bluuebunny/crossref_metadata_2025_split huggingface dataset:
Secretion from permeabilised mast cells is enhanced by addition of gelsolin: contrasting effects of endogenous gelsolin
Authors: Borovikov Yurii S. , Norman James C. , Price Leo S. , Weeds Alan , Koffer Anna 
Published: April 2021

ABSTRACT
Permeabilised rat mast cells were exposed to gelsolin and its N-terminal half (S1-3), proteins that sever actin filaments in a calcium-dependent and independent manner, respectively. Gelsolin and S1-3 induced a decrease in cellular F-actin content and an increase in the extent of the secretory response. The calcium sensitivities of both these effects were consistent with the differential calcium requirements of the two proteins. Segment 1 (S1), which binds G-actin and caps filaments but does not sever them, did not show these effects. Thus, secretion of mast cells is promoted as a consequence of the severing activity of exogenous gelsolin or S1-3.
Most of the endogenous gelsolin remained within permeabilised, washed mast cells and its distribution in resting state was predominantly cortical. Addition of calcium in the absence of MgATP did not reduce the F-actin content; by contrast, calcium with MgATP induced F-actin loss that was unaffected by the presence of anti-gelsolin. Because this antibody inhibits the severing activity of gelsolin, these results indicate that in permeabilised mast cells the severing activity of the remaining endogenous gelsolin is not involved in cortical actin filaments disassembly. Upon exposure to GTP-γ-S in the absence of calcium, the content of cortical gelsolin was reduced. This parallels our previous observation of a GTP-γ-S induced reduction of cortical actin filaments followed by their relocation to the cell’s interior (Norman et al. (1994)J. Cell Biol. 126, 1005-1015) and suggests that actin redistribution may be a consequence of dissociation of gelsolin caps brought about by activation of a GTP-binding protein.
Novel centrosomal protein reveals the presence of multiple centrosomes in turkey (Meleagris Gallopavo) bnbn binucleated erythrocytes
Authors: Woods Catherine M. , Zhu Jian , Coleman Thomas , Bloom Stephen E. , Lazarides Elias 
Published: April 2021

ABSTRACT
The phenotype of the bnbn hemolytic anemia mutation in the domestic turkey is manifested as binucleation specifically in the definitive erythrocyte lineage, most likely as the consequence of anomolous centrosomal activity (Bloom et al., 1970; Searle and Bloom, 1979). Here we have identified in turkey two variants of the novel, centrosomally-associated erythroid-specific protein p23. One variant is Ca2+-sensitive and is highly homologous to its chick counterpart (Zhu et al., 1995, accompanying paper). The other, p21, is a truncated form resulting from a 62 amino acid deletion from the 3′ end and a 40 amino acid insertion at the 5′ end, and appears to lack Ca2+-sensitivity. These proteins are localized at the marginal band, centrosomes and nuclear membrane of differentiated erythrocytes. Anti-p23/p21 immunofluorescence revealed the presence of multiple centrosomes in bnbn eythrocytes. We therefore undertook a detailed genetic analysis to determine whether the p21 variant represented the bn mutation. Initial tests of normal BnBn and mutant bnbn individuals suggested that the p23/p21 proteins might be encoded by the Bn/bn genes. However, further genetic tests demonstrated independent segregation for these two genetic loci. Thus, these proteins are encoded by the heretofore undescribed genes, p23/p21, mapping to an autosomal locus in the turkey genome.
Transverse pattern of microfilament bundles induced in epitheliocytes by cylindrical substrata
Authors: Svitkina Tatyana M. , Rovensky Yury A. , Bershadsky Alexander D. , Vasiliev Jury M. 
Published: April 2021

ABSTRACT
Cylindrical culture substrata are known to induce longitudinal orientation of polarized fibroblasts and corresponding alignment of actin microfilament bundles in these cells. We studied microfilament bundle distribution in two cell types, fibroblasts and epitheliocytes, spread on two kinds of anisotropic substrata, quartz glass cylinders with a diameter 32 μm and narrow (25-40 μm wide) flat glass adhesive strips with non-adhesive borders. Rat embryo and human diploid fibroblasts, as expected, formed predominantly longitudinally aligned bundles on both substrata. In contrast, transverse bundles on cylinders and randomly oriented bundles on flat strips were formed in IAR-2 and MDCK epithelial cells. We interpret these data as showing that the epitheliocyte attempts to override the guiding influence of anisotropic substrata. The microfilament bundle pattern on cylinders depends on the integrity of the microtubules. Colcemid-induced microtubule depolymerization caused formation of longitudinal as well as transverse bundles both in fibroblasts and epitheliocytes, thus diminishing the differences in microfilament bundle patterns in two cell types. These results show that microtubules control the cell-type-specific distribution of microfilament bundles both in polarized fibroblasts and in discoid epitheliocytes. However, the results of this control are opposite: microtubules enhance cell polarization in fibroblasts, but prevent it in epithelial cells.
Relationship between sperm atp content and motility of carp spermatozoa
Authors: Perchec G. , Jeulin C. , Cosson J. , André F. , Billard R. 
Published: April 2021

ABSTRACT
Carp spermatozoa are immotile in seminal plasma or in saline solution of high osmolality (&gt;400 mosmol kg−1). These ‘quiescent’ spermatozoa initiate a progressive forward motility when transferred in freshwater or in saline solution with low osmolality (&lt;160 mosmol kg−1). In this study we investigated ‘in vitro’ the relationship between sperm ATP content (measured by bioluminescence) and sperm motility (analysed by videomicroscopy). Sperm ATP content remained high in the immobilizing medium (200 mM KCl, Tris 30 mM, pH 8.0) where no flagellar movement occurs. Dilution of these spermatozoa in the activating medium (45 mM NaCl, 5 mM KCl, Tris 30 mM, pH 8.0) triggered forward motility which varied with temperature. At 20°C, sperm ATP content decreased rapidly during the progressive forward motility phase from 12 to 4 nmol/108 spermatozoa, concomitantly with decreases in velocity (130 to 10 μm s−1) and the beat frequency (50 to 7 Hz). An inhibitor of mitochondrial respiration (KCN 10 mM) produced a drop in sperm ATP content irrespective of the incubation medium (activating or immobilizing). A second phase of sperm motility in the activating medium was induced following a previous transfer of spermatozoa into a medium of high osmolality for a few minutes prior to the second phase. Within 10 minutes, spermatozoa recover 90% of the initial ATP level as well as forward motility. These results suggest that motility of carp spermatozoa depends on sperm ATP synthesized by mitochondrial respiration mainly stored before activation. In low osmolality conditions, the mitochondrial oxidative phosphorylation is unable to compensate for the ATP hydrolysis required to sustain motility. The increase in osmolality surrounding spermatozoa probably blocks the dynein ATPases and allows an ATP ‘regeneration’ as a result of mitochondrial respiration.
Atp-dependent reorganization of human sperm nuclear chromatin
Authors: Banerjee Subhasis , Smallwood Alan , Hultén Maj 
Published: April 2021

SUMMARY
Chromosomes in terminally differentiated mammalian spermatozoa are extensively condensed by protamines but a small proportion of histones remain. We examined the primary organization of somatic-type chromatin in lysolecithin-permeabilized human sperm nuclei and report that nucleosomes are closely packed with a periodicity of ∼150 bp. Incubation of nuclei in the presence of exogenous Mg2+ and ATP induced chromatin reorganization leading to an increase in spacing of the nucleosomes to ∼190 bp. This ATP-dependent chromatin rearrangement involved phosphorylation of both protamine and histone H2a. Increase in linker length between nucleosomes correlated with the phosphorylation of H2aX, the major H2a variant in human spermatozoa, predominantly at the C-terminal end. Chromatin reorganization was independent of detectable nuclear dispersion, which is an early chromosomal event in male pronuclear formation during fertilization.
Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides
Authors: Rabouille Catherine , Hui Norman , Hunte Felicia , Kieckbusch Regina , Berger Eric G. , Warren Graham , Nilsson Tommy 
Published: April 2021

ABSTRACT
The distribution of β1,2 N-acetylglucosaminyltransferase I (NAGT I), α1,3-1,6 mannosidase II (Mann II), β1,4 galactosyltransferase (GalT), α2,6 sialyltransferase (SialylT) was determined by immuno-labelling of cryo-sections from HeLa cell lines. Antibody labelling in the HeLa cell line was made possible by stable expression of epitope-tagged forms of these proteins or forms from species to which specific antibodies were available. NAGT I and Mann II had the same distribution occupying the medial and trans cisternae of the stack. GalT and SialylT also had the same distribution but they occupied the trans cisterna and the trans-Golgi network (TGN). These results generalise our earlier observations on the overlapping distribution of Golgi enzymes and show that each of the trans compartments of the Golgi apparatus in HeLa cells contains unique mixtures of those Golgi enzymes involved in the construction of complex, N-linked oligosaccharides.
Both cdc2 and cdk2 promote S phase initiation in Xenopus egg extracts
Authors: Chevalier S. , Tassan J.-P. , Cox R. , Philippe M. , Ford C. 
Published: April 2021

ABSTRACT
Xenopus egg extracts induce S phase DNA replication in added sperm pronuclei in a highly regulated manner, similar to events in vivo. Removal of cyclin-dependant kinases (cdks) or cdk2 from these extracts using affinity matrices severely inhibits initiation of S phase. We have used p13suc1 beads to remove both cdk2 and cdc2 proteins from egg extracts and developed a method to replace either protein alone to assess their capacity to initiate DNA replication. Re-addition of either cdk2 or cdc2 proteins to depleted extracts, through translation of their respective mRNAs, restimulated replication, judged by both total synthesis and labelling index. An ATP-binding-site mutant cdk2 mRNA (cdk2.R33) failed to stimulate replication and inhibited S phase initiation in mock-depleted extracts. Both human and Xenopus cdc2 mRNAs rescued replication in this system. Human mutant mRNAs have been used to show that the stimulation induced requires cdc2 catalytic activity, though not its mitotically active form. Rescue of replication by p34cdc2 is also observed in extracts depleted of cdks with a cdk2 antibody, which still retain much of their endogenous cdc2 protein. We conclude that newly synthesised p34cdc2, but not the inherited ‘old’ form, can induce S phase and in this form may overlap in function with p33cdk2.
‘Chromosomal puffing’ in diploid nuclei of Drosophila melanogaster
Authors: Gunawardena Shermali , Heddle Elizabeth , Rykowski Mary C. 
Published: April 2021

ABSTRACT
In situ hybridization has become a powerful technique for dissecting nuclear structure. By localizing nucleic acids with high precision, it is possible to infer the native structure of chromosomes, replication factories and transcript processing complexes. To increase the value of this technique, we have established the limits of resolution of two-color in situ hybridization to chromosomal DNA in diploid chromosomes of Drosophila embryonic nuclei. Using high-resolution 3-dimensional optical microscopy and computational image analysis, we establish that we can distinguish the location of chromosomal sequences that lie 27-29 kb apart within a 40 kb transcription unit with an accuracy of about 100 nm. Contrary to observations made in mammalian tissue culture cells, we find that when the gene is expressed, it assumes an open configuration, and that the extent of decondensation is variable from chromosome to chromosome. Further experiments suggest that variation in gene structure results from asynchrony in transcriptional elongation. We suggest that the phenomenon we observe is the diploid analog to chromosomal puffing that occurs in the transcriptionally active regions of Drosophila polytene chromosomes.
The role of Ca2+ during spore germination in Dictyostelium: autoactivation is mediated by the mobilization of Ca2+ while amoebal emergence requires entry of external Ca2+
Authors: Lydan Michael A. , Cotter David A. 
Published: April 2021

ABSTRACT
One of the developmental pathways used by the social amoeba Dictyostelium discoideum produces dormant spores. As with any temporary resistant stage, these spores must be able to germinate rapidly in response to positive environmental stimuli. One such stimulus is the autoactivator, an endogenous, diffusible molecule that is secreted by spores. Previous work has shown that three phases of germination, autoactivation, spore swelling and amoebal emergence, require the activity of the Ca2+-dependent, regulatory protein calmodulin, implicating Ca2+ as an essential cation during germination. In this study we used a pharmacological approach coupled with the direct measurement of Ca2+ levels in germinating spore populations by atomic adsorption to examine Ca2+-dependent signal transduction during spore activation and germination in D. discoideum. Inhibitors of both phospholipase C and internal Ca2+ release inhibited autoactivation while exogenously added Ins(1,4,5)P3, acted synergistically with the autoactivator. The antagonists specifically affected spore activation as mediated by the autoactivator, since neither had any effect on heat-activated spores. In contrast, La3+, an inhibitor of Ca2+ uptake, had little or no effect on either autoactivation or the swelling of autoactivated spores. However, an inhibition of Ca2+ influx by La3+ inhibited both the swelling of heat-activated spores and amoebal emergence following each period of autoactivation or heat activation.
Ca2+ levels change in the spore population during germination. During activation and swelling, Ca2+ efflux occurs from the spores. Both of the activating stimuli used here, the autoactivator and heat, caused this Ca2+ efflux. The efflux is reversed during emergence when there is a net Ca2+ uptake by the spores and cells from the medium. Together these data provide the first evidence that autoactivation is mediated by Ca2+-dependent signal transduction, leading to Ca2+ efflux, and that the late event of germination, amoebal emergence, requires Ca2+ uptake to proceed. The data also suggest that the responses of the spore to the each of autoactivator and heat, i.e. Ca2+ movements and germination, are mediated by different mechanisms.
CD44 is the major peanut lectin-binding glycoprotein of human epidermal keratinocytes and plays a role in intercellular adhesion
Authors: Hudson David L. , Sleeman Jonathan , Watt Fiona M. 
Published: April 2021

ABSTRACT
Although binding of peanut agglutinin (PNA) to keratinocytes is often used as a marker of terminal differentiation, the identity of the PNA-binding glycoproteins has been unclear. We now show that an antiserum raised against the glycoproteins recognises isoforms of CD44, the most abundant of which could be labelled with [35S]sulphate, indicating the presence of glycosaminoglycan side chains. RT-PCR analysis showed that keratinocytes expressed at least 5 forms of CD44 containing different numbers of exons from the variable region of the extracellular domain and also expressed the standard ‘haemopoietic’ form of CD44 which lacks the variable exons. Standard and variant isoforms of CD44 were expressed both by proliferating keratinocytes and cells undergoing terminal differentiation, although the level of CD44 mRNAs decreased when keratinocytes were placed in suspension to induce differentiation. The role of CD44 in intercellular adhesion was investigated by plating keratinocytes onto a rat pancreatic carcinoma line transfected with different CD44 isoforms. Keratinocyte adhesion to transfectants expressing variant exons 4-7 was greater than to cells expressing standard CD44 and could be inhibited with hyaluronan or digestion with hyaluronidase. These observations confirm earlier predictions that the PNA-binding glycoproteins of keratinocytes play a role in intercellular adhesion.
Formation of stable microspikes containing actin and the 55 kDa actin bundling protein, fascin, is a consequence of cell adhesion to thrombospondin-1: implications for the anti-adhesive activities of thrombospondin-1
Authors: Adams Josephine C. 
Published: April 2021

ABSTRACT
The organisation of the actin cytoskeleton was examined in H9c2 and human intestinal smooth muscle cells adherent on fibronectin or thrombospondin-1. Whereas cells adherent on fibronectin adopted a polygonal shape and rapidly assembled prominent stress fibres and focal contacts, cells adherent on thrombospondin-1 assumed a more irregular morphology with large lamellae containing radial actin microspikes. Focal contacts were not detected in cells adherent on thrombospondin-1, as determined by indirect immunofluorescence staining for vinculin and other focal contact components. Instead, the radial microspikes stained positively for the actin-bundling protein, 55 kDa/fascin, and myosins. In cells adherent on fibronectin, 55 kDa/fascin immunoreactivity was diffuse and tended to be concentrated in the perinuclear region. In long-term adherent cells cultured in serum-containing medium, 55 kDa/fascin was detected in membrane ruffles, in stress fibres and in the perinuclear region. The microspikes formed within 40 minutes of plating cells on thrombospondin-1 and remained present when cells were treated with sodium orthovandate and hydrogen peroxide to increase intracellular phosphotyrosine levels. Indeed, although vanadate-treated cells tended to retract, the microspikes became more prominent and showed an increased intensity of staining for fascin. Under these conditions, a proportion of the microspikes did not appear to be in contact with the substratum: these spikes stained weakly for focal adhesion kinase, talin and vinculin. Cells treated with genistein also spread and formed fascin-containing microspikes which tended to be more slender than those of control cells. In contrast, cells adherent on fibronectin displayed a complex rearrangement of the actin cytoskeleton and a transient enrichment of 55 kDa/fascin-containing structures at the cell surface when treated with sodium orthovanadate and hydrogen peroxide. These observations indicate that cell interactions with fibronectin or thrombospondin-1 send distinct organisational signals to the actin cytoskeleton and may offer a mechanistic framework for further investigations of the anti-adhesive properties of thrombospondin-1.
Role of glycosylation in the expression of human procathepsin D
Authors: Fortenberry Suzanne C. , Schorey Jeffrey S. , Chirgwin John M. 
Published: April 2021

ABSTRACT
Human procathepsin D carries two N-linked glycosylation sites at asparagine residues 70 and 199, widely separated on the surface of the folded protein. We created monoglycosylated procathepsin D molecules by site-directed mutagenesis in vitro of the individual glycosylation sites. With only two exceptions, all 12 mutants of this type were expressed efficiently in mammalian cells. The expressed proteins were stable, targeted to the lysosome, and partially secreted into the medium.
When both glycosylation sites were eliminated, however, the expressed proteins (9 different mutants) were stable but most were not secreted and targeted poorly to the lysosome. Mammalian fibroblasts appear to sort nascent procathepsin D efficiently only if it is N-glycosylated. Procathepsin D monoglycosylated at N70 is readily distinguished from the endogenous protein in transfected human cells and thus provides an excellent substrate for studying lysosomal targeting in an homologous system.
Lysine residues in the C-terminal lobe and lysosomal targeting of procathepsin D
Authors: Schorey Jeffrey S. , Fortenberry Suzanne C. , Chirgwin John M. 
Published: April 2021

ABSTRACT
A major pathway to the lysosome for soluble hydrolases involves the 6-phosphorylation of mannose residues. The initial step in this reaction is catalyzed by a phosphotransferase which recognizes lysosomal precursors. We constructed mutants of human procathepsin D whose targeting to the lysosome could be assayed directly in intact cells. Eight lysine residues were individually converted to glutamic acid on the surface of the carboxyl terminal lobe of the protein. Mutants with as many as four Lys to Glu mutations were normally targeted to the lysosome and processed to the mature form of the enzyme in transfected cells. We conclude that the C-terminal lobe of procathepsin D may not carry a determinant essential for lysosomal targeting in intact fibroblasts.
Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function
Authors: Aitken R. J. , Paterson M. , Fisher H. , Buckingham D. W. , Duin M. van 
Published: April 2021

ABSTRACT
The redox status of human spermatozoa was found to have a profound influence on the fertilizing potential of these cells in association with qualitative and quantitative changes in the patterns of tyrosine phosphorylation. In general, oxidizing conditions enhanced tyrosine phosphorylation and stimulated sperm function, whereas reducing conditions had the opposite effect. Unstimulated human spermatozoa exhibited low levels of spontaneous acrosomal exocytosis and sperm-oocyte fusion and minimal reactive oxygen species generation, while phosphotyrosine expression was largely confined to a single protein of 116 kDa. However, if the spermatozoa were exposed to oxidizing conditions through the addition of exogenous H2O2, or the stimulation of endogenous NADPH-dependent reactive oxygen species generation, then a dramatic increase in tyrosine phosphorylation was observed (major phosphotyrosyl bands at 222 kDa, 200 kDa, 159 kDa, 133 kDa, 116 kDa and 82 kDa) in concert with the functional activation of the spermatozoa. A causal association between reactive oxygen species generation, tyrosine phosphorylation and sperm function was indicated by studies with the ionophore, A23187, which induced high rates of spermoocyte fusion together with enhanced rates of reactive oxygen species production and the increased expression of phosphotyrosyl proteins. This functional response to A23187 could be abrogated, without any concomitant change in sperm motility or viability, by using membrane permeant thiols or catalase to suppress the reactive oxygen species-induced increase in phosphotyrosine expression. The fact that the biological responses of human spermatozoa to biological agonists (recombinant human ZP3 and progesterone) could also be inhibited by catalase indicated the general relevance of these findings. These results suggest that the generation of reactive oxygen species by human spermatozoa plays a key role in the control of sperm function through the redox regulation of tyrosine phosphorylation.