bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of ATP-dependent K+ channels in the electrical excitability of early embryonic stem cell-derived cardiomyocytes
Authors: Gryshchenko O. , Fischer I. R. , Dittrich M. , Viatchenko-Karpinski S. , Soest J. , Böhm-Pinger M. M. , Igelmund P. , Fleischmann B. K. , Hescheler J. 
Published: April 2021

ABSTRACT
Single, murine embryonic stem cell-derived early stage cardiomyocytes dissociated from embryoid bodies expressed two inward rectifier K+ channels, IK1 and the ATP dependent K+ current. IK1 exhibited low density in early stage cardiomyocytes, but increased significantly in late stage cells. In contrast, the ATP dependent K+ current was expressed at similar densities in early and late stage cardiomyocytes. This current was found to be involved in the determination of the membrane potential, since glibenclamide depolarized early cardiomyocytes and exerted a positive chronotropic effect. Some cardiomyocytes displayed a bursting behavior of action potentials, characterized by alternating periods with and without action potentials. During the phases without action potentials, the membrane potential was hyperpolarized, indicating the involvement of K+ channels in the generation of this bursting behavior. Extracellular recording techniques were applied to spontaneously contracting areas of whole embryoid bodies. In 20% of these bursting behavior similar to that seen in the single cells was observed. In regularly beating embryoid bodies, bursting could be induced by reduction of substrates from the extracellular medium as well as by superfusion with the positive chronotropic agents Bay K 8644 or isoproterenol. Perfusion with substrate-reduced medium induced bursting behavior after a short latency, isoproterenol and Bay K 8644 resulted in a positive chronotropic response followed by bursting behavior with longer latencies. The spontaneous bursting was blocked by glibenclamide. These experimental results suggest that intermittent activation of ATP dependent K+ channels underlies the bursting behavior observed in single cardiomyocytes and in the whole embryoid body. Conditions of metabolic stress lead to the rhythmic suppression of action potential generation. Our data indicate that ATP dependent K+ channels play a prominent role in the cellular excitability of early cardiomyocytes.
Leishmania species: models of intracellular parasitism
Authors: Alexander James , Satoskar Abhay R. , Russell David G. 
Published: April 2021

ABSTRACT
Leishmania species are obligate intracellular parasites of cells of the macrophage-dendritic cell lineage. Indeed, the ability to survive and multiply within macrophages is a feature of a surprising number of infectious agents of major importance to public health, including Mycobacterium tuberculosis, Mycobacterium leprae, Listeria monocytogenes, Salmonella typhimurium, Toxoplasma gondii and Trypanosoma cruzi. The relationship between such organisms and their host cells is particularly intriguing because, not only are macrophages capable of potent microbicidal activity, but in their antigen-presenting capacity they can orchestrate the developing immune response. Thus, to initiate a successful infection parasites must gain entry into macrophages, and also withstand or circumvent their killing and degradative functions. However, to sustain a chronic infection, parasites must also subvert macrophage-accessory-cell activities and ablate the development of protective immunity. The leishmanias produce a wide spectrum of disease in mice, and as such they have provided excellent models for studying problems associated with intracellular parasitism. In recent years, largely using these organisms, we have made enormous progress in elucidating the mechanisms by which successful intracellular infection occurs. Furthermore, characterization of immunological pathways that are responsible for resistance or susceptibility to Leishmania has given rise to the Th1/Th2 paradigm of cellular/humoral dominance of the immune response.
PBC68: a nuclear pore complex protein that associates reversibly with the mitotic spindle
Authors: Theodoropoulos Panayiotis A. , Polioudaki Hara , Koulentaki Maria , Kouroumalis Elias , Georgatos Spyros D. 
Published: April 2021

ABSTRACT
Using autoimmune antibodies from a patient with primary biliary cirrhosis we have identified a 68 kDa nuclear envelope protein, termed PBC68. This protein is co-precipitated with a 98 kDa and a 250 kDa polypeptide and is distinct from the nuclear lamins. Immunostaining of digitonin-permeabilized cells indicates that PBC68 is restricted to the inner (nucleoplasmic) face of the nuclear envelope, while indirect immunofluorescence and immunoelectron microscopy show that PBC68 is located on fibrillar structures emanating from the nuclear pore complex. The autoantigen is modified at early prophase and disassembles at prometaphase concurrently with the breakdown of the nuclear envelope. The disassembled material, instead of diffusing throughout the cytoplasm as other nucleoporins, is targeted to the mitotic spindle and remains stably bound to it until anaphase. At telophase PBC68 is released from the mitotic apparatus and reassembles late, after incorporation of LAP2B and B-type lamins, onto the reforming nuclear envelope. The partitioning of PBC68 in dividing cells supports the notion that subsets of nuclear envelope proteins are actively sorted during mitosis by transiently anchoring to spindle microtubules. Furthermore, the data suggest that specific constituents of pore complex are released in a stepwise fashion from their anchorage sites before becoming available for nuclear reassembly.
Characterization of tGLP-1, a Golgi and lysosome-associated, transmembrane glycoprotein of African trypanosomes
Authors: Lingnau Andreas , Zufferey Rachel , Lingnau Maren , Russell David G. 
Published: April 2021

ABSTRACT
Purification of endosomal/lysosomal vesicles of Trypanosoma brucei brucei bloodstream forms and generation of monoclonal antibodies led to the isolation of antibodies directed against an 85 kDa, Golgi and endocytic traffic-associated protein termed tGLP-1, TrypanosomaGolgi/lysosome protein-1. Preliminary immunoelectron microscopical analysis revealed that the protein is present in, but not restricted to, the limiting membrane of multivesicular lysosomes and is more abundant in bloodstream forms compared to the procyclic stage. The corresponding gene was cloned and is present as a single copy. Blast searches did not reveal any homologies to other proteins and genes published. The nucleotide sequence of the gene (1848 base pairs) predicted a type 1 membrane topology with an N-terminal signal sequence (20 aa), a luminal domain with 2 N-glycosylation sites (524 aa), a transmembrane domain (23 aa), and a long cytosolic tail domain (49 aa). Polyclonal antibodies raised against the cytosolic tail confirmed the localization of the gene product to multivesicular lysosomes but revealed that the majority of the protein was in the Golgi apparatus. Colabelling with an antibody against p67, a lysosomal glycoprotein of trypanosomes, revealed extensive overlap between the proteins with opposing relative abundance. Expression of the tGLP-1 open reading frame in Leishmania resulted in Golgi localization, and in Toxoplasma, in localization to both the Golgi and endoplasmic reticulum. These data indicate conservation in the functionality of the Golgi-targeting sequence of tGLP-1.
Volume of liquid below the epithelium of an F9 cell as a signal for differentiation into visceral endoderm
Authors: Miki Kiyoshi 
Published: April 2021

ABSTRACT
When retinoic acid-primed F9 cells are allowed to aggregate, they form embryoid bodies with an outer layer of α-fetoprotein-producing visceral endoderm cells and an internal cavity. I show that maturation of the visceral endoderm is dependent on the size of F9 aggregates. Size fractionation of aggregates of retinoic acid-primed F9 cells on Percoll density gradients revealed that only aggregates with diameters larger than 180 μm developed into embryoid bodies with an endoderm layer secreting α-fetoprotein. Size dependent α-fetoprotein-secretion was also observed when retinoic acid-primed F9 cells were cultured on porous microcarrier beads larger than 185 μm. Retinoic acid-primed F9 cells on flat microporous membranes did not differentiate and secrete α-fetoprotein unless exposed to a limited volume of medium at their basolateral surface. This suggested that maturation of the visceral endoderm is signaled by the volume of liquid phase below the epithelium. I postulate that the epithelial layer of an F9 aggregate encloses liquid and forms a barrier to diffusion of some critical factor(s). The concentration of such a factor may reach a threshold due to enlargement of the liquid phase during growth of the F9 aggregate and thereby signal maturation of the outer layer of cells into visceral endoderm.
A novel protein targeting domain directs proteins to the anterior cytoplasmic face of the flagellar pocket in African trypanosomes
Authors: Hill Kent L. , Hutchings Nathan R. , Russell David G. , Donelson John E. 
Published: April 2021

ABSTRACT
The flagellar pocket of African trypanosomes is a critical sorting station for protein and membrane trafficking, and is considered to be an Achilles’ heel of this deadly pathogen. Although several proteins, including receptors for host-derived growth factors, are targeted specifically to the flagellar pocket, the signals responsible for this restricted subcellular localization are entirely unknown. Using T lymphocyte triggering factor-green fluorescent protein (TLTF1-GFP) fusion proteins, we demonstrate that an internal 144 amino acid domain of TLTF from Trypanosoma brucei is sufficient for directing GFP to the cytoplasmic side of the anterior flagellar pocket. Immuno-gold electron microscopy reveals that the TLTF-GFP fusion protein is located in an electron dense structure that immediately abuts the anterior flagellar pocket membrane. The amino acid sequence of the TLTF targeting domain does not resemble previously characterized protein trafficking signals, and random mutagenesis reveals that flagellar pocket targeting is conferred by a structural motif, rather than a short, contiguous array of amino acids. The aberrant sorting of two mutant proteins into the flagellum, and the targeting of a related human protein to the plus end of the trypanosome’s cytoskeletal microtubules, lead us to suggest that flagellar pocket targeting involves interactions with the trypanosome cytoskeleton. The finding that TLTF-GFP is restricted to the anterior, cytoplasmic face of the flagellar pocket membrane, suggests that there is structural heterogeneity in the membrane of this organelle.
Functional domains of human growth hormone necessary for the adipogenic activity of hGH/hPL chimeric molecules
Authors: Juárez-Aguilar Enrique , Castro-Muñozledo Federico , Guerra-Rodríguez Norma E. , Reséndez-Pérez Diana , Martínez-Rodríguez Herminia G. , Barrera-Saldaña Hugo A. , Kuri-Harcuch Walid 
Published: April 2021

ABSTRACT
Genetic analysis through construction of chimeric genes and their transfection in mammalian cells could provide a better understanding of biological functions of native or modified proteins, and would allow the design of new gene constructs encoding peptides that mimic or block ligand interaction with target tissues. To identify the hGH domains responsible for induction of adipose differentiation we constructed hGH/hPL chimeric molecules using homologous DNA mutagenesis, since hGH, but not human placental lactogen (hPL), promotes adipose differentiation in mouse 3T3-F442A cells. We assayed their adipogenic activity in an autocrine/paracrine biological model consisting of transiently transfected 3T3-F442A cells with the chimeric constructs. Plasmid DNAs carrying these constructs were transfected into growing 3T3-F442A cells, and cultures were further maintained for 7 days to differentiate into adipocytes. Secretion of transfected hGH/hPL chimeric proteins into the medium was in the range of 5-25 ng/ml. Adipogenic activity was a property only of those chimeric proteins that contained hGH exon III together with either hGH exon II or hGH IV. Our results also suggest that hGH binding site-2 is composed of two structural subdomains: subsite 2A encoded by exon II of hGH and subsite-2B encoded by exon IV. We also suggest that full adipogenic activity requires the presence of binding site-1 and any of the subsites of binding site-2. This simple autocrine/paracrine biological model of gene transfection allows the analysis of specific biological activity of products encoded by modified genes.
Translational control of the Cdc25 cell cycle phosphatase: a molecular mechanism coupling mitosis to cell growth
Authors: Daga Rafael R. , Jimenez Juan 
Published: April 2021

ABSTRACT
The eukaryotic translation initiation factor 4A (eIF4A) is an RNA helicase required for translation initiation of eukaryotic mRNAs. By engineering fission yeast mutants with diminished eIF4A activity, we have found that translation of cdc25 mRNAs (a dosage-dependent activator of mitosis in all eukaryotic cells) is particularly sensitive to limitations of protein synthesis mediated by limited eIF4A activity. Genetic and biochemical analysis indicated that a rate-limited translation initiation of cdc25 mRNAs, exerted throughout its unusual 5′ untranslated leader, acts as a molecular sensor to ensure that a minimum cell mass (protein synthesis) is attained before mitosis occurs. The Cdc13 cyclin B is also among the limited pool of proteins whose translation is sensitive to reduced translation initiation activity. Interestingly, the 5′ leader sequences of cdc25 and cdc13 mRNAs have conserved features which are unusual in other yeast mRNAs, suggesting that common mechanisms operate in the expression of these two key mitotic activators at the translational level.
Requirement of nuclear factor κB for the constitutive expression of nitric oxide synthase-2 and cyclooxygenase-2 in rat trophoblasts
Authors: Callejas Nuria A. , Casado Marta , Boscá Lisardo , Martín-Sanz Paloma 
Published: April 2021

ABSTRACT
Recently isolated trophoblasts express nitric oxide synthase 2 (NOS-2) and cyclooxygenase 2 (COX-2), decreasing the levels of the corresponding mRNAs when the cells were maintained in culture. The sustained expression of COX-2 and NOS-2 in trophoblasts was dependent on the activation of nuclear factor κB (NF-κB) since proteasome inhibitors and antioxidants that abrogated NF-κB activity suppressed the induction of both genes. The time-dependent fall of the mRNA levels of NOS-2 and COX-2 paralleled the inhibition of NF-κB, determined by electrophoretic mobility shift assays, and the increase of the IκBα and IκBβ inhibitory proteins. Isolated trophoblasts synthesized reactive oxygen intermediates (ROI), a process impaired after culturing the cells, and that might be involved in the NF-κB activation process. Moreover, treatment of recently isolated cells with ROI scavengers suppressed the expression of COX-2 and NOS-2. Challenge of trophoblasts with interleukin-1β up-regulated the expression of both proteins, an effect that was potentiated by lipopolysaccharide. These results indicate that the physiological expression of NOS-2 and COX-2 in trophoblasts involves a sustained activation of NF-κB which inhibition abrogates the inducibility of both genes.
Assessing the role of the ASP56/CAP homologue of Dictyostelium discoideum and the requirements for subcellular localization
Authors: Noegel Angelika A. , Rivero Francisco , Albrecht Richard , Janssen Klaus-Peter , Köhler Jana , Parent Carole A. , Schleicher Michael 
Published: April 2021

ABSTRACT
The CAP (cyclase-associated protein) homologue of Dictyostelium discoideum is a phosphatidylinositol 4,5-bisphosphate (PIP2) regulated G-actin sequestering protein which is present in the cytosol and shows enrichment at plasma membrane regions. It is composed of two domains separated by a proline rich stretch. The sequestering activity has been localized to the C-terminal domain of the protein, whereas the presence of the N-terminal domain seems to be required for PIP2-regulation of the sequestering activity. Here we have constructed GFP-fusions of N- and C-domain and found that the N-terminal domain showed CAP-specific enrichment at the anterior and posterior ends of cells like endogenous CAP irrespective of the presence of the proline rich region. Mutant cells expressing strongly reduced levels of CAP were generated by homologous recombination. They had an altered cell morphology with very heterogeneous cell sizes and exhibited a cytokinesis defect. Growth on bacteria was normal both in suspension and on agar plates as was phagocytosis of yeast and bacteria. In suspension in axenic medium mutant cells grew more slowly and did not reach saturation densities observed for wild-type cells. This was paralleled by a reduction in fluid phase endocytosis. Development was delayed by several hours under all conditions assayed, furthermore, motile behaviour was affected.
Regulation of the RhoA pathway in human endothelial cell spreading on type IV collagen: role of calcium influx
Authors: Masiero Laura , Lapidos Karen A. , Ambudkar Indu , Kohn Elise C. 
Published: April 2021

ABSTRACT
We have shown that nonvoltage-operated Ca2+ entry regulates human umbilical vein endothelial cell adhesion, migration, and proliferation on type IV collagen. We now demonstrate a requirement for Ca2+ influx for activation of the RhoA pathway during endothelial cell spreading on type IV collagen. Reorganization of actin into stress fibers was complete when the cells where fully spread at 90 minutes. No actin organization into stress fibers was seen in endothelial cells plated on type I collagen, indicating a permissive effect of type IV collagen. CAI, a blocker of nonvoltage-operated Ca2+ channels, prevented development of stress fiber formation in endothelial cells on type IV collagen. This permissive effect was augmented by Ca2+ influx, as stimulated by 0.5 μM thapsigargin or 0.1 μM ionomycin, yielding faster development of actin stress fibers. Ca2+ influx and actin rearrangement in response to thapsigargin and ionomycin were abrogated by CAI. Activated, membrane-bound RhoA is a substrate for C3 exoenzyme which ADP-ribosylates and inactivates RhoA, preventing actin stress fiber formation. Pretreatment of endothelial cells with C3 exoenzyme prevented basal and thapsigargin-augmented stress fiber formation. While regulation of Ca2+ influx did not alter RhoA translocation, it reduced in vitro ADP-ribosylation of RhoA (P2&lt;0.05), suggesting Ca2+ influx is needed for RhoA activation during spreading on type IV collagen; no Ca2+ regulated change in RhoA was seen in HUVECs spreading on type I collagen matrix. Blockade of Ca2+ influx of HUVEC spread on type IV collagen also reduced tyrosine phosphorylation of p190Rho-GAP and blocked thapsigargin-enhanced binding of p190Rho-GAP to focal adhesion kinase. Thus, Ca2+ influx is necessary for RhoA activation and for linkage of the RhoA/stress fiber cascade to the focal adhesion/focal adhesion kinase pathway during human umbilical vein endothelial cell spreading on type IV collagen.
Overexpression of kin17 protein disrupts nuclear morphology and inhibits the growth of mammalian cells
Authors: Kannouche Patricia , Angulo Jaime F. 
Published: April 2021

ABSTRACT
UVC or ionizing radiation of mammalian cells elicits a complex genetic response that allows recovery and cell survival. Kin17 gene, which is highly conserved among mammals, is upregulated during this response. Kin17 gene encodes a 45 kDa protein which binds to DNA and presents a limited similarity with a functional domain of the bacterial RecA protein. Kin17 protein is accumulated in the nucleus of proliferating fibroblasts and forms intranuclear foci. Using expression vectors, we show that overexpression of kin17 protein inhibits cell-cycle progression into S phase. Our results indicate that growth inhibition correlates with disruption of the nuclear morphology which seems to modify the intranuclear network required during the early steps of DNA replication. We report that a mutant encoding a protein deleted from the central domain of kin17 protein enhanced these effects whereas the deletion of the C-terminal domain considerably reduced them. These mutants will be used to elucidate the molecular mechanism by which kin17 protein alters cell growth and DNA replication.
The mitotically phosphorylated form of the transcription termination factor TTF-1 is associated with the repressed rDNA transcription machinery
Authors: Sirri Valentina , Roussel Pascal , Hernandez-Verdun Danièle 
Published: April 2021

ABSTRACT
The transcription termination factor TTF-1 exerts two functions in ribosomal gene (rDNA) transcription: facilitating initiation and mediating termination of transcription. Using HeLa cells, we show that TTF-1 protein is colocalized with the active transcription machinery in the nucleolus and also with the inactive machinery present in certain mitotic nucleolar organizer regions (NORs) when rDNA transcription is repressed. We also show that TTF-1 is specifically phosphorylated during mitosis in a manner dependent on the cdc2-cyclin B kinase pathway and on an okadaic acid-sensitive phosphatase. Interestingly, the mitotically phosphorylated form of TTF-1 appearing at the G2/M transition phase was more easily solubilized than was the interphase form. This indicates that the chromatin-binding affinity of TTF-1 appears to be different in mitotic chromosomes compared to the interphase nucleolus. Correlated with this, the other DNA-binding factor, UBF, which interferes with chromatin conformation in the rDNA promoter, was more strongly bound to rDNA during mitosis than at interphase. The reorganization of the mitotic rDNA promoter might be induced by phosphorylation of certain components of the rDNA transcription machinery and participate in silencing of rDNA during mitosis.
The four populations of myoblasts involved in human limb muscle formation are present from the onset of primary myotube formation
Authors: Edom-Vovard F. , Mouly V. , Barbet J. P. , Butler-Browne G. S. 
Published: April 2021

ABSTRACT
To understand how and when myogenic precursor cells become committed to their particular developmental programs, we have analysed the different populations of myoblasts which grow out from explants of muscle tissue isolated from human limb buds from the beginning of primary fibre formation throughout subsequent development and post-natal growth.
Four phenotypically distinct types of myoblasts were identified on the basis of their expression of desmin, myogenin and myosin heavy chain isoforms (MyHC), and after 5 and 20 divisions, cells were cloned. All four types of myoblasts were present at the beginning of primary myogenesis. Each respective phenotype was stably heritable through cloning and subsequent proliferation. The type 1 clones correspond to a novel class of myoblasts never described during human development, that biochemically differentiates, but does not fuse. Type 2 clones are composed of small myotubes expressing only embryonic MyHC. Type 3 clones are composed of thin and long myotubes expressing both embryonic and fetal MyHCs. The type 4 clones are composed of myotubes that have a phenotype very similar to human satellite cells.
Contrasting with others species, no other population of myoblasts appear during fetal development and only the relative number of these four types changes.