scholarly journals Evidence that myosin does not contribute to force production in chromosome movement.

1982 ◽  
Vol 94 (1) ◽  
pp. 165-178 ◽  
Author(s):  
D P Kiehart ◽  
I Mabuchi ◽  
S Inoué
Keyword(s):  
Gamma Globulin ◽  
Meiotic Chromosome ◽  
Force Production ◽  
Chromosome Movement ◽  
Actomyosin Interaction ◽  
Dividing Cells ◽  
Spindle Elongation ◽  
Chromosome Movements

Antibody against cytoplasmic myosin, when microinjected into actively dividing cells, provides a physiological test for the role of actin and myosin in chromosome movement. Anti-Asterias egg myosin, characterized by Mabuchi and Okuno (1977, J. Cell Biol., 74:251), completely and specifically inhibits the actin activated Mg++ -ATPase of myosin in vitro and, when microinjected, inhibits cytokinesis in vivo. Here, we demonstrate that microinjected antibody has no observable effect on the rate or extent of anaphase chromosome movements. Neither central spindle elongation nor chromosomal fiber shortening is affected by doses up to eightfold higher than those require to uniformly inhibit cytokinesis in all injected cells. We calculate that such doses are sufficient to completely inhibit myosin ATPase activity in these cells. Cells injected with buffer alone, with myosin-absorbed antibody, or with nonimmune gamma-globulin, proceed normally through both mitosis and cytokinesis. Control gamma-globulin, labeled with fluorescein, diffuses to homogeneity throughout the cytoplasm in 2-4 min and remains uniformly distributed. Antibody is not excluded from the spindle region. Prometaphase chromosome movements, fertilization, pronuclear migration, and pronuclear fusion are also unaffected by microinjected antimyosin. These experiments demonstrate that antimyosin blocks the actomyosin interaction thought to be responsible for force production in cytokinesis but has no effect on mitotic or meiotic chromosome motion. They provide direct physiological evidence that myosin is not involved in force production for chromosome movement.

scholarly journals Chromosome movement in lysed mitotic cells is inhibited by vanadate.

1978 ◽  
Vol 79 (2) ◽  
pp. 573-580 ◽  
Author(s):  
W Z Cande ◽  
S M Wolniak
Keyword(s):  
Atpase Activity ◽  
Metabolic Inhibitors ◽  
Chromosome Movement ◽  
Microtubule Polymerization ◽  
Spindle Poles ◽  
Brij 58 ◽  
Spindle Elongation ◽  
Chromosome Movements

Mitotic PtK1 cells, lysed at anaphase into a carbowax 20 M Brij 58 solution, continue to move chromosomes toward the spindle poles and to move the spindle poles apart at 50% in vivo rates for 10 min. Chromosome movements can be blocked by adding metabolic inhibitors to the lysis medium and inhibition of movement can be reversed by adding ATP to the medium. Vanadate at micromolar levels reversibly inhibits dynein ATPase activity and movement of demembranated flagella and cilia. It does not affect glycerinated myofibril contraction or myosin ATPase activty at less than millimolar concentrations. Vanadate at 10--100 micron reversibly inhibits anaphase movement of chromosomes and spindle elongation. After lysis in vanadate, spindles lose their fusiform appearance and become more barrel shaped. In vitro microtubule polymerization is insensitive to vanadate.

The kinesin-like protein CENP-E is kinetochore-associated throughout poleward chromosome segregation during anaphase-A

1996 ◽  
Vol 109 (5) ◽  
pp. 961-969 ◽  
Author(s):  
K.D. Brown ◽  
K.W. Wood ◽  
D.W. Cleveland
Keyword(s):  
Chromosome Segregation ◽  
Chromosome Movement ◽  
Initial Alignment ◽  
Late Prophase ◽  
Nuclear Envelope Breakdown ◽  
Anaphase Onset ◽  
Spindle Elongation ◽  
Anaphase B

The kinesin-like protein CENP-E transiently associates with kinetochores following nuclear envelope breakdown in late prophase, remains bound throughout metaphase, but sometime after anaphase onset it releases and by telophase becomes bound to interzonal microtubules of the mitotic spindle. Inhibition of poleward chromosome movement in vitro by CENP-E antibodies and association of CENP-E with minus-end directed microtubule motility in vitro have combined to suggest a key role for CENP-E as an anaphase chromosome motor. For this to be plausible in vivo depends on whether CENP-E remains kinetochore associated during anaphase. Using Indian muntjac cells whose seven chromosomes have large, easily tracked kinetochores, we now show that CENP-E is kinetochore-associated throughout the entirety of anaphase-A (poleward chromosome movement), relocating gradually during spindle elongation (anaphase-B) to the interzonal microtubules. These observations support roles for CENP-E not only in the initial alignment of chromosomes at metaphase and in spindle elongation in anaphase-B, but also in poleward chromosome movement in anaphase-A.

scholarly journals Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1288
Author(s):  
Wendy Dong ◽  
Boris Kantor
Keyword(s):  
Large Scale ◽  
Lentiviral Vector ◽  
Clinical Applications ◽  
Scale Production ◽  
Base Editing ◽  
Epigenome Editing ◽  
Vector Systems ◽  
Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

Theophylline minimally alters contractile properties of canine diaphragm in vitro

1990 ◽  
Vol 69 (4) ◽  
pp. 1390-1396 ◽  
Author(s):  
E. Derom ◽  
S. Janssens ◽  
V. De Bock ◽  
M. Decramer
Keyword(s):  
High Frequency ◽  
Force Production ◽  
Contractile Properties ◽  
Diaphragm Muscle ◽  
Muscle Bundle ◽  
Time To Peak ◽  
Diaphragm In Vitro ◽  
Tetanic Tension

We examined the effects of theophylline on contractile properties and high-frequency fatigue of canine diaphragm in vitro. Eighteen diaphragm muscle bundles were obtained from 10 anesthetized dogs and equilibrated in oxygenated Krebs solution to 100, 200, or 300 mg/l theophylline. These bundles were compared with 18 matched control bundles from the contralateral hemidiaphragm. No statistically significant differences in twitch tension, tetanic tension, twitch-to-tetanus ratio, time to peak tension, or half-relaxation time were observed. Concentrations of 300 mg/l theophylline, however, significantly (P less than 0.05) increased force production at 10 Hz by 32%. A similar tendency was present at lower concentrations and exhibited a clear dose-response behavior. High-frequency fatigue was similar in control and theophylline-treated bundles. We conclude that supratherapeutic in vitro concentrations of theophylline do not increase maximal tetanic tension and do not protect against muscle fatigue but potentiate relative force production at low stimulation frequencies. This relatively small effect cannot be explained by poor diffusion of the drug in the muscle bundle, because theophylline concentrations in the muscle bath and in the muscle bundle were virtually identical. Moreover, it remains unclear whether this potentially beneficial effect can be achieved at in vivo attainable serum concentrations.

scholarly journals Studies of the antibody-dependent killing of schistosomula of Schistosoma mansoni employing haptenic target antigens. I. Evidence that the loss in susceptibility to immune damage undergone by developing schistosomula involves a change unrelated to the masking of parasite antigens by host molecules.

1980 ◽  
Vol 152 (1) ◽  
pp. 41-53 ◽  
Author(s):  
G Moser ◽  
D L Wassom ◽  
A Sher
Keyword(s):  
Structural Change ◽  
Schistosoma Mansoni ◽  
Immune Evasion ◽  
Gamma Globulin ◽  
Morphological Examination ◽  
Host Molecules ◽  
Minimal Loss ◽  
Effector Mechanisms

A method was developed for coupling a hapten, trinitrophenyl (TNP), to the surface of schistosomula of Schistosoma mansoni which results in a minimal loss in their viability as judged by morphological examination in vitro and survival after injection in vivo. Skin-stage (3-h-old) and lung-stage (5-d-old) schistosomula surface labeled in this manner were then compared for their susceptibility to killing by anti-TNP antibody-dependent effector mechanisms both in vivo and in vitro. TNP skin-stage larvae were readily rejected in mice actively immunized against TNP bovine gamma globulin and were highly susceptible to anti-TNP-dependent killing mediated either by complement or purified human eosinophils in vitro. In contrast, TNP-lung-stage schistosomula, which were shown by microfluorimetry to bind anti-TNP antibody to approximately the same extent as skin-stage schistosomula, were found to be resistant to killing by the same in vivo and in vitro mechanisms. These findings suggest that the insusceptibility of postskin-stage schistosomula to antibody-dependent killing must result at least in part from an intrinsic structural change in the integument of the parasite and cannot be caused solely by the masking of parasite antigens by acquired host molecules, a mechanism of immune evasion previously proposed for schistosomes.

scholarly journals Protein - Quinone Interaction: In Vitro Induction of Indirect Antiglobulin Reactions With Methyldopa

Blood ◽  
1974 ◽  
Vol 43 (1) ◽  
pp. 85-97 ◽  
Author(s):  
Arlan J. Gottlieb ◽  
Harold A. Wurzel
Keyword(s):  
Gamma Globulin ◽  
Red Cell ◽  
Prolonged Incubation ◽  
Chemically Modified ◽  
Latex Beads ◽  
Structural Analogues ◽  
In Vitro Induction ◽  
Equilibrium Centrifugation

Abstract Methyldopa-treated gamma globulin can be demonstrated serologically on either the red cell surface or on latex beads by the indirect antiglobulin reaction. The development of a positive antiglobulin reaction was related to methyldopa concentration and the length and temperature of incubation of methyldopa with protein and could be partially inhibited by the addition of albumin to the incubation mixtures. After more prolonged incubation, antiglobulin positivity also developed with plasma-treated with methyldopa. 14C-methyldopa was covalently bound to gamma globulin. Aggregation of gamma globulin following treatment with methyldopa could be demonstrated by both sedimentation velocity and molecular weight determinations employing low-speed equilibrium centrifugation. Protein aggregation was a function of time, temperature, and methyldopa concentration. Detectability by the antiglobulin reaction, the darkening noted in solutions to which methyldopa or hydroquinone had been added, as well as the aggregation of protein was inhibited by a reducing agent which prevented formation of a quinone from the hydroquinone. Some of the immunologically atypical features of the sensitization of red cells by methyldopa or its structural analogues are explicable by the adherence, in vivo, of chemically modified, nonantibody gamma globulin which renders the red cell directly antiglobulin positive.

scholarly journals Cooperation between PDGF and FGF converts slowly dividing O-2Aadult progenitor cells to rapidly dividing cells with characteristics of O-2Aperinatal progenitor cells.

1992 ◽  
Vol 118 (4) ◽  
pp. 889-900 ◽  
Author(s):  
G Wolswijk ◽  
M Noble
Keyword(s):  
Progenitor Cells ◽  
Adult Brain ◽  
High Rate ◽  
Adult Rat ◽  
Large Numbers ◽  
Dividing Cells ◽  
Rapid Generation ◽  
And Migration

We have shown previously that oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells isolated from adult rat optic nerves can be distinguished in vitro from their perinatal counterparts on the basis of their much slower rates of division, differentiation, and migration when grown in the presence of cortical astrocytes or PDGF. This behavior is consistent with in vivo observations that there is only a modest production of oligodendrocytes in the adult CNS. As such a behavior is inconsistent with the likely need for a rapid generation of oligodendrocytes following demyelinating damage to the mature CNS, we have been concerned with identifying in vitro conditions that allow O-2Aadult progenitor cells to generate rapidly large numbers of progeny cells. We now provide evidence that many slowly dividing O-2Aadult progenitor cells can be converted to rapidly dividing cells by exposing adult optic nerve cultures to both PDGF and bFGF. In addition, these O-2Aadult progenitor cells appear to acquire other properties of O-2Aperinatal progenitor cells, such as bipolar morphology and high rate of migration. Although many O-2Aadult progenitor cells in cultures exposed to bFGF alone also divide rapidly, these cells are multipolar and migrate little in vitro. Oligodendrocytic differentiation of O-2Aadult progenitor cells, which express receptors for bFGF in vitro, is almost completely inhibited in cultures exposed to bFGF or bFGF plus PDGF. As bFGF and PDGF appear to be upregulated and/or released after injury to the adult brain, this particular in vitro response of O-2Aadult progenitor cells to PDGF and bFGF may be of importance in the generation of large numbers of new oligodendrocytes in vivo following demyelination.

scholarly journals Diamond Nanoparticles Downregulate Expression of CycD and CycE in Glioma Cells

Molecules ◽  
2019 ◽  
Vol 24 (8) ◽  
pp. 1549 ◽  
Author(s):  
Marta Grodzik ◽  
Jaroslaw Szczepaniak ◽  
Barbara Strojny-Cieslak ◽  
Anna Hotowy ◽  
Mateusz Wierzbicki ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Glioma Cells ◽  
Migration And Invasion ◽  
Control Group ◽  
Ki 67 ◽  
Normal Cells ◽  
Diamond Nanoparticles ◽  
Dividing Cells

Our previous studies have shown that diamond nanoparticles (NDs) exhibited antiangiogenic and proapoptotic properties in vitro in glioblastoma multiforme (GBM) cells and in tumors in vivo. Moreover, NDs inhibited adhesion, leading to the suppression of migration and invasion of GBM. In the present study, we hypothesized that the NDs might also inhibit proliferation and cell cycle in glioma cells. Experiments were performed in vitro with the U87 and U118 lines of GBM cells, and for comparison, the Hs5 line of stromal cells (normal cells) after 24 h and 72 h of treatment. The analyses included cell morphology, cell death, viability, and cell cycle analysis, double timing assay, and gene expression (Rb, E2F1, CycA, CycB, CycD, CycE, PTEN, Ki-67). After 72 h of ND treatment, the expression level of Rb, CycD, and CycE in the U118 cells, and E2F1, CycD, and CycE in the U87 cells were significantly lower in comparison to those in the control group. We observed that decreased expression of cyclins inhibited the G1/S phase transition, arresting the cell cycle in the G0/G1 phase in glioma cells. The NDs did not affect the cell cycle as well as PTEN and Ki-67 expression in normal cells (Hs5), although it can be assumed that the NDs reduced proliferation and altered the cell cycle in fast dividing cells.

scholarly journals Some Interrelationships of Blood and the Fava Bean Principle in Vitro

Blood ◽  
1952 ◽  
Vol 7 (7) ◽  
pp. 721-728 ◽  
Author(s):  
WILLIAM P. CREGER ◽  
HOUGHTON GIFFORD
Keyword(s):  
Animal Species ◽  
Gamma Globulin ◽  
Test Tube ◽  
Red Cells ◽  
Red Cell ◽  
Fava Bean ◽  
Increased Susceptibility ◽  
Human Red Cells

Abstract 1. Saline suspensions of human red cells, as well as those of several animal species, were agglutinated by normal saline extracts of the Fava bean. 2. This agglutination was potentiated in titer 100-fold in a medium of 10 per cent acacia, as a diluent. 3. The inhibition of the hemagglutination action of the Fava bean extract by human serum was apparently attributable to the gamma globulin fraction. 4. The Fava bean principle could be transferred from cell to cell, as shown by heat-elution and acacia technics. 5. Fava-sensitized red cells did not exhibit increased susceptibility in the test tube to complement, hemolysin, or osmotic or mechanical fragility. 6. The mechanism of in vivo red cell destruction in Favism is as yet unknown, but a special immunologic susceptibility to the action of the bean’s principle is suspected in certain persons. 7. It is suggested that the relation of acacia to Fava-sensitized red cells may form the basis of a diagnostic test for Favism in the early, acute stages of the disease.

The influence of respiratory acid-base changes on muscle performance and excitability of the sarcolemma during strenuous intermittent hand grip exercise

2012 ◽  
Vol 112 (4) ◽  
pp. 571-579 ◽  
Author(s):  
M. Hilbert ◽  
V. Shushakov ◽  
N. Maassen
Keyword(s):  
Force Production ◽  
Respiratory Acidosis ◽  
Respiratory Alkalosis ◽  
Muscle Performance ◽  
Protective Effects ◽  
Acid Base ◽  
M Wave ◽  
Hand Grip

Acidification has been reported to provide protective effects on force production in vitro. Thus, in this study, we tested if respiratory acid-base changes influence muscle function and excitability in vivo. Nine subjects performed strenuous, intermittent hand grip exercises (10 cycles of 15 s of work/45 s of rest) under respiratory acidosis by CO2 rebreathing, alkalosis by hyperventilation, or control. The Pco2, pH, K+ concentration ([K+]), and Na+ concentration were measured in venous and arterialized blood. Compound action potentials (M-wave) were elicited to examine the excitability of the sarcolemma. The surface electromyogram (EMG) was recorded to estimate the central drive to the muscle. The lowest venous pH during the exercise period was 7.24 ± 0.03 in controls, 7.31 ± 0.05 with alkalosis, and 7.17 ± 0.04 with acidosis ( P < 0.001). The venous [K+] rose to similar maximum values in all conditions (6.2 ± 0.8 mmol/l). The acidification reduced the decline in contraction speed ( P < 0.001) but decreased the M-wave area to 73.4 ± 19.8% ( P < 0.001) of the initial value. After the first exercise cycle, the M-wave area was smaller with acidosis than with alkalosis, and, after the second cycle, it was smaller with acidosis than with the control condition ( P < 0.001). The duration of the M-wave was not affected. Acidification diminished the reduction in performance, although the M-wave area during exercise was decreased. Respiratory alkalosis stabilized the M-wave area without influencing performance. Thus, we did not find a direct link between performance and alteration of excitability of the sarcolemma due to changes in pH in vivo.

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