bluuebunny/crossref_metadata_2025_split huggingface dataset:
Okadaic acid induces golgi apparatus fragmentation and arrest of intracellular transport
Authors: Lucocq J. , Warren G. , Pryde J. 
Published: April 2021

ABSTRACT
The specific phosphatase inhibitor okadaic acid (OA) induced fragmentation of the Golgi apparatus in interphase HeLa cells. Immunoelectron microscopy for galactosyltranferase identified a major Golgi fragment composed of a cluster of vesicles and tubules that was morphologically indistinguishable from the ‘Golgi cluster’ previously described in mitotic cells. The presence of homogeneous immunofluorescence staining for galactosyltransferase in OA-treated cells also suggested that isolated Golgi vesicles, previously found in mitotic cells, existed along with the clusters. After removal of OA, both clusters and vesicles appeared to participate in a reassembly pathway that strongly resembled that occurring during telophase.
OA also induced inhibition of intracellular transport, another feature of mitotic cells. OA treatment prevented newly synthesised G protein of vesicular stomatitis virus (VSV) from acquiring resistance to endoglycosidase H and from arriving at the cell surface. In addition, fluid phase endocytosis of horseradish peroxidase (HRP) was reduced to less than 10% of control values. All these effects were dose-dependent and reversible.
OA should be a useful tool to study the Golgi division and membrane traffic.
Human immunodeficiency virus type 1 protease microinjected into cultured human skin fibroblasts cleaves vimentin and affects cytoskeletal and nuclear architecture
Authors: Hüner B. , Shoeman R. L. , Traub P. 
Published: April 2021

ABSTRACT
In human skin fibroblasts microinjected with purified human immunodeficiency virus type 1 protease (HIV-1 PR), stress fibers were lost and alterations in nuclear morphology and condensation of nuclear chromatin were observed. Thereafter, the vimentin intermediate filament (IF) network collapsed. No effect was seen on the microtubules. While complicated by loss of affected cells from the substratum, a minimum estimate of the proportion of cells demonstrating these effects is 50%. Observation of single cells demonstrated that these effects were largely irreversible and were steps leading to the death of the HIV-1 PR-injected cells. After microinjection of various dilutions of the HIV-1 PR, it was observed that the changes in nuclear morphology and chromatin condensation were detectable under conditions where little or no effect was observed on both stress fibers and the IF network. Proteins of cells labelled with [35S]methionine and microinjected with either HIV-1 PR or BSA were subjected to two-dimensional gel electrophoresis. The major differences in the gel patterns were a diminution in the amount of vimentin and the appearance of novel products comigrating with cleavage products obtained after treatment of vimentin with HIV-1 PR in vitro. Thus, the HIV-1 PR is capable not only of cleaving IF subunit proteins in vivo, but also can catalyze alterations in other cellular structures.
Nature and origin of gap filaments in striated muscle
Authors: Trombitás K. , Baatsen P. H. W.W. , Kellermayer M. S. Z. , Pollack G. H. 
Published: April 2021

ABSTRACT
Immunoelectron microscopy was used to study the nature and origin of ‘gap’ filaments in frog semiten-dinosus muscle. Gap filaments are fine longitudinal filaments observable only in sarcomeres stretched beyond thick/thin filament overlap: they occupy the gap between the tips of thick and thin filaments. To test whether the gap filaments are part of the titin-filament system, we employed monoclonal antibodies to titin (T-ll, Sigma) and observed the location of the epitope at a series of sarcomere lengths. At resting sarcomere length, the epitope was positioned in the I-band approximately 50 nm beyond the apparent ends of the thick filament. The location did not change perceptibly with increasing sarcomere length up to 3.6 μm. Above 3.6μm, the span between the epitope and the end of the A-band abruptly increased, and above 4 μm, the antibodies could be seen to decorate the gap filaments. Between 5 and 6μm, the epitope remained approximately in the middle of the gap. Even with this high degree of stretch, the label remained more or less aligned across the myofibril. The abrupt increase of span beyond 3.6 μm implies that the A-band domain of titin is pulled free of its anchor points along the thick filament, and moves toward the gap. Although this domain is functionally inextensible at physiological sarcomere length, the epitope movement in extremely stretched muscle shows that it is intrinsically elastic. Thus, the evidence confirms that gap filaments are clearly part of the titin-filament system. They are derived not only from the I-band domain of titin, but also from its A-band domain.
Activation of Dona sperm motility: phosphorylation of dynein polypeptides and effects of a tyrosine kinase inhibitor
Authors: Dey Chinmoy S. , Brokaw Charles J. 
Published: April 2021

ABSTRACT
A high molecular mass dynein ATPase polypeptide and a 18-20 kDa dynein light chain of Ciona sperm flagella are phosphorylated during in vivo activation of motility or in vitro activation of motility by incubation with cyclic AMP. A similar level of phosphorylation of these proteins is obtained by incubation of washed, demembranated spermatozoa with catalytic subunit of cyclic AMP-dependent protein kinase, under conditions where there is no activation of motility until a supernatant component is added. Therefore, phosphorylation of these dynein polypeptides is not sufficient for activation of motility. Activation of motility in vitro by incubation with cyclic AMP can be completely inhibited by a random copolymer of glutamate and tyrosine that inhibits tyrosine kinase activity. Under these conditions, much of the protein phosphorylation associated with activation of motility is also inhibited. These new results suggest that regulation of motility of these spermatozoa may involve a multicomponent kinase cascade rather than a simple phosphorylation of a protein ‘switch’ by the cyclic AMP-dependent kinase. A 53 kDa axonemal phosphoprotein band, identified as band Ml, shows the strongest correlation with activation of motility in these experiments.
Transfection of the human muc 1 mucin gene into a poorly differentiated human pancreatic tumor cell line, pancl: Integration, expression and ultrastructural changes
Authors: Batra Surinder K. , Kern Horst F. , Worlock Andrew J. , Metzgar Richard S. , Hollingsworth Michael A. 
Published: April 2021

ABSTRACT
Full-length cDNA for the human mucin Muc 1 gene under the control of the β actin promoter was transfected into a morphologically poorly differentiated pancreatic tumor cell line, Pane 1, by the DEAE-dextran method. Integration of the foreign Muc 1 cDNA occurred at multiple sites in the genome of Pane 1. Northern blot analysis showed Muc 1 expression in cells transfected with the Muc 1 cDNA, but not in control cells transfected with vector alone or an antisense Muc 1 cDNA construct. Transfection of Pane 1 with Muc 1 cDNA did not cause any detectable alteration or rearrangements in the Muc 1 gene or cDNA. Western blot analysis of cell lysates from the transfected lines using a monoclonal anti body reactive with the Muc 1 protein (HMFG-2) demonstrated that Muc 1 protein expression correlated with the Northern blot data. Immunoperoxidase staining using HMFG-2 showed that Muc 1 protein was expressed in less than 5% of control Pane 1 cells, whereas greater than 95% of cells transfected with Muc 1 cDNA expressed the protein. Ultrastructural examination of Muc 1-transfected cells demonstrated the formation of dense core granules and increased amounts of rough endoplasmic reticulum.
How individual cells develop from a syncytium: merogony in Theileria parva (Apicomplexa)
Authors: Shaw Michael K. , Tilney Lewis G. 
Published: April 2021

ABSTRACT
The central problem for Theileria parva during merogony is how to form numerous individual, uninucleate merozoites from a syncytial schizont so that each merozoite contains a single nucleus and a prescribed assortment of organelles. The way T. parva packages all the requisite organelles into free merozoites is by binding these organelles to the nuclear envelope, which in turn becomes associated, both directly and through the rhoptry complex, with the schizont plasma membrane. Formation of the merozoites occurs In a synchronous manner by a budding process. The merozoites develop with the rhoptry complex at the apical end by the progressive, outward évagination of the schizont plasma membrane. This évagination of the plasma membrane is associated with, and presumably induced by, the development of an orderly array of tubules that originate from the apical end and progressively form a longitudinal basket enclosing first the rhoptry complex, then the mitochondria and ribosomes, and finally the nucleus.
The process of merogony is compared to sporogony within the tick salivary gland and with the differentiation of the intra-erythrocytic piroplasm stage. Because all three processes occur by a morphologically similar mechanism, the possibility that the parasite uses a single cassette of genes to perform each of these similar processes is discussed.
Immunolocalization of cytoplasmic dynein to lysosomes in cultured cells
Authors: Lin Sharron X. H. , Collins Christine A. 
Published: April 2021

ABSTRACT
Polyclonal antisera have been raised against cytoplasmic dynein purified from calf brain and rat testis. These antibodies reacted most strongly with the 74 kDa dynein intermediate chain, but also recognized the 410 kDa heavy chain, and the 150 and 45 kDa polypeptides previously observed to copurify with cytoplasmic dynein from rat tissues. Localization studies were performed by indirect immunofluorescence microscopy using a fibroblast cell line. Dynein-specific staining appeared vesicular, distributed throughout the cell, but more concentrated near the nucleus. Double-labeling studies using fluorescent markers for membranous organelles indicated a co-localization of dynein with lysosomes. The distribution of the dynein-positive lysosomes was disrupted by treatment of the cells with microtubule-active drugs, and by acidification of the cytoplasm. Comparison of the distribution of lysosomes with peripheral microtubules indicated a high degree of coincidence. These results are consistent with the hypothesis that cytoplasmic dynein is involved in retrograde-directed movement of membranous organelles. In mitotic cells, dynein staining was also apparent along the microtubules of the mitotic apparatus, though vesicular staining was still conspicuous. The presence of dynein on vesicles as well as on spindle microtubules indicates that dynein distribution between these compartments may be regulated by distinct binding proteins.
Parental genome separation in reconstructions of somatic and premeiotic metaphases of hordeum vulgare × h. bulbosum
Authors: Schwarzacher T. , Heslop-Harrison J. S. , Anamthawat-Jónsson K. , Finch R. A. , Bennett M. D. 
Published: April 2021

ABSTRACT
A stable interspecific sexual plant hybrid between Hordeum vulgare cv. Tuleen 346 (barley) × H. bulbosum was shown to have seven chromosomes originating from each parent by genomic in situ hybridization. Electron microscope serial thin-section reconstructions of metaphases and comparison with light micrograph karyotypes enabled chromosomes to be identified from their morphology. The three-dimensional positions of their centromeres were established and analysed in the reconstructions of somatic root tip metaphases and cells at mitotic metaphase near their entry into meiosis. Parental genomes tended to lie in spatially separated domains in both tissues. Although varying in morphology, the two sets of chromosomes had similar mean sizes, so size differences did not cause the separation observed. In the EM, the centromere-associated structures of the chromosomes of the more central genome, originating from H. vulgare, were larger than those of the more peripheral genome of H. bulbosum origin.
The development of a method for the preparation of rat intestinal epithelial cell primary cultures
Authors: Evans G. S. , Flint N. , Somers A. S. , Eyden B. , Potten C. S. 
Published: April 2021

ABSTRACT
We describe a reproducible method for growing small intestinal epithelium (derived from the suckling rat intestine) in short-term (primary) cultures. Optimal culture conditions were determined by quantitative assays of proliferation (i.e. changes in cellularity and DNA synthesis). Isolation of the epithelia and, significantly, preservation of its three-dimensional integrity was achieved using a collagenase/dispase digestion technique. Purification of the epithelium was also facilitated by the use of a simple differential sedimentation method.
The results presented below support the idea that proliferation of normal gut epithelium ex vivo is initially dependent upon the maintenance of the structural integrity of this tissue and upon factors produced by heterologous mesenchymal cells. Proliferation in vitro was also critically dependent upon the quality of the medium and constituents used.
Cultures reached confluence within 10–14 days and consisted of epithelial colonies together with varying amounts of smooth-muscle-like cells. Cultures have been maintained for periods up to one month, but the longer-term potential for growth by sub-culturing has not been examined. Strategies for reducing the proliferation of these non-epithelial cells are also described.
Non-muscle myosin isoforms and cell heterogeneity in developing rabbit vascular smooth muscle
Authors: Giuriato Luca , Scatena Marta , Chiavegato Angela , Tonello Marta , Scannapieco Gianluigi , Pauletto Paolo , Sartore Saverio 
Published: April 2021

ABSTRACT
A panel of monoclonal antibodies specific for cytoskeletal and cytocontractile protein markers has been used to study the expression of vimentin, desmin and α-smooth muscle (SM) actin, as well as non-muscle (NM) and SM myosin isoforms, in developing rabbit aorta. Immunofluorescence experiments show that in the vascular smooth muscle cells (SMC): (1) vimentin and α-actin of SM-type are homogeneously expressed among SMC, since the early stage (day 19, in uterus) of development; (2) desmin is heterogeneously distributed throughout all the developmental stages examined (from day 19, foetal, to day 90, post-natal); and (3) myosin isoform content in pre- and post-natal vascular SM is different when analyzed by anti-SM myosin (SM-E7) and anti-NM myosin (NM-F6, NM-A9 and NM-G2) antibodies. SM myosin in vascular SM is present as early as day 19 in uterus, being especially evident in the region facing the lumen of aortic wall, but not in the outermost layer in which NM myosin is present exclusively. Western blotting and immunofluorescence assays indicate that the foetal aortic SM is specifically labeled by all the three anti-NM myosin antibodies. However, immunoreactivity of aortic SM with NM-F6 and NM-A9 disappears completely around birth. Conversely, NM-G2 binding is maintained during post-natal development up to day 45; between day 45 and day 90 immunoreactivity of aortic SMC with this antibody diminished progressively, without disappearing, in a small number of cells. In aortic SMC cultures from foetal and adult rabbits, NM myosin immunoreactivities appear to be differently distributed, i.e. according to the stress fiber system (NM-F6 and NM-G2), in a diffuse manner (NM-A9) or mainly localized at the level of the cortical cytoplasm (NM-G2).
The fact that a different pattern of NM myosin antigenicity can also be shown in other cell types, such as in the endothelium and the cardiac pericytes as well as in the renal parenchyma, is consistent with the existence of multiple NM myosin in vascular SM isoforms whose expression is developmentally regulated.
DNA replication in cell-free extracts from Xenopus eggs is prevented by disrupting nuclear envelope function
Authors: Cox Lynne S. 
Published: April 2021

ABSTRACT
The lectin, wheat germ agglutinin (WGA), has previously been shown to prevent transport into the cell nucleus. This paper shows that WGA also inhibits nuclear DNA replication, under the same conditions that prevent transport. Although WGA eliminates sperm nuclear DNA replication in a cell-free extract of Xenopus eggs, DNA synthesis on a single-stranded template proceeds normally. Inhibition of nuclear DNA replication is partially reversed by addition of A’-acetylgluco-samine, and completely reversed by triacetylchitotriose. Sensitivity to inhibition by WGA is greatest during the nuclear assembly phase, and nuclear formation on sperm chromatin is blocked. DNA replication in preformed nuclear templates is also sensitive to WGA inhibition. I propose that WGA blocks DNA replication by preventing nuclear transport. The data presented here also indicate that, under certain circumstances, the elongation stage of DNA replication does not proceed in the absence of an intact nuclear envelope. The roles of the nuclear envelope and active nuclear transport in DNA replication are discussed.
DNA-binding domain of RCC1 protein is not essential for coupling mitosis with DNA replication
Authors: Seino Hiroaki , Hisamoto Naoki , Uzawa Satoru , Sekiguchi Takeshi , Nishimoto Takeharu 
Published: April 2021

ABSTRACT
The RCC1 protein that is required for coupling mitosis with the S phase has a DNA-binding domain in the N- terminal region outside the repeat. We found that RCC1 protein without any DNA-binding activity complemented the tsBN2 mutation with the same efficiency as that of intact RCC1 protein. In ts+ transformants of tsBN2 cells transfected with the RCC1 cDNA lacking the DNA-binding domain, an endogenous RCC1 disappeared at 39.5°C, and the deleted RCC1 protein encoded by the transfected cDNA was found in the cytoplasm, but a significant amount of it was also found in the nuclei. This deleted RCC1 protein was eluted from the nuclei with the same concentration of NaCl and DNase I as was used for the intact RCC1 protein in BHK21 cells. Furthermore, the deleted RCC1 protein co-migrated with the nucleosome fraction on sucrose density gradient analysis. These results indicate that the RCC1 protein binds chromatin with the aid of other unknown protein(s). Thus, the DNA-binding domain of RCC1 protein is not essential for coupling between the S and M phases, but was shown instead to function as a nuclear translocation signal.
Antagonistic effects of TGF-β1 and MSF on fibroblast migration and hyaluronic acid synthesis: Possible implications for dermal wound healing
Authors: Ellis Ian , Grey Ann Marie , Schor Ana M. , Schor Seth L. 
Published: April 2021

ABSTRACT
The migration of adult skin fibroblasts into threedimensional collagen gel matrices is differentially affected by cell density, with subconfluent cells displaying a significantly elevated level of migration compared to confluent ones. Fetal fibroblasts differ from adult cells in that they display an elevated level of migration at both subconfluent and confluent cell densities. We have previously reported that this difference in behaviour results from the secretion by fetal fibroblasts of a ‘migration stimulating factor’ (MSF) which is not made by their normal adult counterparts, and that MSF appears to act by stimulating the synthesis of hyaluronic acid (HA). Data presented in this communication indicate that (a) MSF specifically stimulates the synthesis of high molecular weight species of HA, (b) TGF-β inhibits the elevated migration of adult fibroblasts plated at subconfluent cell density, (c) under these conditions, TGF-β1induces a parallel decrease in the synthesis of high molecular weight HA and increase in the synthesis of low molecular weight HA, (d) TGF-β1 is a potent antagonist of MSF, effectively blocking its stimulation of cell migration and synthesis of high molecular weight HA, and (e) the inhibition of fibroblast migration by TGF-β1 does not appear to be a chemotactic response dependent upon the existence of a concentration gradient of the cytokine. Our observations regarding the inhibitory effects of TGF-β1 on fibroblast migration into 3D collagen gels stand in marked contrast to various published reports indicating that this cytokine stimulates the migration of human skin fibroblasts through the pores of polycarbonate filters as used in modified Boyden chamber assays; this discrepancy underscores the importance of the substratum in modulating cellular response to cytokines. Our results are discussed in terms of the possible combined contribution of MSF and TGF-β1 to wound healing.
Cell cycle modification during the transitions between meiotic M-phases in mouse oocytes
Authors: Kubiak J. Z. , Weber M. , Géraud G. , Maro B. 
Published: April 2021

ABSTRACT
When metaphase Il-arrested mouse oocytes (M II) are activated very soon after ovulation, they respond abortively by second polar body extrusion followed by another metaphase arrest (metaphase III, M III; Kubiak, 1989). The M Il/M III transition resembles the natural transition between the first and second meiotic metaphases (M 1/M II). We observed that a similar sequence of events takes place during these two transitions: after anaphase, a polar body is extruded, the microtubules of the midbody disappear rapidly and a new metaphase spindle forms. The MPM-2 monoclonal antibody (which reacts with phosphorylated proteins associated with the centrosome during M-phase) stains discrete foci of peri-centriolar material only in metaphase arrested oocytes; during both transitional periods, a diffuse staining is observed, suggesting that these centrosoma! proteins are dephosphorylated, as in a normal interphase. However, the chromosomes always remain condensed and an interphase network of microtubules is never observed during the transitional periods. Incorporation of 32P into proteins increases specifically during the transitional periods. Pulse-chase experiments, after labeling of the oocytes in M phase with 32P, showed that a 62 kDa phosphoprotein band disappears at the time of polar body extrusion. Histone Hl kinase activity (which reflects the activity of the maturation promoting factor) drops during both transitional periods to the level characteristic of interphase and then increases when the new spindle forms. Both the M 1/M II and M Il/M III transitions require protein synthesis as demonstrated by the effect of puromycin. These results suggest that the two M-phase/M-phase transitions are probably driven by the same molecular mechanism.
Constitutive secretion of a bacterial enzyme by polarized epithelial cells
Authors: Soole Kathleen L. , Hall Judith , Jepson Mark A. , Hazlewood Geoffrey P. , Gilbert Harry J. , Hirst Barry H. 
Published: April 2021

ABSTRACT
The constitutive (or default) pathway for protein secretion was investigated in two epithelial cells, Madin-Darby canine kidney (MDCK) and human colonic adenocarcinoma (Caco-2), using a bacterial enzyme. The choice of a bacterial protein was based on the requirement to identify a protein devoid of sorting signals. The sorting of a bacterial endoglucanase derived from Clostridium thermocellum, endoglucanase E, from stably transfected MDCK and Caco-2 cells was examined. The choice of a bacterial endoglucanase for these studies has advantages of simple, sensitive and quantitative detection, while higher eukaryotic cells do not express endoglucanase activity. Both cell lines secreted a 50 kDa form of the bacterial protein, while smaller intracellular forms were also observed. In polarized layers of MDCK cells the endoglucanase was secreted into both membrane domains in the ratio 62% apical and 38% basolateral. In Caco-2 cells secretion was predominantly, 70%, through the basolateral membrane. These results define the constitutive pathway for protein secretion in these two model epithelial cells.