bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heat shock results in cell cycle delay and synchronisation of mitotic domains in cellularised Drosophila melanogaster embryos
Authors: Maldonado-Codina Gabriela , Llamazares Salud , Glover David M. 
Published: April 2021

ABSTRACT
Cells of Drosophila embryos that are subjected to a 37°C temperature shock whilst undergoing the S-phase of cell cycle 14 arrest with their microtubules in an interphaselike state, and with nuclei showing unusual chromatin condensation. They do not recover from this state within a 30 minute period even though extensive gastrulation movements can occur. Cells of embryos heat shocked in G2-phase are delayed in interphase with high levels of cyclins A and B. Within ten minutes recovery from heat shock, cells enter mitosis throughout the embryo. The degradation of the mitotic cyclins A and B in these synchronised mitotic domains does not follow the normal timing, but is delayed. These findings point to a need for caution when interpreting experiments that use the heat shock promoter to study the expression of cell cycle control genes in Drosophila.
The relaxed crossbridge pattern in isolated rabbit psoas muscle thick filaments
Authors: Kensler Robert W. , Stewart Murray 
Published: April 2021

ABSTRACT
Rabbit muscle is a major source of material for biochemical experiments and spin labelling studies of contraction, and so it is important to establish how closely this material resembles the frog and fish muscles usually used for structural studies. Previous studies have shown that relaxed rabbit muscle thick filaments lose the characteristic order of their crossbridges when they are cooled below about 15-19°C, whereas the order of fish and frog muscles is retained above 0°C. The lack of order has frustrated attempts to examine rabbit thick filament structure and has raised questions about how closely they might resemble other thick filaments. We have therefore developed a procedure for preserving the crossbridge order in isolated filaments. Electron microscopy of these thick filaments after either negative staining or metal shadowing has shown that the crossbridge pattern has a 43 nm axial repeat and is based on three near-helical strands. Computed transforms of either type of image show a series of layer lines confirming that the native relaxed pattern has been preserved, and computer reconstructions show the individual crossbridges lying on a slightly perturbed 3-stranded lattice. These data indicate an unexpectedly high degree of similarity between the rabbit and frog patterns and indicate that, in fully preserved material, there is little structural difference between the two thick filaments at the temperature at which each normally functions. Overall, therefore, our results validate the integration of biochemical data from rabbit muscle with structural data from frog and indicate that the differences between the two muscle types do not result from fundamental structural differences in the arrangement of myosin molecules in their thick filaments.
The bip protein and the endoplasmic reticulum of Schizosaccharomyces pombe: fate of the nuclear envelope during cell division
Authors: Pidoux Alison L. , Armstrong John 
Published: April 2021

ABSTRACT
A polyclonal antibody was raised to the C-terminal region of fission yeast BiP. The use of this antibody for immunoprecipitation, western blotting and immunofluorescence has confirmed and extended the observations made previously with an epitope-tagged BiP molecule. A fraction of BiP protein is glycosylated in Sc h izo s a c charomyces pombe cells. Pulse-chase experiments showed that this modification occurs rapidly upon synthesis and that the extent of glycosylation does not then change with time. BiP protein is induced by elevated temperatures and by treatment with tunicamycin. The antibody cross-reacts with proteins of similar molecular weight in the yeasts Kluyveromyces lactis and Schizosaccharomyces japonicus. Immunofluorescence of BiP has been used to follow the behaviour of the ER and in particular the nuclear envelope through the cell cycle.
Spatial localization of agonist-induced Ca2+ entry in bovine adrenal chromaffin cells: Different patterns induced by histamine and angiotensin II, and relationship to catecholamine release
Authors: Cheek Timothy R. , Morgan Alan , O’Sullivan Antony J. , Moreton Roger B. , Berridge Michael J. , Burgoyne Robert D. 
Published: April 2021

ABSTRACT
The spatial organization of agonist-induced Ca2+ entry in single bovine adrenal chromaffin cells has been investigated using video-imaging techniques to visualize fura2 quenching by the Ca2+ surrogate, Mn 2+.
The potent secretagogue histamine, in addition to releasing Ca2+ from intracellular stores, resulted in a large influx of external Mn2+ that occurred over the entire surface of the cell. The influx of Ca2+ that this mirrors was found to be an obligatory requirement for the triggering of catecholamine release by histamine, which suggests that such a global influx of Ca2+ into the cell probably underlies the ability of this agonist to stimulate a large secretory response. By contrast, the weaker secretagogue angiotensin II, which also acts through the second messenger inositol trisphosphate, produced a localized entry of external Mn2+ in 64% of cells. In these cells, localized Mn2+ entry always occurred at the pole of the cell in which the angiotensin II-induced rise in [Ca2+]i was largest. Since exocytosis in response to angiotensin II has previously been shown to be restricted to this same pole of the cell (Cheek et al. (1989). J. Cell Biol. 109, 1219-1227), these results suggest that localized influx of Ca2+ in response to angiotensin II could underlie the polarized exocytotic response observed with this stimulus. These results directly demonstrate that different agonists can induce different patterns of divalent cation influx in the same cells and, furthermore, suggest how these different patterns can have a direct influence on cellular function.
The control of chondrocyte differentiation during endochondral bone growth in vivo: changes in TGF-β and the proto-oncogene c-myc
Authors: Loveridge N. , Farquharson C. , Hesketh J. E. , Jakowlew S. B. , Whitehead C. C. , Thorp B. H. 
Published: April 2021

ABSTRACT
The expression of transforming growth factor-β and the c-myc proto-oncogene was studied in situ in the chondrocytes of the tibial growth plate of normal chicks and those with avian tibial dyschondroplasia in which the chondrocytes are developmentally arrested in the transitional phase between proliferation and differentiation. This results in an accumulation of unmineralised and avascular cartilage. Dyschondroplastic chicks showed reduced c-myc expression in the transitional chondrocytes but unaltered levels in the proliferating chondrocytes. Transforming growth factor-β expression was reduced in the transitional chondrocytes of dyschondroplastic chicks. In areas where the lesion was being repaired there was evidence of increased expression of both c-myc protein and transforming growth factor-β. Addition of 1,25-dihydroxyvitamin D to the diet, which is known to reduce the incidence of dyschondroplasia, resulted in an increase in c-myc production. These results suggest that both transforming growth factor-β and the proto-oncogene c-myc may be important elements of the cascade of events that lead to chondrocyte differentiation, hypertrophy and mineralisation.
Quantification and immunolocalisation of porcine articular and growth plate cartilage collagens
Authors: Wardale R. John , Duance Victor C. 
Published: April 2021

ABSTRACTThe collagens of growth plate and articular cartilage from 5-6 month old commercial pigs were characterised. Growth plate cartilage was found to contain less total collagen than articular cartilage as a proportion of the dry weight. Collagen types I, II, VI, IX and XI are present in both growth plate and articular cartilage whereas type X is found exclusively in growth plate cartilage. Types III and V collagen could not be detected in either cartilage.Type I collagen makes up at least 10% of the collagenous component of both cartilages. There are significant differences in the ratios of the quantifiable collagen types between growth plate and articular cartilage. Collagen types I, II, and XI were less readily extracted from growth plate than from articular cartilage following pepsin treatment, although growth plate cartilage contains less of the mature collagen cross-links, hydroxylysyl-pyridinoline and lysyl-pyridinoline. Both cartilages contain significant amounts of the divalent reducible collagen cross-links, hydroxylysyl-ketonorleucine and dehydro-hydroxylysinonorleucine.Immunofluorescent localisation indicated that type I collagen is located predominantly at the surface of articular cartilage but is distributed throughout the matrix in growth plate. Types II and XI are located in the matrix of both cartilages whereas type IX is predominantly pericellular in the calcifying region of articular cartilage and the hypertrophic region of the growth plate. Collagen type VI is located primarily as a diffuse area at the articular surface.
The mouse brown (b) locus protein has dopachrome tautomerase activity and is located in lysosomes in transfected fibroblasts
Authors: Winder Alison J. , Wittbjer Anna , Rosengren Evald , Rorsman Hans 
Published: April 2021

ABSTRACT
Many genes mapping to pigmentation loci are involved in the regulation of melanin synthesis in the mouse. The brown (b) locus controls black/brown coat coloration, and its product has significant homology to the key melanogenic enzyme tyrosinase. This has led to suggestions that the b-protein is itself a melanogenic enzyme. In order to investigate its function, we have established lines of mouse fibroblasts stably expressing the b-protein by co-transfection of a b-protein expression vector and a plasmid conferring resistance to the antibiotic G418. The b-protein synthesised by these cells has the expected molecular mass of 75 kDa and reacts with three different anti-b-protein antibodies. We were unable to confirm previous reports that the b-protein has tyrosinase or catalase activity, but detected stereospecific dopachrome tautomerase activity in b-proteinexpressing fibroblasts. This dopachrome tautomerase binds to Concanavalin A-Sepharose, and the major product of its action on L-dopachrome is 5,6-di-hydroxyindole-2-carboxylic acid. Since this activity is not present in untransfected cells we conclude that the b-protein has dopachrome tautomerase activity. Fibroblasts do not contain melanosomes, the specialised organelles in which the b-protein is located in melanocytes. Nevertheless, indirect immunofluorescence localisation of the b-protein in transfected fibroblasts produces a distinctive pattern of intense juxtanuclear staining combined with punctate cytoplasmic staining. Double-labelling shows co-localisation of the b-protein with the late endosomal/lysosomal markers β-glucuronidase and LAMP-1, both in transfected fibroblasts and in mouse melanoma cells. These findings are consistent with the hypothesis that melanosomes are closely related to lysosomes.
Synthesis and assembly of fibrillin by fibroblasts and smooth muscle cells
Authors: Kielty Cay M. , Shuttleworth C. Adrian 
Published: April 2021

ABSTRACT
The expression and assembly of the microfibrillar glycoprotein fibrillin has been investigated in cultures of nuchal ligament fibroblasts, skin fibroblasts and vascular smooth muscle cells. The level of fibrillin expression varied with the cell type and growth conditions. Higher levels of synthesis were recorded in quiescent post-confluent cells than in actively dividing subconfluent cultures. Nuchal ligament fibroblasts consistently synthesized the highest levels of fibrillin. Growth of cells in the presence of ascorbate resulted in an increased proportion of newly synthesized fibrillin retained within cell layers. Fibrillin was immunoprecipitated from medium and cell layer extracts in the form of monomers and high-Mr disulphide-bonded aggregates. Rotary shadowing electron microscopy of cell layer extracts and collagen gels provided direct evidence for the assembly of extensive intact microfibrils by smooth muscle cells and fibroblast cultures. Gel filtration chromatography of medium and cell layer extracts, in combination with immunoprecipitation of column fractions, provided a means of analysing the size distribution and assembly of newly synthesized fibrillin. This cell culture approach provides an opportunity to evaluate normal and aberrant synthesis and assembly of fibrillin in a wide range of cell types.
Retinoic acid mediates post-transcriptional regulation of keratin 19 mrna levels
Authors: Crowe David L. 
Published: April 2021

ABSTRACT
Stratified squamous epithelia have been shown to preferentially express a site-specific pattern of keratin intermediate filaments. Retinoic acid (RA) is known to modulate expression of the basal cell keratins K19 and K5. Expression of these genes is dependent on extracellular RA concentration. We have found that K19 mRNA levels increase over time in cultured keratinocytes exposed to elevated concentrations of RA. K5 mRNA levels decrease in response to RA in a similar fashion. The observed changes in K5 message are primarily the result of RA-induced alterations in gene transcription. However, the RA-mediated induction of K19 mRNA is not the result of increased transcription but is primarily due to enhanced mRNA stability. These results suggest that an RA-dependent post-transcriptional mechanism modulates K19 intermediate filament expression in stratified squamous epithelia.
The origin of parasitophorous vacuole membrane lipids in malaria-infected erythrocytes
Authors: Ward Gary E. , Miller Louis H. , Dvorak James A. 
Published: April 2021

ABSTRACT
During invasion of an erythrocyte by a malaria merozoite, an indentation develops in the erythrocyte surface at the point of contact between the two cells. This indentation deepens as invasion progresses, until the merozoite is completely surrounded by a membrane known as the parasitophorous vacuole membrane (PVM). We incorporated fluorescent lipophilic probes and phospholipid analogs into the erythrocyte membrane, and followed the fate of these probes during PVM formation with low-light-level video fluorescence microscopy. The concentration of probe in the forming PVM was indistinguishable from the concentration of probe in the erythrocyte membrane, suggesting that the lipids of the PVM are continuous with and derived from the host cell membrane during invasion. In contrast, fluorescently labeled erythrocyte surface proteins were largely excluded from the forming PVM. These data are consistent with a model for PVM formation in which the merozoite induces a localized invagination in the erythrocyte lipid bilayer, concomitant with a localized restructuring of the host cell cytoskeleton.
Prp2o, the Saccharomyces cerevisiae homolog of the regulator of chromosome condensation, rcc1, interacts with double-stranded dna through a multi-component complex containing gtp-binding proteins
Authors: Lee Arianna , Tam Rose , Belhumeur Pierre , DiPaolo Tiziano , Clark Michael W. 
Published: April 2021

ABSTRACT
Prp20, a homolog to the mammalian negative regulator of chromosome condensation, RCC1, is retained on double-stranded (ds) DNA-cellulose when extracts are prepared from asynchronously growing wild-type yeast strains. Conversely, neither Prp20 from ts mutant cell extracts nor wt yeast Prp20 produced in Escherichia coli, bind to dsDNA-cellulose. In vitro reconstitution assays using E. coli-expressed Prp20 and inactivated ts mutant extracts of prp20-1 reveal that the Prp20 protein requires the assistance of other proteins in the cell extract to promote its binding to dsDNA. Immunoprecipitations and sizing-column-chromatography indicate that the Prp20 protein binds to the dsDNA column through a multicomponent complex composed of six to seven proteins, which has a collective molecular mass greater than 150,000 Da. At least three of the members of this Prp20 complex will bind GTP in vitro. Moreover, the Prp20 complex is shown to specifically lose its ability to bind dsDNA during the DNA replication phase of the cell cycle. This loss of dsDNA binding during the S phase of the cell cycle does not affect the proper organization of the nucleoplasm and appears to be reversed before the cell enters mitosis.
Adhesion of cho cells to fibronectin is mediated by functionally and structurally distinct adhesion plaques
Authors: Tranqui Léone , Usson Yves , Marie Christiane , Block Marc R. 
Published: April 2021

ABSTRACT
We have investigated the dynamics between free fibronectin receptors and clusters of them organized into adhesion plaques on CHO cells using the ability of these free integrins to be endocytosed and recycled to the plasma membrane. Indirect inhibition of the endo- cytic cycle by monensin resulted in the subsequent internalization of free receptors, which we followed by indirect immunostaining and confocal microscopy. Consequently, all the adhesive structures that were in equilibrium with free integrins became progessively disorganized. The cellular morphological changes were analyzed and correlated with the distribution of cellsubstratum contacts viewed by confocal images obtained after immunostaining with antibodies raised against the fibronectin receptor, talin, vinculin and actin. After cell adhesion to fibronectin, blockage of the endocytic cycle induced disruption of the adhesion plaques that were mainly localized at the cell periphery, and disappearance of the stress fibers. However, the cells remained firmly attached to the substratum through focal contacts localized in the central part of the cell. These central focal contacts, but not the peripheral adhesion plaques, could form when the vesicular traffic was blocked prior to adhesion and they allowed the cells to attach and flatten onto the substratum. Whereas both adhesive structures contained the same receptors linked to talin and vinculin, the central adhesive structures were attached to a short stretch of actin but never permitted the organization of stress fibers.
Selective distributions of proteoglycans and their ligands in pericellular matrix of cultured fibroblasts: implications for their roles in cell-substratum adhesion
Authors: Yamagata Masahito , Saga Shinsuke , Kato Masato , Bernfield Merton , Kimata Koji 
Published: April 2021

ABSTRACT
We showed previously that a large chondroitin sulfate proteoglycan, PG-M (also known as versican), inhibits cell-substratum adhesion, while basement membrane heparan sulfate proteoglycan (recently named perlecan) does not (Yamagata et al. (1989) J. Biol. Chem. 264, 8012-8018). To extend our understanding of the adhesive function of these proteoglycans, we examined the pericellular localization of the proteoglycans and their ligands and also that of some matrix receptors and cytoskeletal molecules in various fibroblast culture systems. PG-M was abundant in the subcellular space of fibroblasts, but was excluded selectively from focal contacts where vinculin, integrins and fibronectin were localized. Hyaluronan, CD44 and tenascin were distributed similarly as PG-M. In contrast, perlecan was associated with fibronectin and was included in focal contacts. Syndecan-1, a membrane heparan sulfate/chon-droitin sulfate proteoglycan, was associated with fibronectin at the cell surface, partly at focal contacts and in association with stress fibers. Thus, complexes of PG-M with hyaluronan, tenascin and CD44, are not involved in focal contacts. On the other hand, perlecan and syndecan-1 together with fibronectin may participate in focal contacts. The difference in localization between these proteoglycans may be related to their glycosaminoglycan content and to their distinctive roles in cell-substratum adhesion.
Mutations in the Drosophila melanogaster gene three rows permit aspects of mitosis to continue in the absence of chromatid segregation
Authors: Philp Alastair Valentine , Axton J. Myles , Saunders Robert D. C. , Glover David M. 
Published: April 2021

ABSTRACT
We have cloned the three rows (thr) gene, by a combination of chromosome microdissection and P element tagging. We describe phenotypes of embryos homozygous for mutations at the thr locus. Maternal mRNA and protein appear to be sufficient to allow 14 rounds of mitosis in embryos homozygous for thr mutations. However, a small percentage of cells in syncytial blastoderm stage thr embryos sink into the interior of the embryo as if they have failed to divide properly. Following cellularisation all cells complete mitosis 14 normally. All cells become delayed at mitosis 15 with their chromosomes remaining aligned on the spindle in a metaphase-like configuration, even though both cyclins A and B have both been degraded. As cyclin B degradation occurs at the metaphase-anaphase transition, subsequent to the microtubule integrity checkpoint, the delay induced by mutations at the thr locus defines a later point in mitotic progression. Chromosomes in the cells of thr embryos do not undertake anaphase separation, but remain at the metaphase plate. Subsequently they decondense. A subset of nuclei go on to replicate their DNA but there is no further mitotic division.
Chediak-higashi syndrome is not due to a defect in microtubule-based lysosomal mobility
Authors: Perou Charles M. , Kaplan Jerry 
Published: April 2021

ABSTRACT
Chediak-Higashi Syndrome is an autosomal recessive disorder that affects intracellular vesicle formation. The diagnostic feature of Chediak-Higashi Syndrome is the presence of ‘giant’ lysosomes clustered near the nucleus. Lysosome morphology in macrophages is maintained by microtubules and microtubule-based motors, such as kinesin. Dramatic changes in lysosome morphology can be induced by lowering cytoplasmic pH or by adding phorbol esters. When macrophages from beige mice (a murine homolog of Chediak-Higashi Syndrome) were subjected to these protocols they were able to alter their lysosomal distribution and morphology to the same degree as macrophages from control mice. These results indicate that lysosomes in Chediak cells are capable of interacting with the microtubule-based motor system, suggesting that the defective gene product is not an altered microtubular element involved in lysosomal movement.
Calcium homeostasis in plants
Authors: Gilroy Simon , Bethke Paul C. , Jones Russell L. 
Published: April 2021

ABSTRACT
Many aspects of Ca2+ homeostasis in plants are similar to those in animals and fungi (Poovaiah and Reddy, 1989), but an understanding of how Ca2+ transport and function are integrated from the level of the whole plant to the sub-cellular level remains elusive. At the whole-plant level, a constant supply of Ca2+ in the range 1-10 mM is required to maintain normal growth and development (Epstein, 1972; Clarkson and Hanson, 1980). Calcium uptake by roots leads to millimolar concentrations of Ca2+ in plant tissues, and in most plants Ca is the second most abundant metal and the fifth most abundant element, after C, H, O and K (Epstein, 1972). At the subcellular level, certain organelles, such as the large central vacuole, may have similarly high Ca concentrations, but cytoplasmic Ca2+ levels are three to four orders of magnitude lower. Despite the abundance of Ca in plant tissues and the small amounts required for most cellular processes, the supply of Ca to the plant must be uninterrupted. Removal of Ca from the nutrient supply results in rapid death of cells in the apical meristem and a cessation of growth (Epstein, 1972). Just why this calcium starvation occurs when Ca levels in the plant are so high is not fully understood, but the low mobility of Ca within the plant body must be at least partially responsible.