidic carboxy-terminal regions of α- and β-tubulin subunits are currently thought to be centrally involved in microtubule stability and in microtubule association with a variety of proteins (MAPs) such as MAP2 and tau proteins.
Here, pure tubulin microtubules were exposed to subtilisin to produce polymers composed of cleaved tubulin subunits lacking carboxy termini. Polymer exposure to subtilisin was achieved in buffer conditions compatible with further tests of microtubule stability. Microtubules composed of normal α-tubulin and cleaved β-tubulin were indistinguishable from control microtubules with regard to resistance to dilution-induced disassembly, to cold temperature-induced disassembly and to Ca2+-induced disassem-bly. Microtubules composed of cleaved α- and β-tubulins showed normal sensitivity to dilution-induced disassembly and to low temperature-induced disassembly, but marked resistance to Ca2+-induced disassembly. Polymers composed of normal α-tubulin and cleaved β-tubulin or of cleaved α- and β-tubulins were stabilized in the presence of added MAP2, myelin basic protein and histone H1. Cleavage of tubulin carboxy termini greatly potentiated microtubule stabilization by tau proteins. We show that this potentiation of polymer stabilization can be ascribed to tau-induced microtubule bundling. In our working con-ditions, such bundling upon association with tau proteins occurred only in the case of microtubules composed of cleaved α- and β-tubulins and triggered apparent micro-tubule cross-stabilization among the bundled polymers.
These results, as well as immunofluorescence analysis, which directly showed interactions between subtilisin-treated microtubules and MAPs, suggest that the carboxy termini of α- and β-tubulins are not primarily involved in the binding of MAPs onto microtubules. However, inter-actions between tubulin carboxy termini and MAPs remain possible and might be involved in the regulation of MAP-induced microtubule bundling.
Novel tools for the study of development, migration and turnover of nematocytes (cnidarian stinging cells)
Authors: Weber Jakob 
Published: April 2021

ABSTRACT
The rhodamine derivatives tetramethyl-rhodamine-5/6-maleimide (TROMI) and tetramethyl-rhodamine-6-iso-thiocyanate (TRITC) were allowed to react with living Hydra vulgaris. The two fluorescent dyes stain the polyps to different degrees, apparently without impairing their viability and behaviour. Concerning nematocytes, TROMI preferentially couples to cytoskeletal elements only of mounted nematocytes whereas TRITC selectively reacts with structural components of cysts of late nematoblasts, which thereafter develop apparently normally into mature nematocytes. Hence TROMI-labelling indicates that nematocytes are mounted and ready for discharge; TRITC-labelling can be used as a tool to investigate the final mat-uration, migration and installation of nematocytes in Hydra.
Together with a new non-fixative method to dissociate Hydra polyps into single, identifiable cells, the two labelling methods allow direct quantitative dynamic studies of nematocyte turnover and open new possibilities of investigating the regulation and the mechanisms of nematocyte supply and migration.
The 190 kda centrosome-associated protein of Drosophila melanogaster contains four zinc finger motifs and binds to specific sites on polytene chromosomes
Authors: Whitfield W. G. F. , Chaplin M. A. , Oegema K. , Parry H. , Glover D. M. 
Published: April 2021

ABSTRACT
Microinjection of a bacterially expressed, TRITC labelled fragment of the centrosome-associated protein CP190 of Drosophila melanogaster, into syncytial Drosophila embryos, shows it to associate with the centrosomes during mitosis, and to relocate to chromatin during interphase. Indirect immunofluorescence staining of salivary gland chromosomes of third instar Drosophila larvae, with anti-bodies specific to CP190, indicate that the protein is asso-ciated with a large number of loci on these interphase polytene chromosomes. The 190 kDa CP190 protein is encoded by a 4.1 kb transcript with a single, long open reading frame specifying a polypeptide of 1,096 amino acids, with a molecular mass of 120 kDa, and an isoelectric point of 4.5. The central region of the predicted amino acid sequence of the CP190 protein contains four CysX2CysX12HisX4His zinc-finger motifs which are similar to those described for several well characterised DNA binding proteins. The data suggest that the function of CP190 involves cell cycle dependent associations with both the centrosome, and with specific chromosomal loci.
Phalloidin unzips nebulin from thin filaments in skeletal myofibrils
Authors: Ao Xiaolei , Lehrer Sherwin S. 
Published: April 2021

ABSTRACT
Fluorescent phallotoxins such as rhodamine-phalloidin take hours to bind uniformly to thin filaments of skeletal myofibrils, after fast initial binding to both ends of thin filaments. Observation of this process in skeletal and cardiac myofibrils and of the resulting re-distribution of nebulin using anti-nebulin antibody showed that: (1) rhodamine-phalloidin binds uniformly to actin in cardiac myofibrils within minutes, in contrast to skeletal myofib-rils; (2) overnight pre-incubation of skeletal myofibrils with phalloidin results in uniform initial binding of rhodamine-phalloidin and a changed nebulin localization; (3) pre-incu-bation of skeletal myofibrils with Ca2+-calmodulin results in uniform initial binding of rhodamine-phalloidin; (4) the binding of rhodamine-phalloidin to actin in skeletal myofibrils is unidirectional, i.e. the fluorescence of incor-porated rhodamine-phalloidin moves from the pointed ends where it is bound initially toward the barbed end at the Z-band; (5) the unidirectional binding of rhodamine-phalloidin results in redistribution of nebulin, i.e. the initial fluorescent bands associated with the epitopes of bound nebulin antibody change to a single band located close to Z-line. These results indicate that nebulin inhibits rhodamine-phalloidin binding to actin and suggests that the unidirectional rhodamine-phalloidin binding may be due to cooperative competitive binding, i.e. phalloidin ‘unzips’ nebulin starting from the pointed ends of the thin filaments.
The roles of Ca2+ and plasma membrane ion channels in hyphal tip growth of Neurospora crassa
Authors: Levina Natalia N. , Lew Roger R. , Hyde Geoffrey J. , Heath I. Brent 
Published: April 2021

ABSTRACT
Growing hyphae of the ascomycete fungus Neurospora crassa contained a tip-high gradient of cytoplasmic Ca2+, which was absent in non-growing hyphae and was insen-sitive to Gd3+in the medium. Patch clamp recordings in the cell-attached mode, from the plasma membrane of these hyphae, showed two types of channel activities; spon-taneous and stretch activated. The spontaneous channels were identified as inward K+channels based on inhibition by tetraethylammonium. The stretch activated channels had increased amplitudes in response to elevated Ca2+in the pipette solution, and thus are permeable to Ca2+and mediate inward Ca2+movement. Gd3+, which is an inhibitor of some stretch activated channels, incompletely inhibited stretch activated channel activity. Both tetraethylammonium and Gd3+only transiently reduced the rates of tip growth without changing tip morphology, thus indicating that the channels are not absolutely essential for tip growth. Furthermore, in contrast to the hyphae of another tip growing organism, Saprolegnia ferax, tip-high gradients of neither spontaneous nor stretch activated channels were found. Voltage clamping of the apical plasma membrane potential in the range from −300 to +150 mV did not affect the rates of hyphal elongation. Collectively, these data suggest that ion transport across the plasma membrane at the growing tip in Neurospora is not obligatory for the maintainance of tip growth, but that a gradient of Ca2+, possibly generated from internal stores in an unknown way, is required.
Yeast ypt51p and mammalian rab5: Counterparts with similar function in the early endocytic pathway
Authors: Singer-Krüger Birgit , Stenmark Harald , Zerial Marino 
Published: April 2021

ABSTRACT
Ypt51p, a small GTPase of Saccharomyces cerevisiae, has been previously identified as a structural homolog of mammalian Rab5. Although disruption analysis revealed that the protein is required for endocytic transport and for vacuolar protein sorting, the precise step controlled by Ypt51p was not determined. In this work we show that by heterologous expression in animal cells Ypt51p was targeted to Rab5-positive early endosomes and stimulated endocytosis. Furthermore, two Ypt51p mutants induced similar morphological alterations as the corresponding Rab5 mutants. Also in yeast cells Ypt51p was found to be required at an early step in endocytic membrane traffic, since α-factor accumulated in an early endocytic inter-mediate in the absence of Ypt51p. Cell fractionation analysis revealed cofractionation of Ypt51p with endocytic intermediates, while no association with the late Golgi com-partment could be detected. Indirect immunofluorescence microscopy allowed us to morphologically identify the Ypt51p-containing compartment. Similar to the mammalian system larger Ypt51p-positive structures were revealed upon expression of Ypt51p Q66L. These structures were also positive for α-factor receptor and for car-boxypeptidase Y, thus providing direct evidence for their endocytic nature and for the convergence of the vacuolar biosynthetic and endocytic pathways.