bluuebunny/crossref_metadata_2025_split huggingface dataset:
An in vitro assay for neural crest cell migration through the somites
Authors: Guillory Georgia , Bronner-Fraser Marianne 
Published: April 2021

ABSTRACT
Neural crest cells in the trunk of the avian embryo come into contact with the somites and neural tube during the course of their migration. However, the relationship between the somites and the early migratory routes followed by these cells is not yet completely understood. Here, we use a tissue culture assay to examine if avian neural crest cells migrate through the somites. Cultures of quail somites were prepared from four adjacent regions along the neural axis in the trunk. Each region had four pairs of consecutive somites with region I being most anterior and region IV containing the last four segments. Within each region, the somites were separated from other tissues by enzymatic digestion and plated onto collagen-coated dishes. Immunocytochemical techniques were used to confirm that no neural crest cells, recognized by the HNK-1 antibody, were present on the surface of the somites at the time of explantation. After several days in culture, the explanted somites were screened to identify pigment cells. Because neural crest cells give rise to all of the melanocytes in the trunk, the presence of pigment cells indicated that neural crest precursors were contained within the initial explant. After 5–11 days in vitro, the percentage of somite cultures containing pigment cells in regions I through IV, respectively, was 36%, 51%, 31% and 1 %. These results suggest that neural crest cells migrate through the somitic mesenchyme and first enter the somites between 5 to 9 segments rostral to the most recently formed somite.
Binding of zona pellucida proteins to a boar sperm polypeptide ofMr53000 and identification of zona moieties involved
Authors: Brown C. R. , Jones R. 
Published: April 2021

ABSTRACTExperiments have been carried out to identify proteins on boar spermatozoa that bind to components of the zona pellucida. Polypeptides in sodium deoxycholate extracts of boar spermatozoa and in whole seminal plasma have been separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose sheet by electroblotting and probed with 125I-labelled heat-solubilized zona pellucida from pig oocytes or ovulated eggs. Zona proteins bound avidly and consistently to a polypeptide of Mr 53 000 on blots of capacitated and noncapacitated sperm and weakly to polypeptides of Mr 67 000, 38 000 and 18000. On blots of seminal plasma the 125I-labelled probes bound to two polypeptides of Mr 65 000 and 19-24 000. Identification of the zona proteins that were binding to the aforementioned proteins on blots showed that all the major zona pellucida glycoproteins were involved, including those acquired from oviduct secretions. Binding of 125I-ovulated zona pellucida to the polypeptide of Mr 53 000 also occurred in extracts of testicular and epididymal boar spermatozoa. The results are discussed in relation to sperm-egg recognition in the pig.
The spatial and temporal pattern of collagens I and II and keratan sulphate in the developing chick metatarsophalangeal joint
Authors: Craig Fiona M. , Bentley George , Archer Charles W. 
Published: April 2021

ABSTRACTBoth intrinsic and extrinsic factors are known to be involved in the morphogenesis of diarthrodial joints. The use of specific antibodies to collagens I and II and keratan-sulphate-containing proteoglycans (KSPG) has enabled the distributions of these macromolecules to be followed during the development of the third metatarsophalangeal joint in the chicken embryo.Our study shows that cartilage differentiation occurs as a continuous rod, which is then subsequently divided into separate elements. Further development also reveals that, unlike the matrix of the cartilaginous elements, there is a differential distribution of collagen (type II) and KSPG in the presumptive joint region. It is proposed that a decrease in KSPG in the presumptive joint region at stages 28/30 may be involved in the mechanism for the flattening of cells in formation of the interzone. Whereas, a decrease in collagen across the joint interzone region may provide an area of weakness, which might allow forces produced by the developing musculature to cause cavitation.
Rescue of developmental lens abnormalities in chimaeras of noncataractous and congenital cataractous mice
Authors: Muggleton-Harris A. L. , Hardy K. , Higbee N. 
Published: April 2021

AbstractIn the study of the lens of a congenital cataractous mouse mutant (CAT), it has been shown that a loss of growth regulation at the cellular level causes gross lens abnormalities. The phenotypic characteristics of the cataractous mouse lens are similar to those seen in human congenital cataract and thus serves as a model system for medical research.In this present investigation, we have demonstrated that the abnormalities of the congenital cataractous lens can be rescued by forming chimaeras between DBA/2 (a noncataractous strain of mouse) and the CAT mutant. This report describes the histological, cellular and biochemical analysis of the resultant chimaeric eyes, and discusses possible mechanisms by which these results were achieved.
A molecular analysis of mouse development from 8 to 10 dayspost coitumdetects changes only in embryonic globin expression
Authors: Wilkinson David G. , Bailes Juliet A. , Champion Janet E. , Mcmahon Andrew P. 
Published: April 2021

AbstractThe pattern of protein synthesis in 8-, 9- and 10-day post coitum (p.c.) mouse embryos was examined by 2-D gel electrophoresis of [35S]methionine-labelled proteins. Of the 600-800 polypeptides detected only one, a 14×103Mr (14 K) protein, was found to accumulate over this period. To isolate cDNA clones that potentially encode this protein, 32P-labelled cDNA was synthesized from 9 and 10 days p.c. embryo poly(A)+RNA, and used for the differential screening of an 8·5-day p.c. mouse embryo cDNA library cloned in λgt10. Six clones that hybridized strongly to the 10-day probe were purified and their inserts subcloned into plasmid vectors. Cross hybridization and restriction mapping of these inserts indicate that they fall into four distinct groups. Each of these hybridize with transcripts of approximately 600 nucleotides, which accumulate in the embryo from 9 to 10 days p.c. Expression was barely detectable in adult tissues and restricted to liver and spleen. Expression of one of these clones, 10.1, was examined by in situ hybridization of 35S-labelled RNA probes to 8·5-12·5 day p.c. embryo sections. Strong hybridization was observed in yolk sac blood islands, fetal liver and embryonic erythrocytes, suggesting that 10.1A encodes an erythrocyte-specific protein. DNA sequence analysis indicates that the four classes of cDNA were derived from transcripts of the α 1, ζ, βh1 and ϵ globin genes. Labelling of 10-day p.c. erythrocyte proteins with [35S]methionine, followed by 2-D gel electrophoresis, clearly demonstrates that the most abundant polypeptide migrates to the same position as the 14 K protein which accumulates from 8 to 10 days p.c. Thus the only abundant transcripts and corresponding proteins that change over a period of profound morphogenetic change correspond to globins of the newly established blood system.
The specificity of motor innervation of the chick wing does not depend upon the segmental origin of muscles
Authors: Keynes R. J. , Stirling R. V. , Stern C. D. , Summerbell D. 
Published: April 2021

AbstractIn vertebrate embryos, motor axons originating from a particular craniocaudal position in the neural tube innervate limb muscles derived from myoblasts of the same segmental level. We have investigated whether this relationship is important for the formation of specific nerve-muscle connections, by altering the segmental origin of muscles and examining their resulting innervation. First, by grafting quail wing somites to a new craniocaudal position opposite the chick wing, we established that the segmental origin of a muscle can be altered: presumptive muscle cells migrated according to their new, rather than their original, somitic level, colonizing a different subset of muscles. However, after reversal of a length of brachial somitic mesoderm along the craniocaudal axis, or exchange or shift of brachial somites, the craniocaudal position of wing muscle motoneurone pools within the spinal cord was undisturbed, despite the new segmental origin of the muscles themselves. While not excluding the possibility that muscles and their motor nerves are labelled segmentally, we conclude that specific motor axon guidance in the wing does not depend upon the existence of such labels.
Observations on scale production in prymnesium parvum
Authors: Manton Irene 
Published: April 2021

Abstract
Information is provided regarding certain structural and developmental aspects of the single Golgi body present in the brown flagellate, Prymnesium parvum. Probably the most important new observation is the demonstration of scale production within certain Golgi cisternae. Different developmental states from immature to mature can be found in successive cisternae on one side of the Golgi centre, the scales themselves being all similarly oriented with respect to the inner and outer faces of the cisternae in which they are formed. A deep pit found in a relatively constant position attached to the plasmalemma beside each flagellar base is interpreted as being possibly the site of scale liberation. These findings are discussed in a preliminary way.
Characterizing titin’s I-band Ig domain region as an entropic spring
Authors: Linke Wolfgang A. , Stockmeier Marc R. , Ivemeyer Marc , Hosser Hiltraud , Mundel Peter 
Published: April 2021

ABSTRACT
The poly-immunoglobulin domain region of titin, located within the elastic section of this giant muscle protein, determines the extensibility of relaxed myofibrils mainly at shorter physiological lengths. To elucidate this region’s contribution to titin elasticity, we measured the elastic properties of the N-terminal I-band Ig region by using immunofluorescence/immunoelectron microscopy and myofibril mechanics and tried to simulate the results with a model of entropic polymer elasticity. Rat psoas myofibrils were stained with titin-specific antibodies flanking the Ig region at the N terminus and C terminus, respectively, to record the extension behaviour of that titin segment. The segment’s end-to-end length increased mainly at small stretch, reaching ∼90% of the native contour length of the Ig region at a sarcomere length of 2.8 µm. At this extension, the average force per single titin molecule, deduced from the steady-state passive length-tension relation of myofibrils, was ∼ 5 or 2.5 pN, depending on whether we assumed a number of 3 or 6 titins per half thick filament. When the force-extension curve constructed for the Ig region was simulated by the wormlike chain model, best fits were obtained for a persistence length, a measure of the chain’s bending rigidity, of 21 or 42 nm (for 3 or 6 titins/half thick filament), which correctly reproduced the curve for sarcomere lengths up to 3.4 µm. Systematic deviations between data and fits above that length indicated that forces of &gt;30 pN per titin strand may induce unfolding of Ig modules. We conclude that stretches of at least 5-6 Ig domains, perhaps coinciding with known super repeat patterns of these titin modules in the I-band, may represent the unitary lengths of the wormlike chain. The poly-Ig regions might thus act as compliant entropic springs that determine the minute levels of passive tension at low extensions of a muscle fiber.
Transient ER retention as stress response: Conformational repair of heat damaged proteins to secretion-competent structures
Authors: Saris Nina , Makarow Marja 
Published: April 2021

ABSTRACT
Mechanisms to acquire tolerance against heat, an important environmental stress condition, have evolved in all organisms, but are largely unknown. When Saccharomyces cerevisiae cells are pre-conditioned at 37°C, they survive an otherwise lethal exposure to 48-50°C, and form colonies at 24°C. We show here that incubation of yeast cells at 48-50°C, after pre-conditioning at 37°C, resulted in inactivation of exocytosis, and in conformational damage and loss of transport competence of proteins residing in the endoplasmic reticulum (ER). Soon after return of the cells to 24°C, membrane traffic was resumed, but cell wall invertase, vacuolar carboxypeptidase Y and a secretory β-lactamase fusion protein remained in the ER for different times. Thereafter their transport competence was resumed very slowly with widely varying kinetics. While the proteins were undergoing conformational repair in the ER, their native counterparts, synthesized after shift of the cells to 24°C, folded normally, by-passed the heat- affected copies and exited rapidly the ER. The Hsp70 homolog Lhs1p was required for acquisition of secretion competence of heat-damaged proteins. ER retention and refolding of heat-denatured glycoproteins appear to be part of the cellular stress response.
GTPγS-induced actin polymerisation in vitro: ATP- and phosphoinositide independent signalling via Rho-family proteins and a plasma membrane- associated guanine nucleotide exchange factor
Authors: Katanaev Vladimir L. , Wymann Matthias P. 
Published: April 2021

ABSTRACT
In a cell-free system from neutrophil cytosol GTPγS can induce an increase in the number of free filament barbed ends and massive actin polymerisation and cross-linking. GTPγS stimulation was susceptible to an excess of GDP, but not Bordetellapertussis toxin and could not be mimicked by aluminium fluoride, myristoylated GTPγS·xGiα2 or Gβ1γ2 subunits of trimeric G proteins. In contrast, RhoGDI and Clostridium difficile toxin B (inactivating Rho family proteins) completely abrogated the effect of GTPγS. When recombinant, constitutively activated and GTPγS-loaded Rac1, RhoA, or Cdc42 proteins alone or in combination were probed at concentrations &gt;100 times the endogenous, however, they were ineffective. Purified Cdc42/Racinteractive binding (CRIB) domain of WASP or C3 transferase did not prevent actin polymerisation by GTPγS. The action of GTPγS was blocked by mM [Mg2+], unless a heat- and trypsin-sensitive component present in neutrophil plasma membrane was added. Liberation of barbed ends seems therefore to be mediated by a toxin B- sensitive cytosolic Rho-family protein, requiring a membrane-associated guanine nucleotide exchange factor (GEF) for its activation by GTPγS under physiologic conditions.
The inefficiency of various protein kinase and phosphatase inhibitors (staurosporine, genistein, wortmannin, okadaic acid and vanadate) and removal of ATP by apyrase, suggests that phosphate transfer reactions are not required for the downstream propagation of the GTPγS signal. Moreover, exogenously added phosphoinositides failed to induce actin polymerisation and a PtdIns(4,5)P2-binding peptide did not interfere with the response to GTPγS. The speed and simplicity of the presented assay applicable to protein purification techniques will facilitate the further elucidation of the molecular partners involved in actin polymerisation.
High Proliferative Potential-Quiescent cells: a working model to study primitive quiescent hematopoietic cells
Authors: Fortunel Nicolas , Batard Pascal , Hatzfeld Antoinette , Monier Marie-Noëlle , Panterne Béatrice , Lebkowski Jane , Hatzfeld Jacques 
Published: April 2021

ABSTRACT
Human adult hematopoietic stem cells are mostly quiescent or slow cycling. We have previously demonstrated that blocking of transforming growth factor-β1 (TGF-β1) is able to activate, in the presence of cytokines, primitive quiescent hematopoietic multipotent progenitors which could not grow in a two week semi-solid culture assay (short term culture). We have also shown that anti-TGF-β1 can up-modulate c-KIT, the receptor of the stem cell factor (steel factor). To elucidate whether TGF-β1 plays a central role in controlling the quiescence of hematopoietic primitive cells, it was necessary to demonstrate, as detailed in this study, that: (1) whatever the cytokine combination tested, addition of anti-TGF-β1 releases from quiescence multipotent progenitors with a significantly higher hematopoietic potential than those activated by cytokines alone. (2) Other important cytokine receptors controlling the most primitive hematopoietic cells such as FLT3 and the IL6 receptor (IL6-R) are down-modulated by TGF-β1 but rapidly up-modulated by anti-TGF-β1. (3) Anti-TGF-β1-sensitive multipotent and high proliferative potential progenitors express these cytokine receptors at a low level (FLT3low and IL6-Rlow).
According to these results, we propose the working model of ‘High Proliferative Potential-Quiescent cells’ to refer to these primitive hematopoietic multipotent progenitors that are highly sensitive to the growth inhibitory effect of TGF-β1. This model could be valid not only to study the human hematopoietic quiescent progenitors but also for other somatic stem cell systems.
Molecular organization of the desmoglein-plakoglobin complex
Authors: Chitaev Nikolai A. , Averbakh Alexander Z. , Troyanovsky Regina B. , Troyanovsky Sergey M. 
Published: April 2021

ABSTRACT
Different epithelial intercellular junctions contain distinct complexes incorporating plakoglobin. In adherens junctions, plakoglobin interacts with two molecules, the transmembrane adhesion protein of the cadherin family (e.g. E-cadherin) and α-catenin. The latter is thought to anchor the cadherin-plakoglobin complex to the cortical actin cytoskeleton. In desmosomes, plakoglobin forms a complex with desmosomal cadherins, either desmoglein (Dsg) or desmocollin (Dsc), but not with α-catenin. To further understand the structure and assembly of the plakoglobin-cadherin complexes we analyzed amino acid residues involved in plakoglobin-Dsg interactions using alanine scanning mutagenesis. Previously, we have shown that plakoglobin interacts with a 72 amino acid-long cytoplasmic domain (C-domain) that is conserved among desmosomal and classic cadherins. In this paper, we show that a row of the large hydrophobic residues located at the C-terminal portion of the Dsg C-domain is indispensable for interaction with plakoglobin. To study a reciprocal site we expressed plakoglobin (MPg) or its mutants tagged by 6 myc epitope in epithelial A-431 cells. Using sucrose gradient centrifugation and subsequent coimmunoprecipitation, MPg was found to be efficiently incorporated into the same type of complexes as endogenous plakoglobin. A major pool of Dsg-plakoglobin complexes sedimented at 8S and exhibited a 1:1 stoichiometry. Using alanine scanning mutagenesis and the co-immunoprecipitation assay we identified nine hydrophobic amino acids within the arm repeats 1-3 of plakoglobin, that are required for binding to Dsg and Dsc. Eight of these amino acids also participate in the interaction with α-catenin. No mutations were found to reduce the affinity of plakoglobin binding to E-cadherin. These data provide direct evidence that the same hydrophobic plakoglobin surface is essential for mutually exclusive interaction with distinct proteins such as α-catenin and desmosomal cadherins.
PDGF induces reorganization of vimentin filaments
Authors: Valgeirsdóttir Sigrĺdur , Claesson-Welsh Lena , Bongcam-Rudloff Erik , Hellman Ulf , Westermark Bengt , Heldin Carl-Henrik 
Published: April 2021

ABSTRACT
In this study we demonstrate that stimulation with platelet-derived growth factor (PDGF) leads to a marked reorganization of the vimentin filaments in porcine aortic endothelial (PAE) cells ectopically expressing the PDGF β-receptor. Within 20 minutes after stimulation, the well-spread fine fibrillar vimentin was reorganized as the filaments aggregated into a dense coil around the nucleus. The solubility of vimentin upon Nonidet-P40-extraction of cells decreased considerably after PDGF stimulation, indicating that PDGF caused a redistribution of vimentin to a less soluble compartment. In addition, an increased tyrosine phosphorylation of vimentin was observed. The redistribution of vimentin was not a direct consequence of its tyrosine phosphorylation, since treatment of cells with an inhibitor for the cytoplasmic tyrosine kinase Src, attenuated phosphorylation but not redistribution of vimentin. These changes in the distribution of vimentin occurred in conjunction with reorganization of actin filaments. In PAE cells expressing a Y740/751F mutant receptor that is unable to bind and activate phosphatidylinositol 3′-kinase (PI3-kinase), the distribution of vimentin was virtually unaffected by PDGF stimulation. Thus, PI3-kinase is important for vimentin reorganization, in addition to its previously demonstrated role in actin reorganization. The small GTPase Rac has previously been shown to be involved downstream of PI3-kinase in the reorganization of actin filaments. In PAE cells overexpressing dominant negative Rac1 (N17Rac1), no change in the fine fibrillar vimentin network was seen after PDGF-BB stimulation, whereas in PAE cells overexpressing constitutively active Rac1 (V12Rac1), there was a dramatic change in vimentin filament organization independent of PDGF stimulation. These data indicate that PDGF causes a reorganization of microfilaments as well as intermediate filaments in its target cells and suggest an important role for Rac downstream of PI3-kinase in the PDGF stimulated reorganization of both actin and vimentin filaments.
cyk-1: a C. elegans FH gene required for a late step in embryonic cytokinesis
Authors: Swan Kathryn A. , Severson Aaron F. , Carter J. Clayton , Martin Paula R. , Schnabel Heinke , Schnabel Ralf , Bowerman Bruce 
Published: April 2021

ABSTRACT
A maternally expressed Caenorhabditis elegans gene called cyk-1 is required for polar body extrusion during meiosis and for a late step in cytokinesis during embryonic mitosis. Other microfilament- and microtubule-dependent processes appear normal in cyk-1 mutant embryos, indicating that cyk-1 regulates a specific subset of cytoskeletal functions. Because cytokinesis initiates normally and cleavage furrows ingress extensively in cyk-1 mutant embryos, we propose that the wild-type cyk-1 gene is required for a late step in cytokinesis. Cleavage furrows regress after completion of mitosis in cyk-1 mutants, leaving multiple nuclei in a single cell. Positional cloning and sequence analysis of the cyk-1 gene reveal that it encodes an FH protein, a newly defined family of proteins that appear to interact with the cytoskeleton during cytokinesis and in the regulation of cell polarity. Consistent with cyk-1 function being required for a late step in embryonic cytokinesis, we show that the CYK-1 protein co-localizes with actin microfilaments as a ring at the leading edge of the cleavage furrow, but only after extensive furrow ingression. We discuss our findings in the context of other studies suggesting that FH genes in yeast and insects function early in cytokinesis to assemble a cleavage furrow.