Comparative effects of phalloidin and cytochalasin B on motility and morphogenesis in Allium

1980 ◽  
Vol 58 (7) ◽  
pp. 773-785 ◽  
Author(s):  
Barry A. Palevitz
Keyword(s):  
Cytochalasin B ◽  
Cell Plate ◽  
Wall Thickening ◽  
Animal Cells ◽  
Host Fungus ◽  
Guard Mother Cells ◽  
Mother Cells ◽  
Chromosome Motion

Cytochalasin B (CB), thought to disaggregate F-actin in animal cells, and phalloidin (Phal), known to stabilize F-actin in vivo and in vitro, have nearly identical effects on cotyledon epidermal cells of Allium cepa. Both drugs rapidly induce cessation of streaming and both, by preventing normal telophase reorientation movement, lead to abnormal division planes in dividing guard mother cells. Neither, however, prevents normal microtubule deposition, wall thickening, and cellulose orientation during guard cell differentiation. Furthermore, both drugs have no effect on spindle formation and anaphase chromosome motion. Examination of Nitella and Chara cells, in which streaming had been stopped by either agent, shows that microfilament cables are still present. With both drugs, the minimum effective concentrations were routinely used (CB, 2 μM; Phal, 100–200 μM). Our results are discussed in terms of the mode of action of these drugs and their possible role in host-fungus interactions. Implications for the mechanisms underlying cell plate alignment, cellulose orientation, and cytoplasmic streaming are discussed.

scholarly journals Metabolic inhibitors block anaphase A in vivo.

1986 ◽  
Vol 102 (6) ◽  
pp. 1995-2005 ◽  
Author(s):  
P K Hepler ◽  
B A Palevitz
Keyword(s):  
Metabolic Inhibitors ◽  
Pole Motion ◽  
Carbonyl Cyanide ◽  
Transport Inhibitors ◽  
Spindle Microtubules ◽  
Guard Mother Cells ◽  
Mother Cells ◽  
Anaphase B ◽  
Chromosome Motion

Anaphase in dividing guard mother cells of Allium cepa and stamen hair cells of Tradescantia virginiana consists almost entirely of chromosome-to-pole motion, or anaphase A. Little or no separation of the poles (anaphase B) occurs. Anaphase is reversibly blocked at any point by azide or dinitrophenol, with chromosome motion ceasing 1-10 min after application of the drugs. Motion can be stopped and restarted several times in the same cell. Prometaphase, metaphase, and cytoplasmic streaming are also arrested. Carbonyl cyanide m-chlorophenyl hydrazone also stops anaphase, but its effects are not reversible. Whereas the spindle collapses in the presence of colchicine, the chromosomes seem to "freeze" in place when cells are exposed to respiratory inhibitors. Electron microscope examination of dividing guard mother cells fixed during azide and dinitrophenol treatment reveals that spindle microtubules are still present. Our results show that chromosome-to-pole motion in these cells is sensitive to proton ionophores and electron transport inhibitors. They therefore disagree with recent reports that anaphase A does not require a continuous supply of energy. It is possible, however, that anaphase does not directly use ATP but instead depends on the energy of chemical and/or electrical gradients generated by cellular membranes.

Factors affecting the time of formation of the mouse blastocoele

Development ◽  
1977 ◽  
Vol 41 (1) ◽  
pp. 79-92
Author(s):  
Rosita Smith ◽  
Anne McLaren
Keyword(s):  
Cytochalasin B ◽  
Critical Factor ◽  
Cell Number ◽  
Experimental Manipulation ◽  
Factors Affecting ◽  
Elapsed Time ◽  
Developmental Age ◽  
Culture Formation

In normal mouse embryos developing in vivo, the first appearance of the blastocyst cavity was found to be associated more closely with developmental age, judged by cell number, than with chronological age, i.e. elapsed time since ovulation. When development was slowed by in vitro culture, formation of the blastocoele was delayed. However, cell number itself was not a critical factor, since the number of cells per embryo could be doubled or tripled or halved by experimental manipulation without substantially affecting the timing of blastocoele formation. Experiments in which one cell division was suppressed with cytochalasin-B, leading to tetraploidy, showed that the number of cell divisions since fertilization was also not critical. A possible role is suggested either for nucleocytoplasmic ratio, or for the number of nuclear or chromosomal divisions or DNA replications since fertilization, all of which increase during cleavage.

scholarly journals Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

2019 ◽  
Author(s):  
Cassandra K. Hayne ◽  
Casey A. Schmidt ◽  
A. Gregory Matera ◽  
Robin E. Stanley
Keyword(s):  
Animal Cells ◽  
Trna Splicing ◽  
Polynucleotide Kinase ◽  
Genes Encoding ◽  
Intron Removal ◽  
And Function ◽  
Insight Into ◽  
Negative Modulator

ABSTRACTThe splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34, and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

scholarly journals Development of A Three-Dimensional Tissue Construct from Dental Human Ectomesenchymal Stem Cells: In Vitro and In Vivo Study

2012 ◽  
Vol 6 (1) ◽  
pp. 226-234 ◽  
Author(s):  
Daniela Guzmán-Uribe ◽  
Keila Neri Alvarado Estrada ◽  
Amaury de Jesús Pozos Guillén ◽  
Silvia Martín Pérez ◽  
Raúl Rosales Ibáñez
Keyword(s):  
Three Dimensional ◽  
Human Tooth ◽  
Animal Cells ◽  
Dental Tissue ◽  
Membrane Markers ◽  
Tissue Organization ◽  
Specific Membrane ◽  
Tissue Construct

Application of regenerative medicine technology provides treatment for patients with several clinical problems, like loss of tissue and its function. The investigation of biological tooth replacement, dental tissue engineering and cell culture, scaffolds and growth factors are considered essential. Currently, studies reported on the making of threedimensional tissue constructs focused on the use of animal cells in the early stages of embryogenesis applied to young biomodels. The purpose of this study was the development and characterization of a three-dimensional tissue construct from human dental cells. The construct was detached, cultured and characterized in mesenchymal and epithelial cells of a human tooth germ of a 12 year old patient. The cells were characterized by specific membrane markers (STRO1, CD44), making a biocomplex using Pura Matrix as a scaffold, and it was incubated for four days and transplanted into 30 adult immunosuppressed male Wistar rats. They were evaluated at 6 days, 10 days and 2 months, obtaining histological sections stained with hematoxylin and eosin. Cell cultures were positive for specific membrane markers, showing evident deviations in morphology under phase contrast microscope. Differentiation and organization were noted at 10 days, while the constructs at 2 months showed a clear difference in morphology, organization and cell type. It was possible to obtain a three-dimensional tissue construct from human dental ectomesenchymal cells achieving a degree of tissue organization that corresponds to the presence of cellular stratification and extracellular matrix.

BS I-B4 isolectin as a probe for an investigation of membrane alterations and transformation phenotypes of mouse L cells

1981 ◽  
Vol 50 (1) ◽  
pp. 79-88
Author(s):  
W.S. Stanley ◽  
E.H. Chu
Keyword(s):  
Cell Aggregation ◽  
3T3 Cells ◽  
Animal Cells ◽  
Binding Studies ◽  
L Cells ◽  
Cell Surface Structures ◽  
Variant Clone ◽  
Fluorescence Binding

BS I-B4, an alpha-D-galactopyranosyl-binding isolectin from Bandeiraea simplicifolia seeds, was found to interact differently with transformed mouse L cells and non-transformed mouse 3T3 cells. The lectin induces detachment of 3T3 cells but increases adhesiveness and clustering of L cells. However, the induced cell aggregation does not lead to cell fusion. A variant clone of L cells, resistant to BS I-B4, which had lost the capacity for agglutination in the presence of the lectin, was isolated. Fluorescence binding studies of this variant suggest a lesion involving alpha and beta-D-galactopyranosyl units on its cell-surface structures. Although the variant cells form colonies in a methylcellulose medium, they do not produce tumours, as do the parental cells, when transplanted in athymic nude mice. The results demonstrate that alterations in cell membrane glyco-conjugates play an important role in tumourigenesis of animal cells, but anchorage-independent growth in vitro, as one of the transformation phenotypes, cannot be correlated absolutely with tumourigenicity in vivo.

scholarly journals Granulocyte aggregometry: a sensitive technique for the detection of C5a and complement activation

Blood ◽  
1980 ◽  
Vol 55 (6) ◽  
pp. 898-902 ◽  
Author(s):  
DE Hammerschmidt ◽  
TK Bowers ◽  
CJ Lammi-Keefe ◽  
HS Jacob ◽  
PR Craddock
Keyword(s):  
Lupus Erythematosus ◽  
Cytochalasin B ◽  
Normal Subjects ◽  
Standard Methods ◽  
Sensitive Technique ◽  
Fresh Serum ◽  
Induced Aggregation ◽  
Transfusion Reactions

Abstract We have previously shown that complement (C) activated plasma causes granulocyte (PMN) aggregation in vitro and that C5a is responsible. The C-induced aggregation of PMNs treated with cytochalasin-B (CB) is markedly enhanced and irreversible, and the magnitude of the response is proportional to the log (concentration of activated plasma), allowing use of this technique to detect C5a and hence C-activation. To compare the sensitivity of granulocyte aggregometry to that of more standard methods of detecting C-activation, we produced graded C- activation in vitro by treating fresh serum with varying amounts of zymosan. Aggregometry was the most sensitive index of C-activation, detecting C-activation, produced by 0.02 mg zymosan/ml of serum--1/10 that required to produce C-activation detectable by C3 immunoelectrophoresis (the next most sensitive technique). Granulocyte aggregometry may also be used to detect in vivo C-activation. We have found aggregating activity in plasmas from patients with systemic lupus erythematosus, immune vasculitis, transfusion reactions, and other conditions associated with in vivo C-activation, but not in the plasmas of normal subjects.

Degranulation enhances release of a stable contractile factor from rabbit polymorphonuclear leukocytes

1998 ◽  
Vol 274 (5) ◽  
pp. H1545-H1551
Author(s):  
Justin R. Hamilton ◽  
Joanne L. Hart ◽  
Owen L. Woodman
Keyword(s):  
Slow Rate ◽  
Polymorphonuclear Leukocytes ◽  
Cytochalasin B ◽  
Contractile Activity ◽  
90 C 10 ◽  
Isolated Aorta ◽  
Aortic Contraction ◽  
Factor Release

We investigated the release of a stable contractile factor(s) from rabbit isolated polymorphonuclear leukocytes (PMNs; 108cells/ml) incubated in Tyrode buffer at 37°C. PMNs were untreated, stimulated with N-formylmethionyl-leucyl-phenylalanine (FMLP; 0.1 μM), or degranulated with cytochalasin B (1 μM) in combination with FMLP (0.1 μM). Products from unstimulated PMNs incubated for 60 min caused significantly greater contraction of rabbit isolated aorta (0.56 ± 0.12 g, n = 8) than did products released from PMNs during a 5-min incubation (0.32 ± 0.07 g, n = 11, P < 0.05). Stimulation alone did not affect contractile factor release; however, products released from degranulated PMNs caused significantly greater aortic contraction (0.48 ± 0.08 g, n = 5) than products from nondegranulated PMNs (0.24 ± 0.04 g, n = 5, P < 0.05) after a 5-min incubation. The contractile activity of PMN-derived products was virtually abolished by heat (90°C, 10 min) or protease (trypsin; 166 U/ml, 5 h) treatment. These findings suggest a PMN-derived protein vasoconstrictor(s) is spontaneously released at a slow rate in vitro and that degranulation can enhance this rate of release. Because PMN degranulation in vivo is associated with inflammation, these results support suggestions that PMN-derived contractile factors may contribute to the impaired blood flow observed during postischemic reperfusion.

scholarly journals Control of Wheat Fusarium Head Blight by Heat-Stable Antifungal Factor (HSAF) from Lysobacter enzymogenes

Plant Disease ◽  
2019 ◽  
Vol 103 (6) ◽  
pp. 1286-1292 ◽  
Author(s):  
Yangyang Zhao ◽  
Chao Cheng ◽  
Tianping Jiang ◽  
Huiyong Xu ◽  
Yun Chen ◽  
...  
Keyword(s):  
Fusarium Head Blight ◽  
Fungal Pathogens ◽  
Ultrastructural Changes ◽  
Wall Thickening ◽  
Head Blight ◽  
Lysobacter Enzymogenes ◽  
Heat Stable ◽  
Wide Range

Heat-stable antifungal factor (HSAF), which belongs to the polycyclic tetramate macrolactam family, was isolated from Lysobacter enzymogenes fermentations and exhibited inhibitory activities against a wide range of fungal pathogens. In this study, the antifungal activity of HSAF against Fusarium graminearum in vitro and in vivo was investigated. A total of 50% of mycelial growth of F. graminearum was suppressed with 4.1 μg/ml of HSAF (EC50 value). HSAF treatment resulted in abnormal morphology of the hyphae, such as curling, apical swelling, and depolarized growth. Furthermore, HSAF adequately inhibited conidial germination and conidiation of F. graminearum with an inhibition rate of 100% when 1 and 6 μg/ml of HSAF were applied, respectively. HSAF caused ultrastructural changes of F. graminearum, including cell wall thickening and plasmolysis. Moreover, the application of HSAF significantly controlled Fusarium head blight in wheat caused by F. graminearum in the field. Overall, these results indicate that HSAF has potential for development as a fungicide against F. graminearum.

Effects of colchicine or cytochalasin B on pulmonary macrophage endocytosis in vivo

1981 ◽  
Vol 50 (3) ◽  
pp. 621-629 ◽  
Author(s):  
P. A. Valberg ◽  
J. D. Brain ◽  
D. Kane
Keyword(s):  
Colloidal Gold ◽  
Cytochalasin B ◽  
Cell Function ◽  
Isotonic Saline ◽  
Reticuloendothelial System ◽  
Carrier Fluid ◽  
Particle Uptake ◽  
Pulmonary Macrophages

Pulmonary macrophages defend lung surfaces by ingesting deposited particles. We investigated to what extent this uptake of particles can be modulated in vivo by two drugs, colchicine and cytochalasin B (CytB). 198Au colloidal gold, in isotonic saline carrier fluid, was intratracheally instilled into male Syrian golden hamsters. The uptake of these particles by pulmonary macrophages was measured when the carrier fluid contained only colloidal gold and when this test particle was combined with graded doses of either drug. We found that macrophage uptake of the particles 1 h after instillation was depressed 37% when colchicine was added to the instillate (150 micrograms/100 g BW). Depression of particle uptake was also seen with CytB at 15 micrograms/100 g BW. Experiments with tritiated colchicine and CytB showed that both drugs were rapidly cleared from the lungs early in the 1 h phagocytic period. The effect of intravenous colchicine and CytB on the clearance of intravenously injected gold colloid by the liver and spleen reticuloendothelial system was negligible. The results of these experiments, in conjunction with in vitro effects of colchicine and CytB, provide insight into the components of cell function active in particle uptake in situ.

scholarly journals Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage

2020 ◽  
Vol 48 (14) ◽  
pp. 7609-7622 ◽  
Author(s):  
Cassandra K Hayne ◽  
Casey A Schmidt ◽  
Maira I Haque ◽  
A Gregory Matera ◽  
Robin E Stanley
Keyword(s):  
Animal Cells ◽  
Trna Splicing ◽  
Polynucleotide Kinase ◽  
Genes Encoding ◽  
Intron Removal ◽  
And Function ◽  
Insight Into ◽  
Negative Modulator

Abstract The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34 and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

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