bluuebunny/crossref_metadata_2025_split huggingface dataset:
Preferential mitochondrial and plastid dna synthesis before multiple cell divisions in Nicotiana tabacum
Authors: Suzuki Takeshi , Kawano Shigeyuki , Sakai Atsushi , Fujie Makoto , Kuroiwa Haruko , Nakamura Hiromu , Kuroiwa Tsuneyoshi 
Published: April 2021

ABSTRACT
Organelle DNA synthesis in root meristem and cultured cell line BY-2, both derived from Nicotiana tabacum cv. Bright Yellow 2, was examined by immunofluorescence microscopy of Technovit sections with antibody against 5-bromodeoxyuridine (BrdU) and co-fluorescent staining with 4′,6-diamidino-2-phenylindole (DAPI) and quantitative Southern hybridization. In the root meristem, the mitochondrial DNAs (mtDNAs) were synthesized in a specific region near to the quiescent center, where a low frequency of DNA synthesis of cell nuclei was observed. The mitochondrial nuclei (nucleoids) changed morphologically from long ellipsoids with a high frequency of DNA synthesis, in the region just above the quiescent center, to granules with a low frequency of DNA synthesis, as cell distance from the quiescent center increased. Similar patterns were observed in the cultured tobacco cell line (BY-2), in which large amounts of preferential synthesis of DNA of both mitochondria and plastids occurred prior to cell nuclear DNA synthesis just after stationary phase cells were transferred to fresh medium. Granular mitochondria which vigorously synthesized mtDNA were observed in both lag phase and logarithmic growth phase cells. However, long, ellipsoidal mitochondria which showed a low frequency of mtDNA synthesis were observed in stationary phase cells. Morphological changes of plastids were more conspicuous than those of mitochondria. After the medium was renewed, spherical plastids became extremely elongated and string-like, for 24 h, but were divided into small pieces after the third day. Vigorous synthesis of plastid DNA (ptDNA) occurred during this period of plastids elongation.
Expression of wild-type and nuclear localization-deficient human lamin a in chick myogenic cells
Authors: Lourim David , Lin Jim Jung-Ching 
Published: April 2021

ABSTRACT
Previous analysis of chick embryonic muscle (CEM) differentiation in vivo and in ovo demonstrated that lamin A accumulation to steady-state levels preceded the accumulation of muscle-specific proteins. These observations have suggested the appearance of A-type lamins may be important for differentiation. To test this hypothesis, we have temporally and quantitatively altered the expression of A-type lamins in CEM cells by transient transfection of wild-type (wt; pHLA) or nuclear localization-deficient (NLd; pHLA-del) human lamin A expression plasmids. Transfected CEM cells synthesized the wt and NLd human lamin As to high levels, both of which were resistant to high-salt extraction. The wt human lamin A localized to the nucleus, whereas the NLd protein showed cytoplasmic staining patterns, as well as time-dependent nuclear localization. The presence of endogenous chicken lamins A and B2 in NLd human lamin A cytoplasmic structures suggested the interspecies lamin copolymerization. Thus, this approach may provide a possible method for analysis of lamin-lamin or lamin-lamina component interactions in vivo. With regard to muscle differentiation, CEM cells transfected with either pHLA or pHLA-del demonstrated moderate and transient increased levels of the muscle-specific myosin heavy chain and creatine kinase activity. These increases appeared temporally and quantitatively to reflect the transient accumulation of the human lamin As. In contrast, β-tubulin and skeletal tropomyosin protein accumulations appeared unaffected. On the basis of these results, we suggest that nuclear lamina content and structure may play a limited, permissive and indirect role in the temporally regulated expression of the myogenic phenotype.
Flagellar, cellular and organismal polarity in Volvox carteri
Authors: Hoops Harold J. 
Published: April 2021

ABSTRACT
It has previously been shown that the flagellar apparatus of the mature Volvox carteri somatic cell lacks the 180° rotational symmetry typical of most unicellular green algae. This asymmetry has been postulated to be the result of rotation of each half of the flagellar apparatus. Here it is shown that V. carteri axonemes contain polarity markers that are similar to those found in Chlamydomonas, except that in V. carteri the number one doublets do not face each other as they do in Chlamydomonas but are oriented in parallel and at approximately right angles to the line that connects the flagella. Thus, the rotational orientations of the axonemes are consistent with the postulate that the flagella of V. carteri have rotated in opposite directions, as was predicted earlier from the positions of the basal fibers and microtubular rootlets. Moreover, high-speed cinephotomicrographic analysis shows that the V. carteri flagellar effective strokes are also oriented in approximately the same direction, and in parallel planes. These results suggest that the direction of the effective stroke in both Chlamydomonas and Volvox is fixed, and that rotation of the axoneme is the cause of the differences in flagellar motility observed between Chlamydomonas and Volvox. These differences are probably essential for effective organismal motility. Cellular polarity of V. carteri can be related to that of Chlamy domonas after taking into account the developmental reorientation of flagellar apparatus components. This reorientation also results in the movement of the eyespot from a position nearer one of the flagellar bases to a position approximately equidistant between them. By analogy to Chlamydomonas, the anti side of the V. car teri somatic cell faces the spheroid anterior, the syn side faces the spheroid posterior. The cis side of the cell is to the cell’s left (the right to an outside observer), although it cannot be described solely on the basis of eyespot position as it can in Chlamydomonas, while the trans side is to the cell’s right. It follows that if the direction of the effective flagellar stroke is specified by structural features, then effective organismal motility in V. carteri, will be accomplished only if the cells are held in the proper orientation with respect to one another. The simplest arrangement that will yield both progression and rotation in ovoid or spherical colonies composed of biflagellate isokont cells is one in which the cells are arranged with rotational symmetry about the anteriorposterior axis of the spheroid. Analysis of the polarity of somatic cells from throughout the spheroid shows that it is constructed with just such symmetry. This symmetry probably originates with the very first divisions.
A survey of interactions made by the giant protein titin
Authors: Soteriou A. , Gamage M. , Trinick J. 
Published: April 2021

ABSTRACT
A simple solid-phase binding assay was used to screen for interactions that the giant myofibrillar protein titin makes with other sarcomeric proteins. The titin used in the tests was purified by a modified procedure that results in isolation of ∼20 mg relatively undegraded protein in &lt;24 h. In addition to the ∼3 MDa polypeptide, bands at ∼160 kDa and ∼100 kDa were also consistently seen on gels. Binding of titin to myosin, C-protein, X-protein and AMP-deaminase was observed. The interaction with myosin appears to be with the light meromyosin part of the molecule.
Microinjection of a monoclonal antibody against SPN antigen, now identified by peptide sequences as the NuMA protein, induces micronuclei in PtK2 cells
Authors: Kallajoki Markku , Harborth Jens , Weber Klaus , Osborn Mary 
Published: April 2021

ABSTRACT
Several high molecular mass proteins which relocate from the interphase nucleus to the spindle poles during mitosis have been defined by antibodies. Microinjection experiments have shown that at least the antigen defined by SPN antibody plays a functional role during mitosis. Recently the cDNA sequence for human NuMA antigen was established and epitopes for antibodies to centrophilin, and to 1F1 and 1H1 antigens were found to be included in the NuMA protein. Here we show that immunoprecipitated SPN antigen reacts with an autoimmune human NuMA serum. In addition three peptides derived from immunoprecipitated human SPN by cyanogen bromide cleavage and covering more than fifty amino acids show a perfect fit with the sequence predicted for NuMA protein. Thus SPN antigen and NuMA are the same protein. Injection of SPN-3 antibody into interphase or mitotic PtK2 cells results in cells with micronuclei. For cells injected in prophase, prometaphase or metaphase 90%, 78% and 77% display defective cytokinesis or yield daughter cells with micronuclei. In contrast only 16% of cells injected in anaphase are abnormal. Thus SPN/NuMA antigen may be required during early, but not during later, stages of mitosis. Surprising parallels are seen between the effects of microinjecting SPN-3 antibody and treatment with colcemid and taxol of PtK2 and HeLa cells. Our results identify an important role during mitosis for the SPN/NuMA antigen.
Lytic granules from cytotoxic T cells exhibit kinesin-dependent motility on microtubules in vitro
Authors: Burkhardt J. K. , McIIvain J. M. , Sheetz M. P. , Argon Y. 
Published: April 2021

ABSTRACT
One major mechanism of cell-mediated cytolysis is the polarized secretion of lytic granules, a process which is highly dependent on microtubules. We isolated lytic granules from murine cytotoxic T cells and tested their ability to bind to and move along microtubules in vitro. In the presence of a motor-containing supernatant, the granules bound to the microtubules and moved along them at an average maximal rate of 1 m/second. Virtually every granule could bind to microtubules, and about half translocated within a few seconds of binding. Motility required exogenous cytosolic motors, hydrolyzable nucleotides, and an intact granule membrane. Although the motor preparation used to support granule movement contains both plus- and minus-enddirected motor proteins, granule movement was strongly biased toward microtubule plus-ends. Inactivation of cytoplasmic dynein had little effect on granule binding and movement, but immuno-depletion of kinesin from the motor preparation inhibited granule binding by 50%. These results indicate that most granule movement in this assay is mediated by kinesin. The speed and direction of granule movement in vitro are sufficient to account for the release of lytic granules in the intact T cell. This model system should be valuable for studying the interactions of secretory granules with microtubules, and for identifying the regulatory factors involved.
Haemopoietic stem cell development to neutrophils is associated with subcellular redistribution and differential expression of protein kinase C subspecies
Authors: Shearman M. S. , Heyworth C. M. , Dexter T. M. , Haefner B. , Owen P. J. , Whetton A. D. 
Published: April 2021

ABSTRACT
Multipotential FDCP-Mix A4 (A4) cells can be induced either to self-renew or to differentiate and develop into mature neutrophils in liquid culture, depending on the haemopoietic growth factors with which they are cultured. When cultured in low concentrations of interleukin 3 (IL-3, 1 unit/ml)) plus Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Granulocyte-CSF (G-CSF), A4 cells proliferate with accompanying development to form cells which resemble mature, postmitotic neutrophils. The presence of high concentrations of IL-3 (100 units/ml) blocks the development of A4 cells even in the presence of GM-CSF plus G-CSF. A4 cell development to neutrophils is accompanied by major changes in the expression of protein kinase C (PKC) subspecies in these cells. The predominant subspecies present in multipotent A4 cells, as judged by direct chromatographic analysis, was the type III enzyme (α) subspecies, whereas in mature A4 cell neutrophils, the type II (βI + βII) enzymes were predominant. Phorbol esters added to immature A4 cells resulted in a proliferative response, but when added to postmitotic A4 cells resembling neutrophils they elicited a large increase in reactive oxygen intermediate production. This suggests that the type III (α) subspecies may mediate proliferative responses in stem cells, whilst the type II (βI + βII) enzymes are more important for the mature cell functions of postmitotic neutrophils. In cultures containing IL-3 (100 units/ml) both the type III, and also the type II subspecies were predominantly membrane-associated for prolonged periods (&gt;24 hours). The addition of IL-3 (100 units/ml) to FDCP-Mix A4 cells starved of haemopoietic growth factors led to the rapid translocation of protein kinase C from the cytosol to the membrane; no such effect was observed with GM-CSF or 1 unit/ml IL-3. Under conditions where differentiation and development were induced (1 unit/ml of IL-3 plus GM-CSF and G-CSF), there was a redistribution of all PKC subspecies to the cytosol from the membrane. Thus, IL-3 preserves the multipotential nature of A4 cells and translocates PKC to the membrane. As GM-CSF cannot stimulate the translocation of protein kinase C, the differential biochemical and developmental effects of these growth factors on multipotent cells may in part be mediated by the activation of protein kinase C.
Chemotactic antagonists of cAMP inhibit Dictyostelium phospholipase C
Authors: Bominaar Anthony A. , Haastert Peter J. M. Van 
Published: April 2021

ABSTRACT
In Dictyostelium discoideum extracellular cAMP induces chemotaxis via a transmembrane signal transduction cascade consisting of surface cAMP receptors, G-proteins and effector enzymes including adenylyl cyclase, guanylyl cyclase and phospholipase C. Previously it was demonstrated that some cAMP derivatives such as 3′-deoxy-3′ -aminoadenosine 3′ :5′ -monophosphate (3 NH-cAMP) bind to the receptor and induce normal activation of adenylyl cyclase and guanylyl cyclase. However these analogues do not induce chemotaxis, probably because the signal is transduced in an inappropriate manner.
We have now studied the regulation of phospholipase C by cAMP and these chemotactic antagonists. cAMP induced the two-fold activation of phospholipase C leading to a transient increase of Ins(1,4,5)P3 levels. In contrast, the analogues induced a rapid decrease of intracellular Ins(1,4,5)P3 levels, due to the inhibition of phospholipase C activity. In a transformed cell-line lacking the G-protein that mediates phospholipase C inhibition, 3′ NH-cAMP did not decrease phospholipase C activity and was no longer an antagonist of chemotaxis. These results suggest that inhibition of phospholipase C leads to aberrant chemotaxis.
cDNA-derived molecular characteristics and antibodies to a new centrosome-associated and G2/m phase-prevalent protein
Authors: Rothbarth Karsten , Petzelt Christian , Lu Xiang , Todorov Ivan T. , Joswig Gaby , Pepperkok Rainer , Ansorge Wilhelm , Werner Dieter 
Published: April 2021

ABSTRACT
Differential screening of a murine RNA-based cDNA library with cell cycle phase-specific transcripts released a cDNA clone (CCD41) to a mRNA (1.349 kb) which, according to the mode of its detection, increases as expected during the cell cycle. The molecular characteristics of the protein (27 103Mr) encoded by this mRNA were deduced from the cDNA sequence and antibodies were prepared against the recombinant protein. Immunofluorescence studies performed with PtK2 cells revealed that the amount of the antigen specified by the CCD41 sequence increases during the cell cycle out of proportion with the DNA content. In G1 phase cells, the antigen is exclusively located at the site of the centrosome. During cell cycle progression the antigen becomes also detectable in perinuclear vesicles that increase in number and size, reaching a maximum in G2 phase cells. The centrosomal location of the CCD41 antigen was investigated in relation to another centrosomal antigen, centrosomin A. Since the latter antigen is detected by a monoclonal antibody reacting specifically and permanently with the centrosomes in PtK2 cells throughout the cell cycle it was possible to investigate the relative positions of the two proteins at the site of the centrosome and to add new information about the general architecture of the organelle and its changes during the cell cycle. While the centrosomin A antibody detects the pronounced cell cycle stage-dependent shape changes of the centrosome, the CCD41-encoded protein appears to be localized as a compact structure inside the centrosome. Its epitopes are exposed throughout the cell cycle except during a brief period immediately after the formation of the daughter centrosome.
A study of the structure, function and distribution of β5 integrins using novel anti-β5 monoclonal antibodies
Authors: Pasqualini Renata , Bodorova Jana , Ye Song , Hemler Martin E. 
Published: April 2021

ABSTRACT
Here, we have utilized six new anti-human β5 mono-clonal antibodies to perform a detailed investigation of the structure, function and distribution of β5 integrins. Monoclonal anti-β5 specificity was confirmed by reac-tivity with β5-transfected CHO cells, by direct binding to the β5 subunit (immunoblotting), and by immunode-pletion experiments using polyclonal anti-β5 serum. The β5 subunit was predominantly associated with the av subunit, although on some cell lines, the level of β5 exceeded that of αv for unknown reasons. Cell adhesion studies showed that the adhesive function of β5 could be stimulated, inhibited or unaltered by different anti-β5 monoclonal antibodies. The β5 subunit was involved in adhesion to both vitronectin and fibronectin and, at least for K562 cells, fibronectin appeared to be the preferred ligand. Flow-cytometry studies showed that the β5 subunit was expressed at moderate to high levels on all adherent cell lines examined, was absent from all lym-phoid cell lines, and was only weakly expressed on myeloid cell lines. Staining of thymic sections showed the distribution of β5 on blood vessels, Hassal’s corpus-cles, cortical and medullary stromal cells, and basement membranes. Skin sections showed β5 on the basal layer of the epidermis and on some dermal blood vessel walls, and kidney sections showed staining of glomerular regions, juxta glomerular apparatus, proximal convo-luted tubules and collecting tubules, and at least one anti-β5 antibody also stained epithelial cells of proximal tubules.
Inhibition of transcription blocks cell cycle progression of NIH3T3 fibroblasts specifically in G1
Authors: Adolph Sabine , Brüsselbach Sabine , Müller Rolf 
Published: April 2021

ABSTRACT
We have analysed the role of RNA polymerase II-depen-dent transcription in cell cycle progression. Time-lapse video recording and cytogenetic analysis were used to determine the sensitivity of NIH3T3 cells to the RNA polymerase II inhibitor -amanitin at different stages of the cell cycle. Our results show that -amanitin blocks cells specifically in G1, irrespective of the concentration within the range of 3 to 30 g/ml. This indicates that transcription in G1 is required to overcome a restriction point located in this phase of the cell cycle. In agree-ment with this conclusion is the requirement for an uninhibited protein synthesis during G1 progression. In addition, the insensitivity of S-phase cells to RNA poly-merase II inhibition suggests that the transcription of genes thought to be normally induced during S/G2 is not required for the completion of an ongoing cell cycle. S/G2 progression was however clearly dependent on protein synthesis. This suggests that cells exposed to -amanitin are able to complete their cell cycle because sufficiently high levels of mRNA are present in S/G2 due to basal level transcription, or are left from preceding cell cycles. It is therefore unlikely that transcriptional regulation in S or G2 plays a crucial role in the control of cell cycle progression in NIH3T3 cells.
Identification of seven new cut genes involved in Schizosaccharomyces pombe mitosis
Authors: Samejima Itaru , Matsumoto Tomohiro , Nakaseko Yukinobu , Beach David , Yanagida Mitsuhiro 
Published: April 2021

ABSTRACT
Fission yeast cut mutants cause cytokinesis in the absence of normal nuclear division. These mutants show abnormal uncoupled mitosis and are known to be the result of mutations in the genes encoding DNA topoiso-merase II, proteins related to spindle pole duplication, and a kinesin-related mitotic motor. We have screened 717 temperature-sensitive (ts) mutants by individually observing their cytological phenotypes at the restrictive temperature, and have newly isolated 25 cut mutants. Genetic analyses indicate that 14 of them fall into five previously identified loci, namely, top2, cut1, cut5, cut7 and cut9, whereas nine have been mapped onto seven new loci, designated cut13 to cut19. The cytological phe-notypes of the newly identified cut mutants can be clas-sified into three groups. One group consists of mutants in which a portion of the nuclear chromatin is stretched by the elongated spindle but the entire nucleus is not separated, reminiscent of, but not identical to, the phe-notypes of top2 and cut1; mutants cut14-208, cut15-85, cut16-267 and cut17-275 display such a phenotype. Another group exhibits non-disjunctioned and con-densed chromosomes in the presence of the spindle; cut13-131 belongs to this group. The cut19-708 mutant has also been found to have condensed chromosomes. The remaining group has a mixed phenotype of the above two groups; namely, stretched chromatin and condensed chromosomes; cut18-447 exhibits such a phenotype. The isolation and characterization of the mutated genes will be the subjects of future investiga-tions.
Stress-relaxation of fibroblasts in collagen matrices triggers ectocytosis of plasma membrane vesicles containing actin, annexins II and VI, and β1 integrin receptors
Authors: Lee Tien-Ling , Lin Ying-Chun , Mochitate Katsumi , Grinnell Frederick 
Published: April 2021

ABSTRACT
To learn about the effects of tension on fibroblast func-tion, we have been studying initial cellular responses to stress-relaxation. Human foreskin fibroblasts were cul-tured in anchored collagen matrices for 2 days, during which time mechanical stress developed. Subsequently, the matrices were dislodged; thereby allowing stress to dissipate. Within 5 min after initiating stress-relaxation, fibroblasts retracted their pseudopodia. At this time, we observed the disappearance of cellular stress fibers and the formation of actin clusters along the cell margins. The actin was found to be located inside 200 nm diam-eter vesicles that were budding from the cell surface. Vesicles isolated from the matrix after stress-relaxation contained prominent 24 kDa, 36 kDa (doublet), 45 kDa, and 135 kDa polypeptides. The 45 kDa polypeptide was the major component in the Triton-insoluble vesicle fraction and appeared to be actin. The 36 kDa (doublet) polypeptide, which was found predominantly in the Triton-soluble vesicle fraction, was identified as annexin II. Vesicles also contained annexin VI and 11 integrin receptors but not tubulin, vimentin, vinculin or annexin I. The results suggest that stress-relaxation of fibrob-lasts induces a novel ectocytotic process involving tran-sient budding of intact, plasma membrane vesicles from the cell cortex. On the basis of their morphological and biochemical features, these vesicles may be analogous to the ‘matrix vesicles’ released by chondrocytes and could play a role in extracellular matrix remodeling after wound contraction.