bluuebunny/crossref_metadata_2025_split huggingface dataset:
Islands of acetylated histone H4 in polytene chromosomes and their relationship to chromatin packaging and transcriptional activity
Authors: Turner B. M. , Franchi L. , Wallace H. 
Published: April 2021

ABSTRACT
The four histones of the nucleosome core particle are all subject to enzyme-catalysed, post-translational acetylation at defined lysine residues in their amino-terminal domains. Much circumstantial evidence suggests a role for this process in modifying chromatin structure and function, but detailed mechanisms have not been defined. To facilitate studies on the functional significance of histone acetylation, we have prepared antibodies specific for the acetylated isoforms of histone H4. Because of the extreme evolutionary conservation of H4, these antisera can be applied to a wide variety of organisms and experimental systems. In the present study we have used polytene chromosomes from the salivary glands of larvae of the midge Chironomus to examine the distribution of acetylated H4 in interphase chromatin. By indirect immunofluorescence, antisera to acetylated H4 labelled the four Chironomus chromosomes with reproducible patterns of sharply defined, fluorescent bands. An antiserum to non-acetylated H4 gave a completely different, more-diffuse labelling pattern. Thus, there are defined regions, or islands, in the interphase genome that are enriched in acetylated H4. Double-labelling experiments with two antisera specific for H4 molecules acetylated at different sites, showed that each antiserum gave the same banding pattern. Immunolabelling patterns were not dependent on the pattern of phase-dense bands characteristic of these chromosomes; strongly labelled regions could correspond to phase-dense bands (i.e. condensed chromatin), to interbands or, frequently, to band-interband junctions. Immunogold electron microscopy confirmed the immunofluorescence results and showed further that regions of relatively high labelling could be either transcriptionally active or quiescent, as judged by the presence or absence of ribonucleoprotein particles. Two rapidly transcribed genes on chromosome 4 of Chironomus form characteristic ‘puffs’, the Balbiani rings BRb and BRc. The antiserum to non-acetylated H4 gave diffuse labelling throughout these puffs, demonstrating the continued presence of this histone in these transcriptionally active regions. Antisera to acetylated H4 strongly labelled the boundaries of BRb and BRc, and revealed clearly defined islands of increased H4 acetylation just within the expanded chromatin of the puffs. Labelling within the central region of each puff was much less intense. A similar pattern was observed in puffs on other chromosomes. Thus, increased H4 acetylation is not found throughout actively transcribed chromatin but occurs only at defined sites, possibly in the non-transcribed flanking regions. H4 acetylation is clearly not required for the passage of RNA polymerase through the nucleosome and we speculate that its role may be to facilitate the binding to DNA of polymerases and other proteins prior to the onset of transcription and possibly replication.
Mapping Map-2
Authors: Harrison Alistair , Hyams Jeremy S. 
Published: April 2021

ABSTRACT
Microtubule-associated proteins (MAPs) are an important class of cytoskeletal proteins that are presumed to confer specific functional characteristics on microtubules. The best-characterised is the heat-stable brain MAP, MAP-2. MAP-2 consists of three subunits MAP-2a, MAP-2b (both having an apparent Mrof∼270K; K=103Mr) and MAP-2c (Mr 70K) (Vallee et al. 1981; Garner et al. 1988a). The first insight into the structure of the MAP-2 molecule emerged from the chymotryptic digestion of the protein (Vallee, 1980). Fragments with Mr values in the range of 32 to 39K were found to bind to, and promote the assembly of, microtubules, whilst a larger 240K fragment had no interaction and probably represented an arm domain that projected from the surface of the microtubule and interacted in some way with other cell components, or, indeed, other microtubules (Vallee and Borisy, 1977; Vallee, 1980).
The response of the centrosome to heat shock and related stresses in a Drosophila cell line
Authors: Debec Alain , Courgeon Anne-Marie , Maingourd MichÈle , Maisonhaute Claude 
Published: April 2021

ABSTRACT
The centrosome of Drosophila melanogaster cells cultured in vitro has been followed by immunofluorescence techniques with the Bx63 antibody of Frasch and Saumweber. After a heat shock, the centrosome labelling becomes very small and finally disappears after 30 min. Other heat-shock protein (hsp) inducers such as ethanol, arsenite and ecdysterone lead to the same disappearance. Moreover, the functional ability of centrosomes to nucleate microtubule assembly is inhibited by these treatments, particularly by heat shock, ethanol and ecdysterone. Two other hsp inducers, cadmium chloride and hydrogen peroxide, do not affect the centrosome seriously. With the exception of cadmium, the rapidity and the intensity of hsp induction are in good agreement with the kinetics of alteration of the organelle. We propose that a close link exists between the heat-shock response and the centrosome and that the physiological induction of hsps could be reinterpreted in terms of cell division control.
Fodrin in the human polymorphonuclear leucocyte: Redistribution induced by the chemotactic peptide
Authors: Fujimoto Toyoshi , Ogawa Kazuo 
Published: April 2021

ABSTRACT
Fodrin, a membrane skeletal protein, was found to accumulate in the posterior portion of human neutrophils polarized morphologically after stimulation by the chemotactic peptide, IV-formylmethionyl-leucylphenylalanine (FMLP). In most (&gt;90%) unstimulated neutrophils, the distribution of fodrin was found to be uniform by immunofluorescence microscopy. When FMLP (10−8M) was applied at 25°C, fodrin became polarized in about 40% of cells by Imin, about 70% by 2 min, and about 80% by 10 min. The cells with polarized distribution decreased thereafter to about 60% of the cells at 20 min and about 20% at 60 min. Using the under-agarose system, it was confirmed that the concentration of fodrin occurred in the region opposite to the direction of chemoattraction in moving cells.
By immunoelectron microscopy, most of the labeling for fodrin was observed in the filamentous cell cortex and not associated with the plasma membrane itself. In cells polarized morphologically by FMLP, the fodrin labeling became concentrated in the posterior portion of the cell; the labeling was found most densely in the granule-rich cytoplasm, while the filamentous tail region was not labeled intensely. The lamellipodium in the head region was also labeled only sparsely.
The results indicate that in human neutrophils fodrin exists as a cytoskeletal protein rather than as a membrane protein and that the protein accumulates in the endoplasm of the posterior portion in migrating cells. The rearrangement is likely to modulate the organization of the actin-rich cell cortex for cell locomotion.
Characterization of Chinese hamster ovary cells with impaired spreading properties on fibronectin
Authors: Joseph L. B. , Kreutzer D. L. , Tanzer M. L. 
Published: April 2021

ABSTRACT
The development of receptor-defective or -deficient mutants can be applied to the investigation of cellmatrix interactions including cell adherence and spreading. In the present study we developed a series of ethyl methyl sulfonate (EMS)-induced Chinese hamster ovary (CHO) cell mutants, which adhere to fibronectin but have impaired spreading characteristics. Using morphometric analysis, a significant suppression in the degree of cell spreading between the wild-type and the mutant cells (PcO.OOl) was seen. This inability of the mutant cells to spread adequately on fibronectin also resulted in a decreased number and diameter of stress fibers as compared to wild-type cells. The decreased cell spreading of the mutant cells was not due to inherent differences in cell size or volume, as determined by fluorescence-activated cell sorter (FACS) analysis. Since integrins, specifically the fibronectin receptor (α FN/β1), are important in cell adhesion and cell spreading, we carried out a comparative immunochemical analysis, using a monoclonal antibody to the β subunit of integrin (7E2). Western blot analysis of cell extracts and cell membranes indicated that both wild-type and mutant cells expressed the α and β subunits of the fibronectin receptor; the mutant cells displayed reduced levels of the subunit. Immunohistochemical analysis indicated that, despite the presence of the receptor in both cell types, their patterns of localization and aggregation were different. The wild-type cells showed a needle-like distribution of the receptor, in contrast to the clumped appearance in the mutants. Furthermore, FACS analysis of antibody-labelled cells showed that the mutant cells had threefold less labelling of the fibronectin receptor by the 7E2 antibody than the wildtype cells. These data suggest that, in the mutant cells, the fibronectin receptor is present, but it is less abundant at the cell surface. This decrease in surface fibronectin receptors may be responsible for decreased cell spreading of the mutant cells on the fibronectin substratum. Thus, the linkage between adhesion and spreading potentially may be explored by studying these mutant cells.
Analysis of wound healing in an in vitro model: Early appearance of laminin and a 125×103M
r polypeptide during adhesion complex formation
Authors: Kurpakus Michelle A. , Stock E. Lee , Jones Jonathan C. R. 
Published: April 2021

ABSTRACT
The adhesion complex, which plays an important role in cell-substratum attachment, consists of a cellular hemidesmosomal plaque, anchoring filaments, the basement membrane zone and anchoring fibrils. An analysis of the temporal sequence of assembly of the adhesion complex was undertaken in an in vitro model of epithelial cell wound healing by immunofluorescence and electron microscopy. A monoclonal antibody directed against a 125K (K= 103Mr) polypeptide (mAbHD), bullous pemphigoid (BP) autoantibodies, antibodies directed against collagen type VII and laminin antibodies were used as markers for anchoring filaments, the hemidesmosome, anchoring fibrils and the laminin component of the basement membrane zone, respectively. Fluorescence labeling could be detected with mAbHD before labeling with BP autoantibodies or collagen type VII antibodies. Laminin fluorescence was detected at the same time as mAbHD. Furthermore, the 125K polypeptide and laminin were located extracellularly prior to the appearance of BP antigen and collagen type VII. The appearance of the hemidesmosomal plaque at the electron microscope level succeeded the localization of BP antigen in basal cells detected by immunofluorescence microscopy. No evidence for the coordinated appearance of BP antigen, collagen type VII and laminin was observed in this model. We discuss the possibility that the 125K protein and laminin may play roles in the initiation of complex formation. Furthermore, although basement membrane zone components were detected early in adhesion complex re-formation, formation of the lamina densa region of the basement membrane zone followed the appearance of the hemidesmosomal plaque, indicating a role for the hemidesmosomal plaque in organizing the structure of the lamina densa.
Biochemical and immunochemical analysis of the arrangement of connexin43 in rat heart gap junction membranes
Authors: Laird D. W. , Revel J.-P. 
Published: April 2021

ABSTRACT
A 43 × 10 3 M r protein (designated connexin 43 or Cx43) is a major constituent of heart gap junctions. The understanding of its arrangement in junctional membranes has been extended by means of site-directed antibodies raised against synthetic peptides of Cx43. These represent part of the first extracellular loop (EL-46), the cytoplasmic loop (CL-100), the second extracellular loop (EL-186) and carboxy-terminal sequences (CT-237 and CT-360). All of the antibodies raised reacted with their respective peptides and the Cx43 protein on Western blots. By immunoelectron microscopy two of the antibodies (CL-100 and CT-360) were shown to label the cytoplasmic surface of isolated gap junction membranes. Immunofluor-escent labeling at locations of neonatal cardiac myocyte-myocyte apposition required an alkali/urea treatment when the EL-46 and EL-186 antibodies were used. Immunoblot analysis of endoproteinase Lys-C-digested gap junctions revealed that the Cx43 protein passed through the lipid bilayer four times. Alkaline phosphatase digestion of isolated junctions was used to show that the CT-360 antibody recognized many phosphorylated forms of Cx43. Our results unequivocally confirm models of the organization of Cx43 that were based on a more limited set of data and a priori considerations of the sequence.
Failure of platelet-derived growth factor plus insulin to stimulate sustained proliferation of swiss 3T3 cells Requirement for hydrocortisone, prostaglandin E1; lipoproteins, fibronectin and an unidentified component derived from serum
Authors: Brooks Robert F. , Howard Mark , Leake David S. , Riddle Peter N. 
Published: April 2021

ABSTRACT
Platelet-derived growth factor (PDGF) has been reported to be a potent mitogen for Swiss 3T3 cells made quiescent by growth to saturation density in high serum, and its activity is further potentiated by high levels of insulin, which alone have little effect. We show here that this is not the case for sparse 3T3 cells made quiescent by plating in low serum. Under these conditions, insulin alone is at least as effective as PDGF and frequently more so. Together, the response is no more than additive at best, and in many cases less than additive, the combined effect being no greater than for insulin alone. Instead, we find that optimal mitogenic stimulation requires the additional presence, besides PDGF and insulin, of hydrocortisone, prostaglandin Ei and an unidentified, non-dialysable component contained in serum treated with dithiothreitol (DTT) to inactivate endogenous growth factors. Interestingly, overnight pretreatment of the cells with hydrocortisone alone potentiates the subsequent response to PDGF+insu-lin, i.e. pretreatment induces a long-term memory that persists after the removal of the hydrocortisone from the medium. In short-term (24 h) thymidine incorporation assays, the combination of PDGF, insulin, hydrocortisone, prostaglandin E, and DTT-serum, is as effective as optimal levels of whole serum, but is unable to sustain longer-term proliferation (measured over 6 days). For this, high-and low-density lipoproteins, fibronectin and, to some extent epidermal growth factor (EGF), are also necessary.
Kinetic analysis of mitotic spindle elongation in vitro
Authors: Baskin Tobias I. , Cande W. Z. 
Published: April 2021

ABSTRACT
Studies of mitotic spindle elongation in vitro using populations of diatom spindles visualized with immunofluorescence microscopy have shown that the two interdigitating half-spindles are driven apart by an ATP-dependent process that generates force in the zone of overlap between half-spindles. To characterize further the system responsible for spindle elongation, we observed spindle elongation directly with polarized light or phase-contrast video-microscopy. We report that the kinetics of spindle elongation versus time are linear. A constant rate of spindle elongation occurs despite the continuous decrease in length of the zone of overlap between half-spindles. The average rate of spindle elongation varies as a function of treatment, and rates measured match spindle elongation rates measured in vivo. When spindles elongated in the presence of polymerizing tubulin (from bovine brain), the extent of elongation was greater than the original zone of halfspindle overlap, but the rate of elongation was constant. No component of force due to tubulin polymerization was found. The total elongation observed in the presence of added tubulin could exceed a doubling of original spindle length, matching the elongation in the intact diatom. The linear rate of spindle elongation in vitro suggests that the force transducer for anaphase B is a mechanochemical ATPase, analogous to dynein or myosin, and that the force for spindle elongation does not arise from stored energy, e.g. in an elastic matrix in the midzone. Additionally, on the basis of observations described here, we conclude that the force-transduction system for spindle elongation must be able to remain in the zone of microtubule overlap during the sliding apart of half-spindles, and that the transducer can generate force between microtubules that are not strictly antiparallel.
Mitotic and meiotic spindles from two insect orders, lepidoptera and diptera, differ in terms of microtubule and membrane content
Authors: Wolf Klaus Werner 
Published: April 2021

ABSTRACT
Spindles from the gonads of five insect species were examined after conventional preparation for electron microscopy. The aim of the study was to determine (1) the range of variation of the spindle membranes between mitotic and meiotic cells and (2) the correlation of possible differences with the microtubule content of the spindles. The study involved four moth species, Ephestia kuebniella, Pbragmato-bia fuliginosa, Orgyia tbyellina, Orgyia antiqua, and one fly, Megaselia scalaris. Somatic and gonial mitoses in all species examined showed a sparse spindle membrane inventory. In contrast, spermatocytes consistently had a multi-layered spindle envelope. In spermatocytes of all Lepidoptera species examined, but not in those of M. scalaris, diverse forms of intraspindle membranes existed in addition to the spindle envelope. Microtubule counts in serially cross-sectioned spindles of E. kuebniella revealed an about 6-fold increase in the mass of polymerized tubulin during the transition from spermatogonia to primary spermatocytes. The increase was 3.3-fold in O. tbyellina and less than 3-fold in Af. scalaris. The density of intraspindle membranes in E. kuebniella was higher than in O. tbyellina by fac tors of 1.8 to 3.0. The correlation between the amount of spindle membranes and the microtubule content of the spindle indicates a functional relationship. Spindle membranes are believed to influence microtubule stability via the regulation of the Ca2+ concentration within the spindle area. The high microtubule mass in spindles from Lepidoptera spermatocytes may result from the membrane-dependent lowering of the Ca2+ level within the spindles. Finally, an unconventional idea on the role of intraspindle membranes is offered. This concept is not intended to challenge the function of spindle-associated membranes as Ca2+-sequestrating compartments. Intraspindle membranes are considered as stuffing material in sheathed spindles. Membranous compartments reduce the free volume within the spindle. Thereby, monomeric tubulin is concentrated and the formation of microtubules is favoured.
Positional order and cellular handedness
Authors: Frankel Joseph 
Published: April 2021

ABSTRACT
A major problem in thinking about asymmetrical patterns in organisms is that of obtaining ‘big hands from little hands’ (Harrison, 1979). The big hands are our own, whereas the little hands are asymmetrical organic molecules. Since the molecules that make up our left and right hands are of identical asymmetry, the different handedness of these big hands must be specified at some more complex level of organization. For Harrison (1979) and, more recently, Brown and Wolpert (1990), the link between the levels is a tethered macromolecule or macromolecular aggregate, implying a pre-existing structural system within which the tethering takes place.
Tailless keratins assemble into regular intermediate filaments in vitro
Authors: Hatzfeld Mechthild , Weber Klaus 
Published: April 2021

ABSTRACT
To study the influence of the non a-helical tail domain of keratins in filament formation, we prepared a truncated keratin 8 mutant, K8/tailless. Using site-directed in vitro mutagenesis we introduced a stop codon in the position coding for amino acid number 417 of the K8/wild-type sequence, thereby deleting 86 amino acids of the non α-helical tail domain but leaving the consensus sequence at the end of the rod domain intact. Expression of the truncated keratin 8 in Escherichia coli allowed us to purifiy the protein by a two-step procedure.
The filament–forming capacity of the truncated K8 with wild–type K18 and K19 was analyzed using in vitro reconstitution. The in vitro assembly studies with K8/tailless and K18 wild-type indicate that the C-terminal tail domain of a type II keratin, including the homologous subdomain H2, is not required for filament formation. Moreover, reconstitution experiments with K8/tailless and K19, a naturally occuring tailless keratin I, show that the tail domains of type I as well as type II keratins are not an essential requirement for in vitro filament formation. Our results suggest that in vitro filament elongation does not depend on interactions between head and tail domains, although the tail domain might have a role in stabilization of intermediate filaments arising from certain keratin pairs.
Senescent human diploid fibroblasts are able to support DNA synthesis and to express markers associated with proliferation
Authors: Kill Ian R. , Shall Sydney 
Published: April 2021

ABSTRACT
The characteristic limited reproductive life-span of normal human fibroblasts in culture is due to a steadily decreasing fraction of cells able to proliferate in the standard rich growth media. We have observed that restricting the growth factor supply to old cells for variable lengths of time in culture increases the fraction of cells that can enter S-phase; although these cells do not go on to divide. Thus, it seems that there is a transient phase between the proliferating state and the irreversibly post-mitotic,senescent state. Perhaps a ‘quiescent-G0’ state, which can be maintained in the presence of growth factors, is a stage on the pathway to mortalization and senescence.
Structure of a Plasmodium yoelii gene-encoded protein homologous to the Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum
Authors: Murakami Kenji , Tanabe Kazuyuki , Takada Suehisa 
Published: April 2021

ABSTRACT
A cation-transporting ATPase gene of Plasmodium yoelii was cloned from the parasite genomic library using an oligonucleotide probe derived from a conserved amino acid sequence of the phosphorylation domain of the aspartyl phosphate family of ATPases. The complete nucleotide sequence was determined and it predicts a 126717Afr encoded protein composed of 1115 amino acids. Northern blot analysis revealed that the gene is transcribed during the asexual stages of parasite development. The P. yoelii protein contains functional and structural features common to the family of aspartyl phosphate cation-transporting ATPases. The parasite protein shows the highest overall homology in amino acid sequence (42%) to the Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum. Homologies to other aspartyl phosphate cation-transporting ATPases including a plasma membrane Ca2+-ATPase were between 13 and 24 %. The structure predicted from a hydropathy plot also shows 10 transmembrane domains, the number and location of which correlated well with the sarcoplasmic reticulum Ca2+- ATPase. On the basis of these results, we conclude that the parasite gene encodes an organellar, but not plasma membrane, Ca2+-ATPase. The P. yoelii protein, furthermore, contains all six amino acid residues in the transmembrane domains that were recently identified as comprising a high-affinity Ca2+-binding site. It follows that organellar Ca2+- ATPases of rabbit and Plasmodium conserve functionally important amino acid residues, even though they are remote from each other phylogenetically.